WO1999042589A2 - Insecticidal toxins from photorhabdus - Google Patents

Abstract

Novel nucleic acid sequences isolated from Photorhabdus luminescens, whose expression results in novel insecticidal toxins, are disclosed herein. The invention also discloses compositions and formulations containing the insecticidal toxins that are capable of controlling insect pests. The invention is further drawn to methods of making the toxins and to methods of using the nucleotide sequences, for example in microorganisms to control insect pests or in transgenic plants to confer insect resistance.

Description

INSECTICIDAL TOXINS FROM PHOTORHABDUS

The invention relates to novel toxins from Photorhabdus luminescens, 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, about $7.7 billion 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 home owners.

Insect pests are mainly controlled by intensive applications 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 partially alleviated 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 like d-endotoxins, have also been applied with satisfactory results, offering an alternative or a complement to chemical insecticides. Recently, the genes coding for some of these d-endotoxins have been isolated and their expression in heterologous hosts have been shown to provide another tool for the control of economically important insect pests. In particular, the expression of insecticidal toxins in transgenic plants, such as Bacillus thuringiensis d- endotoxins, has provided efficient protection against selected insect pests, and transgenic plants expressing such toxins have been commercialized, allowing farmers to reduce applications of chemical insect control agents. Yet, even in this case, the development of resistance remains a possibility and only a few specific insect pests are controllable. Consequently, there remains a long-felt but unfulfilled need to discover new and effective insect control agents that provide an economic benefit to farmers and that are environmentally acceptable.

The present invention addresses the need for novel insect control agents. Particularly needed are control agents that are targeted to economically important insect pests and that efficiently control insect strains resistant to existing insect control agents. Furthermore, agents whose application minimizes the burden on the environment are desirable.

In the search of novel insect control agents, certain classes of nematodes from the genera Heterorhabdus and Steinernema are of particular interest because of their insecticidal properties. They kill insect larvae and their offspring feed in the dead larvae. Indeed, the insecticidal activity is due to symbiotic bacteria living in the nematodes. These symbiotic bacteria are Photorhabdus in the case of Heterorhabdus and Xenorhabdus in the case of Steinernema.

The present invention is drawn to nucleic acid sequences isolated from Photorhabdus luminescens, and sequences substantially similar thereto, whose expression results in toxins that are highly toxic to economically important insect pests, particularly insect pests that infest plants. The invention is further drawn to the toxins resulting from the expression of the nucleic acid sequences, and to compositions and formulations containing the toxins, which are capable of inhibiting the ability 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 toxins and to methods of using the nucleic acid sequences, for example in microorganisms to control insects or in transgenic plants to confer insect resistance, and to a method of using the toxins, and compositions and formulations comprising the toxins, for example applying the toxins or compositions or formulations to insect-infested areas, or to prophylactically treat insect-susceptible areas or plants to confer protection or resistance to the insects.

The novel toxins are highly active against insects. For example, a number of economically important insect pests, such as the Lepidopterans Plutella xylostella (Diamondback Moth), Trichoplusia ni (Cabbage Looper), Ostrinia nubilalis (European Corn Borer), Heliothis virescens (Tobacco Budworm), Helicoverpa zea (Corn Earworm), Manduca sexta (Tobacco Hornworm), Spodoptera exigua (Beet Armyworm), and Spodoptera frugiperda (Fall Armyworm), as well as the Coleopterans Diabrotica virgifera virgifera (Western Corn Rootworm), Diabrotica undecimpunctata howardi (Southern Corn Rootworm), and Leptinotarsa decimlineata (Colorado Potato Beetle) can be controlled by one or more of the toxins. The 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 an isolated nucleic acid molecule comprising: (a) a nucleotide sequence substantially similar to a nucleotide sequence selected from the group consisting of: nucleotides 412-1665 of SEQ ID NO:1 , nucleotides 1686-2447 of SEQ ID NO:1 , nucleotides 2758-3318 of SEQ ID NO:1 , nucleotides 3342-4118 of SEQ ID NO:1 , nucleotides 4515-9269 of SEQ ID NO:1 , nucleotides 15,171 -18,035 of SEQ ID NO:11 , and nucleotides 31 ,393-35,838 of SEQ ID NO:11 ; (b) a nucleotide sequence comprising nucleotides 23,768-31 ,336 of SEQ ID NO:11 ; or (c) a nucleotide sequence isocoding with the nucleotide sequence of (a) or (b); wherein expression of the nucleic acid molecule results in at least one toxin that is active against insects.

In one embodiment of this aspect, the nucleotide sequence is isocoding with a nucleotide sequence substantially similar to nucleotides 412-1665 of SEQ ID NO:1 , nucleotides 1686-2447 of SEQ ID NO:1 , nucleotides 2758-3318 of SEQ ID NO:1 , nucleotides 3342-41 18 of SEQ ID NO:1 , or nucleotides 4515-9269 of SEQ ID NO:1. Preferably, the nucleotide sequence is substantially similar to nucleotides 412-1665 of SEQ ID NO:1 , nucleotides 1686-2447 of SEQ ID NO:1 , nucleotides 2758-3318 of SEQ ID NO:1 , nucleotides 3342-41 18 of SEQ ID NO:1 , or nucleotides 4515-9269 of SEQ ID NO:1. More preferably, the nucleotide sequence encodes an amino acid sequence selected from the group consisting of SEQ ID NOs:2-6. Most preferably, the nucleotide sequence comprises nucleotides 412-1665 of SEQ ID NO:1 , nucleotides 1686-2447 of SEQ ID NO:1 , nucleotides 2758-3318 of SEQ ID NO:1 , nucleotides 3342-4118 of SEQ ID NO:1 , or nucleotides 4515- 9269 of SEQ ID NO:1.

In another embodiment of this aspect, the nucleotide sequence is isocoding with a nucleotide sequence substantially similar to nucleotides 15,171 -18,035 of SEQ ID NO:1 1. Preferably, the nucleotide sequence is substantially similar to nucleotides 15,171 -18,035 of SEQ ID NO:11. More preferably, the nucleotide sequence encodes the amino acid sequence set forth in SEQ ID NO:12. Most preferably, the nucleotide sequence comprises nucleotides 15,171 -18,035 of SEQ ID NO:11.

In still another embodiment of this aspect, the nucleotide sequence is isocoding with a nucleotide sequence substantially similar to nucleotides 31 ,393-35,838 of SEQ ID NO:1 1. Preferably, the nucleotide sequence is substantially similar to nucleotides 31 ,393-35,838 of SEQ ID NO:1 1. More preferably, the nucleotide sequence encodes the amino acid sequence set forth in SEQ ID NO: 14. Most preferably, the nucleotide sequence comprises nucleotides 31 ,393-35,838 of SEQ ID NO:1 1. In yet another embodiment of this aspect, the nucleotide sequence encodes the amino acid sequence set forth in SEQ ID NO:13, and preferably comprises nucleotides 23,768-31 ,336 of SEQ ID NO:1 1 .

In one embodiment, the nucleotide sequence of the invention comprises the approximately 9.7 kb DNA fragment harbored in E. coll strain DH5a, designated as NRRL accession number B-21835.

In another embodiment, the nucleotide sequence of the invention comprises the approximately 38 kb DNA fragment harbored in E. coil strain DH5a, designated as NRRL accession number B-30077.

In still another embodiment, the nucleotide sequence of the invention comprises the approximately 22.2 kb DNA fragment harbored in E. coli strain DH5a, designated as NRRL accession number B-30078.

According to one embodiment of the invention, the toxins resulting from expression of the nucleic acid molecules of the invention have activity against Lepidopteran insects. Preferably, according to this embodiment, the toxins have activity against Plutella xylostella (Diamondback Moth), Trichoplusia ni (Cabbage Looper), Ostrinia nubilalis (European Corn Borer), Heliothis virescens (Tobacco Budworm), Helicoverpa zea (Corn Earworm), Spodoptera exigua (Beet Armyworm), and Spodoptera frugiperda (Fall Armyworm).

According to another embodiment of the invention, the toxins resulting from expression of the nucleic acid molecule of the invention have activity against Lepidopteran and Coleopteran insects. Preferably, according to this embodiment, the toxins have insecticidal activity against Plutella xylostella (Diamondback Moth), Ostrinia nubilalis (European Corn Borer), and Manduca sexta (Tobacco Homworm), Diabrotica virgifera virgifera (Western Corn Rootworm), Diabrotica undecimpunctata howardi (Southern Corn Rootworm), and Leptinotarsa decimlineata (Colorado Potato Beetle).

In another aspect, the present invention provides an isolated nucleic acid molecule comprising a 20 base pair nucleotide portion identical in sequence to a consecutive 20 base pair nucleotide portion of a nucleotide sequence selected from the group consisting of: nucleotides 412-1665 of SEQ ID NO:1 , nucleotides 1686-2447 of SEQ ID NO:1 , nucleotides 2758-3318 of SEQ ID NO:1 , nucleotides 3342-4118 of SEQ ID NO:1 , nucleotides 4515- 9269 of SEQ ID NO:1 , nucleotides 15,171 -18,035 of SEQ ID NO:1 1 , and nucleotides 31 ,393-35,838 of SEQ ID NO:1 1 , wherein expression of the nucleic acid molecule results in at least one toxin that is active against insects. ln one embodiment of this aspect, the isolated nucleic acid molecule of the invention comprises a 20 base pair nucleotide portion identical in sequence to a consecutive 20 base pair nucleotide portion of nucleotides 412-1665 of SEQ ID NO:1 , nucleotides 1686-2447 of SEQ ID NO:1 , nucleotides 2758-3318 of SEQ ID NO:1 , nucleotides 3342-41 18 of SEQ ID NO:1 , or nucleotides 4515-9269 of SEQ ID NO:1.

In another embodiment of this aspect, the isolated nucleic acid molecule of the invention comprises a 20 base pair nucleotide portion identical in sequence to a consecutive 20 base pair nucleotide portion of nucleotides 15,171 -18,035 of SEQ ID NO:11.

In still another embodiment of this aspect, the isolated nucleic acid molecule of the invention comprises a 20 base pair nucleotide portion identical in sequence to a consecutive 20 base pair nucleotide portion of nucleotides 31 ,393-35,838 of SEQ ID NO:1 1.

In a further aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence from Photorhabdus luminescens selected from the group consisting of: nucleotides 412-1665 of SEQ ID NO:1 , nucleotides 1686-2447 of SEQ ID NO:1 , nucleotides 2758-3318 of SEQ ID NO:1 , nucleotides 3342-4118 of SEQ ID NO:1 , nucleotides 4515-9269 of SEQ ID NO:1 , nucleotides 66-1898 of SEQ ID NO:11 , nucleotides 2416-9909 of SEQ ID NO:11 , the complement of nucleotides 2817-3395 of SEQ ID NO:11 , nucleotides 9966-14,633 of SEQ ID NO:11 , nucleotides 14,699-15,007 of SEQ ID NO:11 , nucleotides 15,171 -18,035 of SEQ ID NO:1 1 , the complement of nucleotides 17,072-17,398 of SEQ ID NO:1 1 , the complement of nucleotides 18,235- 19,167 of SEQ ID NO:11 , the complement of nucleotides 19,385-20,1 16 of SEQ ID NO:11 , the complement of nucleotides 20,217-20,963 of SEQ ID NO:1 1 , the complement of nucleotides 22,172-23,086 of SEQ ID NO:11 , nucleotides 23,768-31 ,336 of SEQ ID NO:11 , nucleotides 31 ,393-35,838 of SEQ ID NO:1 1 , the complement of nucleotides 35,383- 35,709 of SEQ ID NO:1 1 , the complement of nucleotides 36,032-36,661 of SEQ ID NO:1 1 , and the complement of nucleotides 36,654-37,781 of SEQ ID NO:1 1.

The present invention also provides a chimeric gene comprising a heterologous promoter sequence operatively linked to the nucleic acid molecule of the invention. Further, the present invention provides a recombinant vector comprising such a chimeric gene. Still further, the present invention provides a host cell comprising such a chimeric gene. A host cell according to this aspect of the invention may be a bacterial cell, a yeast cell, or a plant cell, preferably a plant cell. Even further, the present invention provides a plant comprising such a plant cell. Preferably, the plant is maize.

In yet another aspect, the present invention provides toxins produced by the expression of DNA molecules of the present invention.

According to one embodiment, the toxins of the invention have activity against Lepidopteran insects, preferably against Plutella xylostella (Diamondback Moth), Trichoplusia ni (Cabbage Looper), Ostrinia nubilalis (European Corn Borer), Heliothis virescens (Tobacco Budworm), Helicoverpa zea (Corn Earworm), Spodoptera exigua (Beet Armyworm), and Spodoptera frugiperda (Fall Armyworm).

According to another embodiment, the toxins of the invention have activity against Lepidopteran and Coleopteran insects, preferably against Plutella xylostella (Diamondback Moth), Ostrinia nubilalis (European Corn Borer), and Manduca sexta (Tobacco Homworm), Diabrotica virgifera virgifera (Western Corn Rootworm), Diabrotica undecimpunctata howardi (Southern Corn Rootworm), and Leptinotarsa decimlineata (Colorado Potato Beetle).

In one embodiment, the toxins are produced by the E. coli strain designated as NRRL accession number B-21835.

In another embodiment, the toxins are produced by E. coli strain designated as NRRL accession number B-30077.

In still another embodiment, the toxins are produced by E. coli strain designated as NRRL accession number B-30078.

In one embodiment, a toxin of the invention comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs:2-6.

In another embodiment, a toxin of the invention comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs:12-14.

The present invention also provides a composition comprising an insecticidally effective amount of a toxin according to the invention.

In another aspect, the present invention provides a method of producing a toxin that is active against insects, comprising: (a) obtaining a host cell comprising a chimeric gene, which itself comprises a heterologous promoter sequence operatively linked to the nucleic acid molecule of the invention; and (b) expressing the nucleic acid molecule in the cell, which results in at least one toxin that is active against insects. ln a further aspect, the present invention provides a method of producing an insect- resistant plant, comprising introducing a nucleic acid molecule of the invention into the plant, wherein the nucleic acid molecule is expressible in the plant in an effective amount to control insects. According to one embodiment, the insects are Lepidopteran insects, preferably selected from the group consisting of: Plutella xylostella (Diamondback Moth), Trichoplusia ni (Cabbage Looper), Ostrinia nubilalis (European Corn Borer), Heliothis virescens (Tobacco Budworm), Helicoverpa zea (Corn Earworm), Spodoptera exigua (Beet Armyworm), and Spodoptera frugiperda (Fall Armyworm). According to another embodiment, the insects are Lepidopteran and Coleopteran insects, preferably selected from the group consisting of: Plutella xylostella (Diamondback Moth), Ostrinia nubilalis (European Corn Borer), and Manduca sexta (Tobacco Homworm), Diabrotica virgifera virgifera (Western Corn Rootworm), Diabrotica undecimpunctata howardi (Southern Corn Rootworm), and Leptinotarsa. decimlineata (Colorado Potato Beetle).

In still a further aspect, the present invention provides a method of controlling insects comprising delivering to the insects an effective amount of a toxin according to the present invention. According to one embodiment, the insects are Lepidopteran insects, preferably selected from the group consisting of: Plutella xylostella (Diamondback Moth), Trichoplusia ni (Cabbage Looper), Ostrinia nubilalis (European Corn Borer), Heliothis virescens (Tobacco Budworm), Helicoverpa zea (Corn Earworm), Spodoptera exigua (Beet Armyworm), and Spodoptera frugiperda (Fall Armyworm). According to another embodiment, the insects are Lepidopteran and Coleopteran insects, preferably selected from the group consisting of: Plutella xylostella (Diamondback Moth), Ostrinia nubilalis (European Corn Borer), and Manduca sexta (Tobacco Hornworm), Diabrotica virgifera virgifera (Western Corn Rootworm), Diabrotica undecimpunctata howardi (Southern Corn Rootworm), and Leptinotarsa decimlineata (Colorado Potato Beetle). Preferably, the toxin is delivered to the insects orally.

Yet another aspect of the present invention is the provision of a method for mutagenizing a nucleic acid molecule according to the present invention, wherein the nucleic acid molecule has been cleaved into population of double-stranded random fragments of a desired size, comprising: (a) adding to the population of double-stranded random fragments one or more single- or double-stranded oligonucleotides, wherein the oligonucleotides each comprise an area of identity and an area of heterology to a double- stranded template polynucleotide; (b) denaturing the resultant mixture of double-stranded random fragments and oligonucleotides into single-stranded fragments; (c) incubating the resultant population of single-stranded fragments with a polymerase under conditions which result in the annealing of the single-stranded fragments at the areas of identity to form pairs of annealed fragments, the areas of identity being sufficient for one member of a pair to prime replication of the other, thereby forming a mutagenized double-stranded polynucleotide; and (d) repeating the second and third steps for at least two further cycles, wherein the resultant mixture in the second step of a further cycle includes the mutagenized double-stranded polynucleotide from the third step of the previous cycle, and wherein the further cycle forms a further mutagenized double-stranded polynucleotide.

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

DEFINITIONS

"Activity" of the toxins of the invention is meant that the toxins function as orally 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 delivered to the insect, the result is typically death of the insect, or the insect does not feed upon the source that makes the toxin available to the insect.

"Associated with / operatively linked" refer to two nucleic acid sequences that are related physically or functionally. 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 will affect the expression level of the coding or structural DNA sequence.

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 normally operatively linked 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.

To "control" insects means to inhibit, through a toxic effect, the ability 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.

To "deliver" 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 delivered in many recognized ways, e.g., orally 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 delivery system.

"Expression cassette" as used herein means a nucleic acid sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically 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 naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, 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 naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell 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 cell is exposed to some particular external stimulus. In the case of a multicellular organism, such as a plant, the promoter can also be specific to a particular tissue, or organ, or stage of development.

A "gene" is a defined region that is located within a genome and that, besides the aforementioned coding nucleic acid sequence, comprises other, primarily regulatory, nucleic acid sequences responsible for the control of the expression, that is to say the transcription and translation, of the coding portion. A gene may also comprise other 5' and 3' untranslated sequences and termination sequences. Further elements that may be present are, for example, introns.

"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 capability. The "gene of interest" may also be one that is transferred to plants for the production of commercially valuable enzymes or metabolites in the plant.

A "heterologous" nucleic acid sequence is a nucleic acid sequence not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleic acid sequence.

A "homologous" nucleic acid sequence is a nucleic acid sequence naturally associated with a host cell into which it is introduced.

"Homologous recombination" is the reciprocal exchange of nucleic acid fragments between homologous nucleic acid molecules.

"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 cell.

A "nucleic acid molecule" or "nucleic acid sequence" is a linear segment of single- or double-stranded DNA or RNA that can be isolated from any source. In the context of the present invention, the nucleic acid molecule is preferably a segment of DNA.

"ORF" means open reading frame.

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

A "plant cell" is a structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, plant tissue, a plant organ, or a whole plant. "Plant cell culture" means cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development.

"Plant material" refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell 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 cells 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 cells 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 listed 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 cell without a cell wall or with only parts of the cell wall.

"Regulatory elements" refer to sequences involved in controlling the expression of a nucleotide sequence. Regulatory elements comprise a promoter operably linked to the nucleotide sequence of interest and termination signals. They also typically encompass sequences required for proper translation of the nucleotide sequence.

In its broadest sense, the term "substantially similar", when used herein with respect to a nucleotide sequence, means a nucleotide sequence corresponding to a reference nucleotide sequence, wherein the corresponding sequence encodes a polypeptide having substantially the same structure and function as the polypeptide encoded by the reference nucleotide sequence, e.g. where only changes in amino acids not affecting the polypeptide function occur. Desirably the substantially similar nucleotide sequence encodes the polypeptide encoded by the reference nucleotide sequence. The percentage of identity between the substantially similar nucleotide sequence and the reference nucleotide sequence desirably is at least 80%, more desirably at least 85%, preferably at least 90%, more preferably at least 95%, still more preferably at least 99%. A nucleotide sequence "substantially similar" to reference nucleotide sequence hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0₄, 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 NaP0₄, 1 mM EDTA at 50°C with washing in 1 X SSC, 0.1% SDS at 50°C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0₄, 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 NaP0₄, 1 mM EDTA at 50°C with washing in 0.1 X SSC, 0.1% SDS at 50°C, more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaP0₄, 1 mM EDTA at 50°C with washing in 0.1 X SSC, 0.1% SDS at 65°C.

"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 cell or organism. In particular, "transformation" means the stable integration of a DNA molecule into the genome of an organism of interest.

'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-replicating. Transformed cells, 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 wild-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 following standard abbreviations: adenine (A), cytosine (C), thymine (T), and guanine (G). Amino acids are likewise indicated by the following 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), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). Furthermore, (Xaa; X) represents any amino acid. BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING

SEQ ID NO:1 is the sequence of the approximately 9.7 kb DNA fragment comprised in pCIB9359-7 which comprises the following ORFs at the specified nucleotide positions:

Name Start End orf1 412 1665 orf2 1686 2447 orf3 2758 3318 orf4 3342 41 18 orfδ 4515 9269

SEQ ID NO:2 is the sequence of the -46.4 kDa protein encoded by orfl of SEQ ID NO:1. SEQ ID NO:3 is the sequence of the -28.1 kDa protein encoded by orf2 of SEQ ID NO:1. SEQ ID NO:4 is the sequence of the -20.7 kDa protein encoded by orf3 of SEQ ID NO:1. SEQ ID NO:5 is the sequence of the -28.7 kDa protein encoded by orf4 of SEQ ID NO:1. SEQ ID NO:6 is the sequence of the -176 kDa protein encoded by orfδ of SEQ ID NO:1. SEQ ID NOs:7-10 are oligonucleotides.

SEQ ID NO:11 is the sequence of the approximately 38 kb DNA fragment comprised in pNOV2400, which comprises the following ORFs at the specified nucleotide positions (descending numbers and "C" indicates that the ORF is on the complementary strand):

Name Start End orf7 66 1898 ( (pPartial sequence) hph3 2416 9909 orfl 8 3395 2817 C orf4 9966 14,633 orfl 9 14,699 15,007 orfδ 15,171 18,035 orf22 17,398 17,072 C orflO 19,167 18,235 C orfl 4 20,116 19,385 C orfl 3 20,963 20,217 C orfM 23,086 22,172 C hph2 23,768 31 ,336 orf2 31 ,393 35,838 orf21 35,709 35,383 C orfl 6 36,661 36,032 C orfδ 37,781 36,654 C

SEQ ID NO:1 1 also includes the following restriction sites, some of which are used in the subcloning steps set forth in Example 17:

Restriction Site Nucleotide Position(s)

Λcclll 2835

SamHI 18,915

Bsm \ 11 ,350

BstΛ 1071 29,684

Eagl 13,590; 31 ,481

Eco721 34,474

Mlυ\ 2444; 5116; 9327; 26,204

Λ/ofl 13,589

Pad 9915; 23,3δ3; 37,888

Pvu\ 8816

Sapl 35,248

SeλAI 28,946

Sgf\ 8815

Spel 2167; 3769; 7831 ; 11 ,168

Sph\ 755

Stu\ 35,690

Tf/7111 I 21 ,443

SEQ ID NO:12 is the sequence of the protein encoded by orfδ of SEQ ID NO:1 1. SEQ ID NO:13 is the sequence of the protein encoded by hph2 of SEQ ID NO:11 . SEQ ID NO:14 is the sequence of the protein encoded by orf2 of SEQ ID NO:1 1 . SEQ ID NOs:15-22 are oligonucleotides.

DEPOSITS

The following material has been deposited with the Agricultural Research Service, Patent Culture Collection (NRRL), 1815 North University Street, Peoria, Illinois 61604, under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. All restrictions on the availability of the deposited material will be irrevocably removed upon the granting of a patent.

Clone Accession Number Date of Deposit pCIB9359-7 NRRL B-21836 September 17, 1997 pNOV2400 NRRL B-30077 December 3, 1998 pNOV1001 NRRL B-30078 December 3, 1998

Novel Nucleic Acid Sequences whose Expression Results in Insecticidal Toxins

This invention relates to nucleic acid sequences whose expression results in novel toxins, and to the making and using of the toxins to control insect pests. The nucleic acid sequences are derived from Photorhabdus luminescens, a member of the Enterobacteriaceae family. P. luminescens is a symbiotic bacterium of nematodes of the genus Heterorhabditis. The nematodes colonize insect larva, kill them, and their offspring feed on the dead larvae. The insecticidal activity is actually produced by the symbiotic P. luminescens bacteria. The inventors are the first to isolate the nucleic acid sequences of the present invention from P. luminescens (ATCC strain number 29999). The expression of the nucleic acid sequences of the present invention results in toxins that can be used to control Lepidopteran insects such as Plutella xylostella (Diamondback Moth), Trichoplusia ni (Cabbage Looper), Ostrinia nubilalis (European Corn Borer), Heliothis virescens (Tobacco Budworm), Helicoverpa zea (Corn Earworm), Manduca sexta (Tobacco Hornworm), Spodoptera exigua (Beet Armyworm), and Spodoptera frugiperda (Fall Armyworm), as well as Coleopteran insects such as Diabrotica virgifera virgifera (Western Corn Rootworm), Diabrotica undecimpunctata howardi (Southern Corn Rootworm), Diabrotica longicornis barberi (Northern Corn Rootworm), and Leptinotarsa decimlineata (Colorado Potato Beetle).

In one preferred embodiment, the invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence substantially similar to the approximately 9.7 kb nucleic acid sequence set forth in SEQ ID NO:1 , whose expression results in insect control activity (further illustrated in Examples 1 -11 ). Five open reading frames (ORFs) are present in the nucleic acid sequence set forth in SEQ ID NO:1 , coding for proteins of predicted sizes 45 kDa, 28 kDa, 21 kDA, 29 kDa, and 176 kDa. The five ORFs are arranged in an operon- like structure. When expressed in a heterologous host, the - 9.7 kb DNA fragment from P. luminescens results in insect control activity against Lepidopterans such as Plutella xylostella (Diamondback Moth), Trichoplusia ni (Cabbage Looper), Ostrinia nubilalis (European Corn Borer), Heliothis virescens (Tobacco Budworm), Helicoverpa zea (Corn Earworm), Spodoptera exigua (Beet Armyworm), and Spodoptera frugiperda (Fall Armyworm), showing that expression of the - 9.7 kb nucleotide sequence set forth in SEQ ID NO:1 is necessary and sufficient for such insect control activity. In a preferred embodiment, the invention encompasses a DNA molecule, whose expression results in an insecticidal toxin, which is deposited in the E. coli strain pCIB9359-7 (NRRL accession number B-2183δ).

In another preferred embodiment, the invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence substantially similar to the approximately 38 kb nucleic acid fragment set forth in SEQ ID NO:11 and deposited in the E. coli strain pNOV2400 (NRRL accession number B-30077), whose expression results in insect control activity (see Examples 12-18). In a more preferred embodiment, the invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence substantially similar to the - 22 kb DNA fragment deposited in the E. coli strain pNOV1001 (NRRL accession number B-30078), whose expression results in insect control activity. In a most preferred embodiment, the invention encompasses isolated nucleic acid molecules comprising nucleotide sequences substantially similar to the three ORFs corresponding to nucleotides 23,768-31 ,336 (hph2), 31 ,393-36,838 (orf2), and 16,171 -18,035 (oι15) of the DNA fragment set forth in SEQ ID NO:11 , as well as the proteins encoded thereby. When co-expressed in a heterologous host, these three ORFs result in insect control activity against Lepidopterans such as Plutella xylostella (Diamondback Moth), Ostrinia nubilalis (European Corn Borer), and Manduca sexta (Tobacco Hornworm), as well as against Coleopterans such as Diabrotica virgifera virgifera (Western Corn Rootworm), Diabrotica undecimpunctata howardi (Southern Corn Rootworm), and Leptinotarsa decimlineata (Colorado Potato Beetle), showing that co-expression of these three ORFs (hph2, orf2, and orf5) is necessary and sufficient for such insect control activity.

The present invention also 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 cell capable of expressing the nucleotide sequences. Such regulatory elements usually comprise promoter and termination signals and preferably also comprise elements allowing efficient translation of polypeptides encoded by the nucleic acid sequences of the present invention. Vectors comprising the nucleic acid sequences are usually capable of replication in particular host cells, preferably as extrachromosomal molecules, and are therefore used to amplify the nucleic acid sequences of this invention in the host cells. In one embodiment, host cells for such vectors are microorganisms, such as bacteria, in particular E.coli. In another embodiment, host cells for such recombinant vectors are endophytes or epiphytes. A preferred host cell for such vectors is a eukaryotic cell, such as a yeast, a plant cell, or an insect cell. Plant cells such as maize cells are most preferred host cells. In another preferred embodiment, such vectors are viral vectors and are used for replication of the nucleotide sequences in particular host cells, e.g. insect cells or plant cells. Recombinant vectors are also used for transformation of the nucleotide sequences of this invention into host cells, whereby the nucleotide sequences are stably integrated into the DNA of such host cells. In one, such host cells are prokaryotic cells. In a preferred embodiment, such host cells are eukaryotic cells, such as yeast cells, insect cells, or plant cells. In a most preferred embodiment, the host cells are plant cells, such as maize cells.

In preferred embodiments, the insecticidal toxins of the invention each comprise at least one polypeptide encoded by a nucleotide sequence of the invention. In another preferred embodiment, the insecticidal toxins are produced from a purified strain of P. luminescens, such the strain with ATTC accession number 29999. The toxins of the present invention have insect control activity when tested against insect pests in bioassays; and these properties of the insecticidal toxins are further illustrated in Examples 1 -18. The insecticidal toxins desribed in the present invention are further characterized in that their molecular weights are larger than 6,000, as found by size fractionation experiments. The insecticidal toxins retain full insectidical activity after being stored at 4°C for 2 weeks. One is also shown to retain its full insecticidal activity after being freeze-dried and stored at 22°C for 2 weeks. However, the insecticidal toxins of the invention lose their insecticidal activity after incubation for 5 minutes at 100°C.

In further embodiments, the nucleotide sequences of the invention can be modified by incorporation of random mutations in a technique known as in-vitro recombination or DNA shuffling. This technique is described in Stemmer et al., Nature 370: 389-391 (1994) and US Patent 6,605,793, which are incorporated herein by reference. Millions of mutant copies of a nucleotide sequence are produced based on an original nucleotide sequence of this invention and variants with improved properties, such as increased insecticidal activity, enhanced stability, or different specificity or range of target insect pests are recovered. The method encompasses forming a mutagenized double-stranded polynucleotide from a template double-stranded polynucleotide comprising a nucleotide sequence of this invention, wherein the template double-stranded polynucleotide has been cleaved into double-stranded-random fragments of a desired size, and comprises the steps of adding to the resultant population of double-stranded random fragments one or more single or double-stranded oligonucleotides, wherein said oligonucleotides comprise an area of identity and an area of heterology to the double-stranded template polynucleotide; denaturing the resultant mixture of double-stranded random fragments and oligonucleotides into single-stranded fragments; incubating the resultant population of single-stranded fragments with a polymerase under conditions which result in the annealing of said single- stranded fragments at said areas of identity to form pairs of annealed fragments, said areas of identity being sufficient for one member of a pair to prime replication of the other, thereby forming a mutagenized double-stranded polynucleotide; and repeating the second and third steps for at least two further cycles, wherein the resultant mixture in the second step of a further cycle includes the mutagenized double-stranded polynucleotide from the third step of the previous cycle, and the further cycle forms a further mutagenized double-stranded polynucleotide. In a preferred embodiment, the concentration of a single species of double- stranded random fragment in the population of double-stranded random fragments is less than 1 % by weight of the total DNA. In a further preferred embodiment, the template double-stranded polynucleotide comprises at least about 100 species of polynucleotides. In another preferred embodiment, the size of the double-stranded random fragments is from about 5 bp to 5 kb. In a further preferred embodiment, the fourth step of the method comprises repeating the second and the third steps for at least 10 cycles.

Expression of the Nucleotide Sequences in Heterologous Microbial Hosts

As biological insect control agents, the insecticidal toxins are produced by expression of the nucleotide sequences in heterologous host cells capable of expressing the nucleotide sequences. In a first embodiment, P. luminescens cells comprising modifications of at least one nucleotide sequence of this invention at its chromosomal location are described. Such modifications encompass mutations or deletions of existing regulatory elements, thus leading to altered expression of the nucleotide sequence, or the incorporation of new regulatory elements controlling the expression of the nucleotide sequence. In another embodiment, additional copies of one or more of the nucleotide sequences are added to P. luminescens cells either by insertion into the chromosome or by introduction of extrachromosomally replicating molecules containing the nucleotide sequences.

In another embodiment, at least one of the nucleotide sequences of the invention is inserted into an appropriate expression cassette, comprising a promoter and termination signals. Expression of the nucleotide sequence is constitutive, or an inducible promoter responding to various types of stimuli to initiate transcription is used. In a preferred embodiment, the cell in which the toxin is expressed is a microorganism, such as a virus, a bacteria, or a fungus. In a preferred embodiment, a virus, such as a baculovirus, contains a nucleotide sequence of the invention in its genome and expresses large amounts of the corresponding insecticidal toxin after infection of appropriate eukaryotic cells that are suitable for virus replication and expression of the nucleotide sequence. The insecticidal toxin thus produced is used as an insecticidal agent. Alternatively, baculoviruses engineered to include the nucleotide sequence are used to infect insects in-vivo and kill them either by expression of the insecticidal toxin or by a combination of viral infection and expression of the insecticidal toxin.

Bacterial cells are also hosts for the expression of the nucleotide sequences of the invention. In a preferred embodiment, non-pathogenic symbiotic bacteria, which are able to live and replicate within plant tissues, so-called endophytes, or non-pathogenic symbiotic bacteria, which are capable of colonizing the phyllosphere or the rhizosphere, so-called epiphytes, are used. Such bacteria include bacteria of the genera Agrobacterium, Alcaligenes, Azospirillum, Azotobacter, Bacillus, Clavibacter, Enterobacter, Erwinia, Flavobacter, Klebsiella, Pseudomonas, Rhizobium, Serratia, Streptomyces and Xanthomonas. Symbiotic fungi, such as Trichoderma and Gliocladium are also possible hosts for expression of the inventive nucleotide sequences for the same purpose.

Techniques for these genetic manipulations are specific for the different available hosts and are known in the art. For example, the expression vectors pKK223-3 and pKK223-2 can be used to express heterologous genes in E. coli, either in transcriptional or translational fusion, behind the tac or trc promoter. For the expression of operons encoding multiple ORFs, the simplest procedure is to insert the operon into a vector such as pKK223- 3 in transcriptional fusion, allowing the cognate ribosome binding site of the heterologous genes to be used. Techniques for overexpression in gram-positive species such as Bacillus are also known in the art and can be used in the context of this invention (Quax et al. In.: Industπal Microorganisms: Basic and Applied Molecular Genetics, Eds. Baltz et al., American Society for Microbiology, Washington (1993)). Alternate systems for overexpression rely for example, on yeast vectors and include the use of Pichia, Saccharomyces and Kluyveromyces (Sreekπshna, In- Industrial microorganisms basic and applied molecular genetics, Baltz, Hegeman, and Skatrud eds., American Society for Microbiology, Washington (1993); Dequin & Barre, Biotechnology 12/173-177 (1994); van den Berg er a/., Biotechnology 8:135-139 (1990)).

In another preferred embodiment, at least one of the described nucleotide sequences is transferred to and expressed in Pseudomonas fluorescens strain CGA267356 (described in the published application EU 0 472 494 and in WO 94/01561 ) which has biocontrol characteristics. In another preferred embodiment, a nucleotide sequence of the invention is transferred to Pseudomonas aureofaαens strain 30-84 which also has biocontrol characteristics. Expression in heterologous biocontrol strains requires the selection of vectors appropriate for replication in the chosen host and a suitable choice of promoter. Techniques are well known in the art for expression in gram-negative and gram- positive bacteria and fungi.

Expression of the Nucleotide Sequences in Plant Tissue

In a particularly preferred embodiment, at least one of the insecticidal toxins of the invention is expressed in a higher organism, e.g , 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 will deter the insect from further biting into the plant tissue or may even harm or kill the insect. A nucleotide sequence of the present invention is inserted into an expression cassette, which is then preferably stably integrated in the genome of said plant. In another preferred embodiment, the nucleotide sequence is included in a non-pathogenic self- replicating virus. 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, broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, squash, pumpkin, hemp, zucchini, apple, pear, quince, melon, 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

A nucleotide sequence of this invention is preferably expressed in transgenic plants, thus causing the biosynthesis of the corresponding toxin in the transgenic plants In this way, transgenic plants with enhanced resistance to insects are generated. 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 all 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, while maintaining the ammo 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 illegitimate splice sites that may cause message truncation All changes required to be made within the nucleotide sequences such as those described above are made using well known techniques of site directed mutagenesis, PCR, and synthetic gene construction using the methods described in the published patent applications EP 0 385 962 (to Monsanto), EP 0 359 472 (to Lubπzol, and WO 93/07278 (to Ciba-Geigy)

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 (whilst leaving the second amino acid unmodified), or alternatively up to and including the GTC subsequent to the ATG (with the possibility 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 will 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, ideally 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 cell.

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 chemically 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 detailed in the published application EP 0 332 104 (to Ciba- Geigy) and US patent 5,614,395. A preferred promoter for chemical induction is the tobacco PR-1a 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. Ideally, such a promoter should only be active locally at the sites of infection, and in this way the insecticidal toxins only accumulate in cells which need to synthesize the insecticidal toxins to kill the invading insect pest. Preferred promoters of this kind include those described by Stanford et al. Mol. Gen. Genet. 215: 200-208 (1989), Xu ef al. Plant Molec. Biol. 22: 573-588 (1993), Logemann ef al. Plant Cell 1: 151 -158 (1989), Rohrmeier & Lehle, Plant Molec. Biol. 22: 783-792 (1993), Firek ef al. Plant Molec. Biol. 22: 129-142 (1993), and Warner ef al. Plant J. 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 that described by de Framond (FEBS 290: 103-106 (1991 ); EP 0 462 269 to Ciba-Geigy). A preferred stem specific promoter is that described in US patent δ,62δ,136 (to Ciba-Geigy) and which drives expression of the maize trpA gene.

Especially 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 available and known in the art (e.g. tm1 from CaMV, E9 from rbcS). Any available 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 Adh1 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 cellular localizations in the plant. In some cases, localization in the cytosol may be desirable, whereas in other cases, localization in some subcellular organelle may be preferred. Subcellular localization of transgene encoded enzymes is undertaken using techniques well known in the art. Typically, the DNA encoding the target peptide from a known organelle-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 cells. Techniques to achieve this are well-known in the art.

Vectors suitable for plant transformation are described elsewhere in this specification. For

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 >4grøJbacfet/^'um-mediated transfer, transformation is usually (but not necessarily) undertaken with a selectable marker which may provide resistance to an antibiotic (kanamycin, hygromycin or methotrexate) or a herbicide (basta). The choice of selectable marker is not, however, critical to 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 generally 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. 6,451 ,513, 5,545,817, and 5,545,818, in PCT application no. WO 95/16783, and in McBride etal. (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 biolistics or protoplast transformation (e.g., calcium chloride or PEG mediated transformation). The 1 to 1.5 kb flanking regions, termed targeting sequences, facilitate homologous recombination with the plastid genome and thus allow the replacement or modification of specific regions of the plastome. Initially, point mutations in the chloroplast 16S rRNA and rps12 genes conferring resistance to spectinomycin and/or streptomycin are utilized as selectable markers for transformation (Svab, Z., Hajdukiewicz, P., and Maliga, P. (1990) Proc. Natl. Acad. Sci. USA 87, 8526-8530; Staub, J. M., and Maliga, P. (1992) Plant Cell 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 allowed creation of a plastid targeting vector for introduction of foreign genes (Staub, J.M., and Maliga, 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 Maliga, P. (1993) Proc. Natl. Acad. Sci. USA 90, 913-917). Previously, this marker had been used successfully 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. Typically, approximately 16-20 cell division cycles following transformation are required to reach a homoplastidic state. Plastid expression, in which genes are inserted by homologous recombination into all of the several thousand copies of the circular plastid genome present in each plant cell, takes advantage of the enormous copy number advantage over nuclear- expressed genes to permit expression levels that can readily 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 preferentially capable of high expression of the nucleotide sequence.

Formulation of Insecticidal Compositions

The invention also includes compositions comprising at least one of the insecticidal toxins of the present invention. In order to effectively control insect pests such compositions preferably contain sufficient amounts of toxin. Such amounts vary depending on the crop to be protected, on the particular pest to be targeted, and on the environmental conditions, such as humidity, temperature or type of soil. In a preferred embodiment, compositions comprising the insecticidal toxins comprise host cells expressing the toxins without additional purification. In another preferred embodiment, the cells expressing the insecticidal toxins are lyophilized prior to their use as an insecticidal agent. In another embodiment, the insecticidal toxins are engineered to be secreted from the host cells. In cases where purification of the toxins from the host cells in which they are expressed is desired, various degrees of purification of the insecticidal toxins are reached.

The present invention further embraces the preparation of compositions comprising at least one insecticidal toxin of the present invention, which is homogeneously mixed with one or more compounds or groups of compounds described herein. The present invention also relates to methods of treating plants, which comprise application of the insecticidal toxins or compositions containing the insecticidal toxins, to plants. The insecticidal toxins can be applied to the crop area in the form of compositions or plant to be treated, simultaneously or in succession, with further compounds. These compounds can be both fertilizers or micronutrient donors or other preparations that influence plant growth. They can also be selective herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides or mixtures of several of these preparations, if desired together with further carriers, surfactants or application-promoting adjuvants customarily employed in the art of formulation. Suitable carriers and adjuvants can be solid or liquid and correspond to the substances ordinarily employed in formulation technology, e.g. natural or regenerated mineral substances, solvents, dispersants, wetting agents, tackifiers, binders or fertilizers.

A preferred method of applying insecticidal toxins of the present invention is by spraying to the environment hosting the insect pest like the soil, water, or foliage of plants. The number of applications and the rate of application depend on the type and intensity of infestation by the insect pest. The insecticidal toxins can also penetrate the plant through the roots via the soil (systemic action) by impregnating the locus of the plant with a liquid composition, or by applying the compounds in solid form to the soil, e.g. in granular form (soil application). The insecticidal toxins may also be applied to seeds (coating) by impregnating the seeds either with a liquid formulation containing insecticidal toxins, or coating them with a solid formulation. In special cases, further types of application are also possible, for example, selective treatment of the plant stems or buds. The insecticidal toxins can also be provided as bait located above or below the ground.

The insecticidal toxins are used in unmodified form or, preferably, together with the adjuvants conventionally employed in the art of formulation, and are therefore formulated in known manner to emulsifiable concentrates, coatable pastes, directly sprayable or dilutable solutions, dilute emulsions, wettable powders, soluble powders, dusts, granulates, and also encapsulations, for example, in polymer substances. Like the nature of the compositions, the methods of application, such as spraying, atomizing, dusting, scattering or pouring, are chosen in accordance with the intended objectives and the prevailing circumstances.

The formulations, compositions or preparations containing the insecticidal toxins and, where appropriate, a solid or liquid adjuvant, are prepared in known manner, for example by homogeneously mixing and/or grinding the insecticidal toxins with extenders, for example solvents, solid carriers and, where appropriate, surface-active compounds (surfactants).

Suitable solvents include aromatic hydrocarbons, preferably the fractions having 8 to 12 carbon atoms, for example, xylene mixtures or substituted naphthalenes, phthalates such as dibutyl phthalate or dioctyl phthalate, aliphatic hydrocarbons such as cyclohexane or paraffins, alcohols and glycols and their ethers and esters, such as ethanol, ethylene glycol monomethyl or monoethyl ether, ketones such as cyclohexanone, strongly polar solvents such as N-methyl-2-pyrrolidone, dimethyl sulfoxide or dimethyl formamide, as well as epoxidized vegetable oils such as epoxidized coconut oil or soybean oil or water.

The solid carriers used e.g. for dusts and dispersible powders, are normally natural mineral fillers such as calcite, talcum, kaolin, montmorillonite or attapulgite. In order to improve the physical properties it is also possible to add highly dispersed silicic acid or highly dispersed absorbent polymers. Suitable granulated adsorptive carriers are porous types, for example pumice, broken brick, sepiolite or bentonite; and suitable nonsorbent carriers are materials such as calcite or sand. In addition, a great number of pregranulated materials of inorganic or organic nature can be used, e.g. especially dolomite or pulverized plant residues.

Suitable surface-active compounds are nonionic, cationic and/or anionic surfactants having good emulsifying, dispersing and wetting properties. The term "surfactants" will also be understood as comprising mixtures of surfactants. Suitable anionic surfactants can be both water-soluble soaps and water-soluble synthetic surface-active compounds.

Suitable soaps are the alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts of higher fatty acids (chains of 10 to 22 carbon atoms), for example the sodium or potassium salts of oleic or stearic acid, or of natural fatty acid mixtures which can be obtained for example from coconut oil or tallow oil. The fatty acid methyltaurin salts may also be used.

More frequently, however, so-called synthetic surfactants are used, especially fatty suifonates, fatty sulfates, sulfonated benzimidazole derivatives or alkylarylsulfonates.

The fatty suifonates or sulfates are usually in the form of alkali metal salts, alkaline earth metal salts or unsubstituted or substituted ammonium salts and have a 8 to 22 carbon alkyl radical which also includes the alkyl moiety of alkyl radicals, for example, the sodium or calcium salt of lignonsulfonic acid, of dodecylsulfate or of a mixture of fatty alcohol sulfates obtained from natural fatty acids. These compounds also comprise the salts of sulfuric acid esters and sulfonic acids of fatty alcohol/ethylene oxide adducts. The sulfonated benzimidazole derivatives preferably contain 2 sulfonic acid groups and one fatty acid radical containing 8 to 22 carbon atoms. Examples of alkylarylsulfonates are the sodium, calcium or triethanolamine salts of dodecylbenzenesulfonic acid, dibutylnapthalenesulfonic acid, or of a naphthalenesulfonic acid/formaldehyde condensation product. Also suitable are corresponding phosphates, e.g. salts of the phosphoric acid ester of an adduct of p-nonylphenol with 4 to 14 moles of ethylene oxide.

Non-ionic surfactants are preferably polyglycol ether derivatives of aliphatic or cycloaliphatic alcohols, or saturated or unsaturated fatty acids and alkylphenols, said derivatives containing 3 to 30 glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms in the alkyl moiety of the alkylphenols.

Further suitable non-ionic surfactants are the water-soluble adducts of polyethylene oxide with polypropylene glycol, ethylenediamine propylene glycol and alkylpolypropylene glycol containing 1 to 10 carbon atoms in the alkyl chain, which adducts contain 20 to 250 ethylene glycol ether groups and 10 to 100 propylene glycol ether groups. These compounds usually contain 1 to 5 ethylene glycol units per propylene glycol unit.

Representative examples of non-ionic surfactants are nonylphenolpolyethoxyethanols, castor oil polyglycol ethers, polypropylene/polyethylene oxide adducts, tributylphenoxypolyethoxyethanol, polyethylene glycol and octylphenoxyethoxyethanol. Fatty acid esters of polyoxyethylene sorbitan and polyoxyethylene sorbitan trioleate are also suitable non-ionic surfactants.

Cationic surfactants are preferably quaternary ammonium salts which have, as N- substituent, at least one C8-C22 alkyl radical and, as further subεtituents, lower unsubstituted or halogenated alkyl, benzyl or lower hydroxyalkyl radicals. The salts are preferably in the form of halides, methylsulfates or ethylsulfates, e.g. stearyltrimethylammonium chloride or benzyldi(2-chloroethyl)ethylammonium bromide.

The surfactants customarily employed in the art of formulation are described, for example, in "McCutcheon's Detergents and Emulsifiers Annual," MC Publishing Corp. Ringwood, New Jersey, 1979, and Sisely and Wood, "Encyclopedia of Surface Active Agents," Chemical Publishing Co., Inc. New York, 1980.

EXAMPLES

The invention will be further described by reference to the following detailed examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Ausubel (ed.), Current Protocols in Molecular Biology, John Wiley 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).

A. Isolation Of Nucleotide Sequences Whose Expression Results In Toxins Active Against Lepidopteran Insects

Example 1 : Construction of Cosmid Library from Photorhabdus luminescens

Photorhabdus luminescens strain ATCC 29999 is grown in nutrient broth at 25°C for three days as described in the ATCC protocol for bioassay. The culture is grown for 24 hours for DNA isolation. Total DNA is isolated by treating freshly grown cells resuspended in 100 mM Tris pH 8, 10 mM EDTA with 2 mg/ml lysozyme for 30 minutes at 37°C. Proteinase K is added to a final concentration of 100 mg/ml, SDS is added to a final concentration of 0.5% SDS and the sample is incubated at 45°C. After the solution becomes clear and viscous, the SDS concentration is raised to 1 %, and 300 mM NaCI and an equal volume of phenol-chloroform-isoamyl alcohol are added, mixed gently for 5 minutes and centrifuged at 3K. The phenol-chloroform-isoamyl alcohol extraction is repeated twice. The aqueous phase is mixed with 0.7 volumes isopropanol, and the sample is centrifuged. The pellet is washed 3 times with 70% ethanol and the nucleic acids are gently resuspended in 0.5X TE.

The DNA is treated with 0.3 units of Sau3A per mg DNA at 37°C for 3.5 minutes in 100 ml volume containing a total of 6 mg DNA. The reaction is then heated for 30 minutes at 65°C to inactivate the enzyme. Then 2 units of Calf Intestinal Alkaline Phosphatase are added and incubated for 30 minutes at 37°C. The sample is mixed with an equal volume of phenol-chloroform-isoamyl alcohol and centrifuged. The aqueous phase is removed, precipitated with 0.7 volume isopropanol and centrifuged. The supernatant is transferred to a fresh tube, precipitated with ethanol, and the nucleic acids are resuspended in O.δX TE at a concentration of 100 hg/ml.

SuperCos cosmid vector (Stratagene, La Jolla, CA) is prepared as described by the supplier utilizing the BamHI cloning site. Prepared SuperCos at 100 hg/ml is ligated with the Sau3A digested P. luminescens DNA at a molar ratio of 2:1 in a 5 ml volume overnight at 6°C. The ligation mixture is packaged using Gigapack XL III (Stratagene), as described by the supplier. Packaged phages are used to infect XL-1 MR (Stratagene) cells as described by the supplier. The cosmid library is plated on L-agar with 50 mg/ml kanamycin and incubated 16 hours at 37°C. 500 colonies are patched onto fresh L-kan plates at 50 colonies per plate. From the other plates the cells are washed off with L broth and mixed with 20% glycerol and frozen at -80°C.

Example 2: Insect Bioassays

Plutella xylostella bioassays are performed by aliquoting of 50 μl of the E. coli culture on the solid artificial Plutella xylostella diet (Biever and Boldt, Annals of Entomological Society of America, 1971 ; Shelton et al., J. Ent. Sci. 26:17). 4 ml of the diet is poured into 1 oz. clear plastic cups (Bioserve product #9051 ). 5 neonate P. xylostella from a diet adapted lab colony are placed in each diet-containing cup and then covered with a white paper lid (Bioserve product #9049). 10 larvae are assayed per concentration. Trays of cups are placed in an incubator for 3 days at 72°F with a 14:10 (hours) lighfdark cycle. Then, the number of live larvae in each cup is recorded. Bioassays for other insects are performed as described for Plutella xylostella, but using the diet required by the insect to be tested.

The broth of P. luminescens undiluted and diluted 1 :100 gives 100% mortality against P. xylostella. The broth of P. luminescens also gives 100% mortality against Diabrotica virgifera virgifera. Three clones with activity against P. xylostella and Heliothis virescens are obtained after screening 500 E. coli clones by insect bioassay. These cosmid clones are given the numbers pC!B9349, pCIB9350, and pCIB9351. Example 3: Isolation of the Nucleotide Sequence Responsible for insect Control Activity from Clones pCIB9349, pCIB9350, and pCIB9351

The three clones pCIB9349, pCIB9350 and pCIB9361 are found to be overlapping cosmids by restriction enzyme mapping. After digestion with Pad, clones pCIB9349 and pCIB9351 give two DNA fragments each, and pCIB9350 gives three DNA fragments. Each fragment is isolated and is self-hgated. The enzyme Pad does not cut the SuperCos vector, therefore, only fragments linked to it are re-isolated. The ligation mixtures are transformed into DH5 E. coli cells. Isolated transformed bacterial colonies are grown in L broth with 50 μg/ml kanamycin, and plasmid DNA is isolated by using the alkaline mmiprep protocol as described in Sambrook, et al. DNA is digested with Notl/Pacl and two clones, pCIB93δδ and pCIB9356, are found by bioassay to still contain the insecticidal activity Clone pCIB935δ is digested with Λ/ofl and a 17 kb and a 4 kb DNA fragment are generated. The 17 kb fragment is isolated and hgated into Bluescnpt vector previously cut with Λ/ofl and transformed into DHδα E. coli cells. The isolated transformed bacterial colonies are grown as described and plasmid DNA is isolated by the alkaline mmiprep protocol. A clone containing the 17 kb insert is named pCIB93δ9 and tested by bioassay The results are shown in Example δ. 3 μg of the 17 kb insert is isolated and treated with 0 3 unit of Sau3A per μg DNA for 4, 6, and 8 minutes at 37°C, heated at 7δ°C for 15 minutes The samples are pooled and hgated into pUC19 previously cut with BamHI and treated with calf intestinal alkaline phosphatase The ligation is transformed into DH5α cells and plated on L agar with Xgal/Amp as described in Sambrook et al. and grown overnight at 37°C. White colonies are picked and grown in L broth with 100 μg/ml and plasmid DNA is isolated as previously described. DNA is digested with EcoRI/Hindlll and novel restriction patterns are sequenced Sequencing primers are ordered from Genosys Biotechnologies (Woodlands, TX) Sequencing is performed using the dideoxy chain-termination method Sequencing is completed using Applied Biosystems Inc. model 377 automated DNA sequencer (Foster City, CA) Sequence is assembled using 3.0 from Gene Codes Corporation (Ann Arbor, Ml) Example 4: Subcloning of the 9.7 kb EcoRI/Xbal Fragment From pCIB9359

pCIB9359 is digested with EcoRl and Xbal and the DNA is run on a 0.8% Seaplaque/TBE gel. The 9.7 kb fragment (SEQ ID NO:1) is isolated and ligated into pUC19 previously digested with EcoRl and Xbal. The ligation mixture is transformed into DH5α E. coli cells. Transformed bacteria are grown and plasmid DNA is isolated as previously described. The vector containing the 9.7 kb fragment in pUC19 is designated pCIB9359-7 and bioassay results are shown in Example 5.

Example 5: Bioassay Results for Cosmid Clones pCIB9359 and pCIB9359-7

Cultures of E. coli strains 9359 and 9359-7 containing clones pCIB9359 and pCIB9369-7, respectively, are tested for insecticidal activity against the following insects in insect bioassays:

na = not active

+ = significant growth inhibition

++ = >40% mortality, but less than 100%

+++ = 100% mortality The clones show insecticidal activity against P. xylostella, H. virescens, H. zea, T. ni, and O. nubilalis, and significant insect control activity against S. exigua and S. frugiperda.

Example 6: Identification of Active Region of pCIB9359-7 By Subcloning

Cultures of E. coli strains containing subclones of pCIB9359-7 are tested for insecticidal activity in insect bioassays against P. xylostella.

Restriction Nucleotide Position Relative to 9.7 kb Insecticidal Activity Against

Fragment EcoRV Xba\ fragment (SEQ ID NO:1) Plutella xylostella from pCIB9539-7 and Size in kb

EcoRI/Xbal 1 to 9712 9.7 kb +++

EcoRV (-912) to 2309 3.2 kb na

Hindlll 665 to 5438 4.7 kb na

Kpnl 1441 to 8137 6.9 kb na

Sacl/Xbal 2677 to 9712 7.0 kb na

na = not active

+ = significant growth inhibition

++ = >40% mortality, but less than 100%

+++ = 100% mortality

Example 7: Characterization of pCIB9359-7 Insect Control Activity By Titration

Dilutions of a culture of E.coli strain 9369-7 containing pCIB9369-7 are tested for insecticidal activity in insect bioassays. Dilutions are prepared in a culture of E.coli XL-1 in a total volume of 100 μl and are transferred to diet cups with δ insects per cup. The results show the percentage (%) of insect mortality. μl 9359-7 Culture Px Hv Hz Tn

100 100 72 48 100

60 100 84 68 92

25 100 62 32 100

12.5 96 52 36 68

6.25 88 20 4 32

0 36 20 24 0

Px= P. xylostella, Hv= H. virescens, Hz= H. zea, Tn = T. ni.

Cultures of E. coli 9359-7 still show substantial insecticidal activity after dilution.

Example 8: Stability of pCIB9359-7 Activity

The stability of the toxins is tested after storage for 2 weeks at different temperatures and conditions. 300 ml of Luria broth containing 100 (μg/ml ampicillin is inoculated with E. coli strain 9359-7 and grown overnight at 37°C. Samples are placed in sterile 15 ml screw cap tubes and stored at 22°C and 4°C. Another sample is centrifuged; the supernatant is removed, freeze dried and stored at 22°C. The samples are stored under these conditions for 2 weeks and then a bioassay is conducted against P. xylostella. The freeze dried material is resuspended in the same volume as before. All samples are resuspended by vortexing.

Conditions Results

22°C (2 weeks) +++

4°C (2 weeks) +++

Freeze Dried (2 weeks) +++

na = not active; + = significant growth inhibition; ++ = >40% mortality, but less than 100%; +++ = 100% mortality This demonstrates that the toxins retain their activity for at least two weeks at 22°C, 4°C, and freeze-dπed, and are therefore very stable

Example 9: Size Fraction of pCIB9359-7 Activity

The approximate sizes of the insecticidal toxins are determined. P luminescens cosmid clones pCIB9359-7 and pUC19 in E. coli host DHδα are grown in media consisting of 60% Terrific broth and 60% Luna broth, supplemented with 60 μg/ml ampicillin Cultures (three tubes of each strain) are inoculated into 3 ml of the above media in culture tubes and incubated on a roller wheel overnight at 37°C. Cultures of each strain are combined and sonicated using a Branson Model 460 Sonicator, micro tip, for approximately six 10 second cycles with cooling on ice between cycles. The sonicates are centrifuged in a Sorvall SS34 rotor at 6000 RPM for 10 minutes The resultant supernatants are filtered through a 0.2 μ filter The 3 ml fractions of the filtrates are applied to Bio-Rad Econo-Pac 10DG columns that have been previously equilibrated with 10 ml of δOmM NaCI, 25 mM Tns base, pH 7.0 The flow through collected during sample loading is discarded The samples are fractionated with two subsequent additions of 4 ml each of the NaCI - Tns equilibration buffer The two four ml fractions are saved for testing The first fraction contains all material above about 6,000 mol. wt; the second fraction contains material smaller than 6,000 mol wt. A sample of the whole culture broth, the sonicate, and the filtered supernatant on the sonicate are tested along with the three fractions from the 10DG column for activity on P xylostella neonates in bioassays.

The culture, the sonicate, and the filtered supernatant of the sonicate, and the first column fraction from the 9359-7 sample are highly active on P. xylostella. The second column fraction from 9359-7 is slightly active (some stunting only) No activity is found in the third fraction from 9359-7 The sample from DH5-pUC19 does not have any activity This indicates that the molecular weights of the toxins are above 6,000

Example 10: Heat Inactivitation of pCIB9359-7 Activity

The heat stability of the toxins is determined Overnight cultures of the E coli strain pCIB93δ9-7 are grown in a 60 50 mixture of Luna broth and Terrific broth Cultures are grown at 37°C in culture tubes on a tube roller A one ml sample of the culture is placed in a 1.5 mi eppendorf tube and placed in a boiling water bath. The sample is removed after five minutes and allowed to cool to room temperature. This sample along with an untreated portion of the culture is assayed on P. xylostella. 50μl of sample of sample is spread on diet, allowed to dry and neonate larvae P. xylostella applied to the surface. The assay is incubated for δ days at room temperature.

The untreated sample causes 100% mortality. The heat treated sample and a diet alone control do not cause any observable mortality, showing the toxins are heat sensitive.

Example 11 : Leaf Dip Bioassay of pCIB9359-7

Insecticidal activity of the toxins is tested in a leaf dip bioassay. Six leaves approximately 2cm in diameter each are cut from seedlings of turnip and placed in a 1 oz. plastic cup (Jet Plastica) with 4ml-5ml of the resuspended toxin, covered tightly, and shaken until thoroughly wetted. The treated leaves are placed in 50mm petri dishes (Gelman Sciences) on absorbent pads moistened with 300μl of water. The dish covers are left open until the leaf surface appears dry and then placed on tightly so that the leaves do not dry out.

Ten neonate P. xylostella larvae are placed in each petri dish arena. Also, a treatment of 0.1 % Bond spreader/sticker with no toxin is set up as a control. The arenas are monitored daily for signs of drying leaves, and water is added or leaves replaced if necessary. After 3 days the leaves and arenas are examined under a dissecting microscope, and the number of live larvae in each arena is recorded.

100% mortality is found for 9359-7 and none in the no-toxin control, showing that the toxins are also insecticidal in a leaf dip assay.

B. Isolation Of Nucleic Acid Sequences Whose Expression Results In Toxins Active Against Lepidopteran and Coleopteran Insects

Example 12: Total DNA Isolation from Photorhabdus luminescens

Photorhabdus luminescens strain ATCC 29999 is grown 14-18 hours in L broth. Total DNA is isolated from 1.5 mis of culture resuspended in 0.5% SDS, 100μg/ml proteinase K, TE to a final volume of 600 μl. After a 1 hour incubation at 37°C, 100μl 5M NaCI and 80μl CTAB/NaCI are added and the culture is incubated at 65°C for 10 minutes. An equal volume of chloroform is added; the culture is mixed gently and spun. The aqueous phase is extracted once with phenol and once with chloroform. The nucleic acids are treated with 10 μg RNase A for 30 minutes at room temperature. The aqueous phase is mixed with 0.6 volumes isopropanol and the sample is centrifuged. The pellet is washed once with 70% ethanol and the nucleic acids are gently resuspended in 100-200ul TE.

Example 13: PCR Amplification of Probes

Two probes are PCR amplified from Photorhabdus luminescens strain ATCC 29999 genomic DNA using oligos δ'-ACACAGCAGGTTCGTCAG-3' (SEQ ID NO:7) and 5'- GGCAGAAGCACTCAACTC-3' (SEQ ID NO:8) to amplify probe #1 and oligos δ'- ATTGATAGCACGCGGCGACC-3' (SEQ ID NO:9) and δ'-

TTGTAACGTGGAGCCGAACTGG-3' (SEQ ID NO:10) to amplify probe #2. The oligos are ordered from Genosys Biotechnologies, Inc. (Texas). Approximately 10-50 ng of genomic DNA is used as the template. 0.8μM of oligos, 200μM of dNTPs, 1 X Taq DNA Polymerase buffer and 2.5 units of Taq DNA Polymerase are included in the reaction. The reaction conditions are as follows:

94°C - 1 minute

94°C - 30 seconds / 60°C - 30 seconds / 72°C - 30 seconds (25 cycles)

72°C - 5 minutes

4°C - indefinite soak The reactions are preferably carried out in a PCR System 9600 (Perkin Elmer) thermocycler.

Example 14: Probing a Photorhabdus luminescens Library

600 clones from the P. luminescens cosmid library described in Example 1 are patched to L-amp plates in duplicate. The colonies are grown overnight then moved to 4°C. The colonies are lifted onto Colony/Plaque Screen Hybridization Transfer Membranes (Biotechnology Systems NEN Research Products). The membranes are incubated 2-3 minutes in 0.7δml O.δN NaOH twice. The membranes are then incubated 2-3 minutes in 0.75ml 1.0M Tris-HCI, pH 7.5 twice. The membranes are allowed to dry at room temperature.

Probe #1 and probe #2 described in Example 13 are labeled using the DECAprime II Kit as described by the manufacturer (Ambion cat# 1455). Unincorporated nucleotides are removed from the labeled probes using Quick Spin Columns as described by the manufacturer (Boehringer Mannheim cat #1273973). The labeled probes are measured for incorporated radioactivity and the specific activity is 10,000,000 cpm. Membranes are prewetted with 2X SSC and hybridized with the probes for 12-16 hours at 65°C. One set of colony lifts is hybridized with probe #1 and the other set is hybridized with probe #2. The membranes are washed with wash CHURCH solutions 1 and 2 (Church and Gilbert, Proc. Natl. Acad. Sci. USA 81 :1991 -1995 (1984)) and exposed to Kodak film.

Twenty one clones are identified that hybridize to probe #1 and seven clones are identified that hybridize to probe #2. The gene in the clones isolated with probe #1 is named hphl and the gene in the clones isolated with probe #2 is named hph2.

Example 15: Insect Bioassays

The clones identified in Example 14 are tested for insecticidal activity against the following insects in insect bioassays: Diabrotica virgifera virgifera (Western Corn Rootworm (WCR)), Diabrotica undecimpunctata howardi (Southern Corn Rootworm (SCR)), Ostrinia nubilalis (European Corn Borer (ECB)), and Plutella xylostella (Diamondback Moth (DBM)).

Diabrotica virgifera virgifera (Western Corn Rootworm) and Diabrotica undecimpunctata howardi (Southern Corn Rootworm) assays are performed using a diet incorporation method. 500μl of an overnight culture of the cosmid library in XL-1 Blue MR cells (Stratagene) is sonicated and then mixed with δOOμl of diet. Once the diet solidifies, it is dispensed in a petri dish and 20 larvae are introduced over the diet. Trays of dishes are placed in an incubator for 3-δ days, and percent mortality is recorded at the end of the assay period.

Ostrinia nubilalis (European Corn Borer) and Plutella xylostella (Diamondback Moth) assays are performed by a surface treatment method. The diet is poured in the petri dish and allowed it to solidify. The E. coli culture of 200 -300μl volume is dispensed over the diet surface and entire diet surface is covered to spread the culture with the help of bacterial loop. Once the surface is dry, 10 larvae are introduced over the diet surface. Trays of dishes are placed in an incubator for 3-δ days. The assay with European Corn Borer is incubated at 30°C in complete darkness; the assay with Diamondback Moth is incubated at 72°F with a 14:10 (hours) lighfdark cycle. Percent mortality is recorded at the end of the assay period.

Cosmids containing hph2 are identified with a range of activities, including: WCR only; SCR only; WCR and SCR; SCR and ECB; WCR, SCR, and ECB; or WCR, SCR, ECB, and DBM activity.

In addition to probing the P. luminescens cosmid library with DNA probes, 600 clones are screened by Western Corn Rootworm bioassay. A clone is identified with activity against Western Corn Rootworm. This clone hybridizes with probe #2.

From these bioassays, cosmid 614, having activity against WCR, SCR, ECB, and DBM, is selected for sequencing.

Example 16: Sequencing of Cosmid 514

Cosmid 514 is sequenced using dye terminator chemistry on an ABI 377 instrument. The nucleotide sequence of cosmid 514 is set forth as SEQ ID NO:11. Cosmid 514 is designated pNOV2400 and deposited with the NRRL in E. coli DHδα and assigned accession no. B-30077.

Example 17: Subcloning Insecticidal Regions of Cosmid 514

514a

An 9011 base pair fragment within cosmid 514 (SEQ ID NO:11 ) is removed by digesting the cosmid with the restriction endonuclease Spel (New England Biolabs (Massachusetts), and ligating (T4 DNA Ligase, NEB) the remainder of 514. Subclone 614a consists of cosmid 614 DNA from base pairs 1 -2167 ligated to base pairs 1 1 ,169-37,948.

H2Q2/PET34 hph2 and orf2 (SEQ ID NO:11 , base pairs 23,768-36,838) are cloned into pET34b (Novagen, Wisconsin). Restriction sites are engineered on both ends of each gene to facilitate cloning. PCR is used to add the restriction sites to the genes. A SamHI site is on the 5' end of hph2 immediately upstream of the ATG of hph2, and a Sad site is added to the 3' end of hph2 immediately following the DNA triplet encoding the stop codon. A guanidine is added between the SamHI site and the start codon of hph2 to put the hph2 gene in frame with the Cellulose Binding Domain tag in pET34b. Orf2 has a Sad site upstream of the 56 base pairs between the stop codon of hph2 and the start codon of orf2. The 56 base pairs are included in the hph2-orf2 construct to mimic their setup in the 514 cosmid. Orf2 has an Xho\ site on the 3' end immediately following the stop codon. The oligos used to add the restriction sites to hph2 and orf2 are as follows: hph2-A 5'-CGGGATCCGATGATTTTAAAAGG-3' (SEQ ID NO:1 δ) hph2-B 5'-GCGCCATTGATTTGAG-3' (SEQ ID NO:16) hph2-C 5'-CATTAGAGGTCGAACGTAC-3' (SEQ ID NO:17) hph2-D δ'-GAGCGAGCTCTTACTTAATGGTGTAG-3' (SEQ ID NO:18) orf2-A3 δ'-CAGCGAGCTCCATGCAGAATTCACAGAC-3' (SEQ ID NO:19) oι12-B δ'-GGCAATGGCAGCGATAAG-3' (SEQ ID NO:20) orf2-C 5'-CATTAACGCAGGAAGAGC-3' (SEQ ID NO:21 ) orf2-D 5'-GACCTCGAGTTACACGAGCGCGTCAG-3' (SEQ ID NO:22) The SamHI-Sa 7683 base pair fragment, corresponding to the hph2 gene, and the Sad-Xho\ 4602 base pair orf2 (including the 56 base pairs between hph2 and orf2 open reading frames), corresponding to orf2, are hgated with BamHI-Xftol-digested vector DNA pET34b.

The 5325 base pair Λ/ofl-SamHI fragment of cosmid 514 is cloned into pBS-SK using Afl\\\-NoΑ (415 bp) and BamYW-AfhW (2530 bp) fragments of pBS-SK.

05-H2-Q2 The 12,031 base pair BamY\\-Xho\ fragment of H202/pET34 is cloned into the 8220 base pair Xho\-BamH\ fragment of Orf5/pBS.

Oδ1011 H2O2 A 7298 base pair Bam λ\-Mlu\ fragment from subclone 514a is hgated (T4 DNA Ligase, NEB) with 9588 bp Mlu\-Xho\ and 8220 bp Xho\-Bam \ fragments of subclone 05- H2-02. The resulting - 22 kb subclone O5101 1 H202, which has activity against WCR and ECB, is designated pNOV1001 and deposited with the NRRL in E. coli DH5α and assigned accession no. B-30078.

AKH2Q2

A 12,074 base pair

fragment of H202/pET34 is ligated (T4 DNA Ligase, NEB) into pK184 Nhe\-Bam \ fragment (2228 bp), generating a clone containing hph2 and orf2 in a plδa origin of replication, kanamycin-resistant vector.

Example 18: Insecticidal Activity of Subciones

Bioassays as described above are performed with E. coli cultures that express the above subciones, both singly and in combination. Coexpressing AKH202 and Orfδ/pBS in E. coli, for example in DH5α or HB101 , is found to give insecticidal activity against the Lepidopterans Plutella xylostella (Diamondback Moth), Ostrinia nubilalis (European Corn Borer), and Manduca sexta (Tobacco Hornworm), as well as against the Coleopterans Diabrotica virgifera virgifera (Western Corn Rootworm), Diabrotica undecimpunctata howardi (Southern Corn Rootworm), and Leptinotarsa decimlineata (Colorado Potato Beetle). Thus, coexpression of hph2 (SEQ ID NO:11 , base pairs 23,768-31 ,336), orf2 (SEQ ID NO:11 , base pairs 31 ,393-35,838), and orfδ (SEQ ID NO:1 1 , base pairs 15,171 - 18,035) is sufficient to control these insects, in addition, expression of each of these three ORFs on separate plasmids gives insect control activity, demonstrating that they do not have to be genetically linked to be active, so long as all three gene products are present.

C. Expression of the Nucleic Acid Sequences of the Invention in Heterologous

Microbial Hosts

Microorganisms which are suitable for the heterologous expression of the nucleotide sequences of the invention are all microorganisms which are capable of colonizing plants or the rhizosphere. As such they will be brought into contact with insect pests. These include gram-negative microorganisms such as Pseudomonas, Enterobacter and Serratia, the gram-positive microorganism Bacillus and the fungi Trichoderma, Gliocladium, and Saccharomyces cerevisiae. Particularly preferred heterologous hosts are Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas cepacia, Pseudomonas aureofaciens, Pseudomonas aurantiaca, Enterobacter cloacae, Serratia marscesens, Bacillus subtilis, Bacillus cereus, Trichoderma viride, Trichoderma harzianum, Gliocladium virens, and Saccharomyces cerevisiae.

Example 19: Expression of the Nucleotide Sequences in E. coli and Other Gram- Negative Bacteria

Many genes have been expressed in gram-negative bacteria in a heterologous manner. Expression vector pKK223-3 (Pharmacia catalogue # 27-4935-01) allows expression in E. coli. This vector has a strong tac promoter (Brosius, J. et al., Proc. Natl. Acad. Sci. USA 81) regulated by the lac repressor and induced by IPTG. A number of other expression systems have been developed for use in E. coli. The thermoinducible expression vector pPι_ (Pharmacia #27-4946-01) uses a tightly regulated bacteriophage λ promoter which allows for high level expression of proteins. The lac promoter provides another means of expression but the promoter is not expressed at such high levels as the tac promoter. With the addition of broad host range replicons to some of these expression system vectors, expression of the nucleotide sequence in closely related gram negative- bacteria such as Pseudomonas, Enterobacter, Serratia and Erwinia is possible. For example, pLRKD211 (Kaiser & Kroos, Proc. Natl. Acad. Sci. USA 81 : 5816-5820 (1984)) contains the broad host range replicon ori T which allows replication in many gram-negative bacteria.

In E. coli, induction by IPTG is required for expression of the tac (i.e. trp-lac) promoter. When this same promoter (e.g. on wide-host range plasmid pLRKD211 ) is introduced into Pseudomonas it is constitutively active without induction by IPTG. This trp- lac promoter can be placed in front of any gene or operon of interest for expression in Pseudomonas or any other closely related bacterium for the purposes of the constitutive expression of such a gene. Thus, a nucleotide sequence whose expression results in an insecticidal toxin can therefore be placed behind a strong constitutive promoter, transferred to a bacterium which has plant or rhizosphere colonizing properties turning this organism to an insecticidal agent. Other possible promoters can be used for the constitutive expression of the nucleotide sequence in gram-negative bacteria. These include, for example, the promoter from the Pseudomonas regulatory genes gafA and lemA (WO 94/01561 ) and the Pseudomonas savastanoi IAA operon promoter (Gaffney ef al., J. Bacteriol. 172: 5593-6601 (1990).

Example 20: Expression of the Nucleotide Sequences in Gram-Positive Bacteria

Heterologous expression of the nucleotides sequence in gram-positive bacteria is another means of producing the insecticidal toxins. Expression systems for Bacillus and Streptomyces are the best characterized. The promoter for the erythromycin resistance gene (ermR) from Streptococcus pneumoniae has been shown to be active in gram-positive aerobes and anaerobes and also in E.coli (Theu-Cuot et al., Nucl Acids Res 18: 3660 (1990)). A further antibiotic resistance promoter from the thiostreptone gene has been used in Streptomyces cloning vectors (Bibb, Mol Gen Genet 199: 26-36 (1985)). The shuttle vector pHT3101 is also appropriate for expression in Bacillus (Lereclus, FEMS Microbiol Lett 60: 211-218 (1989)). A significant advantage of this approach is that many gram- positive bacteria produce spores which can be used in formulations that produce insecticidal agents with a longer shelf life. Bacillus and Streptomyces species are aggressive colonizers of soils

Example 21 : Expression of the Nucleotide Sequences in Fungi

Trichoderma harzianum and Gliocladium virens have been shown to provide varying levels of biocontrol in the field (US 5,165,928 and US 4,996,157, both to Cornell Research Foundation). A nucleotide sequence whose expression results in an insecticidal toxin could be expressed in such a fungus. This could be accomplished by a number of ways which are well known in the art. One is protoplast-mediated transformation of the fungus by PEG or electroporation-mediated techniques. Alternatively, particle bombardment can be used to transform protoplasts or other fungal cells with the ability to develop into regenerated mature structures. The vector pAN7-1 , originally developed for Aspergillus transformation and now used widely for fungal transformation (Curragh ef al., Mycol. Res. 97(3): 313-317 (1992;; Tooley et al., Curr. Genet. 21: 55-60 (1992); Punt et al., Gene 66: 1 17-124 (1987)) is engineered to contain the nucleotide sequence. This plasmid contains the E. coli the hygromycin B resistance gene flanked by the Aspergillus nidulans gpd promoter and the trpC terminator (Punt et al., Gene 66: 1 17-124 (1987)). ln a preferred embodiment, the nucleic acid sequences of the invention are expressed in the yeast Saccharomyces cerevisiae. Each of the three ORF's of SEQ ID NO:1 1 (hph2, orf2 and orfδ), which together confer insecticidal activity, are cloned into individual vectors with the GAL1 inducible promoter and the CYC1 terminator. Each vector has ampicillin resistance and the 2 micron replicon. The vectors differ in their yeast growth markers. hph2 is cloned into p424 (TRP1 , ATCC 87329), orf2 into p423 (HIS3, ATCC 87327), and orfδ into p42δ (LEU2, ATCC 87331 ). The three constructs are transformed into S. cerevisiae independently and together. The three ORFs are expressed together and tested for protein expression and insecticidal activity.

D. Expression of the Nucleotide Sequences in Transgenic Plants

The nucleic acid sequences described in this application can be incorporated into plant cells using conventional recombinant DNA technology. Generally, this involves inserting a coding sequence of the invention into an expression system to which the coding sequence is heterologous (i.e., not normally 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 λgth , λgtIO and Charon 4; plasmid vectors such as pBI121 , pBR322, pACYC177, pACYC184, pAR series, pKK223-3, pUC8, pUC9, pUC18, pUC19, pLG339, pRK290, pKC37, pKC101 , pCDNAII; and other similar systems. The components of the expression system may also be modified to increase expression. For example, truncated sequences, nucleotide substitutions or other modifications may be employed. The expression systems described herein can be used to transform virtually any crop plant cell under suitable conditions. Transformed cells can be regenerated into whole plants such that the nucleotide sequence of the invention confer insect resistance to the transgenic plants.

Example 22: Modification of Coding Sequences and Adjacent Sequences

The nucleotide sequences described in this application can be modified for expression in transgenic plant hosts. A host plant expressing the nucleotide sequences and which produces the insecticidal toxins in its cells 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 which encode separate enzymes but which 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 individually 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 still 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 cells. 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, Gaffney 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 successfully without modification of the coding sequence and with x bp of the Pseudomonas gene upstream of the ATG still attached, and y bp downstream of the STOP codon still attached to the nahG ORF. Preferably as little 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 availability of restriction sites.

In other cases, the expression of genes derived from microbial sources may provide problems in expression. These problems have been well 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 well known in the art. The following 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) will enable an identification of the codons within the ORF which should preferably be changed. Typically 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 illegitimate splicing will be overcome.

2. GC/AT Content.

Plant genes typically 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 believed to cause destabilization of messages and are found at the 3' end of many short-lived mRNAs. Secondly, the occurrence of polyadenyiation signals such as AATAAA at inappropriate positions within the message is believed to cause premature truncation of transcription. In addition, monocotyledons may recognize AT-rich sequences as splice 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 believed that ribosomes attach to the 5' end of the message and scan for the first available ATG at which to start translation. Nevertheless, it is believed 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), incorporated 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 following results:

Position Before the Initiating ATG in 14 Maize Genes:

-10 -9 -8 -7 -6 -5 -4 -3 -2 -1 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 incorporate the preferred nucleotides.

4. Removal of Illegitimate Splice 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' splice sites, and be cleaved, thus generating truncated or deleted messages. These sites can be removed using the techniques well known in the art.

Techniques for the modification of coding sequences and adjacent sequences are well 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 accomplished according to methods well known in the art. These are, for example, described in the published patent disclosures EP 0 386 962 (to Monsanto), EP 0 369 472 (to Lubrizol) and WO 93/07278 (to Ciba-Geigy), all 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 well known in the art) prior to their transfer to transgenic plants. Example 23: 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 organelles and cell compartments. These expression cassettes can then be easily transferred to the plant transformation vectors described below The following is a description of various components of typical expression cassettes.

1. Promoters

The selection of the promoter used in expression cassettes will determine the spatial and temporal expression pattern of the transgene in the transgenic plant. Selected promoters will express transgenes in specific cell types (such as leaf epidermal cells, mesophyll cells, root cortex cells) or in specific tissues or organs (roots, leaves or flowers, for example) and the selection will 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., ability to promote transcription. Depending upon the host cell system utilized, any one of a number of suitable promoters can be used, including the gene's native promoter. The following are non- hmiting 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 cell 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 - Chnstensen ef al. Plant Molec. Biol. 12: 619- 632 (1989); and Arabidopsis - Norns 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 publication EP 0 342 926 (to Lubnzol) which is herein incorporated by reference Taylor et al (Plant Cell Rep. 12: 491-495 (1993)) describe a vector (pAHC2δ) that comprises the maize ubiquitin promoter and first intron and its high activity in cell suspensions of numerous monocotyledons when introduced via microprojectile 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 pAHC2δ or any of the transformation vectors described in this application, 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 published patent application EP 0 392 225 (Example 23), which is hereby incorporated by reference. pCGN1761 contains the "double" CaMV 35S promoter and the tml transcriptional terminator with a unique EcoRl site between the promoter and the terminator and has a pUC-type backbone. A derivative of pCGN1761 is constructed which has a modified polyiinker which includes Notl and Xhol sites in addition to the existing EcoRl site. This derivative is designated pCGN1761 ENX. pCGN1761 ENX is useful for the cloning of cDNA sequences or coding sequences (including microbial ORF sequences) within its polyiinker for the purpose of their expression under the control of the 35S promoter in transgenic plants. The entire 35S promoter-coding sequence-fm/ terminator cassette of such a construction can be excised by Hindlll, Sphl, Sail, and Xbal sites δ' 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 36S promoter fragment can be removed by δ' excision with Hindlll, Sphl, Sail, Xbal, or Pstl, and 3' excision with any of the polyiinker restriction sites (EcoRl, 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, pCGN1761 ENX may be modified by optimization of the translational initiation site as described in Example 37 of U.S. Patent No. 5,639,949, incorporated 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 Cell 2: 163-171 (1990)). A 1.3kb fragment of the promoter was found to contain all the regulatory elements required for expression in rice protoplasts. Furthermore, numerous expression vectors based on the Actl promoter have been constructed specifically for use in monocotyledons (McElroy et al. Mol. Gen. Genet. 231/ 160-160 (1991 )). These incorporate the cf/-intron 1 , Adhl 5' flanking sequence and Adhl-Mron 1 (from the maize alcohol dehydrogenase gene) and sequence from the CaMV 36S promoter. Vectors showing highest expression were fusions of 36S 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 : 160-160 (1991 )) can be easily 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 pCGN1761 ENX, which is then available 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 cells (Chibbar et al. Plant Cell Rep. 12: 506-509 (1993)).

d. Inducible Expression, the PR-1 Promoter:

The double 35S promoter in pCGN1761 ENX may be replaced with any other promoter of choice that will result in suitably high expression levels. By way of example, one of the chemically regulatable promoters described in U.S. Patent No. 5,614,395 may replace the double 35S promoter. The promoter of choice is preferably excised from its source by restriction enzymes, but can alternatively be PCR-amplified using primers that carry appropriate terminal restriction sites. Should PCR-a plification 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 chemically/pathogen regulatable tobacco PR- l a promoter is cleaved from plasmid pCIB1004 (for construction, see example 21 of EP 0 332 104, which is hereby incorporated by reference) and transferred to plasmid pCGN1761 ENX (Uknes et al., 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-1 a promoter- containing fragment is gel purified and cloned into pCGN1761 ENX from which the double 35S promoter has been removed. This is done by cleavage with Xhol and blunting with T4 polymerase, followed by cleavage with Hindlll and isolation of the larger vector-terminator containing fragment into which the pCIB1004 promoter fragment is cloned. This generates a pCGN1761 ENX derivative with the PR-1 a promoter and the tml terminator and an intervening polyiinker with unique EcoRl and Notl sites. The selected coding 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 salicylic acid compounds disclosed in U.S. Patent Nos. 5,523,311 and 5,614,395.

e. Inducible Expression, an Ethanol-lnducible 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 purposes 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 well known in the art.

f. Inducible Expression, a Glucocorticoid-lnducible 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 application of a glucocorticoid, for example a synthetic glucocorticoid, preferably dexamethasone, preferably at a concentration ranging from 0.1 mM to 1 mM, more preferably from 10mM to 100mM. For the purposes 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 well known in the art. The trans-acting factor comprises the GAL4 DNA-binding domain (Keegan et al. (1986) Science 231 : 699-704) fused to the transactivating domain of the herpes 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 controlled 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 described by de Framond (FEBS 290: 103-106 (1991 )) and also in the published patent application EP 0 452 269, which is herein incorporated by reference. This promoter is transferred to a suitable vector such as pCGN1761 ENX 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-lnducible 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 Cell 1: 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 all are suitable for use with the instant invention. Logemann et al. describe the 5' upstream sequences of the dicotyledonous potato wunl gene. Xu ef 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. Similar, Firek ef al. and Warner ef al. have described a wound- induced gene from the monocotyledon Asparagus officinalis, which is expressed at local wound and pathogen invasion sites. Using cloning techniques well 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 Application WO 93/07278, which is herein incorporated by reference, describes the isolation of the maize trpA gene, which is preferentially expressed in pith cells. 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 utility in transgenic plants.

j. Leaf-Specific Expression:

A maize gene encoding phosphoenol carboxylase (PEPC) has been described by Hudspeth & Grula (Plant Molec Biol 12: 679-589 (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. Pollen-Specific Expression:

WO 93/07278 describes the isolation of the maize calcium-dependent protein kinase (CDPK) gene which is expressed in pollen cells. 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 pollen-specific manner.

2. Transcriptional Terminators

A variety of transcriptional terminators are available 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 cells. For example, the introns of the maize Ad hi gene have been found to significantly enhance the expression of the wild-type gene under its cognate promoter when introduced into maize cells. Intron 1 was found to be particularly effective and enhanced expression in fusion constructs with the chloramphenicol acetyltransferase gene (Cal s et al., Genes Develop. 1: 1183-1200 (1987)). In the same experimental system, the intron from the maize bronzet gene had a similar effect in enhancing expression. Intron sequences have been routinely incorporated into plant transformation vectors, typically 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 cells. Specifically, 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. Galhe et al. Nucl. Acids Res. 15: 8693-8711 (1987), Skuzeski et al. Plant Molec. Biol. 16: 65-79 (1990)).

4. Targeting of the Gene Product Within the Cell

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 detail. For example, the targeting of gene products to the chloroplast is controlled by a signal sequence found at the ammo terminal end of various proteins which is cleaved during chloroplast import to yield the mature protein (e.g. Comai ef al. J. Biol. Chem. 263' 15104-16109 (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 organelles such as the mitochondrion and the peroxisome (e.g. Unger ef 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 organelles. Examples of such sequences are the nuclear-encoded ATPases and specific aspartate amino transferase isoforms for mitochondria. Targeting cellular protein bodies has been described by Rogers ef 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 cell compartments. Amino terminal sequences are responsible for targeting to the ER, the apoplast, and extracellular secretion from aleurone cells (Koehler & Ho, Plant Cell 2: 769-783 (1990)). Additionally, 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 organelle or cell 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 fulfilled by the addition of a small 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 followed by in vitro chloroplast uptake using techniques described by Bartlett ef 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 well known in the art and are equally applicable to mitochondria and peroxisomes. The above-described mechanisms for cellular 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 cell-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 24: Construction of Plant Transformation Vectors

Numerous transformation vectors available for plant transformation are known to those of ordinary skill 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 will 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 Cell Biol 4: 2929- 2931 ), and the dhfr gene, which confers resistance to methatrexate (Bourouis et al., EMBO J. 2(7): 1099-1104 (1983)), and the EPSPS gene, which confers resistance to glyphosate (U.S. Patent Nos. 4,940,935 and 5,188,642).

1. Vectors Suitable for Agrobacterium Transformation

Many vectors are available for transformation using Agrobacterium tumefaciens. These typically 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 pclB200 and pCIB2001 are used for the construction of recombinant vectors for use with Agrobacterium and are constructed in the following manner. pTJS75kan is created by Narl digestion of pTJS75 (Schmidhauser & Helinski, J. Bacteriol. 164: 446-466 (1985)) allowing excision of the tetracycline-resistance gene, followed by insertion of an Accl fragment from pUC4K carrying an NPTII (Messing & Vierra, 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 ligated 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 polyiinker (Rothstein et al., Gene 53: 153-161 (1987)), and the Xhol- digested fragment are cloned into Sa//-digested pTJS75kan to create pCIB200 (see also EP 0 332 104, example 19). pCIB200 contains the following unique polyiinker restriction sites: EcoRl, Sstl, Kpnl, Bglll, Xbal, and Sail. pCIB2001 is a derivative of pCIB200 created by the insertion into the polyiinker of additional restriction sites. Unique restriction sites in the polyiinker of pCIB2001 are EcoRl, 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-meόiated transformation, the RK2-derived trfA function for mobilization between E. coli and other hosts, and the Or/Tand OriV functions also from RK2. The pCIB2001 polyiinker is suitable for the cloning of plant expression cassettes containing their own regulatory signals.

b. pCIBIO and Hygromycin Selection Derivatives thereof:

The binary vector pCIBIO contains a gene encoding kanamycin resistance for selection in plants and T-DNA right and left border sequences and incorporates sequences from the wide host-range plasmid pRK252 allowing it to replicate in both E coli and Agrobacterium. Its construction is described by Rothstein et al. (Gene 53: 153-161 (1987)). Various derivatives of pCIBIO are constructed which incorporate the gene for hygromycin B phosphotransferase described by Gritz et al. (Gene 25: 179-188 (1983)). These derivatives enable selection of transgenic plant cells 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. pCIB3064: 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 published application 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 Pvull. 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 pCIB246 is designated pCIB3025. The GUS gene is then excised from pCIB3025 by digestion with Sail and Sacl, the termini rendered blunt and religated to generate plasmid pCIB3060. The plasmid pJIT82 is obtained from the John Innes Centre, Norwich and the a 400 bp Smal fragment containing the oat gene from Streptomyces viridochromogenes is excised and inserted into the Hpal site of pCIB3060 (Thompson et al. EMBO J 6: 2519-2623 (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 ampicillin resistance (for selection in E. coli) and a polyiinker 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 Adh1 gene (-550 bp) and 18 bp of the GUS untranslated leader sequence from pSOGI O. 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 nopaline 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 nopaline 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 ampicillin resistance and have Hindlll, Sphl, Pstl and EcoRl sites available for the cloning of foreign substances.

Example 25: Transformation

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

1. Transformation of Dicotyledons

Transformation techniques for dicotyledons are well known in the art and include >4grabacfeπ^'um-based techniques and techniques that do not require Agrobacterium. Non- Agrobacterium techniques involve the uptake of exogenous genetic material directly by protoplasts or cells. This can be accomplished by PEG or electroporation mediated uptake, particle bombardment-mediated delivery, 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 ef al., Nature 327: 70-73 (1987). In each case the transformed cells are regenerated to whole plants using standard techniques known in the art.

Agrobacterium-mediated transformation is a preferred technique for transformation of dicotyledons because of its high efficiency of transformation and its broad utility with many different species. Agrobacterium transformation typically involves the transfer of the binary vector carrying the foreign DNA of interest (e.g. pCIB200 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 chromosomally (e.g. strain CIB542 for pCIB200 and pCIB2001 (Uknes ef al. Plant Cell 5: 159-169 (1993)). The transfer of the recombinant binary vector to Agrobacterium is accomplished 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 usually involves co-cultivation of the Agrobacterium with explants from the plant and follows protocols well 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 cells with a gene involves propelling inert or biologically active particles at plant tissues and cells. This technique is disclosed in U.S. Patent Nos. 4,945,050, 5,036,006, and 5,100,792 all to Sanford et al. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and afford incorporation within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the desired gene. Alternatively, the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles (e.g., dried yeast cells, dried bacterium or a bacteriophage, each containing DNA sought to be introduced) can also be propelled into plant cell tissue.

2. Transformation of Monocotyledons

Transformation of most monocotyledon species has now also become routine. Preferred techniques include direct gene transfer into protoplasts using PEG or electroporation techniques, and particle bombardment into callus 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 Applications EP 0 292 435, EP 0 392 225, and WO 93/07278 describe techniques for the preparation of callus and protoplasts from an elite inbred line of maize, transformation of protoplasts using PEG or electroporation, and the regeneration of maize plants from transformed protoplasts. Gordon-Kamm ef al. (Plant Cell 2: 603-618 (1990)) and Fromm et al. (Biotechnology 8: 833-839 (1990)) have published 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 elite inbred lines of maize by particle bombardment. This technique utilizes immature maize embryos of 1.5-2.5 mm length excised from a maize ear 14-15 days after pollination and a PDS-1000He Biolistics 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 Japoπ/^'ca-types and /noVca-types (Zhang ef al. Plant Cell 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 Application EP 0 332 581 describes techniques for the generation, transformation and regeneration of Pooideae protoplasts. These techniques allow the transformation of Dactylis and wheat. Furthermore, wheat transformation has been described by Vasil et al. (Biotechnology 10: 667-674 (1992)) using particle bombardment into cells of type C long-term regenerable callus, and also by Vasil et al. (Biotechnology U/ 1553-1558 (1993)) and Weeks ef al. (Plant Physiol. 102: 1077-1084 (1993)) using particle bombardment of immature embryos and immature embryo-derived callus. A preferred technique for wheat transformation, however, involves the transformation of wheat by particle bombardment of immature embryos and includes either a high sucrose or a high maltose step prior to gene delivery. 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/l 2,4-D for induction of somatic embryos, which is allowed 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. duction medium with sucrose or maltose added at the desired concentration, typically 15%). The embryos are allowed 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 Biohstics® helium 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 (still 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 callus are transferred to regeneration medium (MS + 1 mg/ ter NAA, 6 mg/hter GA), further containing the appropriate selection agent (10 mg/l basta in the case of pCIB3064 and 2 mg/l methotrexate in the case of pSOG35). After approximately one month, developed shoots are transferred to larger sterile 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. 6,591 ,616, both of which are incorporated herein by reference.

E. Breeding and Seed Production

Example 26: 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 list of agronomically important target crops set forth supra. The expression of a gene of the present invention in combination with other characteristics important for production and quality can be incorporated into plant lines through breeding. Breeding approaches and techniques are known in the art. See, for example, Welsh J R , Fundamentals of Plant Genetics and Breeding, John Wiley & Sons, NY (1981 ), Crop Breeding, Wood D. R. (Ed.) American Society of Agronomy Madison, Wisconsin (1983), Mayo O , The Theory of Plant Breeding, Second Edition, Clarendon Press, Oxford (1987), Singh, D P , Breeding for Resistance to Diseases and Insect Pests, Springer-Verlag, NY (1986); and 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. Generally said maintenance and propagation make use of known agricultural methods developed to fit specific purposes such as tilling, sowing or harvesting. Specialized processes such as hydroponics or greenhouse technologies can also be applied. As the growing crop is vulnerable to attack and damages caused by insects or infections as well 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 tillage of the soil or removal of weeds and infected plants, as well as the application 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 breeding steps are characterized by well-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 well 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 sterile plants by mechanical, chemical, or biochemical means. Cross pollination of a male sterile plant with pollen of a different line assures that the genome of the male sterile but female fertile plant will uniformly obtain properties of both parental lines. Thus, the transgenic seeds and plants according to the invention can be used for the breeding of improved plant lines, that for example, increase the effectiveness of conventional methods such as herbicide or pestidice treatment or allow 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 quality than products that were not able to tolerate comparable adverse developmental conditions.

Example 27: Seed Production

In seed production, germination quality and uniformity of seeds are essential product characteristics, whereas germination quality 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 seedbome diseases, and to produce seed with good germination, fairly extensive and well-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 quality standards instead of using seed harvested from his own crop. Propagation material to be used as seeds is customarily treated with a protectant coating comprising herbicides, insecticides, fungicides, bactericides, nematicides, molluscicides, or mixtures thereof. Customarily used protectant coatings comprise compounds such as captan, carboxin, thiram (TMTD^®), methalaxyl (Apron^®), and pirimiphos-methyl (Actellic^®). If desired, these compounds are formulated together with further carriers, surfactants or application- promoting adjuvants customarily employed in the art of formulation to provide protection against damage caused by bacterial, fungal or animal pests. The protectant coatings may be applied by impregnating propagation material with a liquid formulation or by coating with a combined wet or dry formulation. Other methods of application are also possible such as treatment directed at the buds or the fruit.

It is a further aspect of the present invention to provide new agricultural methods, such as the methods examplified above, which are characterized by the use of transgenic plants, transgenic plant material, or transgenic seed according to the present invention.

The seeds may be provided in a bag, container or vessel comprised of a suitable packaging material, the bag or container capable of being closed to contain seeds. The bag, container or vessel may be designed for either short term or long term storage, or both, of the seed. Examples of a suitable packaging material include paper, such as kraft paper, rigid or pliable plastic or other polymeric material, glass or metal. Desirably the bag, container, or vessel is comprised of a plurality of layers of packaging materials, of the same or differing type. In one embodiment the bag, container or vessel is provided so as to exclude or limit water and moisture from contacting the seed. In one example, the bag, container or vessel is sealed, for example heat sealed, to prevent water or moisture from entering. In another embodiment water absorbent materials are placed between or adjacent to packaging material layers. In yet another embodiment the bag, container or vessel, or packaging material of which it is comprised is treated to limit, suppress or prevent disease, contamination or other adverse affects of storage or transport of the seed. An example of such treatment is sterilization, for example by chemical means or by exposure to radiation. Comprised by the present invention is a commercial bag comprising seed of a transgenic plant comprising a gene of the present invention that is expressed in said transformed plant at higher levels than in a wild type plant, together with a suitable carrier, together with label instructions for the use thereof for conferring broad spectrum disease resistance to plants.

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Claims

What is claimed is:

1. An isolated nucleic acid molecule comprising:

(a) a nucleotide sequence substantially similar to a nucleotide sequence selected from the group consisting of: nucleotides 412-1665 of SEQ ID NO:1 , nucleotides 1686-2447 of SEQ ID NO:1 , nucleotides 2758-3318 of SEQ ID NO:1 , nucleotides 3342-4118 of SEQ ID NO:1 , nucleotides 4515-9269 of SEQ ID NO:1 , nucleotides 15,171-18,035 of SEQ ID NO:11 , and nucleotides 31 ,393-35,838 of SEQ ID NO:11 ;

(b) a nucleotide sequence comprising nucleotides 23,768-31 ,336 of SEQ ID NO:11 ; or

(c) a nucleotide sequence isocoding with the nucleotide sequence of (a) or (b); wherein expression of said nucleic acid molecule results in at least one toxin that is active against insects.

2. An isolated nucleic acid molecule comprising a 20 base pair nucleotide portion identical in sequence to a consecutive 20 base pair nucleotide portion of a nucleotide sequence selected from the group consisting of: nucleotides 412-1665 of SEQ ID NO:1 , nucleotides 1686-2447 of SEQ ID NO:1 , nucleotides 2758-3318 of SEQ ID NO:1 , nucleotides 3342-4118 of SEQ ID NO:1 , nucleotides 4515-9269 of SEQ ID NO:1 , nucleotides 15,171-18,035 of SEQ ID NO:11 , and nucleotides 31 ,393-35,838 of SEQ ID NO:1 1 , wherein expression of said nucleic acid molecule results in at least one toxin that is active against insects.

3. An isolated nucleic acid molecule comprising a nucleotide sequence from Photorhabdus luminescens selected from the group consisting of: nucleotides 412-1665 of SEQ ID NO:1 , nucleotides 1686-2447 of SEQ ID NO:1 , nucleotides 2758-3318 of SEQ ID NO:1 , nucleotides 3342-41 18 of SEQ ID NO:1 , nucleotides 4515-9269 of SEQ ID NO:1 , nucleotides 66-1898 of SEQ ID NO:11 , nucleotides 2416-9909 of SEQ ID NO:1 1 , the complement of nucleotides 2817-3395 of SEQ ID NO:1 1 , nucleotides 9966-14,633 of SEQ ID NO:11 , nucleotides 14,699-15,007 of SEQ ID NO:1 1 , nucleotides 15,171 -18,035 of SEQ ID NO:11 , the complement of nucleotides 17,072-17,398 of SEQ ID NO:11 , the complement of nucleotides 18,235-19,167 of SEQ ID NO:1 1 , the complement of nucleotides 19,385- 20,116 of SEQ ID NO:1 1 , the complement of nucleotides 20,217-20,963 of SEQ ID NO:1 1 , the complement of nucleotides 22,172-23,086 of SEQ ID NO:11 , nucleotides 23,768-31 ,336 of SEQ ID NO:1 1 , nucleotides 31 ,393-35,838 of SEQ ID NO:11 , the complement of nucleotides 35,383-35,709 of SEQ ID NO:11 , the complement of nucleotides 36,032-36,661 of SEQ ID NO:1 1 , and the complement of nucleotides 36,654-37,781 of SEQ ID NO:11.

4. An isolated nucleic acid molecule according to claim 1 , wherein said nucleotide sequence is substantially similar to nucleotides 412-1665 of SEQ ID NO:1 , nucleotides 1686-2447 of SEQ ID NO:1 , nucleotides 2758-3318 of SEQ ID NO:1 , nucleotides 3342- 4118 of SEQ ID NO:1 , or nucleotides 4515-9269 of SEQ ID NO:1.

5. An isolated nucleic acid molecule according to claim 1 , wherein said nucleotide sequence encodes an amino acid sequence selected from the group consisting of SEQ ID NOs:2-6.

6. An isolated nucleic acid molecule according to claim 1 , wherein said nucleotide sequence comprises nucleotides 412-1665 of SEQ ID NO:1 , nucleotides 1686-2447 of SEQ ID NO:1 , nucleotides 2758-3318 of SEQ ID NO:1 , nucleotides 3342-4118 of SEQ ID NO:1 , or nucleotides 4515-9269 of SEQ ID NO:1.

7. An isolated nucleic acid molecule according to claim 1 , wherein said nucleotide sequence is substantially similar to nucleotides 15,171 -18,035 or 31 ,393-35,838 of SEQ ID NO:11 .

8. An isolated nucleic acid molecule according to claim 1 , wherein said nucleotide sequence encodes the amino acid sequence set forth in SEQ ID NOs:12-14.

9. An isolated nucleic acid molecule according to claim 1 , wherein said nucleotide sequence comprises nucleotides 15,171 -18,035; 23,768-31 ,336; or 31 ,393-35,838 of SEQ ID NO:11.

10. An isolated nucleic acid molecule according to claim 2, comprising a 20 base pair nucleotide portion identical in sequence to a consecutive 20 base pair nucleotide portion of nucleotides 412-1665 of SEQ ID NO:1 , nucleotides 1686-2447 of SEQ ID NO:1 , nucleotides 2758-3318 of SEQ ID NO:1 , nucleotides 3342-4118 of SEQ ID NO:1 , or nucleotides 4515- 9269 of SEQ ID NO: 1.

11. An isolated nucleic acid molecule according to claim 2, comprising a 20 base pair nucleotide portion identical in sequence to a consecutive 20 base pair nucleotide portion of nucleotides 15,171-18,035 or 31 ,393-35,838 of SEQ ID NO:11.

12. A chimeric gene comprising a heterologous promoter sequence operatively linked to the nucleic acid molecule of claim 1 or claim 2.

13. A recombinant vector comprising the chimeric gene of claim 12.

14. A host cell comprising the chimeric gene of claim 12.

15. A host cell according to claim 14, which is a bacterial cell.

16. A-host cell according to claim 14, which is a yeast cell.

17. A host cell according to claim 14, which is a plant cell.

18 A plant comprising the plant cell of claim 17

19. A plant according to claim 18, which is maize.

20. A toxin produced by the expression of a DNA molecule according to claim 1 or claim 2.

21. A toxin according to claim 20, wherein said toxin has activity against Lepidopteran insects.

22. A toxin according to claim 21 , wherein said toxin has activity against Plutella xylostella (Diamondback Moth), Trichoplusia ni (Cabbage Looper), Ostrinia nubilalis (European Corn Borer), Heliothis virescens (Tobacco Budworm), Helicoverpa zea (Corn Earworm), Spodoptera exigua (Beet Armyworm), and Spodoptera frugiperda (Fall Armyworm).

23. A toxin according to claim 20, wherein said toxin has activity against Lepidopteran and Coleopteran insects.

24. A toxin according to claim 23, wherein said toxin has insecticidal activity against Plutella xylostella (Diamondback Moth), Ostrinia nubilalis (European Corn Borer), and Manduca sexta (Tobacco Hornworm), Diabrotica virgifera virgifera (Western Corn Rootworm), Diabrotica undecimpunctata howardi (Southern Corn Rootworm), and Leptinotarsa decimlineata (Colorado Potato Beetle).

25. A toxin according to claim 20, wherein said toxin comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs:2-6.

26. A toxin according to claim 20, wherein said toxin comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs:12-14.

27. A composition comprising an insecticidally effective amount of a toxin according to claim 20.

28. A method of producing a toxin that is active against insects, comprising:

(a) obtaining the host cell of claim 14; and

(b) expressing the nucleic acid molecule in said cell, which results in at least one toxin that is active against insects.

29. A method of producing an insect-resistant plant, comprising introducing a nucleic acid molecule according to claim 1 into said plant, wherein said nucleic acid molecule is expressible in said plant in an effective amount to control insects.

30. A method of controlling insects comprising delivering to the insects an effective amount of a toxin according to claim 44.

31. The method of claim 29 or claim 30, wherein the insects are Lepidopteran insects. - 16 -

32. The method of claim 31 , wherein the insects are selected from the group consisting of: Plutella xylostella (Diamondback Moth), Trichoplusia ni (Cabbage Looper), Ostrinia nubilalis (European Corn Borer), Heliothis virescens (Tobacco Budworm), Helicoverpa zea (Corn Earworm), Spodoptera exigua (Beet Armyworm), and Spodoptera frugiperda (Fall Armyworm).

33. The method of claim 29 or claim 30, wherein the insects are Lepidopteran and Coleopteran insects.

34. The method of claim 33, wherein the insects are selected from the group consisting of: Plutella xylostella (Diamondback Moth), Ostrinia nubilalis (European Corn Borer), and Manduca sexta (Tobacco Hornworm), Diabrotica virgifera virgifera (Western Corn Rootworm), Diabrotica undecimpunctata howardi (Southern Corn Rootworm), and Leptinotarsa decimlineata (Colorado Potato Beetle).

35. The method of claim 30, wherein the toxin is delivered to the insects orally.

36. A method for mutagenizing a nucleic acid molecule according to claim 1 , wherein the nucleic acid molecule has been cleaved into population of double-stranded random fragments of a desired size, comprising:

(a) adding to the population of double-stranded random fragments one or more single- or double-stranded oligonucleotides, wherein said oligonucleotides each comprise an area of identity and an area of heterology to a double- stranded template polynucleotide;

(b) denaturing the resultant mixture of double-stranded random fragments and oligonucleotides into single-stranded fragments;

(c) incubating the resultant population of single-stranded fragments with a polymerase under conditions which result in the annealing of said single- stranded fragments at said areas of identity to form pairs of annealed fragments, said areas of identity being sufficient for one member of a pair to prime replication of the other, thereby forming a mutagenized double- stranded polynucleotide; and (d) repeating the second and third steps for at least two further cycles, wherein the resultant mixture in the second step of a further cycle includes the mutagenized double-stranded polynucleotide from the third step of the previous cycle, and wherein the further cycle forms a further mutagenized double-stranded polynucleotide.