WO2002015701A2

WO2002015701A2 - Bacillus thuringiensis crystal protein hybrids

Info

Publication number: WO2002015701A2
Application number: PCT/EP2001/009751
Authority: WO
Inventors: Nadine Barbara Carozzi; Scott M. Rabe; Paul J. Miles; Gregory Wayne Warren; Petrus Theodorus De Haan
Original assignee: Syngenta Participations Ag
Priority date: 2000-08-25
Filing date: 2001-08-23
Publication date: 2002-02-28
Also published as: AR030576A1; KR100581163B1; KR20030029858A; WO2002015701A3; DE60111236T2; ATE296539T1; DE60111236D1; AU8590001A; AU2001285900B2; ES2243543T3; EP1311162A2; BR0113500A; CN100353846C; CA2419029A1; JP2004506432A; EP1311162B1; CN1449250A

Abstract

Synthetic nucleotide sequences optimized for expression in plants encode varying forms of the hybrid Bacillus thuringiensis delta-endotoxin H04, the toxin portion of which contains domains I and II of Cry1Ab and domain III of Cry1C. Compositions and formulations containing the insecticidal toxins are capable of controlling insect pests. The invention is further drawn to methods of making the hybrid toxins and to methods of using the nucleotide sequences, for example in microorganisms to control insect pests and in trenasgenic plants to confer insect resistance.

Description

Novel insecticidal toxins derived from Bacillus thuringiensis insecticidal crystal proteins

The invention relates to novel insecticidal toxins derived from Bacillus thuringiensis insecticidal crystal proteins, nucleic acid sequences whose expression results in said toxins, and methods of making and methods of using the toxins and corresponding nucleic acid sequences to control insects.

Insect pests are a major cause of crop losses. Solely in the US, billions of doUars are lost every year due to infestation by various genera of insects. In addition to losses in field crops, insect pests are also a burden to vegetable and fruit growers, to producers of ornamental flowers, and they are a nuisance to gardeners and homeowners. Insect pests are mainly controUed by intensive appUcations of chemical insecticides, which are active through inhibition of insect growth, prevention of insect feeding or reproduction, or death of the insects. Good insect control can thus be reached, but these chemicals can sometimes also affect other, beneficial insects. Another problem resulting from the wide use of chemical pesticides is the appearance of resistant insect varieties. This has been partiaUy aUeviated by various resistance management strategies, but there is an increasing need for alternative pest control agents.

Biological insect control agents, such as Bacillus thuringiensis strains expressing insecticidal toxins have also been appUed with satisfactory results, offering an alternative or a complement to chemical insecticides. Bacillus thuringiensis belongs to the large group of gram-positive, aerobic, endospore-forming bacteria. Unlike other very closely related species of Bacillus such as B. cereus or B. anthracis, the majority of the hitherto known Bacillus thuringiensis species produce in the course of their sporulation a parasporal inclusion body which, on account of its crystalline structure, is generaUy referred to also as a crystalline body. This crystalline body is composed of insecticidaUy active crystalline protoxin proteins, the so- caUed δ-endotoxins. These protein crystals are responsible for the toxicity to insects of Bacillus thuringiensis. The δ-endotoxin does not exhibit its insecticidal activity until after oral intake of the crystalline body, when the latter is dissolved in the intestinal juice of the target insects. In most cases the actual toxic component is released from the protoxin as a result of proteolytic cleavage caused by the action of proteases from the digestive tract of the insects. The δ-endotoxins of the various Bacillus thuringiensis strains are characterized by high specificity with respect to certain target insects, especiaUy with respect to various Lepidoptera, Coleoptera and Diptera larvae, and by a high degree of activity against these larvae. A further advantage in using δ-endotoxins of Bacillus thuringiensis resides in the fact that the toxins are harmless to humans, other mammals, birds and fish.

Based on sequence homology and insecticidal specificity, Bacillus thuringiensis crystal proteins have been categorized into different classes. Best studied are the Cryl class of proteins, which are produced as 140 kDa pro-toxins and are active towards lepidopterans. To some extent the mode of action of crystal proteins has been elucidated. After oral uptake the crystals dissolve in the alkaline environment of the larval midgut. The solubiUzed proteins are subsequently processed by midgut proteinases (e.g. trypsin) to a proteinase-resistant toxic fragment of about 65kDa that binds to receptors on epitheUal ceUs of the insect midgut and penetrates the ceU membrane. This eventuaUy leads to bursting of the ceUs and death of the larvae. The activity spectrum of a particular crystal protein is to a large extent determined by the occurrence of receptors on the midgut epitheUal ceUs of susceptible insects. The spectrum is co-determined by the efficiency of solubiUzation of the crystal protein and its proteolytic activation in vivo. The importance of the binding of the crystal protein to midgut epitheUal receptors is further demonstrated where insects have developed resistance to one of the crystal proteins in that the binding of crystal proteins to midgut epitheUal ceUs in resistant insects is significantly reduced.

In the past several years, the genes coding for some of these crystal proteins have been isolated and their expression in heterologous hosts have been shown to provide another tool for the control of economicaUy important insect pests. In particular, the expression of insecticidal toxins in transgenic plants, such as Bacillus thuringiensis crystal proteins, has provided efficient protection against selected insect pests, and transgenic plants expressing such toxins have been commercialized, aUowing farmers to reduce appUcations of chemical insect control agents. Furthermore, it is also possible to express recombinant toxins that have a chosen combination of functions designed to enhance the degree of insecticidal activity against a particular insect or insect class, or to expand the spectrum of insects against which the toxin protein is active. For example, chimeric insecticidal proteins can be constructed having novel sequences not found in nature by combining the toxin portion from one δ-endotoxin with the protoxin (taU) portion of a different δ-endotoxin. See, for example, WO 98/15170, incorporated herein by reference. Toxic fragments of crystal proteins are thought to be composed of three distinct structural domains. Domain I, the most N-terminal domain, consists of 7 a-heUces and probably is partiaUy or entirely inserted in the target ceU membrane. Domain II comprises 3 β- sheets in a so-caUed Greek key-conformation. Domain II is beUeved by most researchers to interact with receptors and to thereby determine toxin specificity. Indeed, there is much evidence impUcating domain JJ residues in specific toxicity and in high affinity binding. Domain III, the most C-terminal domain, consists of two β-sheets in a so-caUed jeUyroll conformation and has also been impUcated in determining specificity. Swapping domain III between toxins, such as by in vivo recombination between the coding regions, can result in changes in specific activity. Binding experiments using such hybrids have shown that domain HI is involved in binding to putative receptors of target insects, suggesting that domain III may exert its role in specificity through a role in receptor recognition. If projected on Cryl sequences, domain I runs from about amino acid residue 28 to 260, domain II from about 260 to 460 and domain III from about 460 to 600. See, Nakamura et al, Agric. Biol. Chem. 54(3): 715-724 (1990); Li et al, Nature 353: 815-821 (1991); Ge et al, J. Biol. Chem. 266(27): 17954-17958 (1991); and Honee et al, Mol. Microbiol. 5(11): 2799-2806 (1991); each of which are incorporated herein by reference. U.S. Pat. No. 5,736,131, incorporated herein by reference describes Bacillus thuringiensis hybrid toxin fragments comprising at their C-terminus domain DI of a first Cry protein and at its N-terminus domains I and II of a second Cry protein. Such hybrid crystal proteins have altered insecticidal specificity. For example, the H04 hybrid toxin, which is also described in De Maagd et al, Appl Environ. Microbiol. 62(5): 1537-1543 (1996), comprises at its N-terrninus domains I and II of CrylAb and at its C-terminus domain HI of CrylC. H04 is reportedly highly toxic to Spodoptera exigua (beet armyworm) compared with the parental CrylAb toxin and significantly more toxic than the CrylC parental toxin. See also, Bosch et al, FEMS Microbiology Letters 118: 129-134 (1994); Bosch et al, Bio/Technology 12: 915-918 (1994); De Maagd et al, Appl. Environ. Microbiol. 62(8): 2753-2757 (1996); and De Maagd et al, Mol. Microbiol. 31(2): 463-471 (1999); each of which is incorporated herein by reference.

Despite the previous successes realized by incorporation of insect resistant genes through breeding programs and genetic engineering, there remains a long-felt but unfulfiUed need to discover new and effective insect control agents. Particularly needed are control agents that are targeted to economicaHy important insect pests such as European Corn Borer and FaU Army Worm and that efficiently control insect species resistant to existing insect control agents. Furthermore, agents whose appUcation mmimizes the burden on the environment are desirable.

The present invention addresses the aforementioned needs by providing novel gene sequences that encode hybrid Bacillus thuringiensis toxins, including synthetic nucleotide sequences optimized for expression in plants. In preferred embodiments, the novel gene sequences encode varying forms of the hybrid Bacillus thuringiensis delta-endotoxin H04, the toxin portion of which contains domains I and II of CrylAb and domain III of CrylC. The hybrid Bacillus thuringiensis toxins encoded by the novel gene sequences are highly active against economicaUy important insect pests such as faU armyworm, pink boUworm, tobacco budworm, European cornborer, and diamondback moth. The hybrid Bacillus thuringiensis toxins can be used in multiple insect control strategies, resulting in maximal efficiency with minimal impact on the environment.

The invention is further drawn to the hybrid insecticidal toxins resulting from the expression of the nucleotide sequences of the invention, and to compositions and formulations containing the hybrid insecticidal toxins, which are capable of inhibiting the abiUty of insect pests to survive, grow or reproduce, or of limiting insect-related damage or loss in crop plants. The invention is further drawn to a method of making the hybrid toxins and to methods of using the nucleotide sequences, for example in transgenic plants to confer insect resistance, and to methods of using the toxins, and compositions and formulations comprising the toxins, for example applying the toxins, composition, or formulation to insect infested areas, or to prophylacticaUy treat insect susceptible areas or plants to confer protection or resistance against harmful insects. The hybrid toxins can be used in multiple insect control strategies, resulting in maximal efficiency with minimal impact on the environment. According to one aspect, the present invention provides a method for controlling an insect selected from the group consisting of faU armyworm, pink boUworm, tobacco budworm, European cornborer and diamondback moth, comprising deUvering to the insect an effective amount of a hybrid Bacillus thuringiensis toxin comprising domains I and II from a CrylAb toxin joined in the amino to carboxy direction to domain III from a CrylC toxin. In a preferred embodiment, the hybrid Bacillus thuringiensis toxin comprises an amino acid sequence at least 90% identical to SEQ JD NO:2, 4, 6, 8, or 10. In a more preferred embodiment, the hybrid Bacillus thuringiensis toxin comprises SEQ ID NO:2, 4, 6, 8, or 10.

In another embodiment of the above-described method of the invention, the hybrid Bacillus thuringiensis toxin further comprises a C-terminal tail region, such as a CrylC taU region or a CrylAb tail region. The C-terminal tail region may be fuU-length or may be truncated, such as to approximately 40 amino acids in length.

In a preferred embodiment of the above-described method of the invention, deUvering an effective amount of the hybrid Bacillus thuringiensis toxin to the insect comprises feeding or contacting the insect with transgenic plant tissue transformed with recombinant DNA comprising a nucleotide sequence that encodes the hybrid Bacillus thuringiensis toxin, wherein expression of the hybrid Bacillus thuringiensis toxin in said transgenic plant tissue confers resistance to the insect. Preferably, said nucleotide sequence is substantiaUy identical to SEQ ID NO:l, 3, 5, 7, or . According to another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence that encodes a hybrid Bacillus thuringiensis toxin comprising: (a) an N-terminal toxin portion comprising domains I and II from a CrylAb toxin joined in the amino to carboxy direction to domain III from a CrylC toxin; and (b) a C- terminal tail region from a CrylAb toxin. Preferably, the hybrid Bacillus thuringiensis toxin comprises an amino acid sequence at least 90% identical to SEQ ID NO: 6, 8, or 10. More preferably, the hybrid Bacillus thuringiensis toxin comprises SEQ ID NO: 6, 8, or 10. Even more preferably, said nucleotide sequence is at least 90% identical to SEQ ID NO:5, 7, or 9. Most preferably, said nucleotide sequence comprises SEQ ID NO: 5, 7, or 9.

The present invention further provides a chimeric gene comprising a heterologous promoter sequence operatively Unked to a nucleic acid molecule of the invention, as described above; a recombinant vector comprising such a chimeric gene; a transgenic host ceU (e.g., a plant ceU) comprising such a chimeric gene; a transgenic plant (e.g., a maize, cotton, rice, or cabbage plant) comprising such a transgenic plant ceU; and seed from such a transgenic plant.

According to yet another aspect, the present invention provides a method of protecting a plant against insects, comprising expressing a hybrid Bacillus thuringiensis toxin in a plant transformed with a chimeric gene comprising: (a) a nucleic acid promoter sequence that promotes in a plant the transcription of an associated coding sequence at elevated levels, and (b) a nucleic acid molecule according to the invention operatively Unked to said promoter sequence, wherein expression of the hybrid Bacillus thuringiensis toxin in said plant protects said plant against insects. According to still another aspect, the present invention provides a method of producing a hybrid Bacillus thuringiensis toxin that is active against insects, comprising: (a) obtaining a transgenic host ceU according to the invention; and (b) expressing the nucleic acid molecule of the invention in said transgenic host ceU, which results in a hybrid Bacillus thuringiensis toxin that is active against insects. According to stiU another aspect, the present invention provides a method of producing a plant resistant to insects, comprising introducing a nucleic acid molecule according to the present invention into said plant, wherein said nucleic acid molecule is expressible in said plant in an amount effective to control insects.

According to another aspect, the present invention provides an isolated nucleic acid molecule comprising SEQ ID NO:3, 5, 7, 9, 11, 12, 13, 14, 15, 16 or 17; a chimeric gene comprising a heterologous promoter sequence operatively Unked to such a nucleic acid molecules; a recombinant vector comprising such a chimeric gene; a transgenic host ceU (e.g., a plant ceU) comprising such a chimeric gene; a transgenic plant (e.g., a maize, cotton, rice, or cabbage plant) comprising such a transgenic plant ceU; and seed from such a transgenic plant. According to a still further aspect, the present invention provides a hybrid Bacillus thuringiensis toxin, comprising: (a) an N-terminal toxin portion comprising domains I and II from a CrylAb toxin joined in the amino to carboxy direction to domain III from a CrylC toxin; and (b) a C-terminal taU region from a CrylAb toxin. Preferably, the hybrid Bacillus thuringiensis toxin of the invention comprises an amino acid sequence at least 90% identical to SEQ ID NO:6, 8, or 10. More preferably, the hybrid Bacillus thuringiensis toxin of the invention comprises SEQ ID NO:6, 8, or 10. According to a further aspect, the presesnt invention provides a composition comprising the hybrid Bacillus thuringiensis toxin of the invention in an amount effective to control insects.

Other aspects and advantages of the present invention wiU become apparent to those skilled in the art from a study of the foUowing description of the invention and non-limiting examples.

BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING

SEQ ID NO: 1 shows the nucleotide sequence encoding the H04 hybrid toxin described in De Maagd et al, Appl. Environ. Microbiol. 62(5): 1537-1543 (1996), the toxin portion of which comprises at its N-terminus domains I and H of CrylAb and at its C-terminus domain HI of CrylC, plus a fuU-length CrylC tail portion.

SEQ ID NO: 2 shows the amino acid sequence of the H04 hybrid toxin encoded by the nucleotide sequence depicted in SEQ 3D NO:l, comprising toxin domains I and II of CrylAb and toxin domain HI of CrylC, plus a fuU-length CrylC tail portion.

SEQ ID NO:3 shows a synthetic nucleotide sequence encoding the toxin portion of H04 without a tail, as if the trypsin site had been cleaved.

SEQ ID NO:4 shows the amino acid sequence of the H04 toxin portion encoded by the synthetic nucleotide sequence depicted in SEQ ID NO:3. SEQ ID NO: 5 shows a synthetic nucleotide sequence encoding the toxin portion of H04 plus a fuU-length CrylAb tail portion.

SEQ ID NO:6 shows the amino acid sequence of the H04 + CrylAb tail encoded by the synthetic nucleotide sequence depicted in SEQ ID NO:5.

SEQ ID NO:7 shows another synthetic nucleotide sequence encoding the toxin portion of H04 plus a fuU-length CrylAb tail portion.

SEQ ID NO:8 shows the amino acid sequence of the H04 + CrylAb tail encoded by the synthetic nucleotide sequence depicted in SEQ JD NO:7.

SEQ ID NO: 9 shows a synthetic nucleotide sequence encoding the toxin portion of H04 plus the first 40 amino acids of the CrylAb tail. SEQ ID NO: 10 shows the amino acid sequence of the H04 + 40-amino acid truncated

CrylAb tail encoded by the synthetic nucleotide sequence depicted in SEQ ID NO:9. SEQ ID NO: 11 shows the nucleotide sequence of construct pNOV1308, which contains the constitutive maize ubiquitin promoter operatively Unked to the synthetic nucleotide sequence encoding the toxin portion of H04 without a tail, as set forth in SEQ ID NO:3.

SEQ JD NO: 12 shows the nucleotide sequence of construct pNOV1436, which contains the root-preferred maize MTL promoter operatively linked to the synthetic nucleotide sequence encoding the toxin portion of H04 plus a fuU-length CrylAb tail portion, as set forth in SEQ ID NO:5.

SEQ ID NO:13 shows the nucleotide sequence of construct pNOV1441, which contains the constitutive maize ubiquitin promoter operatively linked to the synthetic nucleotide sequence encoding the toxin portion of H04 plus a fuU-length CrylAb tail portion, as set forth in SEQ ID NO:5.

SEQ ID NO: 14 shows the nucleotide sequence of construct pNOV1305, which contains the constitutive maize ubiquitin promoter operatively linked to the synthetic nucleotide sequence encoding the toxin portion of H04 plus a fuU-length CrylAb tail portion, as set forth in SEQ ID NO:7.

SEQ ID NO:15 shows the nucleotide sequence of construct pNOV1313, which contains the constitutive maize ubiquitin promoter operatively linked to the synthetic nucleotide sequence encoding the toxin portion of H04 plus a fuU-length CrylAb tail portion, as set forth in SEQ ID NO:7. SEQ ID NO: 16 shows the nucleotide sequence of construct pNOV1435, which contains the root-preferred maize MTL promoter operatively linked to the synthetic nucleotide sequence encoding the toxin portion of H04 plus the first 40 amino acids of the CrylAb tail, as set forth in SEQ JX>NO:9.

SEQ ID NO: 17 shows the nucleotide sequence of construct pZU578, which contains the Arabidopsis actin-2 promoter operatively linked to the synthetic nucleotide sequence encoding the toxin portion of H04 plus the first 40 -vmino acids of the CrylAb tail, as set forth in SEQ ID NO:9.

DEFINITIONS

"Activity" of the toxins of the invention is meant that the toxins function as oraUy active insect control agents, have a toxic effect, or are able to disrupt or deter insect feeding, which may or may not cause death of the insect. When a toxin of the invention is deUvered to the insect, the result is typicaUy death of the insect, or the insect does not feed upon the source that makes the toxin avaUable to the insect.

"Associated with / operatively Unked" refer to two nucleic acid sequences that are related physicaUy or functionaUy. For example, a promoter or regulatory DNA sequence is said to be "associated with" a DNA sequence that codes for an RNA or a protein if the two sequences are operatively linked, or situated such that the regulator DNA sequence wiU affect the expression level of the coding or structural DNA sequence.

"Binding site" means a site on a molecule wherein the binding between site and toxin is reversible such that the Ka between site and toxin is on the order of at least 10⁴dm³mole^"1. A "chimeric gene" is a recombinant nucleic acid sequence in which a promoter or regulatory nucleic acid sequence is operatively linked to, or associated with, a nucleic acid sequence that codes for an mRNA or which is expressed as a protein, such that the regulator nucleic acid sequence is able to regulate transcription or expression of the associated nucleic acid sequence. The regulator nucleic acid sequence of the chimeric gene is not normaUy operatively Unked to the associated nucleic acid sequence as found in nature.

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

Complementary: "complementary" refers to two nucleotide sequences that comprise antiparaUel nucleotide sequences capable of pairing with one another upon formation of hydrogen bonds between the complementary base residues in the antiparaUel nucleotide sequences.

"Conservatively modified variations" of a particular nucleic acid sequence refers to those nucleic acid sequences that encode identical or essentiaUy identical amino acid sequences, or where the nucleic acid sequence does not encode an amino acid sequence, to essentiaUy identical sequences. Because of the degeneracy of the genetic code, a large number of functionaUy identical nucleic acids encode any given polypeptide. For instance the codons

CGT, CGC, CGA, CGG, AGA, and AGG aU encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded protein. Such nucleic acid variations are "silent variations" which are one species of "conservatively modified variations." Every nucleic acid sequence described herein which encodes a protein also describes every possible sUent variation, except where otherwise noted. One of skill wiU recognize that each codon in a nucleic acid (except ATG, which is ordinarily the only codon for methionine) can be modified to yield a functionaUy identical molecule by standard techniques. Accordingly, each "silent variation" of a nucleic acid which encodes a protein is impUcit in each described sequence.

Furthermore, one of skiU will recognize that individual substitutions deletions or additions that alter, add or delete a single amino acid or a smaU percentage of amino acids (typicaUy less than 5%, more typicaUy less than 1%) in an encoded sequence are "conservatively modified variations," where the alterations result in the substitution of an amino acid with a chemicaUy similar amino acid. Conservative substitution tables providing functionaUy similar amino acids are weU known in the art. The foUowing five groups each contain amino acids that are conservative substitutions for one another: AUphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Sulfur-containing: Methionine (M), Cysteine (C); Basic: Arginine (R), Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q). See also, Creighton (1984) Proteins, W.H. Freeman and Company. In addition, individual substitutions, deletions or additions which alter, add or delete a single amino acid or a smaU percentage of amino acids in an encoded sequence are also "conservatively modified variations."

To "control" insects means to inhibit, through a toxic effect, the abiUty of insect pests to survive, grow, feed, and or reproduce, or to limit insect-related damage or loss in crop plants. To "control" insects may or may not mean killing the insects, although it preferably means killing the insects. Corresponding to: in the context of the present invention, "corresponding to" or

"corresponds to" means that when the nucleic acid coding sequences or amino acid sequences of different δ-endotoxins of Bacillus thuringiensis are aUgned with each other, the nucleic or amino acids that "correspond to" certain enumerated positions are those that ahgn with these positions but that are not necessarUy in these exact numerical positions relative to the particular δ-endotoxin' s respective nucleic acid coding sequence or amino acid sequence. Likewise, when the coding or amino acid sequence of a particular δ-endotoxin (for example, CrylB) is atigned with the coding or amino acid sequence of a reference δ-endotoxin (for example, CrylAb), the nucleic acids or amino acids in the CrylB sequence that "correspond to" certain enumerated positions of the CrylAb sequence are those that aUgn with these positions of the CrylAb sequence, but are not necessarUy in these exact numerical positions of the CrylB toxin's respective nucleic acid coding sequence or amino acid sequence.

To "deUver" a toxin means that the toxin comes in contact with an insect, resulting in toxic effect and control of the insect. The toxin can be deUvered in many recognized ways, e.g., oraUy by ingestion by the insect or by contact with the insect via transgenic plant expression, formulated protein composition(s), sprayable protein composition(s), a bait matrix, or any other art-recognized toxin deUvery system.

"Expression cassette" as used herein means a nucleic acid sequence capable of directing expression of a particular nucleotide sequence in an appropriate host ceU, comprising a promoter operably Unked to the nucleotide sequence of interest which is operably linked to termination signals. It also typicaUy comprises sequences required for proper translation of the nucleotide sequence. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one which is naturaUy occurring but has been obtained in a recombinant form useful for heterologous expression. TypicaUy, however, the expression cassette is heterologous with respect to the host, i.e., the particular nucleic acid sequence of the expression cassette does not occur naturaUy in the host ceU and must have been introduced into the host ceU or an ancestor of the host ceU by a transformation event. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter which initiates transcription only when the host ceU is exposed to some particular external stimulus. In the case of a multiceUular organism, such as a plant, the promoter can also be specific to a particular tissue, or organ, or stage of development.

Gene: the term "gene" is used broadly to refer to any segment of DNA associated with a biological function. Thus, genes include coding sequences and/or the regulatory sequences required for their expression. Genes also include nonexpressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing from known or predicted sequence information, and may include sequences designed to have desired parameters.

"Gene of interest" refers to any gene which, when transferred to a plant, confers upon the plant a desired characteristic such as antibiotic resistance, virus resistance, insect resistance, disease resistance, or resistance to other pests, herbicide tolerance, improved nutritional value, improved performance in an industrial process or altered reproductive capabiUty. The "gene of interest" may also be one that is transferred to plants for the production of commerciaUy valuable enzymes or metaboUtes in the plant. As used herein, "H04" refers to the hybrid Bt toxin described in De Maagd et al., Appl.

Environ. Microbiol. 62(5): 1537-1543 (1996), the toxin fragment of which comprises at its N- terrninus domains I and H of CrylAb and at its C-terminus domain HI of CrylC.

Heterologous nucleic acid sequence: The terms "heterologous nucleic acid [or DNA] sequence", "exogenous nucleic acid [or DNA] segment" or "heterologous gene," as used herein, each refer to a sequence that originates from a source foreign to the particular host ceU or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host ceU includes a gene that is endogenous to the particular host ceU but has been modified through, for example, the use of codon optimization. The terms also includes non-naturaUy occurring multiple copies of a naturaUy occurring sequence. Thus, the terms refer to a nucleic acid segment that is foreign or heterologous to the ceU, or homologous to the ceU but in a position within the host ceU nucleic acid in which the element is not ordinarily found. Exogenous nucleic acid segments are expressed to yield exogenous polypeptides.

A "homologous" nucleic acid [or DNA] sequence is a nucleic acid [or DNA] sequence naturaUy associated with a host ceU into which it is introduced. "Homologous recombination" is the reciprocal exchange of nucleic acid fragments between homologous nucleic acid molecules.

"Homoplastidic" refers to a plant, plant tissue or plant ceU wherein aU of the plastids are geneticaUy identical. This is the normal state in a plant when the plastids have not been transformed, mutated, or otherwise geneticaUy altered. In different tissues or stages of development, the plastids may take different forms, e.g., chloroplasts, proplastids, etioplasts, amyloplasts, chromoplasts, and so forth. The terms "identical" or percent "identity" in the context of two or more nucleic acid or protein sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aUgned for maximum correspondence, as measured using one of the sequence comparison algorithms described below or by visual inspection.

"Insecticidal" is defined as a toxic biological activity capable of controlling insects, preferably by killing them.

A nucleic acid sequence is "isocoding with" a reference nucleic acid sequence when the nucleic acid sequence encodes a polypeptide having the same amino acid sequence as the polypeptide encoded by the reference nucleic acid sequence.

An "isolated" nucleic acid molecule or an isolated enzyme is a nucleic acid molecule or enzyme that, by the hand of man, exists apart from its native environment and is therefore not a product of nature. An isolated nucleic acid molecule or enzyme may exist in a purified form or may exist in a non-native environment such as, for example, a recombinant host ceU. A "juncture" between toxin domains in a hybrid toxin, i.e., between domains II and πi of a hybrid insecticidal toxin according to the invention, is the homologous crossover region or site in the hybrid. Amino acids to the left of the crossover site are from one parental toxin, whereas amino acids to the right of the crossover site are from the other parental toxin.

Mature Protein: protein that is normaUy targeted to a ceUular organeUe and from which the transit peptide has been removed.

Minimal Promoter: promoter elements, particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation. In the presence of a suitable transcription factor, the minimal promoter functions to permit transcription. Native: refers to a gene that is present in the genome of an untransformed ceU.

NaturaUy occurring: the term "naturaUy occurring" is used to describe an object that can be found in nature as distinct from being artificiaUy produced by man. For example, a protein or nucleotide sequence present in an organism (including a virus), which can be isolated from a source in nature and which has not been intentionaUy modified by man in the laboratory, is naturaUy occurring. Nucleic acid: the term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specificaUy limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have simUar binding properties as the reference nucleic acid and are metabolized in a manner simUar to naturaUy occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also impUcitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences and as weU as the sequence expUcitly indicated. SpecificaUy, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or aU) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., I. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al, Mol. Cell. Probes 8: 91-98 (1994)). The terms "nucleic acid" or "nucleic acid sequence" may also be used interchangeably with gene, cDNA, and mRNA encoded by a gene.

"ORF" means Open Reading Frame. By "part" of a protein is meant a peptide comprised by said protein and having at least 80% of the consecutive sequence thereof.

A "plant" is any plant at any stage of development, particularly a seed plant.

A "plant ceU" is a structural and physiological unit of a plant, comprising a protoplast and a ceU waU. The plant ceU may be in form of an isolated single ceU or a cultured ceU, or as a part of higher organized unit such as, for example, plant tissue, a plant organ, or a whole plant.

"Plant ceU culture" means cultures of plant units such as, for example, protoplasts, ceU culture ceUs, ceUs in plant tissues, poUen, poUen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development. "Plant material" refers to leaves, stems, roots, flowers or flower parts, fruits, poUen, egg ceUs, zygotes, seeds, cuttings, ceU or tissue cultures, or any other part or product of a plant.

A "plant organ" is a distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower bud, or embryo. "Plant tissue" as used herein means a group of plant ceUs organized into a structural and functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture and any groups of plant ceUs organized into structural and/or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as Usted above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.

A "promoter" is an untranslated DNA sequence upstream of the coding region that contains the binding site for RNA polymerase II and initiates transcription of the DNA. The promoter region may also include other elements that act as regulators of gene expression.

A "protoplast" is an isolated plant ceU without a ceU waU or with only parts of the ceU waU.

Purified: the term "purified," when appUed to a nucleic acid or protein, denotes that the nucleic acid or protein is essentiaUy free of other ceUular components with which it is associated in the natural state. It is preferably in a homogeneous state although it can be in either a dry or aqueous solution. Purity and homogeneity are typicaUy determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance Uquid chromatography. A protein which is the predominant species present in a preparation is substantiaUy purified. The term "purified" denotes that a nucleic acid or protein gives rise to essentiaUy one band in an electrophoretic gel. Particularly, it means that the nucleic acid or protein is at least about 50% pure, more preferably at least about 85% pure, and most preferably at least about 99% pure.

Two nucleic acids are "recombined" when sequences from each of the two nucleic acids are combined in a progeny nucleic acid. Two sequences are "directly" recombined when both of the nucleic acids are substrates for recombination. Two sequences are "indirectly recombined" when the sequences are recombined using an intermediate such as a cross-over oUgonucleotide. For indirect recombination, no more than one of the sequences is an actual substrate for recombination, and in some cases, neither sequence is a substrate for recombination.

"Regulatory elements" refer to sequences involved in controlling the expression of a nucleotide sequence. Regulatory elements comprise a promoter operably Unked to the nucleotide sequence of interest and termination signals. They also typicaUy encompass sequences required for proper translation of the nucleotide sequence. SubstantiaUy identical: the phrase "substantiaUy identical," in the context of two nucleic acid or protein sequences, refers to two or more sequences or subsequences that have at least 60%, preferably 80%, more preferably 90, even more preferably 95%, and most preferably at least 99% nucleotide or amino acid residue identity, when compared and aUgned for maximum correspondence, as measured using one of the foUowing sequence comparison algorithms or by visual inspection. Preferably, the substantial identity exists over a region of the sequences that is at least about 50 residues in length, more preferably over a region of at least about 100 residues, and most preferably the sequences are substantiaUy identical over at least about 150 residues. In a most preferred embodiment, the sequences are substantiaUy identical over the entire length of the coding regions. Furthermore, substantially identical nucleic acid or protein sequences perform substantiaUy the same function.

For sequence comparison, typicaUy one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

Optimal aUgnment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), by the homology aUgnment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970), by the search for sirr larity method of Pearson & Lipman, Proc. Natl Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally, Ausubel et al, infra). One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al, J. Mol. Biol. 215: 403-410 (1990). Software for performing BLAST analyses is pubUcly avaUable through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aUgned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., 1990). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative aUgnment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative aUgnment score faUs off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue aUgnments, or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the aUgnment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).

In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the simUarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5787 (1993)). One measure of simUarity provided by the BLAST algorithm is the smaUest sum probabiUty (P(N)), which provides an indication of the probabiUty by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered simUar to a reference sequence if the smaUest sum probabiUty in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

Another indication that two nucleic acid sequences are substantiaUy identical is that the two molecules hybridize to each other under stringent conditions. The phrase "hybridizing specificaUy to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total ceUular) DNA or RNA. "Bind(s) substantiaUy" refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence.

"Stringent hybridization conditions" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. Longer sequences hybridize specificaUy at higher temperatures. 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 "Overview of principles of hybridization and the strategy of nucleic acid probe assays" Elsevier, New York. GeneraUy, highly stringent hybridization and wash 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. TypicaUy, under "stringent conditions" a probe wiU hybridize to its target subsequence, but to no other sequences.

The T_m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the T_m for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42°C, with the hybridization being carried out overnight. An example of highly stringent wash conditions is 0.1 5M NaCl at 72°C for about 15 minutes. An example of stringent wash conditions is a 0.2x SSC wash at 65°C for 15 minutes (see, Sambrook, infra, for a description of SSC buffer). Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is lx SSC at 45°C for 15 minutes. An example low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6x SSC at 40°C for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typicaUy involve salt concentrations of less than about 1.0M Na ion, typicaUy about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typicaUy at least about 30°C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific

li hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are still substantiaUy identical if the proteins that they encode are substantiaUy identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. The foUowing are examples of sets of hybridization/wash conditions that may be used to clone homologous nucleotide sequences that are substantiaUy identical to reference nucleotide sequences of the present invention: a reference nucleotide sequence preferably hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C, more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50°C with washing in IX SSC, 0.1% SDS at 50°C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50°C with washing in 0.5X SSC, 0.1% SDS at 50°C, preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50°C with washing in 0.1X SSC, 0.1% SDS at 50°C, more preferably in 1% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50°C with washing in 0. IX SSC, 0.1 % SDS at 65°C.

A further indication that two nucleic acid sequences or proteins are substantiaUy identical is that the protein encoded by the first nucleic acid is immunologicaUy cross reactive with, or specificaUy binds to, the protein encoded by the second nucleic acid. Thus, a protein is typicaUy substantiaUy identical to a second protein, for example, where the two proteins differ only by conservative substitutions.

The phrase "specificaUy (or selectively) binds to an antibody," or "specificaUy (or selectively) immunoreactive with," when referring to a protein or peptide, refers to a binding reaction which is determinative of the presence of the protein in the presence of a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein and do not bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, antibodies raised to the protein with the amino acid sequence encoded by any of the nucleic acid sequences of the invention can be selected to obtain antibodies specificaUy immunoreactive with that protein and not with other proteins except for polymorphic variants. A variety of immunoassay formats may be used to select antibodies specificaUy immunoreactive with a particular protein. For example, soUd-phase ELIS A immunoassays, Western blots, or immunohistochemistry are routinely used to select monoclonal antibodies specificaUy immunoreactive with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor PubUcations, New York "Harlow and Lane"), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. TypicaUy a specific or selective reaction will be at least twice background signal or noise and more typicaUy more than 10 to 100 times background. A "subsequence" refers to a sequence of nucleic acids or amino acids that comprise a part of a longer sequence of nucleic acids or amino acids (e.g., protein) respectively. "Synthetic" refers to a nucleotide sequence comprising structural characters that are not present in the natural sequence. For example, an artificial sequence that resembles more closely the G+C content and the normal codon distribution of dicot and or monocot genes is said to be synthetic.

'Transformation" is a process for introducing heterologous nucleic acid into a host ceU or organism. In particular, "transformation" means the stable integration of a DNA molecule into the genome of an organism of interest. Transformed ceUs, tissues, or insects are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof.

"Transformed / transgenic / recombinant" refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-repUcating. Transformed ceUs, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. A "non-transformed", "non-transgenic", or "non-recombinant" host refers to a wUd-type organism, e.g., a bacterium or plant, which does not contain the heterologous nucleic acid molecule.

Nucleotides are indicated by their bases by the foUowing standard abbreviations: adenine (A), cytosine (C), thymine (T), and guanine (G). Amino acids are likewise indicated by the foUowing standard abbreviations: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gin; Q), glutamic acid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (He; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), protine (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Tip; W), tyrosine (Tyr; Y), and valine (Val; V). Furthermore, (Xaa; X) represents any amino acid.

This invention relates to novel nucleic acid sequences whose expression results in novel toxins, and to the making and using of the toxins to control insect pests. In particular, the present invention concerns synthetic gene sequences optimized for expression in plants that encode varying forms of the hybrid Bacillus thuringiensis delta-endotoxin H04, the toxin portion of which contains domains I and II of CrylAb and domain III of CrylC. The hybrid gene encoding the H04 hybrid toxin, as constructed from the native crylAb and CrylC genes is described in U.S. Pat. No. 5,736,131 and De Maagd et al, Appl. Environ. Microbiol. 62(5): 1537-1543 (1996). The preferred method for constructing the synthetic H04 genes of the invention is set forth in WO 93/07278. The hybrid Bacillus thuringiensis toxins encoded by the novel gene sequences are highly active against economicaUy important insect pests such as faU armyworm, pink boUworm, tobacco budworm, European cornborer, and diamondback moth. The hybrid Bacillus thuringiensis toxins can be used in multiple insect control strategies, resulting in maximal efficiency with minimal impact on the environment.

The present invention encompasses DNA molecules comprising nucleotide sequences that encode the insecticidal toxins of the invention. The present invention further encompasses recombinant vectors comprising the nucleic acid sequences of this invention. In such vectors, the nucleic acid sequences are preferably comprised in expression cassettes comprising regulatory elements for expression of the nucleotide sequences in a host ceU capable of expressing the nucleotide sequences. Such regulatory elements usuaUy comprise promoter and termination signals and preferably also comprise elements aUowing efficient translation of proteins encoded by the nucleic acid sequences of the present invention. Vectors comprising the nucleic acid sequences are usuaUy capable of repUcation in particular host ceUs, preferably as extrachromosomal molecules, and are therefore used to amplify the nucleic acid sequences of this invention in the host ceUs. In one embodiment, host ceUs for such vectors are microorganisms, such as bacteria, in particular Bacillus thuringiensis or E. coli. In another embodiment, host ceUs for such recombinant vectors are endophytes or epiphytes. A preferred host ceU for such vectors is a eukaryotic ceU, such as a plant ceU. Plant ceUs such as maize ceUs are most preferred host ceUs.

In a particularly preferred embodiment, an insecticidal toxin of the invention is expressed in a plant. In this case, transgenic plants expressing effective amounts of the toxins protect themselves from insect pests. When the insect starts feeding on such a transgenic plant, it also ingests the expressed toxins. This wiU deter the insect from further biting into the plant tissue or may even harm or kiU the insect.

The nucleic acid sequences described in this appUcation can be incoφorated into plant ceUs using conventional recombinant DNA technology. GeneraUy, this involves inserting a coding sequence of the invention into an expression system to which the coding sequence is heterologous (i.e., not normaUy present) using standard cloning procedures known in the art. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences. A large number of vector systems known in the art can be used, such as plasmids, bacteriophage viruses and other modified viruses. Suitable vectors include, but are not limited to, viral vectors such as lambda vector systems λgtll, λgtlO and Charon 4; plasmid vectors such as pBI121, pBR322, pACYC177, pACYC184, pAR series, pKK223- 3, pUC8, pUC9, pUC18, pUC19, pLG339, pRK290, pKC37, pKClOl, pCDNAII; and other simUar systems. Transformed ceUs can be regenerated into whole plants such that the nucleotide sequences of the invention confer insect resistance to the transgenic plants. Plants transformed in accordance with the present invention may be monocots or dicots and include, but are not limited to, maize, wheat, barley, rye, sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoU, turnip, radish, spinach, asparagus, onion, garUc, pepper, celery, squash, pumpkin, hemp, zucchini, apple, pear, quince, melon (e.g., watermelon), plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugarcane, sugarbeet, sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, Arabidopsis, and woody plants such as coniferous and deciduous trees. Once a desired nucleotide sequence has been transformed into a particular plant species, it may be propagated in that species or moved into other varieties of the same species, particularly including commercial varieties, using traditional breeding techniques. For their expression in transgenic plants, the nucleotide sequences of the invention may require modification and optimization. Although in many cases genes from microbial organisms can be expressed in plants at high levels without modification, low expression in transgenic plants may result from microbial nucleotide sequences having codons that are not preferred in plants. It is known in the art that aU organisms have specific preferences for codon usage, and the codons of the nucleotide sequences described in this invention can be changed to conform with plant preferences, whUe maintaining the amino acids encoded thereby. Furthermore, high expression in plants is best achieved from coding sequences that have at least 35% about GC content, preferably more than about 45%, more preferably more than about 50%, and most preferably more than about 60%. Microbial nucleotide sequences which have low GC contents may express poorly in plants due to the existence of ATTTA motifs which may destabilize messages, and AATAAA motifs which may cause inappropriate polyadenylation. Although preferred gene sequences may be adequately expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific codon preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. Nucl. Acids Res. 17: 477-498 (1989)). In addition, the nucleotide sequences are screened for the existence of Ulegitimate spUce sites that may cause message truncation. AU changes required to be made within the nucleotide sequences such as those described above are made using weU known techniques of site directed mutagenesis, PCR, and synthetic gene construction using the methods described in the pubhshed patent appUcations EP 0 385 962, EP 0 359 472, and WO 93/07278.

For efficient initiation of translation, sequences adjacent to the initiating methionine may require modification. For example, they can be modified by the inclusion of sequences known to be effective in plants. Joshi has suggested an appropriate consensus for plants (NAR 15: 6643-6653 (1987)) and Clontech suggests a further consensus translation initiator (1993/1994 catalog, page 210). These consensuses are suitable for use with the nucleotide sequences of this invention. The sequences are incorporated into constructions comprising the nucleotide sequences, up to and including the ATG (whUst leaving the second amino acid unmodified), or alternatively up to and including the GTC subsequent to the ATG (with the possibiUty of modifying the second amino acid of the transgene). Expression of the nucleotide sequences in transgenic plants is driven by promoters shown to be functional in plants. The choice of promoter wiU vary depending on the temporal and spatial requirements for expression, and also depending on the target species. Thus, expression of the nucleotide sequences of this invention in leaves, in ears, in inflorescences (e.g. spikes, panicles, cobs, etc.), in roots, and/or seedlings is preferred. In many cases, however, protection against more than one type of insect pest is sought, and thus expression in multiple tissues is desirable. Although many promoters from dicotyledons have been shown to be operational in monocotyledons and vice versa, ideaUy dicotyledonous promoters are selected for expression in dicotyledons, and monocotyledonous promoters for expression in monocotyledons. However, there is no restriction to the provenance of selected promoters; it is sufficient that they are operational in driving the expression of the nucleotide sequences in the desired ceU.

Preferred promoters that are expressed constitutively include promoters from genes encoding actin or ubiquitin and the CaMV 35S and 19S promoters. The nucleotide sequences of this invention can also be expressed under the regulation of promoters that are chemicaUy regulated. This enables the insecticidal toxins to be synthesized only when the crop plants are treated with the inducing chemicals. Preferred technology for chemical induction of gene expression is detaUed in the pubhshed appUcation EP 0 332 104 and US patent 5,614,395. A preferred promoter for chemical induction is the tobacco PR- la promoter. A preferred category of promoters is that which is wound inducible. Numerous promoters have been described which are expressed at wound sites and also at the sites of phytopathogen infection. IdeaUy, such a promoter should only be active locaUy at the sites of infection, and in this way the insecticidal toxins only accumulate in ceUs which need to synthesize the insecticidal toxins to k l the invading insect pest. Preferred promoters of this kind include those described by Stanford et al, Mol. Gen. Genet. 215: 200-208 (1989), Xu et al, Plant Molec. Biol. 22: 573-588 (1993), Logemann et al, Plant Cell 1: 151-158 (1989), Rohrmeier & Lehle, Plant Molec. Biol. 22: 783-792 (1993), Firek et al, Plant Molec. Biol. 22: 129-142 (1993), and Warner et al, Plant I. 3: 191-201 (1993).

Preferred tissue specific expression patterns include green tissue specific, root specific, stem specific, and flower specific. Promoters suitable for expression in green tissue include many which regulate genes involved in photosynthesis and many of these have been cloned from both monocotyledons and dicotyledons. A preferred promoter is the maize PEPC promoter from the phosphoenol carboxylase gene (Hudspeth & Grula, Plant Molec. Biol. 12: 579-589 (1989)). A preferred promoter for root specific expression is the maize metaUothionein-Uke (MTL) promoter described by de Framond (FEBS 290: 103-106 (1991); EP 0 452 269. A preferred stem specific promoter is that described in US patent 5,625,136 sand which drives expression of the maize trpA gene.

EspeciaUy preferred embodiments of the invention are transgenic plants expressing at least one of the nucleotide sequences of the invention in a root-preferred or root-specific fashion. Further preferred embodiments are transgenic plants expressing the nucleotide sequences in a wound-inducible or pathogen infection-inducible manner.

In addition to the selection of a suitable promoter, constructions for expression of an insecticidal toxin in plants require an appropriate transcription terminator to be attached downstream of the heterologous nucleotide sequence. Several such terminators are avaUable and known in the art (e.g. tml from CaMV, E9 from rbcS). Any avaUable terminator known to function in plants can be used in the context of this invention.

Numerous other sequences can be incorporated into expression cassettes described in this invention. These include sequences which have been shown to enhance expression such as intron sequences (e.g. from Adhl and bronzel) and viral leader sequences (e.g. from TMV, MCMV and AMV). It may be preferable to target expression of the nucleotide sequences of the present invention to different ceUular localizations in the plant. In some cases, localization in the cytosol may be desirable, whereas in other cases, localization in some subceUular organeUe may be preferred. SubceUular localization of transgene encoded enzymes is undertaken using techniques weU known in the art. TypicaUy, the DNA encoding the target peptide from a known organeUe-targeted gene product is manipulated and fused upstream of the nucleotide sequence. Many such target sequences are known for the chloroplast and their functioning in heterologous constructions has been shown. The expression of the nucleotide sequences of the present invention is also targeted to the endoplasmic reticulum or to the vacuoles of the host ceUs. Techniques to achieve this are weU-known in the art. Vectors suitable for plant transformation are described elsewhere in this specification.

For Agrøbαcteπwm-mediated transformation, binary vectors or vectors carrying at least one T- DNA border sequence are suitable, whereas for direct gene transfer any vector is suitable and linear DNA containing only the construction of interest may be preferred. In the case of direct gene transfer, transformation with a single DNA species or co-transformation can be used (Schocher et al. Biotechnology 4: 1093-1096 (1986)). For both direct gene transfer and Agrobacterium-mediated transfer, transformation is usuaUy (but not necessarUy) undertaken with a selectable marker which may provide resistance to an antibiotic (kanamycin, hygromycin or methotrexate) or a herbicide (basta). Examples of such markers are neomycin phosphotransferase, hygromycin phosphotransferase, dihydrofolate reductase, phosphinothricin acetyltransferase, 2, 2-dichloroproprionic acid dehalogenase, acetohydroxyacid synthase, 5- enolpy vyl-sliikimate-phosphate synthase, haloarylnitrUase, protoporhyrinogen oxidase, acetyl-coenzyme A carboxylase, dihydropteroate synthase, chloramphenicol acetyl transferase, and β-glucuronidase. Another type of marker providing for positive selection is the mannose- 6-phosphate isomerase (MPI/PMI) gene, which provides the abitity to metabolize mannose mannose-6-phosphate isomerase. The choice of selectable or screenable marker for plant transformation is not, however, critical to the invention.

The recombinant DNA described above can be introduced into the plant ceU in a number of art-recognized ways. Those skiUed in the art will appreciate that the choice of method might depend on the type of plant targeted for transformation. Suitable methods of transforming plant ceUs include microinjection (Crossway et al., BioTechniques 4:320-334 (1986)), electroporation (Riggs et αl, Proc. Nαtl. Acαd. Sci. USA 53:5602-5606 (1986), Agrobαcterium-mediated transformation (Hinchee et al., Biotechnology 6:915-921 (1988); See also, Ishida et al, Nature Biotechnology 14:145-150 (June 1996) for maize transformation), direct gene transfer (Paszkowski et al, EMBO I. 3:2111-2122 (1984); Hayashimoto et al, Plant Physiol. 93:857-863 (1990)(rice)), and baUistic particle acceleration using devices avaUable from Agracetus, Inc., Madison, Wisconsin and Dupont, Inc., Wilmington, Delaware (see, for example, Sanford et al., U.S. Patent 4,945,050; and McCabe et al., Biotechnology (5:923-926 (1988)). See also, Weissinger et al, Annual Rev. Genet. 22:421-411 (1988); Sanford et al, Paniculate Science and Technology 5:27-37 91987)(onion); Svab et al, Proc. Natl Acad. Sci. USA 87: 8526-8530 (1990) (tobacco chloroplast); Christou et al, Plant Physiol. 87:611-614 (1988)(soybean); McCabe et al, Bio Technology f5:923-926 (1988)(soybean); Klein et al, Proc. Natl. Acad. Sci. USA, S5:4305-4309 (1988)(maize); Klein et al, Biotechnology (5:559-563 (1988) (maize); Klein et al, Plant Physiol. 97:440-444 (1988) (maize); Fromm et al, Bio/Technology 5:833-839 (1990); and Gordon-Kamm et al, Plant Cell 2: 603-618 (1990) (maize); Koziel et al, Biotechnology 11: 194-200 (1993) (maize); Shimamoto et al, Nature 338: 274-277 (1989) (rice); Christou et al, Biotechnology 9: 957-962 (1991) (rice); Datta et al, Bio/Technology 8:136-140 (1990) (rice); European Patent AppUcation EP 0 332 581 (orchardgrass and other Pooideae); VasU et al, Biotechnology 11: 1553-1558 (1993) (wheat); Weeks et al, Plant Physiol. 102: 1077-1084 (1993) (wheat); Wan et al, Plant Physiol. 104: 37-48 (1994) (barley); Jahne et al, Theor. Appl. Genet. 59:525-533 (1994)(barley); Umbeck et al, Bio/Technology 5: 263-266 (1987) (cotton); Casas et al, Proc. Natl. Acad. Sci. USA 90:11212-11216 (Dec. 1993) (sorghum); Somers et al, Bio/Technology 70:1589-1594 (Dec. 1992) (oat); Torbert et al, Plant Cell Reports 7^:635-640 (1995) (oat); Weeks et al, Plant Physiol. 702:1077-1084 (1993) (wheat); Chang et al, WO 94/13822 (wheat) and Nehra et al, The Plant Journal 5:285-297 (1994) (wheat). A particularly preferred set of embodiments for the introduction of recombinant DNA molecules into maize by microprojectUe bombardment can be found in Koziel et al, Biotechnology 11: 194-200 (1993), Hill et al, Euphytica 55:119-123 (1995) and Koziel et al, Annals of the New York Academy of Sciences 792:164-171 (1996). An additional preferred embodiment is the protoplast transformation method for maize as disclosed in EP 0 292 435. Transformation of plants can be undertaken with a single DNA species or multiple DNA species (i.e. co-transformation) and both these techniques are suitable for use with a coding sequence of the invention.

In another preferred embodiment, a nucleotide sequence of the present invention is directly transformed into the plastid genome. A major advantage of plastid transformation is that plastids are generaUy capable of expressing bacterial genes without substantial modification, and plastids are capable of expressing multiple open reading frames under control of a single promoter. Plastid transformation technology is extensively described in U.S. Patent Nos. 5,451,513, 5,545,817, and 5,545,818, in PCT appUcation no. WO 95/16783, and in McBride et al (1994) Proc. Natl. Acad. Sci. USA 91, 7301-7305. The basic technique for chloroplast transformation involves introducing regions of cloned plastid DNA flanking a selectable marker together with the gene of interest into a suitable target tissue, e.g., using bioUstics or protoplast transformation (e.g., calcium chloride or PEG mediated transformation). The 1 to 1.5 kb flanking regions, termed targeting sequences, faciUtate homologous recombination with the plastid genome and thus aUow the replacement or modification of specific regions of the plastome. InitiaUy, point mutations in the chloroplast 16S rRNA and rpsl2 genes conferring resistance to spectinomycin and/or streptomycin are utilized as selectable markers for transformation (Svab, Z., Hajdukiewicz, P., and MaUga, P. (1990) Proc. Natl. Acad. Sci. USA 87, 8526-8530; Staub, J. M., and MaUga, P. (1992) Plant CeU 4, 39-45). This resulted in stable homoplasmic transformants at a frequency of approximately one per 100 bombardments of target leaves. The presence of cloning sites between these markers aUowed creation of a plastid targeting vector for introduction of foreign genes (Staub, J.M., and MaUga, P. (1993) EMBO J. 12, 601-606). Substantial increases in transformation frequency are obtained by replacement of the recessive rRNA or r-protein antibiotic resistance genes with a dominant selectable marker, the bacterial aadA gene encoding the spectinomycin-detoxifying enzyme aminoglycoside-3'-adenyltransferase (Svab, Z., and MaUga, P. (1993) Proc. Natl. Acad. Sci. USA 90, 913-917). Previously, this marker had been used successfuUy for high-frequency transformation of the plastid genome of the green alga Chlamydomonas reinhardtii (Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. 19: 4083-4089). Other selectable markers useful for plastid transformation are known in the art and encompassed within the scope of the invention. TypicaUy, approximately 15-20 ceU division cycles foUowing transformation are required to reach a homoplastidic state. Plastid expression, in which genes are inserted by homologous recombination into aU of the several thousand copies of the circular plastid genome present in each plant ceU, takes advantage of the enormous copy number advantage over nuclear-expressed genes to permit expression levels that can readUy exceed 10% of the total soluble plant protein. In a preferred embodiment, a nucleotide sequence of the present invention is inserted into a plastid targeting vector and transformed into the plastid genome of a desired plant host. Plants homoplastic for plastid genomes containing a nucleotide sequence of the present invention are obtained, and are preferentiaUy capable of high expression of the nucleotide sequence.

EXAMPLES The invention wiU be further described by reference to the foUowing detaUed examples. These examples are provided for purposes of iUustration only, and are not intended to be limiting unless otherwise specified. Standard recombinant DNA and molecular cloning techniques used here are weU known in the art and are described by Ausubel (ed.), Current Protocols in Molecular Biology, John WUey and Sons, Inc. (1994); T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, Cold Spring Harbor, NY (1989); and by T.J. Silhavy, M.L. Berman, and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984).

Example 1 : Expression and Purification of an H04 Toxin Fragment

A truncated form of the H04 hybrid toxin gene (described in De Maagd et al, Appl. Environ. Microbiol. 62(5): 1537-1543 (1996), which encodes a Bt toxin consisting essentiaUy of domains I and H of CrylAb and domain HI of CrylC, is cloned into an expression vector such as pBluescript SK-, BaciUus shuttle vector, or pET 21b(+) for overexpression in E. coli. CeUs are grown in LB media containing 50 micrograms/ml ampiciUin for 24 to 48 h at 37°C shaker (250 rpm). CeUs are harvested by centrifugation for 10 min at 7,000 φm. The peUet is sonicated with a Bronson sonifier for 2 min 30 sec with 2 sec pulse. Complete sonication is checked under microscope. Soluble fractions are removed by centrifugation at 10,000 rpm for 10 min. The resulting peUet containing crystal proteins is washed 4-5 times with 2% Triton X-100 containing 0.5 M NaCl. Continuous washing is done with 0.5 M NaCl (4-5 times) and the final peUet is washed with distUled water (2 times). The resulting peUet is solubitized in 50 mM Na₂CO₃ buffer containing 10 mM dithiothreitol at 37°C for 2 h. Solubilized protein is separated from insoluble materials by centrifugation at 12,000 rpm for 10 min. Protein samples are dialyzed with 50 mM Na₂CO₃, pH 9.0 buffer for bioassays.

Example 2: Bioassays LC50's are performed on faU armyworm, pink boUworm, tobacco budworm, and European cornborer using purified truncated H04 protein that is produced, for example, as described above in Example 1. Results are as foUows: LC50 faU armyworm 133 ng/cm² LC50 pink boUworm 691 ng/cm²

LC50 tobacco budworm 299 ng/cm² LC50 European cornborer 31 ng/cm²

Example 3: Synthetic H04 Gene Construction

A synthetic nucleotide sequence encoding the toxin portion of H04 is designed by backtranslating the amino acid sequence of the H04 hybrid toxin fragment described in De Maagd et al, Appl. Environ. Microbiol. 62(5): 1537-1543 (1996) (domains I and H of CrylAb and domain HI of CrylC) using the "Backtranslation" program found in the University of Wisconsin GCG group of programs using a maize preference codon table (Murray et al., Nucl Acids Res. 17:477-498, 1989, incoφorated herein by reference). Preferably, the most frequently used maize codon is used for each amino acid, as described in WO 93/07278.

The synthetic nucleotide sequence encoding the toxin portion of H04 may be constructed in several fragments. Each fragment is constructed by hybridization of ten pairs of oUgomers 60-75 nucleotides in length representing both strands of the gene. An approximately 15 nucleotide overlap is designed between sequential oUgonucleotide pairs for correct orientation and assembly. OUgonucleotides may be synthesized by, for example, Genosys Biotechnologies Inc., TX. Each pair of oUgomers is hybridized and phosphorylated using the enzyme polynucleotide kinase from, e.g., New England Biolabs, Inc., MA using conditions specified by the vendor. Kinased fragment pairs are then hybridized and Ugated into a high copy plasmid vector containing, e.g., an ampiciUin resistance gene and transformed into, e.g., competent DH5 ceUs. The ceUs are plated onto ampiciUin containing media and incubated overnight at 37 C. Colonies are screened for inserted DNA. The DNA is sequenced and clones containing the correct sequence are selected. The fragments are then joined by restriction digestion, Ugation and transformation using unique restriction sites between the fragments. SEQ ID NO: 3 shows the synthetic nucleotide sequence encoding the 631 -amino acid toxin portion of H04 (without a protoxin taU region), and SEQ ID NO:4 shows the amino acid sequence of the H04 toxin encoded by the synthetic nucleotide sequence depicted in SEQ ID NO:3. SEQ ID NO: 11 shows the nucleotide sequence of construct pNOV1308, which contains the constitutive maize ubiquitin promoter operatively linked to the synthetic H04 gene sequence set forth in SEQ ID NO:3.

In addition to the above-described synthetic gene (SEQ ID NO:3) that encodes only the toxin portion of the H04 hybrid (domains I and H of CrylAb and domain HI of CrylC), additional synthetic H04 genes are constructed with aU or a portion of the synthetic crylAb taU region described in U.S. Patent No. 5,625,136 (herein incorporated by reference) fused to the 3' end of the H04 toxin portion. These synthetic H04 gene sequences with crylAb taUs are described below:

SEQ ID NO: 5 shows a synthetic nucleotide sequence encoding the toxin portion of H04 plus a fuU-length CrylAb taU portion, and SEQ ID NO: 6 shows the amino acid sequence of the H04 + CrylAb tail encoded by the synthetic nucleotide sequence depicted in SEQ ID NO:5. SEQ ID NO: 12 shows the nucleotide sequence of construct pNOV1436, which contains the root- preferred maize MTL promoter operatively Unked to the synthetic H04 gene sequence set forth in SEQ ID NO:5. SEQ ID NO: 13 shows the nucleotide sequence of construct pNOV1441, which contains the constitutive maize ubiquitin promoter operatively linked to the synthetic H04 gene sequence set forth in SEQ ID NO:5.

SEQ ID NO:7 shows another synthetic nucleotide sequence encoding the toxin portion of H04 plus a fuU-length CrylAb taU portion, and SEQ ID NO: 8 shows the amino acid sequence of the H04 + CrylAb taU encoded by the synthetic nucleotide sequence depicted in SEQ ID NO:7. SEQ ID NO: 14 shows the nucleotide sequence of construct pNOV1305, which contains the constitutive maize ubiquitin promoter operatively linked to the synthetic H04 gene sequence set forth in SEQ ID NO:7. SEQ ID NO:15 shows the nucleotide sequence of construct pNOV1313, which contains the constitutive maize ubiquitin promoter operatively Unked to the synthetic H04 gene sequence set forth in SEQ ID NO:7.

SEQ ID NO: 9 shows a synthetic nucleotide sequence encoding the toxin portion of H04 plus only the first 40 amino acids of the CrylAb taϋ, and SEQ ID NO: 10 shows the amino acid sequence of the H04 + 40-amino acid truncated CrylAb taU encoded by the synthetic nucleotide sequence depicted in SEQ ID NO:9. SEQ ID NO: 16 shows the nucleotide sequence of construct pNOV1435, which contains the root-preferred maize MTL promoter operatively linked to the synthetic H04 gene sequence set forth in SEQ ID NO:9. SEQ ID NO: 17 shows the nucleotide sequence of construct pZU578, which contains the Arabidopsis actin-2 promoter operatively linked to the synthetic H04 gene sequence set forth in SEQ ID NO:9.

Example 4: Modification of Coding Sequences and Adjacent Sequences

The nucleotide sequences described in this appUcation can be modified for expression in transgenic plant hosts. A host plant expressing the nucleotide sequences and which produces the insecticidal toxins in its ceUs has enhanced resistance to insect attack and is thus better equipped to withstand crop losses associated with such attack.

The transgenic expression in plants of genes derived from microbial sources may require the modification of those genes to achieve and optimize their expression in plants. In particular, bacterial ORFs that encode separate enzymes but that are encoded by the same transcript in the native microbe are best expressed in plants on separate transcripts. To achieve this, each microbial ORF is isolated individuaUy and cloned within a cassette which provides a plant promoter sequence at the 5' end of the ORF and a plant transcriptional terminator at the 3' end of the ORF. The isolated ORF sequence preferably includes the initiating ATG codon and the terminating STOP codon but may include additional sequence beyond the initiating ATG and the STOP codon. In addition, the ORF may be truncated, but stiU retain the required activity; for particularly long ORFs, truncated versions which retain activity may be preferable for expression in transgenic organisms. By "plant promoter" and "plant transcriptional terminator" it is intended to mean promoters and transcriptional terminators which operate within plant ceUs. This includes promoters and transcription terminators which may be derived from non-plant sources such as viruses (an example is the Cauliflower Mosaic Virus).

In some cases, modification to the ORF coding sequences and adjacent sequence is not required. It is sufficient to isolate a fragment containing the ORF of interest and to insert it downstream of a plant promoter. For example, Gaffhey et al. (Science 261: 754-756 (1993)) have expressed the Pseudomonas nahG gene in transgenic plants under the control of the CaMV 35S promoter and the CaMV tml terminator successfuUy without modification of the coding sequence and with x bp of the Pseudomonas gene upstream of the ATG stiU attached, and y bp downstream of the STOP codon stiU attached to the nahG ORF. Preferably as Uttle adjacent microbial sequence should be left attached upstream of the ATG and downstream of the STOP codon. In practice, such construction may depend on the avaUabiUty of restriction sites.

In other cases, the expression of genes derived from microbial sources may provide problems in expression. These problems have been weU characterized in the art and are particularly common with genes derived from certain sources such as Bacillus. These problems may apply to the nucleotide sequence of this invention and the modification of these genes can be undertaken using techniques now weU known in the art. The foUowing problems may be encountered:

1. Codon Usage.

The preferred codon usage in plants differs from the preferred codon usage in certain microorganisms. Comparison of the usage of codons within a cloned microbial ORF to usage in plant genes (and in particular genes from the target plant) wiU enable an identification of the codons within the ORF which should preferably be changed. TypicaUy plant evolution has tended towards a strong preference of the nucleotides C and G in the third base position of monocotyledons, whereas dicotyledons often use the nucleotides A or T at this position. By modifying a gene to incorporate preferred codon usage for a particular target transgenic species, many of the problems described below for GC/AT content and Ulegitimate spUcing wiU be overcome.

2. GC/AT Content. Plant genes typicaUy have a GC content of more than 35%. ORF sequences which are rich in A and T nucleotides can cause several problems in plants. Firstly, motifs of ATTTA are beheved to cause destabilization of messages and are found at the 3' end of many short-Uved mRNAs. Secondly, the occurrence of polyadenylation signals such as AATAAA at inappropriate positions within the message is beUeved to cause premature truncation of transcription. In addition, monocotyledons may recognize AT-rich sequences as spUce sites (see below). 3. Sequences Adjacent to the Initiating Methionine.

Plants differ from microorganisms in that their messages do not possess a defined ribosome binding site. Rather, it is beUeved that ribosomes attach to the 5' end of the message and scan for the first avaUable ATG at which to start translation. Nevertheless, it is beUeved that there is a preference for certain nucleotides adjacent to the ATG and that expression of microbial genes can be enhanced by the inclusion of a eukaryotic consensus translation initiator at the ATG. Clontech (1993/1994 catalog, page 210, incorporated herein by reference) have suggested one sequence as a consensus translation initiator for the expression of the E. coli uidA gene in plants. Further, Joshi (NAR 15: 6643-6653 (1987), incoφorated herein by reference) has compared many plant sequences adjacent to the ATG and suggests another consensus sequence. In situations where difficulties are encountered in the expression of microbial ORFs in plants, inclusion of one of these sequences at the initiating ATG may improve translation. In such cases the last three nucleotides of the consensus may not be appropriate for inclusion in the modified sequence due to their modification of the second AA residue. Preferred sequences adjacent to the initiating methionine may differ between different plant species. A survey of 14 maize genes located in the GenBank database provided the foUowing results:

Position Before the Initiating ATG in 14 Maize Genes: zlQ z9 8 7 6 5 r4 z3 2 l

C 3 8 4 6 2 5 6 0 10 7

T 3 0 3 4 3 2 1 1 1 0

A 2 3 1 4 3 2 3 7 2 3 G 6 3 6 0 6 5 4 6 1 5

This analysis can be done for the desired plant species into which the nucleotide sequence is being incorporated, and the sequence adjacent to the ATG modified to incoφorate the preferred nucleotides.

4. Removal of IUegitimate SpUce Sites. Genes cloned from non-plant sources and not optimized for expression in plants may also contain motifs which may be recognized in plants as 5' or 3' spUce sites, and be cleaved, thus generating truncated or deleted messages. These sites can be removed using the techniques weU known in the art. Techniques for the modification of coding sequences and adjacent sequences are weU known in the art. In cases where the initial expression of a microbial ORF is low and it is deemed appropriate to make alterations to the sequence as described above, then the construction of synthetic genes can be accompUshed according to methods weU known in the art. These are, for example, described in the pubUshed patent disclosures EP 0 385 962, EP 0 359 472 and WO 93/07278, aU of which are incorporated herein by reference. In most cases it is preferable to assay the expression of gene constructions using transient assay protocols (which are weU known in the art) prior to their transfer to transgenic plants.

Example 5: Construction of Plant Expression Cassettes

Coding sequences intended for expression in transgenic plants are first assembled in expression cassettes behind a suitable promoter expressible in plants. The expression cassettes may also comprise any further sequences required or selected for the expression of the transgene. Such sequences include, but are not restricted to, transcription terminators, extraneous sequences to enhance expression such as introns, vital sequences, and sequences intended for the targeting of the gene product to specific organeUes and ceU compartments. These expression cassettes can then be easUy transferred to the plant transformation vectors described below. The foUowing is a description of various components of typical expression cassettes.

1. Promoters

The selection of the promoter used in expression cassettes wiU determine the spatial and temporal expression pattern of the transgene in the transgenic plant. Selected promoters will express transgenes in specific ceU types (such as leaf epidermal ceUs, mesophyU ceUs, root cortex ceUs) or in specific tissues or organs (roots, leaves or flowers, for example) and the selection wiU reflect the desired location of accumulation of the gene product. Alternatively, the selected promoter may drive expression of the gene under various inducing conditions. Promoters vary in their strength, i.e., abitity to promote transcription. Depending upon the host ceU system utilized, any one of a number of suitable promoters can be used, including the gene's native promoter. The foUowing are non-limiting examples of promoters that may be used in expression cassettes.

a. Constitutive Expression, the Ubiquitin Promoter:

Ubiquitin is a gene product known to accumulate in many ceU types and its promoter has been cloned from several species for use in transgenic plants (e.g. sunflower - Binet et al. Plant Science 79: 87-94 (1991); maize - Christensen et al. Plant Molec. Biol. L2: 619-632 (1989); and Arabidopsis - Norris et al, Plant Mol. Biol. 21:895-906 (1993)). The maize ubiquitin promoter has been developed in transgenic monocot systems and its sequence and vectors constructed for monocot transformation are disclosed in the patent pubUcation EP 0 342 926 which is herein incoφorated by reference. Taylor et al (Plant CeU Rep. 12: 491-495 (1993)) describe a vector (pAHC25) that comprises the maize ubiquitin promoter and first intron and its high activity in ceU suspensions of numerous monocotyledons when introduced via microprojectUe bombardment. The Arabidopsis ubiquitin promoter is ideal for use with the nucleotide sequences of the present invention. The ubiquitin promoter is suitable for gene expression in transgenic plants, both monocotyledons and dicotyledons. Suitable vectors are derivatives of pAHC25 or any of the transformation vectors described in this appUcation, modified by the introduction of the appropriate ubiquitin promoter and/or intron sequences.

b. Constitutive Expression, the CaMV 35S Promoter:

Construction of the plasmid pCGN1761 is described in the pubUshed patent appUcation EP 0 392 225 (Example 23), which is hereby incoφorated by reference. pCGN1761 contains the "double" CaMV 35S promoter and the tml transcriptional terminator with a unique EcoRI site between the promoter and the terminator and has a pUC-type backbone. A derivative of pCGN1761 is constructed which has a modified polylinker which includes Notl and Xhol sites in addition to the existing EcoRI site. This derivative is designated pCGN1761ENX. pCGN1761ENX is useful for the cloning of cDNA sequences or coding sequences (including microbial ORF sequences) within its polylinker for the puφose of their expression under the control of the 35S promoter in transgenic plants. The entire 35S promoter-coding sequence- tml terminator cassette of such a construction can be excised by Hindlll, Sphl, Sail, and Xbal sites 5' to the promoter and Xbal, BamHI and Bgll sites 3' to the terminator for transfer to transformation vectors such as those described below. Furthermore, the double 35S promoter fragment can be removed by 5' excision with Hindlll, Sphl, Sail, Xbal, or Pstl, and 3' excision with any of the polylinker restriction sites (EcoRI, Notl or Xhol) for replacement with another promoter. If desired, modifications around the cloning sites can be made by the introduction of sequences that may enhance translation. This is particularly useful when overexpression is desired. For example, pCGN1761ENX may be modified by optimization of the translational initiation site as described in Example 37 of U.S. Patent No. 5,639,949, incoφorated herein by reference.

c. Constitutive Expression, the Actin Promoter:

Several isoforms of actin are known to be expressed in most cell types and consequently the actin promoter is a good choice for a constitutive promoter. In particular, the promoter from the rice Actl gene has been cloned and characterized (McElroy et al. Plant CeU 2: 163- 171 (1990)). A 1.3kb fragment of the promoter was found to contain aU the regulatory elements required for expression in rice protoplasts. Furthermore, numerous expression vectors based on the Actl promoter have been constructed specificaUy for use in monocotyledons (McElroy et al. Mol. Gen. Genet. 231: 150-160 (1991)). These incoφorate the et/-intron 1, Adhl 5' flanking sequence and Adhl-intτon 1 (from the maize alcohol dehydrogenase gene) and sequence from the CaMV 35S promoter. Vectors showing highest expression were fusions of 35S and Actl intron or the Actl 5' flanking sequence and the Actl intron. Optimization of sequences around the initiating ATG (of the GUS reporter gene) also enhanced expression. The promoter expression cassettes described by McElroy et al. (Mol. Gen. Genet. 231: 150-160 (1991)) can be easUy modified for gene expression and are particularly suitable for use in monocotyledonous hosts. For example, promoter-containing fragments is removed from the McElroy constructions and used to replace the double 35S promoter in pCGN1761ENX, which is then avaUable for the insertion of specific gene sequences. The fusion genes thus constructed can then be transferred to appropriate transformation vectors. In a separate report, the rice Actl promoter with its first intron has also been found to direct high expression in cultured barley ceUs (Chibbar et al. Plant CeU Rep. 12: 506-509 (1993)).

d. Inducible Expression, the PR-1 Promoter: The double 35S promoter in pCGN1761ENX may be replaced with any other promoter of choice that wiU result in suitably high expression levels. By way of example, one of the chemicaUy regulatable promoters described in U.S. Patent No. 5,614,395, such as the tobacco PR- la promoter, may replace the double 35S promoter. Alternately, the Arabidopsis PR-1 promoter described in Lebel et al, Plant J. 16:223-233 (1998) may be used. The promoter of choice is preferably excised from its source by restriction enzymes, but can alternatively be PCR-ampUfied using primers that carry appropriate terminal restriction sites. Should PCR- amplification be undertaken, then the promoter should be re-sequenced to check for amplification errors after the cloning of the amplified promoter in the target vector. The chemicaUy/pathogen regulatable tobacco PR- la promoter is cleaved from plasmid pCIB1004 (for construction, see example 21 of EP 0 332 104, which is hereby incoφorated by reference) and transferred to plasmid pCGN1761ENX (Uknes et al, Plant Cell 4: 645-656 (1992)). pCIB1004 is cleaved with Ncol and the resultant 3' overhang of the linearized fragment is rendered blunt by treatment with T4 DNA polymerase. The fragment is then cleaved with Hindlll and the resultant PR- la promoter-containing fragment is gel purified and cloned into pCGN1761ENX from which the double 35S promoter has been removed. This is done by cleavage with Xhol and blunting with T4 polymerase, foUowed by cleavage with Hindlll and isolation of the larger vector-terminator containing fragment into which the pCIB1004 promoter fragment is cloned. This generates a pCGN1761ENX derivative with the PR-la promoter and the tml terminator and an intervening polylinker with unique EcoRI and Notl sites. The selected codmg sequence can be inserted into this vector, and the fusion products (i.e. promoter- gene-terminator) can subsequently be transferred to any selected transformation vector, including those described infra. Various chemical regulators may be employed to induce expression of the selected coding sequence in the plants transformed according to the present invention, including the benzothiadiazole, isonicotinic acid, and saUcyUc acid compounds disclosed in U.S. Patent Nos. 5,523,311 and 5,614,395. e. Inducible Expression, an Ethanol-Inducible Promoter:

A promoter inducible by certain alcohols or ketones, such as ethanol, may also be used to confer inducible expression of a coding sequence of the present invention. Such a promoter is for example the alcA gene promoter from Aspergillus nidulans (Caddick et al. (1998) Nat. Biotechnol 16:177-180). In A. nidulans, the alcA gene encodes alcohol dehydrogenase I, the expression of which is regulated by the AlcR transcription factors in presence of the chemical inducer. For the puφoses of the present invention, the CAT coding sequences in plasmid palcA:CAT comprising a alcA gene promoter sequence fused to a minimal 35S promoter (Caddick et al. (1998) Nat. Biotechnol 16:177-180) are replaced by a coding sequence of the present invention to form an expression cassette having the coding sequence under the control of the alcA gene promoter. This is carried out using methods weU known in the art.

f. Inducible Expression, a Glucocorticoid-Inducible Promoter:

Induction of expression of a nucleic acid sequence of the present invention using systems based on steroid hormones is also contemplated. For example, a glucocorticoid- mediated induction system is used (Aoyama and Chua (1997) The Plant Journal 11: 605-612) and gene expression is induced by appUcation of a glucocorticoid, for example a synthetic glucocorticoid, preferably dexamethasone, preferably at a concentration ranging from 0.1 mM to lmM, more preferably from lOmM to lOOmM. For the puφoses of the present invention, the luciferase gene sequences are replaced by a nucleic acid sequence of the invention to form an expression cassette having a nucleic acid sequence of the invention under the control of six copies of the GAL4 upstream activating sequences fused to the 35S minimal promoter. This is carried out using methods weU known in the art. The trans-acting factor comprises the GAL4 DΝA-binding domain (Keegan et al. (1986) Science 231: 699-704) fused to the transactivating domain of the heφes viral protein VP16 (Triezenberg et al. (1988) Genes Devel. 2: 718-729) fused to the hormone-binding domain of the rat glucocorticoid receptor (Picard et al. (1988) Cell 54: 1073-1080). The expression of the fusion protein is controUed by any promoter suitable for expression in plants known in the art or described here. This expression cassette is also comprised in the plant comprising a nucleic acid sequence of the invention fused to the 6xGAL4/minimal promoter. Thus, tissue- or organ-specificity of the fusion protein is achieved leading to inducible tissue- or organ-specificity of the insecticidal toxin. g. Root Specific Expression:

Another pattern of gene expression is root expression. A suitable root promoter is the promoter of the maize metaUothionein-Uke (MTL) gene described by de Framond (FEBS 290: 103-106 (1991)) and also in U.S. Patent No. 5,466,785, incoφorated herein by reference. This "MTL" promoter is transferred to a suitable vector such as pCGN1761ENX for the insertion of a selected gene and subsequent transfer of the entire promoter-gene-terminator cassette to a transformation vector of interest.

h. Wound-Inducible Promoters:

Wound-inducible promoters may also be suitable for gene expression. Numerous such promoters have been described (e.g. Xu et al. Plant Molec. Biol. 22: 573-588 (1993), Logemann et al. Plant CeU 151-158 (1989), Rohrmeier & Lehle, Plant Molec. Biol. 22: 783- 792 (1993), Firek et al. Plant Molec. Biol. 22: 129-142 (1993), Warner et al. Plant J. 3: 191- 201 (1993)) and aU are suitable for use with the instant invention. Logemann et al. describe the 5' upstream sequences of the dicotyledonous potato wunl gene. Xu et al. show that a wound-inducible promoter from the dicotyledon potato (pin2) is active in the monocotyledon rice. Further, Rohrmeier & Lehle describe the cloning of the maize Wipl cDNA which is wound induced and which can be used to isolate the cognate promoter using standard techniques. SimUar, Firek et al. and Warner et al. have described a wound-induced gene from the monocotyledon Asparagus ojficinalis, which is expressed at local wound and pathogen invasion sites. Using cloning techniques weU known in the art, these promoters can be transferred to suitable vectors, fused to the genes pertaining to this invention, and used to express these genes at the sites of plant wounding.

i. Pith-Preferred Expression:

Patent AppUcation WO 93/07278, which is herein incoφorated by reference, describes the isolation of the maize trpA gene, which is preferentiaUy expressed in pith ceUs. The gene sequence and promoter extending up to -1726 bp from the start of transcription are presented. Using standard molecular biological techniques, this promoter, or parts thereof, can be transferred to a vector such as pCGN1761 where it can replace the 35S promoter and be used to drive the expression of a foreign gene in a pith-preferred manner. In fact, fragments containing the pith-preferred promoter or parts thereof can be transferred to any vector and modified for utitity in transgenic plants.

j. Leaf-Specific Expression:

A maize gene encoding phosphoenol carboxylase (PEPC) has been described by Hudspeth & Grula (Plant Molec Biol 12: 579-589 (1989)). Using standard molecular biological techniques the promoter for this gene can be used to drive the expression of any gene in a leaf-specific manner in transgenic plants.

k. PoUen-Specific Expression:

WO 93/07278 describes the isolation of the maize calcium-dependent protein kinase (CDPK) gene which is expressed in poUen ceUs. The gene sequence and promoter extend up to 1400 bp from the start of transcription. Using standard molecular biological techniques, this promoter or parts thereof, can be transferred to a vector such as pCGN1761 where it can replace the 35S promoter and be used to drive the expression of a nucleic acid sequence of the invention in a poUen-specific manner.

1. Receptor Mediated Transactivation In The Presence Of A Chemical Ligand: U.S. Patent No. 5,880,333, incoφorated herein by reference, describes a system whereby class II hormone receptors such as Ecdysone Receptor (EcR) and Ultraspiracle (USP), which function together as a heterodimer, regulate the expression of a target polypeptide in a plant ceU in the presence of an appropriate chemical Ugand, e.g. tebufenozide.

2. Transcriptional Terminators

A variety of transcriptional terminators are avaUable for use in expression cassettes. These are responsible for the termination of transcription beyond the transgene and its correct polyadenylation. Appropriate transcriptional terminators are those that are known to function in plants and include the CaMV 35S terminator, the tml terminator, the nopaline synthase terminator and the pea rbcS E9 terminator. These can be used in both monocotyledons and dicotyledons. In addition, a gene's native transcription terminator may be used. 3. Sequences for the Enhancement or Regulation of Expression Numerous sequences have been found to enhance gene expression from within the transcriptional unit and these sequences can be used in conjunction with the genes of this invention to increase their expression in transgenic plants.

Various intron sequences have been shown to enhance expression, particularly in monocotyledonous ceUs. For example, the introns of the maize Adhl gene have been found to significantly enhance the expression of the wUd-type gene under its cognate promoter when introduced into maize ceUs. Intron 1 was found to be particularly effective and enhanced expression in fusion constructs with the chloramphenicol acetyltransferase gene (Caltis et al,

Genes Develop. 1: 1183-1200 (1987)). In the same experimental system, the intron from the maize bronzel gene had a simUar effect in enhancing expression. Intron sequences have been routinely incoφorated into plant transformation vectors, typicaUy within the non-translated leader. A number of non-translated leader sequences derived from viruses are also known to enhance expression, and these are particularly effective in dicotyledonous ceUs. SpecificaUy, leader sequences from Tobacco Mosaic Virus (TMV, the "W-sequence"), Maize Chlorotic

Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effective in enhancing expression (e.g. GaUie et al. Nucl. Acids Res. 15: 8693-8711 (1987); Skuzeski et al. Plant Molec. Biol. 15: 65-79 (1990)).

4. Targeting of the Gene Product Within the CeU

Various mechanisms for targeting gene products are known to exist in plants and the sequences controlling the functioning of these mechanisms have been characterized in some detaU. For example, the targeting of gene products to the chloroplast is controUed by a signal sequence found at the amino terminal end of various proteins which is cleaved during chloroplast import to yield the mature protein (e.g. Comai et al. J. Biol. Chem. 263: 15104- 15109 (1988)). These signal sequences can be fused to heterologous gene products to effect the import of heterologous products into the chloroplast (van den Broeck, et al. Nature 313: 358-363 (1985)). DNA encoding for appropriate signal sequences can be isolated from the 5' end of the cDNAs encoding the RUBISCO protein, the CAB protein, the EPSP synthase enzyme, the GS2 protein and many other proteins which are known to be chloroplast localized. See also, the section entitled "Expression With Chloroplast Targeting" in Example 37 of U.S. Patent No. 5,639,949.

Other gene products are localized to other organeUes such as the mitochondrion and the peroxisome (e.g. Unger et al. Plant Molec. Biol. 13: 411-418 (1989)). The cDNAs encoding these products can also be manipulated to effect the targeting of heterologous gene products to these organeUes. Examples of such sequences are the nuclear-encoded ATPases and specific aspartate amino transferase isoforms for mitochondria. Targeting ceUular protein bodies has been described by Rogers et al. (Proc. Natl. Acad. Sci. USA 82: 6512-6516 (1985)). In addition, sequences have been characterized which cause the targeting of gene products to other ceU compartments. Amino terminal sequences are responsible for targeting to the ER, the apoplast, and extraceUular secretion from aleurone ceUs (Koehler & Ho, Plant CeU 2: 769-783 (1990)). AdditionaUy, amino terminal sequences in conjunction with carboxy terminal sequences are responsible for vacuolar targeting of gene products (Shinshi et al. Plant Molec. Biol. 14: 357-368 (1990)).

By the fusion of the appropriate targeting sequences described above to transgene sequences of interest it is possible to direct the transgene product to any organeUe or ceU compartment. For chloroplast targeting, for example, the chloroplast signal sequence from the RUBISCO gene, the CAB gene, the EPSP synthase gene, or the GS2 gene is fused in frame to the amino terminal ATG of the transgene. The signal sequence selected should include the known cleavage site, and the fusion constructed should take into account any amino acids after the cleavage site which are required for cleavage. In some cases this requirement may be fulfiUed by the addition of a smaU number of amino acids between the cleavage site and the transgene ATG or, alternatively, replacement of some amino acids within the transgene sequence. Fusions constructed for chloroplast import can be tested for efficacy of chloroplast uptake by in vitro translation of in vitro transcribed constructions foUowed by in vitro chloroplast uptake using techniques described by Bartlett et al. In: Edelmann et al. (Eds.) Methods in Chloroplast Molecular Biology, Elsevier pp 1081-1091 (1982) and Wasmann et al. Mol. Gen. Genet. 205: 446-453 (1986). These construction techniques are weU known in the art and are equaUy appUcable to mitochondria and peroxisomes. The above-described mechanisms for ceUular targeting can be utilized not only in conjunction with their cognate promoters, but also in conjunction with heterologous promoters so as to effect a specific ceU-targeting goal under the transcriptional regulation of a promoter that has an expression pattern different to that of the promoter from which the targeting signal derives.

Example 6: Construction of Plant Transformation Vectors

Numerous transformation vectors avaUable for plant transformation are known to those of ordinary skiU in the plant transformation arts, and the genes pertinent to this invention can be used in conjunction with any such vectors. The selection of vector wiU depend upon the preferred transformation technique and the target species for transformation. For certain target species, different antibiotic or herbicide selection markers may be preferred. Selection markers used routinely in transformation include the nptll gene, which confers resistance to kanamycin and related antibiotics (Messing & Vierra. Gene 19: 259-268 (1982); Bevan et al,

Nature 304:184-187 (1983)), the bar gene, which confers resistance to the herbicide phosphinothricin (White et al., Nucl. Acids Res 18: 1062 (1990), Spencer et al. Theor. Appl.

Genet 79: 625-631 (1990)), the hph gene, which confers resistance to the antibiotic hygromycin (Blochinger & Diggelmann, Mol CeU Biol 4: 2929-2931), and the dhfr gene, which confers resistance to methatrexate (Bourouis et al., EMBO J. 2{7): 1099-1104 (1983)), the EPSPS gene, which confers resistance to glyphosate (U.S. Patent Nos. 4,940,935 and

5,188,642), and the mannose-6-phosphate isomerase gene, which provides the abitity to metabolize mannose (U.S. Patent Nos. 5,767,378 and 5,994,629).

1. Vectors Suitable for Agrobacterium Transformation

Many vectors are avaUable for transformation using Agrobacterium tumefaciens. These typicaUy carry at least one T-DNA border sequence and include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984)) and pXYZ. Below, the construction of two typical vectors suitable for Agrobacterium transformation is described.

a. pCIB200 and pCIB2001: The binary vectors pCIB200 and pCIB2001 are used for the construction of recombinant vectors for use with Agrobacterium and are constructed in the foUowing manner. pTJS75kan is created by Narl digestion of pTJS75 (Schmidhauser & Helinski, J. Bacteriol. 164: 446-455 (1985)) aUowing excision of the tetracycUne-resistance gene, foUowed by insertion of an Accl fragment from pUC4K carrying an NPTII (Vieira & Messing, Gene 19: 259-268 (1982): Bevan et al, Nature 304: 184-187 (1983): McBride et al., Plant Molecular Biology 14: 266- 276 (1990)). Xhol linkers are Ugated to the EcoRV fragment of PCIB7 which contains the left and right T-DNA borders, a plant selectable nos/nptll chimeric gene and the pUC polylinker (Rothstein et al., Gene 53: 153-161 (1987)), and the X/zoZ-digested fragment are cloned into S /7-digested pTJS75kan to create pCIB200 (see also EP 0 332 104, example 19). pCIB200 contains the foUowing unique polylinker restriction sites: EcoRI, Sstl, Kpnl, Bglll, Xbal, and Sail. pCIB2001 is a derivative of pCIB200 created by the insertion into the polylinker of additional restriction sites. Unique restriction sites in the polylinker of pCIB2001 are EcoRI, Sstl, Kpnl, Bglll, Xbal, Sail, MM, Bell, Avrll, Apal, Hpal, and Stul. pCIB2001, in addition to containing these unique restriction sites also has plant and bacterial kanamycin selection, left and right T-DNA borders for Agrobacterium-τ diaXed transformation, the RK2-derived trfA function for mobilization between E. coli and other hosts, and the OriT and OriV functions also from RK2. The pCD32001 polylinker is suitable for the cloning of plant expression cassettes containing their own regulatory signals.

b. pCIBlO and Hygromycin Selection Derivatives thereof:

The binary vector pCIBlO contains a gene encoding kanamycin resistance for selection in plants and T-DNA right and left border sequences and incoφorates sequences from the wide host-range plasmid pRK252 aUowing it to repUcate in both E. coli and Agrobacterium. Its construction is described by Rothstein et al. (Gene 53: 153-161 (1987)). Various derivatives of pCIBlO are constructed which incoφorate the gene for hygromycin B phosphotransferase described by Gritz et al. (Gene 25: 179-188 (1983)). These derivatives enable selection of transgenic plant ceUs on hygromycin only (pCIB743), or hygromycin and kanamycin (pCIB715, pCIB717).

2. Vectors Suitable for non-Agrobacterium Transformation Transformation without the use of Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector and consequently vectors lacking these sequences can be utilized in addition to vectors such as the ones described above which contain T-DNA sequences. Transformation techniques that do not rely on Agrobacterium include transformation via particle bombardment, protoplast uptake (e.g. PEG and electroporation) and microinjection. The choice of vector depends largely on the preferred selection for the species being transformed. Below, the construction of typical vectors suitable for non-Agrobacterium transformation is described.

a. pCH33064: pCIB3064 is a pUC-derived vector suitable for direct gene transfer techniques in combination with selection by the herbicide basta (or phosphinothricin). The plasmid pCIB246 comprises the CaMV 35S promoter in operational fusion to the E. coli GUS gene and the CaMV 35S transcriptional terminator and is described in the PCT pubUshed appUcation WO 93/07278. The 35S promoter of this vector contains two ATG sequences 5' of the start site. These sites are mutated using standard PCR techniques in such a way as to remove the ATGs and generate the restriction sites Sspl and PvuII. The new restriction sites are 96 and 37 bp away from the unique Sail site and 101 and 42 bp away from the actual start site. The resultant derivative of pCH3246 is designated pCIB3025. The GUS gene is then excised from pCIB3025 by digestion with Sail and Sad, the termini rendered blunt and reUgated to generate plasmid pCH33060. The plasmid pHT82 is obtained from the John Innes Centre, Norwich and the a 400 bp Smal fragment containing the bar gene from Streptomyces viridochromogenes is excised and inserted into the Hpal site of pCH33060 (Thompson et al. EMBO J 6: 2519-2523 (1987)). This generated pCIB3064, which comprises the bar gene under the control of the CaMV 35S promoter and terminator for herbicide selection, a gene for ampiciUin resistance (for selection in E. coli) and a polylinker with the unique sites Sphl, Pstl, Hindlll, and BamHI. This vector is suitable for the cloning of plant expression cassettes containing their own regulatory signals.

b. pSOG19 and pSOG35: pSOG35 is a transformation vector that utilizes the E. coli gene dihydrofolate reductase (DFR) as a selectable marker conferring resistance to methotrexate. PCR is used to amplify the 35S promoter (-800 bp), intron 6 from the maize Adhl gene (-550 bp) and 18 bp of the GUS untranslated leader sequence from pSOGlO. A 250-bp fragment encoding the E. coli dihydrofolate reductase type II gene is also amplified by PCR and these two PCR fragments are assembled with a Sacl-Pstl fragment from pB1221 (Clontech) which comprises the pUC19 vector backbone and the nopaUne synthase terminator. Assembly of these fragments generates pSOG19 which contains the 35S promoter in fusion with the intron 6 sequence, the GUS leader, the DHFR gene and the nopaUne synthase terminator. Replacement of the GUS leader in pSOG19 with the leader sequence from Maize Chlorotic Mottle Virus (MCMV) generates the vector pSOG35. pSOG19 and pSOG35 carry the pUC gene for ampiciUin resistance and have Hindlll, Sphl, Pstl and EcoRI sites avaUable for the cloning of foreign substances.

3. Vector Suitable for Chloroplast Transformation

For expression of a nucleotide sequence of the present invention in plant plastids, plastid transformation vector pPH143 (WO 97/32011, example 36) is used. The nucleotide sequence is inserted into pPH143 thereby replacing the PROTOX coding sequence. This vector is then used for plastid transformation and selection of transformants for spectinomycin resistance. Alternatively, the nucleotide sequence is inserted in pPH143 so that it replaces the aadH gene. In this case, transformants are selected for resistance to PROTOX inhibitors.

Example 7: Transformation

Once a nucleic acid sequence of the invention has been cloned into an expression system, it is transformed into a plant ceU. Methods for transformation and regeneration of plants are weU known in the art. For example, Ti plasmid vectors have been utilized for the deUvery of foreign DNA, as weU as direct DNA uptake, Uposomes, electroporation, micro-injection, and microprojectUes. In addition, bacteria from the genus Agrobacterium can be utilized to transform plant ceUs. Below are descriptions of representative techniques for transforming both dicotyledonous and monocotyledonous plants, as weU as a representative plastid transformation technique.

1. Transformation of Dicotyledons Transformation techniques for dicotyledons are weU known in the art and include

Agrobacterium-hased techniques and techniques that do not require Agrobacterium. Non- Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or ceUs. This can be accompUshed by PEG or electroporation mediated uptake, particle bombardment-mediated deUvery, or microinjection. Examples of these techniques are described by Paszkowski et al, EMBO J 3: 2717-2722 (1984), Potrykus et al, Mol. Gen. Genet. 199: 169-177 (1985), Reich et al, Biotechnology 4: 1001-1004 (1986), and Klein et al, Nature 327: 70-73 (1987). In each case the transformed ceUs are regenerated to whole plants using standard techniques known in the art.

Agrobαcter wm-mediated transformation is a preferred technique for transformation of dicotyledons because of its high efficiency of transformation and its broad utUity with many different species. Agrobacterium transformation typicaUy involves the transfer of the binary vector carrying the foreign DNA of interest (e.g. pCH3200 or pCIB2001) to an appropriate Agrobacterium strain which may depend of the complement of vir genes carried by the host Agrobacterium strain either on a co-resident Ti plasmid or chromosomaUy (e.g. strain CD3542 for pCIB200 and pCffi2001 (Uknes et al. Plant CeU 5: 159-169 (1993)). The transfer of the recombinant binary vector to Agrobacterium is accompUshed by a triparental mating procedure using E. coli carrying the recombinant binary vector, a helper E. coli strain which carries a plasmid such as pRK2013 and which is able to mobilize the recombinant binary vector to the target Agrobacterium strain. Alternatively, the recombinant binary vector can be transferred to Agrobacterium by DNA transformation (Hofgen & Willmitzer, Nucl. Acids Res. 16: 9877 (1988)).

Transformation of the target plant species by recombinant Agrobacterium usuaUy involves co-cultivation of the Agrobacterium with explants from the plant and foUows protocols weU known in the art. Transformed tissue is regenerated on selectable medium carrying the antibiotic or herbicide resistance marker present between the binary plasmid T- DNA borders. Another approach to transforming plant ceUs with a gene involves propelling inert or biologicaUy active particles at plant tissues and ceUs. This technique is disclosed in U.S. Patent Nos. 4,945,050, 5,036,006, and 5,100,792. GeneraUy, this procedure involves propelling inert or biologicaUy active particles at the ceUs under conditions effective to penetrate the outer surface of the ceU and afford incoφoration within the interior thereof. When inert particles are utUized, the vector can be introduced into the ceU by coating the particles with the vector containing the desired gene. Alternatively, the target ceU can be surrounded by the vector so that the vector is carried into the ceU by the wake of the particle. BiologicaUy active particles (e.g., dried yeast ceUs, dried bacterium or a bacteriophage, each containing DNA sought to be introduced) can also be propeUed into plant cell tissue.

2. Transformation of Monocotyledons

Transformation of most monocotyledon species has now also become routine. Preferred techniques mclude direct gene transfer into protoplasts using PEG or electroporation techniques, and particle bombardment into caUus tissue. Transformations can be undertaken with a single DNA species or multiple DNA species (i.e. co-transformation) and both these techniques are suitable for use with this invention. Co-transformation may have the advantage of avoiding complete vector construction and of generating transgenic plants with unlinked loci for the gene of interest and the selectable marker, enabling the removal of the selectable marker in subsequent generations, should this be regarded desirable. However, a disadvantage of the use of co-transformation is the less than 100% frequency with which separate DNA species are integrated into the genome (Schocher et al. Biotechnology 4: 1093-1096 (1986)).

Patent AppUcations EP 0 292 435, EP 0 392 225, and WO 93/07278 describe techniques for the preparation of caUus and protoplasts from an eUte inbred line of maize, transformation of protoplasts using PEG or electroporation, and the regeneration of maize plants from transformed protoplasts. Gordon-Kamm et al. (Plant CeU 2: 603-618 (1990)) and Fromm et al. (Biotechnology 8: 833-839 (1990)) have pubUshed techniques for transformation of A188- derived maize line using particle bombardment. Furthermore, WO 93/07278 and Koziel et al. (Biotechnology 11: 194-200 (1993)) describe techniques for the transformation of eUte inbred Unes of maize by particle bombardment. This technique utilizes Unmature maize embryos of 1.5-2.5 mm length excised from a maize ear 14-15 days after pollination and a PDS-lOOOHe BioUstics device for bombardment.

Transformation of rice can also be undertaken by direct gene transfer techniques utilizing protoplasts or particle bombardment. Protoplast-mediated transformation has been described for Japonica-types and Indica-tyip s (Zhang et al. Plant CeU Rep 7: 379-384 (1988); Shimamoto et al. Nature 338: 274-277 (1989); Datta et al. Biotechnology 8: 736-740 (1990)). Both types are also routinely transformable using particle bombardment (Christou et al. Biotechnology 9: 957-962 (1991)). Furthermore, WO 93/21335 describes techniques for the transformation of rice via electroporation. Patent AppUcation EP 0 332 581 describes techniques for the generation, transformation and regeneration of Pooideae protoplasts. These techniques aUow the transformation of Dactylis and wheat. Furthermore, wheat transformation has been described by VasU et al. (Biotechnology 10: 667-674 (1992)) using particle bombardment into ceUs of type C long-term regenerable caUus, and also by VasU et al. (Biotechnology H: 1553-1558 (1993)) and Weeks et al. (Plant Physiol. 102: 1077-1084 (1993)) using particle bombardment of immature embryos and immature embryo-derived caUus. A preferred technique for wheat transformation, however, involves the transformation of wheat by particle bombardment of immature embryos and mcludes either a high sucrose or a high maltose step prior to gene deUvery. Prior to bombardment, any number of embryos (0.75-1 mm in length) are plated onto MS medium with 3% sucrose (Murashiga & Skoog, Physiologia Plantarum 15: 473-497 (1962)) and 3 mg/1 2,4-D for induction of somatic embryos, which is aUowed to proceed in the dark. On the chosen day of bombardment, embryos are removed from the induction medium and placed onto the osmoticum (i.e. induction medium with sucrose or maltose added at the desired concentration, typicaUy 15%). The embryos are aUowed to plasmolyze for 2-3 h and are then bombarded. Twenty embryos per target plate is typical, although not critical. An appropriate gene-carrying plasmid (such as pCIB3064 or pSG35) is precipitated onto micrometer size gold particles using standard procedures. Each plate of embryos is shot with the DuPont BioUstics® heUum device using a burst pressure of -1000 psi using a standard 80 mesh screen. After bombardment, the embryos are placed back into the dark to recover for about 24 h (stiU on osmoticum). After 24 hrs, the embryos are removed from the osmoticum and placed back onto induction medium where they stay for about a month before regeneration. Approximately one month later the embryo explants with developing embryogenic caUus are transferred to regeneration medium (MS + 1 mg/titer NAA, 5 mg/titer GA), further containing the appropriate selection agent (10 mg/1 basta in the case of pCIB3064 and 2 mg/1 methotrexate in the case of pSOG35). After approximately one month, developed shoots are transferred to larger sterUe containers known as "GA7s" which contain half-strength MS, 2% sucrose, and the same concentration of selection agent.

Tranformation of monocotyledons using Agrobacterium has also been described. See, WO 94/00977 and U.S. Patent No. 5,591,616, both incoφorated herein by reference.

3. Transformation of Plastids

Seeds of Nicotiana tabacum c.v. 'Xanthi nc' are germinated seven per plate in a 1" circular array on T agar medium and bombarded 12-14 days after sowing with l jum tungsten particles (M10, Biorad, Hercules, CA) coated with DNA from plasmids pPH143 and pPH145 essentiaUy as described (Svab, Z. and MaUga, P. (1993) PNAS 90, 913-917). Bombarded seedUngs are incubated on T medium for two days after which leaves are excised and placed abaxial side up in bright Ught (350-500 μmol photons/m²/s) on plates of RMOP medium (Svab, Z., Hajdukiewicz, P. and MaUga, P. (1990) PNAS 87, 8526-8530) containing 500 μg/ml spectinomycin dihydrochloride (Sigma, St. Louis, MO). Resistant shoots appearing underneath the bleached leaves three to eight weeks after bombardment are subcloned onto the same selective medium, aUowed to form caUus, and secondary shoots isolated and subcloned. Complete segregation of transformed plastid genome copies (homoplasmicity) in independent subclones is assessed by standard techniques of Southern blotting (Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor). BamHI/EcoRI-digested total ceUular DNA (Mettler, I. J. (1987) Plant Mol Biol Reporter 5, 346-349) is separated on 1 % Tris-borate (TBE) agarose gels, transferred to nylon membranes (Amersham) and probed with ³²P-labeled random primed DNA sequences corresponding to a 0.7 kb BamHI/Ηindlll DNA fragment from pC8 containing a portion of the rps7/12 plastid targeting sequence. Homoplasmic shoots are rooted asepticaUy on spectinomycin-containing MS/IB A medium (McBride, K. E. et al. (1994) PNAS 91, 7301- 7305) and transferred to the greenhouse. Example 8: Breeding

The plants obtained via tranformation with a nucleic acid sequence of the present invention can be any of a wide variety of plant species, including those of monocots and dicots; however, the plants used in the method of the invention are preferably selected from the Ust of agronomicaUy important target crops set forth supra. The expression of a gene of the present invention in combination with other characteristics important for production and quaUty can be incoφorated into plant Unes through breeding. Breeding approaches and techniques are known in the art. See, for example, Welsh J. R., Fundamentals of Plant Genetics and Breeding, John WUey & Sons, NY (1981); Crop Breeding, Wood D. R. (Ed.) American Society of Agronomy Madison, Wisconsin (1983); Mayo O., The Theory of Plant Breeding, 2nd Edition, Clarendon Press, Oxford (1987); Singh, D.P., Breeding for Resistance to Diseases and Insect Pests, Springer- Verlag, NY (1986); Wricke and Weber, Quantitative Genetics and Selection Plant Breeding, Walter de Gruyter and Co., Berlin (1986). The genetic properties engineered into the transgenic seeds and plants described above are passed on by sexual reproduction or vegetative growth and can thus be maintained and propagated in progeny plants. GeneraUy said maintenance and propagation make use of known agricultural methods developed to fit specific puφoses such as tilling, sowing or harvesting. Specialized processes such as hydroponics or greenhouse technologies can also be appUed. As the growing crop is vulnerable to attack and damages caused by msects or mfections as weU as to competition by weed plants, measures are undertaken to control weeds, plant diseases, insects, nematodes, and other adverse conditions to improve yield. These include mechanical measures such a tiUage of the soU or removal of weeds and infected plants, as weU as the appUcation of agrochemicals such as herbicides, fungicides, gametocides, nematicides, growth regulants, ripening agents and insecticides.

Use of. the advantageous genetic properties of the transgenic plants and seeds according to the invention can further be made in plant breeding, which aims at the development of plants with improved properties such as tolerance of pests, herbicides, or stress, improved nutritional value, increased yield, or improved structure causing less loss from lodging or shattering. The various breedmg steps are characterized by weU-defined human intervention such as selecting the lines to be crossed, directing pollination of the parental lines, or selecting appropriate progeny plants. Depending on the desired properties, different breeding measures are taken. The relevant techniques are weU known in the art and include but are not limited to hybridization, inbreeding, backcross breeding, multiline breeding, variety blend, interspecific hybridization, aneuploid techniques, etc. Hybridization techniques also include the sterilization of plants to yield male or female sterUe plants by mechanical, chemical, or biochemical means. Cross pollination of a male sterUe plant with poUen of a different line assures that the genome of the male sterUe but female fertile plant wiU uniformly obtain properties of both parental Unes. Thus, the transgenic seeds and plants according to the invention can be used for the breeding of improved plant Unes, that for example, increase the effectiveness of conventional methods such as herbicide or pestidice treatment or aUow one to dispense with said methods due to their modified genetic properties. Alternatively new crops with improved stress tolerance can be obtained, which, due to their optimized genetic "equipment", yield harvested product of better quaUty than products that were not able to tolerate comparable adverse developmental conditions.

Example 9: Seed Production

In seed production, germination quaUty and uniformity of seeds are essential product characteristics, whereas germination quaUty and uniformity of seeds harvested and sold by the farmer is not important. As it is difficult to keep a crop free from other crop and weed seeds, to control seedborne diseases, and to produce seed with good germination, fairly extensive and weU-defined seed production practices have been developed by seed producers, who are experienced in the art of growing, conditioning and marketing of pure seed. Thus, it is common practice for the farmer to buy certified seed meeting specific quaUty standards instead of using seed harvested from his own crop. Propagation material to be used as seeds is customarUy treated with a protectant coating comprising herbicides, insecticides, fungicides, bactericides, nematicides, moUuscicides, or mixtures thereof. CustomarUy used protectant coatings comprise compounds such as captan, carboxin, thiram (TMTD^®), methalaxyl (Apron^®), and pirimiphos-methyl (ActeUic^®). If desired, these compounds are formulated together with carriers, surfactants or appUcation-promoting adjuvants customarUy employed in formulation art to protect against damage caused by bacterial, fungal or animal pests. The protectant coatmgs may be appUed by impregnating propagation material with a Uquid formulation or by coating with a combined wet or dry formulation. Other methods of appUcation are also possible such as treatment directed at the buds or the fruit.

Example 10: Maize Plant Analysis

Maize plants transformed with plasmids pNOV1436, pNOV1441, and pNOV1313 via Agrobacterium-mediated transformation give 100% mortahty against European cornborer and faU armyworm. ELISA data is set forth below:

Event Plasmid ProMaize T0/T1 ELISA (ng/mg)

Number moter Genotype leaf silk husk pith rind

3275-2 pNOV1436 MTL A188 125/299 4465/1913 4351/2611

3277-2 pNOV1436 MTL A188 218/234 136 798 743/3251 613/3055

3279-1 pNOV1436 MTL A188 108/398 1566/2505 1457/2514

3309-6 pNOV1436 MTL A188 168/326 1164/1017 1527/2391

3324-1 pNOV1436 MTL A188 192 0 203 1068 1437

3330-2 pNOV1436 MTL A188 262/800 0 542 5565 3366

3331-1 pNOV1436 MTL A188 236/347 1010 1341

3338-1 pNOV1436 MTL A188 287/457 13 4578 1795

3357-1 pNOV1436 MTL A188 349/551 61 780 3968 2022

3360-1 pNOV1436 MTL A188 300/428 0 392 2026 1764

3717-2 pNOV1441 Mz Ubi Hi ll 2142 374 1719 NS NS

3723-5 pNOV1441 Mz Ubi Hi ll 2302 13757 7215

3838-1 pNOV1441 Mz Ubi Hi ll 2188 24013 13564

3847-2 pNOV1441 Mz Ubi Hi π 741 699 3707 NS NS

3877-1 pNOV1441 Mz Ubi Hi ll 991 436 1349 15105 10904

3720-1 pNOV1441 Mz Ubi Hi H 1437 3854 2719

3833-3 pNOV1441 Mz Ubi Hi ll 878 166 799

4013-5 pNOV1441 Mz Ubi Hi H 944 174 1918

4029-4 pNOV1441 Mz Ubi Hi ll 1661

4708-1 pNOV1313 Mz Ubi HiU 832

4709-2 pNOV1313 Mz Ubi HiU 581

4710-5 pNOV1313 Mz Ubi HiU 625

4711-2 pNOV1313 Mz Ubi Hill 570

4713-2 pNOV1313 Mz Ubi HiU 962

4717-1 pNOV1313 Mz Ubi HiU 881

MTL = maize metaUothionein-like Mz Ubi = maize ubiquitin Example 11. Rice Plant Analysis

Rice plants transformed with plasmid pNOV1305 via Agrobacterium-mediated transformation give 100% mortaUty against European cornborer and faU armyworm. ELISA data is set forth below:

Event TO

Number Plasmid Promoter ELISA

(ng mg) Leaf

639 pNOV1305 MTL 294

640 pNOV1305 MTL 241

643 pNOV1305 MTL 153

650 pNOV1305 MTL 149

847 pNOV1305 MTL 173

871 pNOV1305 MTL 244

872 pNOV1305 MTL 252

886 pNOV1305 MTL 185

888 pNOV1305 MTL 160

893 pNOV1305 MTL 168

1148 pNOV1305 MTL 1816

1149 pNOV1305 MTL 224

1152 pNOV1305 MTL 173

1154 pNOV1305 MTL 142

1163 pNOV1305 MTL 139

1164 pNOV1305 MTL 138

1167 pNOV1305 MTL 284

1168 pNOV1305 MTL 137

1177 pNOV1305 MTL 167

1349 pNOV1305 MTL 164

1350 pNOV1305 MTL 115

1357 pNOV1305 MTL 132

1363 pNOV1305 MTL 119

1497 pNOV1305 MTL 94

MTL = maize metaUothionein-like Example 12. Cabbage Plant Analysis

Cabbage plants transformed with plasmid pZU578 (SEQ ID NO: 17) via Agrobacterium- mediated transformation were tested against Plutella xylostella (Diamondback moth). Transgenic and control plants were infested with 16 larvae (1-3 instar), 4 on each of 4 leaves transferred with a paint brush from a caged Plutella culture (with cabbage plants). Infested plants were transferred to Ixlxlm cages for the duration of the test. Control plants included non-transformed cabbage plants (susceptible control) and non-transformed cabbage plants sprayed with the commercial Bt pesticide Dipel (resistant control). Scoring (after 2 weeks) was: - = no damage (or only tiny holes = resistant); + = large holes on plant (= susc); ++ many large holes, plant heavUy damaged (= susc). Dipel plants always scored -, susceptible controls scored ++. Insect damage ratings for transgenic and control plants and ELISA data is set forth below.

Event ProDamage TO ELISA

Number Plasmid moter Rating (ng mg)

Leaf

04-05-01-01 pZU578 Act2 ++ 0

04-05-01-02 pZU578 Act2 ++ 0 7-11-01 pZU578 Act2 - 921

10-25-05 pZU578 Act2 ++ 0

10-39-06 pZU578 Act2 - 270 04-F-07 pZU578 Act2 - 04-F-l l pZU578 Act2 - 04-F-15 pZU578 Act2 - 04-F-16 pZU578 Act2 - 04-F-38 pZU578 Act2 - 04-g-07 pZU578 Act2 - 04-g-08 pZU578 Act2 - 04-g-12 pZU578 Act2 - 04-g-21 pZU578 Act2 - 304-g-24 pZU578 Act2

304-H-01 pZU578 Act2

304-H-08 pZU578 Act2

304-H-09 pZU578 Act2

304-H-34 pZU578 Act2

304-H-35 pZU578 Act2

391-J-08 pZU578 Act2

394-F-5 pZU578 Act2

394-H-12 pZU578 Act2

Act2 = Arabidopsis actin 2

The above disclosed embodiments are Ulustrative. This disclosure of the invention will place one skUled in the art in possession of many variations of the invention. All such obvious and foreseeable variations are intended to be encompassed by the present invention.

Claims

What is claimed is:

1. A method for controlling an insect selected from the group consisting of faU armyworm, pink boUworm, tobacco budworm, European cornborer, and diamondback moth comprising deUvering to the insect an effective amount of a hybrid Bacillus thuringiensis toxin comprising domains I and II from a CrylAb toxin joined in the amino to carboxy direction to domam III from a CrylC toxin.

2. The method of claim 1 , wherein the hybrid Bacillus thuringiensis toxin comprises an amino acid sequence at least 90% identical to SEQ ID NO:2, 4, 6, 8, or 10.

3. The method of claim 2, wherein the hybrid Bacillus thuringiensis toxin comprises SEQ ID NO:2, 4, 6, 8, or 10.

4. The method of claim 1, wherein the hybrid Bacillus thuringiensis toxin further comprises a C-terminal taU region.

5. The method of claim 4, wherein the C-terminal taU region is a CrylC taU region.

6. The method of claim 4, wherein the C-terminal taU region is a CrylAb taU region.

7. The method of claim 4, wherein the C-terminal taU region is approximately 40 amino acids in length.

8. The method of claim 1, wherein deUvering an effective amount of the hybrid Bacillus thuringiensis toxin to the insect comprises feeding or contacting the insect with transgenic plant tissue transformed with recombinant DNA comprising a nucleotide sequence that encodes the hybrid Bacillus thuringiensis toxin, wherein expression of the hybrid Bacillus thuringiensis toxin in said transgenic plant tissue confers resistance to the insect.

9. The method of claim 8, wherein said nucleotide sequence is substantiaUy identical to SEQ ID NO: 1, 3, 5, 7, or 9.

10. An isolated nucleic acid molecule comprising a nucleotide sequence that encodes a hybrid Bacillus thuringiensis toxin comprising:

(a) an N-terminal toxin portion comprising domains I and II from a CrylAb toxin joined in the amino to carboxy direction to domain III from a CrylC toxin; and

(b) a C-terminal taU region from a CrylAb toxin.

11. The nucleic acid molecule of claim 10, wherein the hybrid Bacillus thuringiensis toxin comprises an amino acid sequence at least 90% identical to SEQ ID NO:6, 8, or 10.

12. The nucleic acid molecule of claim 11, wherein the hybrid Bacillus thuringiensis toxin comprises SEQ ID NO:6, 8, or 10.

13. The nucleic acid molecule of claim 10, wherein said nucleotide sequence is at least 90% identical to SEQ ID NO:5, 7, or 9.

14. The nucleic acid molecule of claim 13, wherein said nucleotide sequence comprises SEQ ID NO:5, 7, or 9.

15. A chimeric gene comprising a heterologous promoter sequence operatively Unked to the nucleic acid molecule of claim 10.

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

17. A transgenic host ceU comprising the chimeric gene of claim 15.

18. A transgenic host ceU according to claim 17, which is a plant ceU.

19. A transgenic plant comprising the transgenic plant ceU of claim 18.

20. A transgenic plant according to claim 19, which is a maize, cotton, rice, or cabbage plant.

21. Seed from the transgenic plant of claim 19.

22. A method of protecting a plant against insects, comprising expressing a hybrid Bacillus thuringiensis toxin in a plant transformed with a chimeric gene comprising:

(a) a nucleic acid promoter sequence that promotes in a plant the transcription of an associated coding sequence at elevated levels, and

(b) a nucleic acid molecule according to claim 10 operatively linked to said promoter sequence, wherein expression of the hybrid Bacillus thuringiensis toxin in said plant protects said plant against insects.

23. A method of producing a hybrid Bacillus thuringiensis toxin that is active against insects, comprising:

(a) obtaining a transgenic host ceU accordmg to claim 17; and

(b) expressing the nucleic acid molecule in said transgenic host ceU, which results in a hybrid Bacillus thuringiensis toxin that is active against insects.

24. A method of producing a plant resistant to insects, comprising introducing a nucleic acid molecule according to claim 10 into said plant, wherein said nucleic acid molecule is expressible in said plant in an amount effective to control insects.

25. An isolated nucleic acid molecule comprising SEQ ID NO:3, 5, 7, 9, 11, 12, 13, 14, 15, 16, or 17.

26. The nucleic acid molecule of claim 25, comprising SEQ ID NO:3, 5, 7, or 9.

27. The nucleic acid molecule of claim 25, comprising SEQ ID NO: 11, 12, 13, 14, 15, 16, or 17.

28. A chimeric gene comprising a heterologous promoter sequence operatively Unked to the nucleic acid molecule of claim 26.

29. A recombinant vector comprising the chimeric gene of claim 28.

30. A transgenic host ceU comprising the chimeric gene of claim 28.

31. A transgenic host ceU according to claim 30, which is a plant ceU.

32. A transgenic plant comprising the transgenic plant ceU of claim 31.

33. A transgenic plant according to claim 32, which is a maize, cotton, rice, or cabbage plant.

34. Seed from the transgenic plant of claim 33.

35. A transgenic plant ceU comprising the DNA molecule of claim 27.

36. A transgenic plant comprising the transgenic plant ceU of claim 35.

37. A transgenic plant according to claim 36, which is a maize, cotton, rice, or cabbage plant.

38. Seed from the transgenic plant of claim 36.

39. A hybrid Bacillus thuringiensis toxm, comprismg:

(a) an N-terminal toxin portion comprising domams I and II from a CrylAb toxin joined in the amino to carboxy direction to domain III from a CrylC toxin; and (b) a C- terminal taU region from a CrylAb toxin.

40. The hybrid Bacillus thuringiensis toxin of claim 39, comprising an amino acid sequence at least 90% identical to SEQ ID NO:6, 8, or 10.

41. The hybrid Bacillus thuringiensis toxin of claim 40, comprising SEQ ID NO:6, 8, or 10.

42. A composition comprising the hybrid Bacillus thuringiensis toxin of claim 39 in an amount effective to control insects.