ZA200005662B - Novel insecticidal toxins from xenorhabdus nematophilus and nucleic acid sequences coding therefor. - Google Patents

Description

NOVEL INSECTICIDAL TOXINS FROM XEENORHABDUS NEMATOPHILUS AND

NUCLEIC ACID SEQUENCE S CODING THEREFOR

The invention relates to novel toxins from Xenorhabdus nematophilus, Xenorhabdus

Joy poinarii, and Photorhabdus luminescens, nucleic acid sequences whose expression results

A 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 Bosses. 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 intexnsive 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, bemeficial insects. Another problem resulting from the wide use of chemical pesticides is th € 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 $-endotoxins, have also been applied with satisfactory results, of fering an alternative or a complement to chemical insecticides. Recently, the genes coding for some of these §-endotoxins have been isolated and their expression in heterologous hosts have been shown to provide another tool for the control of economically i mportant insect pests. In particular, the expression of insecticidal toxins in transgenic plants, such as Bacillus thuringiensis §- endotoxins, has provided efficient protection ag ainst 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 ecoromic benefit to farmers and that are environmentally acceptable.

The present invention addresses the long-standing 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. PRY _ in the search for nove | insect control agents, certain classes of nematodes from the A genera Heterorhabdus and Steinernema are of particular interest because of their Ll 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 nucleotide sequences isolated from Xenorhabdus nematophilus, and nucleotid e sequences substantially similar thereto, whose expression result in insecticidal toxins th at are highly toxic to economically important pests, particularly plant pests. The invention is further drawn to the insecticidal toxin resulting from the : expression of the nucleotide sequence, and to compositions and formulations containing ’ the insecticidal toxin, that ares capable of inhibiting the ability of insect pests to survive, grow or reproduce, or of limiting i nsect-related damage or loss in crop plants. The invention is further drawn to a method of making the toxin and to methods of using the nucleotide sequence, for example in microorganisms to control insects or in transgenic plants to conter _insect resistance, and to a method of using the toxin, and compositions and formulations comprising the toxin, for example applying the toxin, composition or formulation to insect infested areas, or to prophylactically treat insect susceptible areas or plants to confer protection or resistance agat nst harmtul insects.

The novel toxin is highly insecticidal against Plutella xylostelia (diamondback moth), an economically important insect pest. The toxin can be used in multiple insect control strategies, resulting in maxirnal efficiency with minimal impact on the environment.

According to one aspect, the present invention provides an isolated nucleic acid molecule comprising: (a) @ nucleotide sequence substantially similar to a nucleotide sequence selected from the group consisting of: nucleotides 569-979 of SEQ ID NO:1, nucleotides 1045-2334 of S EQ ID NO:1, SEQ ID NO:4, SEQ 1D NO:6, SEQ ID NO8, SEQ

ID NO:10, SEQ ID NO:12, and SEQ ID NO:14; or (b) a nucleotide sequence isocoding with the nucleotide sequence of (a); wherein expression of said 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 nucleotides sequence substantially similar to nucleotides 569-979 of SEQ ID NO:1, nucleotides 1@045-2334 of SEQ ID NO:1, SEQ ID

NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14. ° Preferably, the nucleotide sequence is substantially si milar to nucleotides 569-979 of SEQ

ID NO:1, nucleotides 1045-2334 of SEQ ID NO:1, SEEQ ID NO:4, SEQ ID NO:6, SEQ ID ) . NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO :14. More preferably, the nucleotide sequence encodes an amino acid sequence selected from the group consisting of SEQ ID

NOs:2, 3, 5, 7, 9, 11, 13, and 15. Most preferably, the nucleotide sequence comprises nucleotides 569-979 of SEQ ID NO:1, nucleotides 14045-2334 of SEQ ID NO:1, SEQ ID

NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14. In another embodiment, the nucleotide sequence comp-rises the approximately 3.0 kb DNA fragment comprised in pCiB9369 (NRRL B-21883). : i According to a preferred embodiment, the toxins resulting from expression of the «nucleic acid molecules of the invention have activity aggainst Plutelfa xylostelia. 3 : In another aspect, the present invention providees an isolated nucleic acid molecule ‘ comprising a 20, 25, 30, 35, 40, 45, or 50 (prefera bly 20) base pair nucleotide portion identical in sequence to a respective consecutive 20, 25, 30, 35, 40, 45, or 50 (preferably 20) base pair nucleotide portion of a nucleotide sequence selected from the group . consisting of: nucleotides 569-979 of SEQ ID NO:1 , nucleotides 1045-2334 of SEQ ID

NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, and SEQ

ID NO:14, wherein expression of said nucleic acid mokecule results in at least one toxin that is active against insects.

The present invention also provides a chimemic gene comprising a heterologous promoter sequence operatively linked to a nucleic ac id molecule of the invention. Further, the present invention provides a recombinant vector c omprising such a chimeric gene. Still further, the present invention provides a host cell comaprising such a chimeric gene. A host cell according to this aspect of the invention may be aa 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 inventior provides toxins produced by the expression of DNA molecules of the present irmvention. According to a preferred embodiment, the toxins of the invention have activity a_gainst Piutella xylostella.

’ in one embodirnent, the toxins are produced by the E. coli strain designated as NRR L oe accession number B -21883. in another embodiment, a toxin of the invention comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs:2, 3, 5,7, 9, 11, 13, and 15. oo Also provided is a composition comprising an insecticidally effective amount of a toxan - oe of 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, whic h 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. ) in 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 reucleic acid molecule is expressible in the plant in an effective amount to control insects. According to a preferred embodiment, the insects are Plutella xylostella. in a still furthear aspect, the present invention provides a method of controlling insects 8 comprising deliverimg to the insects an effective amount of a toxin according to the present invention. Accordimg to a preferred embodiment, the insects are Plutelia xyiosteria.

Preferably, the toxir 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 mole<cule has been cleaved into population of double-stranded random fragments of a dessired size, comprising: (a) adding to the population of double-stranded random fragments one or more single- or double-stranded oligonucieotides, wherein the oligonucleotides ezach comprise an area of identity and an area of heterology to a double- stranded template polynucleotide; (b) denaturing the resultant mixture of double-strancded random fragments and oligonucleotides into single-stranded fragments; (c) incubating the resultant populatiom of single-stranded fragments with a polymerase under conditions wheich result in the annea ling of the single-stranded fragments at the areas of identity to form pairs of annealed fragmments, the areas of identity being sufficient for one member of a pair to prime replication of the other, thereby forming a mutagenized double-strancied 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 formss a further mutagenized double-stranded polynucleotide.

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

DEFINITIONS

. Activity” of the toxirs 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 it 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. : CL 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 serquence. The regulator nucleic acid sequence of the chimeric . gene is not normally opera tively 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 eftect, 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 ire 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 th e 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 J expression cassette may also be onc 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 h ost, 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 a Iso 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 sequeences. Further elements that may be present are, for example, introns. "Gene of interest" refers to any gene which, when transterred 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 seguence naturally associated with a host cell into which it is introduced.

“Homologous recombination” is the reciprocal exchange of nuclezic acid fragments betwee n homologous nucleic acid molecules. “Insecticidal” is defined as a toxic biological activity capable of controlling insects, prefera bly by killing them. ” A nucleic acid sequence is ‘isocoding with” a reference nucleic ac id sequence when the nucleic acid sequence encodes a polypeptide having the same amino acid sequence as " the pol=ypeptide 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 erwironment and is therefore not a product of nature. An isolated nucleic acid molecule or emzyme may exist in a purified form or may exist in a non-native environment such ass, for example, a recomioinant 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 presenst 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 o r 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 cumlture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and ermbryos 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 imcluded. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture and any geoups of plant cells organized into structural and/or functional units. The use of this term i n 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 Il 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 isclated plant cell without a cell wall or wiith only parts of the cell wall. . “Regulatory elements” refer to sequences involved in control fing 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 uased herein with respect to a nucleotide sequence, means a nucleotide sequence corresponding to a reference n ucleotide sequence, wherein the corresponding sequence encodes a polypeptide having s ubstantially the same structure and function as the polypeptide ermcoded by the reference n ucleotide sequence, e.g. where only changes in amino acids not emffecting the polypeptide ’ function occur. Desirably the substantially similar nucleotide sequence encodes the polypeptide encoded by the reference nucleotide sequence. Thez percentage of identity b»etween the substantially similar nucleotide sequence and thee reference nucleotide sequence desirably is at least 80%, more desirably at least 85%, gporeferably at least 90%, ranore preferably at least 95%, still more preferably at least 99%. A nucleotide sequence » substantially similar" to reference nucleotide sequence hybriclizes to the reference rucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO,, 1 mM EDTA at 50°C with washing in 2X SSC, 0.1% SDS at 50°C, more desirably in 7% sodium dodecyl ssulfate (SDS), 0.5 M NaPO,, 1 mM EDTA at 50°C with washing ire 1X SSC, 0.1% SDS at 550°C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 NA NaPO,, 1 mM EDTA at 50°C with washing in 0.6X SSC, 0.1% SDS at 50°C, preferably in 7% sodium dodecyl! sulfate (SDS), 0.5 M NaPO,, 1 mM EDTA at 50°C with washing in €.1X SSC, 0.1% SDS at 50°C, more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 Mi NaPO,, 1 mM EDTA at £0°C with washing in 0.1X SSC, 0.1% SDS at 65°C. "Synthetic" refers to a nucleotide sequence comprising structural characters that are rot present in the natural sequence. For example, an artificial sequence that resembles rmore 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 erad product of a transformation process, but also transgenic progeny thereof. A “non-transformed”, “non-transgenic”, or “non- recombinant” host refers to a wild-type organisrm, 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:

E adenine (A), cytosine (C), thymine (T), and gua nine (G). Amino acids are likewise indicated d by the following standard abbreviations: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; ro N), aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gin; Q), glutamic acid (Glu; E), ! glycine (Gly; G), histidine (His; H), isoleucine (lle; 1), 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 3.0 kb DNA fragment comprised in

Xenorhabdus nematophilus clone pClB9369, which comprises the following ORFs at the specified nucleotide positions:

Name Start End ort1 569 979 orf2 1045 2334

SEQ ID NO:2 is the sequence of the ~15 kDa protein encoded by orft of clone pCiB9369.

SEQ ID NO:3 is the sequence of the ~47.7 kDa Juvenile Hormone Esterase-like protein encoded by orf2 of clone pCIB9369.

SEQ ID NO:4 is the DNA sequence of orf1 of Xenorhabdus nematophilus clone pCIB9381.

SEQ ID NO:5 is the sequence of the protein emcoded by orf1 of clone pCiB9381.

SEQ ID NO:6 is the DNA sequence of orf2 of Xenorhabdus nematophilus clone pCIB9381.

SEQ ID NO:7 is the sequ ence of the Juvenile Hormone Esterase-like protein encoded by orf2 of clone pCIB9381.

SEQ ID NO:8 is the DNA sequence of orf1 of Xenorhabdus poinarii clone pCiB9354.

SEQ ID NO:9 is the seque=nce of the protein encoded by orf1 of clone pCIB9354. ’

SEQ ID NO:10 is the DNA. sequence of orf2 of Xenorhabdus poinarii clone pCIBS354.

SEQ ID NO:11 is the sequence of the Juvenile Hormone Esterase-like protein encoded by . orf2 of clone pCiB9354.

SEQ ID NO:12 is the DNA. sequence of orf1 of Photorhabdus luminescens clone pCiB9383-21.

SEQ ID NO:13 is the sequience of the protein encoded by orf1 of clone pCiB9383-21.

SEQ ID NO: 14 is the DNA sequence of orf2 of Photorhabdus luminescens clone pCIB9383-21.

SEQ ID NO:15 is the sequence of the Juvenile Hormone Esterase-like protein encoded by orf2 of clone pCiB9383-271.

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 P urposes of Patent Procedure. All restrictions on the availability ot the deposited material willl be irrevocably removed upon the granting of a patent.

Clone Accession Number Date of Deposit pCliBS369 NRRL B-21883 November 12, 1997 pCIiB9354 NRRL B-30109 February 25, 1999 pClB9381 NRRL B-30110 February 25, 1999 pCIB9383-21 NRRL B-30111 February 25, 1999

Nove! Nucleic Acidl 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 isolated from Xenorhabdus nematophilus, Xenorhabdus poinarii, and

Photorhabdus luminescens, members of the Enterobacteriaceae family. Xenorhabdus are symbiotic bacteria of nematodes of the genus Steinemema. Photorhabdus are symbiotic bacteria of nematodes of the genus Heterorhabditiss. The nematodes colonize insect larva, kill them, and their offspring feed on the dead lanae. The insecticidal activity is actually produced by the symbiotic Xenorhabdus and Photorhabdus bacteria. The inventors are the first to isolate the nucleic acid sequences of the praesent invention. The expression of the ‘ nucleic acid sequences of the present invention reseults in toxins that can be used to control

Lepidopteran insects such as Plutella xylostella (Dia mondback Moth). . A nucleotide sequence of the present inven-tion in clone pCIB9369 is characterized by an approximately 3.0 kb DNA fragment deposited pursuant to the Budapest Treaty for

Patent Deposits under Accession Number NRRL B-21883. The sequence of this DNA fragment is set forth in SEQ ID NO:1. Two open reading frames (ORF) are present in SEQ

ID NO:1 (nucleotides 569-979 and nucleotides 1045-2334, respectively), coding for proteins of predicted sizes of 15 kDa and 47.7 kDa (SEQ | D NOs:2 and 3, respectively). The two

ORFs are arranged in an operon-like structure. A. search for known sequences showing homology to each individual ORF using the UWGACG Blast and Gap programs does not reveal any significant match for ORF #1 and revezals 21% identity between ORF #2 and o- Bacillus thuringensis cry3A protein, which is not considered to be significant in the an. A

Gap analysis of the protein encoded by ORF ##2 of pCIB9369 by the Blast program identifies 30.6% AA identity and 44.1% AA similaritwy to a juvenile hormone esterase-related . protein (GenBank accession 2921553; Henikoff et al., PNAS USA 89. 10915-10919 : SEES (1992)). The nucleotide sequence of the present invention is also compared to known

Xenorhabdus nematophilus sequences encodimg the insecticidal toxin toxb4 (WO 95/00647), but no significant homology is foun-d. The 3.0 kb DNA fragment is also compared to the nucleotide sequence published im WO 98/08388. Twenty-two sequences of 60 nucleotides each (60-mers) from the 38.2 kb DNA fragment described in WO 98/08388 are compared to the 3.0 kb DNA fragrment of the present invention using the

UWGCG Gap program. The nucleotide sequence «of the first 60-mer starts at base 1 of the 38.2 kb DNA fragment and the other 60-mers are Bocated at approximately 2.0 kb intervals.

Each of the 22 sequences as well as their complementary sequences are tested. The highest percent of identity between the 3.0 kb DNBA fragment of the present invention and one of these 60-mers is 53%, which is not a significant homology. Furthermore, five different DNA fragments of the 38.2 kb sequence are tested for hybridization to the 3.0 kb fragment of the present invention by Southern blot: analysis. None of them reveal a positive hybridization signal.

The nucleotides sequences of pCIB9381, pClB9354, and pCIB9383-21 also reeveal two open reading fra mes in each of these clones. The nucleotide sequences of the two : ORFs in each of pCIE39381 and pCIB9383-21 are highly homologous to those in pCIBS369. : Hence, the ORF #2 proteins of pClB9381 and pCIiB9383-21 have essentially the same homology to the juve nile hormone esterase-related protein as does the ORF #2 protein of ’ pClB9369. The nucleotide sequence of ORF #1 of pCIB9354 is 77% identical tao the nucleotide sequence of ORF #1 of pCIB9369, and the nucleotide sequence of ORF #2 of . ~ pCiB9354 is 79% identical to the nucleotide sequence of ORF #2 of pCiB9362. The ORF #2 protein of pCIB9354 also has homology to the juvenile hormone esterase-related p rotein (29.2% AA identity ard 42.2% AA similarity). : In a preferred embodiment, the invention encompasses a nucleotide sequience - substantially similar te nucleotides 569-979 of SEQ ID NO:1, nuclectides 1045-2334 oft SEQ : ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, and

SEQ ID NO:14, whosse expression results in an insecticidal toxin. The present inventiomn also encompasses recom binant vectors comprising the nucleic acid sequences of this inveantion.

In such vectors, the nucleic acid sequences are preferably comprised in expression cassettes comprising reguiatory elements for expression of the nucieotide sequence s ina host cell capable of expressing the nucleotide sequences. Such regulatory elements umsually comprise promoter a.nd termination signals and preferably also comprise elements allowing efficient translation of polypeptides encoded by the nucleic acid sequences of the p resent _ invention. Vectors comprising the nucleic acid sequences are usually capable of replication in particular host cel Is, 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 a nother preferred embodimeznt, 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, ssuch host cells are prokaryotic cells. In a preferred embodiment, such host cells are eukawyotic cells, such as yeast cells, insect cells, or plant cells. In za most preferred embodimeznt, the host cells are plant cells, such as maize cells.

The nucleotide sequences of the invention can be isolated using the techniques described in the exa mples below, or by PCR using the sequences set forth in the sequenace listing as the basis for constructing PCR primers. For example, oligonucleotides having the sequence of approx imately the first and last 20-25 consecutive nucleotides of orf1 of SEEQ a ID NO:1 (e.g., nucleotides 569-588 and 957-976 of SEQ ID NO:1) can be used as PCR primers to amplify tree ori1 coding sequence (nucleotides 569-976 of SEQ ID NO:1) directly . from the source strain (Xenorhabdus nematophilus strain ATCC 19061). The other geme sequences of the invention can likewise be amplified by PCR from the respective sour-ce strains using the ends of the coding sequences set forth in the sequence listing as the bassis for PCR primers.

In another preferred embodiment, the insecticidal toxins comprise at least o-ne polypeptide encoded by a nucleotide sequence of the invention. The molecular weight of an insecticidal toxin according to the invention is larger than 6,000, as determined by s ize ot fractionation experiments. After treatment with proteinase K, only a minimal decrease in : o insecticidal activity is observed in the insect bioassay, indicating that the insecticidal tox ins

Con are substantially resistant to proteinase K treatment. The insecticidal toxins retain their ‘ insecticidal activity after being stored at 22°C or at 4°C for 2 weeks. They also retains their insecticidal activity after being freeze dried and stored at 22°C for 2 weeks. The insectici dal toxins are also still active after incubation for 5 minutes at 60°C, but they loses teir ) insecticidal activity after incubation for 5 minutes at 100°C or 80°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 recombinatiorm or

DNA shuffling. This technique is described in Stemmer et al., Nature 370: 389-391 (1994) and US Patent 5,605,793, 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 wariants with improved properties, such as increased insecticidal actiwity, enhanced stability, or different specificity or range of target insect pests are recovered. “The method encompasses forming a mutagenized double-stranded polynucleotide froran 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 addin gto 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 doub le-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 cycies. _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, Xenorhabdus nematophilus, Xenorhabdus poinarii, or

Photorhabdus 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 Xenorhabdus nematophilus, Xenorhabdus poinarii, or Photorhabdus 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 transcrip®ion is used. in a preferred embodiment, the cell in which the toxin is expressed is a mic roorganism, 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 ex presses large amounts of the : corresponding insecticidal toxin after infection of appropriate eukaryotic cells that are suitable for virus replication and expression of the nucleotisde 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 th e nucleotide sequences of the invention. In a preferred embodiment, non-pathogenic symbi otic bacteria, which are able to live and replicate within plant tissues, so-called endophytes, or non-pathogenic symbiotic 0 bacteria, which are capable of colonizing the phyllosphere= or the rhizosphere, so-called ok . epiphytes, are used. Such bacteria include bacteria of the genera Agrobacterium, - Alcaligenes, Azospirillum, Azotobacter, Bacillus, Clavibaacter, Enterobacter, Erwinia,

Flavobacter, Klebsislla, 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 expr-ession 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 operor 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-peositive species such as Bacillus are also known in the art and can be used in the context ost this invention (Quax et al. In.:

Industrial Microorganisms: Basic and Applied Molecular Genetics, Eds. Baltz et al,

American Society for Microbiology, Washington (199 3)). Alternate systems for overexpression rely for example, on yeast vectors an«d include the use of Pichia,

Saccharomyces and Kluyveromyces (Sreekrishna, In: Indusstrial microorganisms: basic and applied molecular genetics, Baltz, Hegeman, and Skatriud eds., American Society for

"Microbiology, Washington (1993); Dequin & Barre, Biotechnology 12:173-177 ( 1994); van "den Berg etal, Biotechnology 8:135-139 (1990)). in anotter preferred embodiment, at least one of the described nucleotide sequences is transferred to and expressed in Pseudomonas fluorescens strain &GA267356 (described in the published application EU 0 472 494 and in WO 94/01561) which has ’ biocontrol characteristics. In another preferred embodiment, a nucleotide sequeance of the invention is transferred to Pseudomonas aureofaciens strain 30-84 which also has . biocontrol characteristics. Expression in heterologous biocontrol strains resquires the ) selection of vectors appropriate for replication in the chosen host and a suitabl € choice of promoter. Tech niques 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 exgoressed 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 fe eding on such a transgenic plant, it also ingests the expressed toxins. This will deter the imsect from further biting into the plant tissue or may even harrm 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— [n another © preferred embodiment, the nucleotide sequence is included in a non-pathegenic self- replicating vinds. Plants transformed in accordance with the present inventkon may be monocots or clicots and include, but are not limited to, maize, wheat, barley , rye, sweet potato, bean, pea, chicory, lettuce, cabbage, cauliflower, broccoli, tumip, radish, spinach, asparagus, orion, garlic, pepper, celery, squash, pumpkin, hemp, zucchini, apple, pear, quince, melor, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, sorghum, sugarcane, stagarbeet, sunflower, rapeseed, clover, tobacco, carrot, cotton, alfalfa, rice, potato, eggplant, cucumber, Arabidopsis, and woody plants such as coruiferous and deciduous trees.

Once a desired nucleotide sequence has been transformed into a pa rticular 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 texchniques.

A nucleotide sequence of this invention is preferably expressed in transgenic plants, thus causing the b iosynthesis 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 amino acids encoded thereby. Furthermore, high expression in plants is best achieved from coding sequences that have at least 35% about GC content, preferably more than about 45%, more preferably more than about 50%, and most preferably more than about 60%. Microbial nucleotide : sequences which have low GC contents may express poorly in plants due to the existence . of ATTTA motifs vwhich may destabilize messages, and AATAAA motifs which may cause : ; inappropriate poly adenylation. 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 o r 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 Lubrizol, and WO 93/07278 (to

Ciba-Geigy).

For efficiert initiation of translation, sequences adjacent to the initiating methionine may require modif ication. 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 asmino acid of the transgene).

Expression of the nucleotide sequences in trarisgenic 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, efc.), 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 morvocotyledons. 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 publish ed 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 w hich 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 sh ould 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 in sect pest. Preferred promoters of this kind include those described by Stanford et al. Mol. Gen. Genet. 215: 200-208 (1989), Xu et al. Plant Molec. Biol. 22: 573-588 (1993), Logemanm et al. Plant Cell 1: 151-158 (1989),

Rohrmeier & Lehle, Plant Molec. Biol. 22: 783-792 (1993), Firek et al. Plant Molec. Biol. 22: 129-142 (1993), and Warner et al. Plant 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 dicotwledons. A preferred promoter is the maize PEPC promoter from the phosphoenol carboxyl ase gene (Hudspeth & Grula, Plant

Molec. Biol. 12: 579-589 (1989)). A preferred promoter for root specific expression is that : described by de Framond (FEBS 280: 103-106 (1991 ); EP 0 452 269 to Ciba-Geigy). A preferred stem specific promoter is that described in LJS patent 5,625,136 (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 transgermic plants expressing the nucleotide sequences in a wound-inducible or pathogen infection-i nducible manner. in addition to the selection of a suitable promoter, constructions for expression of an insecticidal toxin in plants require an appropriate trarscription terminator to be attached : downstream of the heterologous nucleotide sequen ce. Several such terminators are : available and known in the ant (e.g. tm1 from CaNhAV, EQ 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 A«h? 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 transge=ne 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 manipeulated and fused upstream of the nucleotide sequence. Many such target sequences ar-e known for the chloroplast and their functioning in heterologous constructions has beemn 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 achi eve this are well-known in the art.

Vectors suitable for plant transformation are described elsewhere in this specification. For Agrobacterium-mediated transto-rmation, 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 spec ies or - co-transformation «an be used (Schocher et al. Biotechnology 4: 1093-1096 (1986)) . For both direct gene transfer and Agrobacterium-mediated transfer, transformation is u sually : (but not necessarily) undertaken with a selectable marker which may provide resistarce to an antibiotic (kana mycin, hygromycin or methotrexate) or a herbicide (basta). Examples ot ) such markers are neomycin phosphotransferase, hygromycin phosphotransferase, dihydrofolate reductase, phosphinothricin acetyltransterase, 2, 2-dichloroproprionic acid : dehalogenase, acetohydroxyacid synthase, 5-enolpyruvyl-shikimate-phosphate synthase, - haloarylnitrilase, protoporhyrinogen oxidase, acetyl-coenzyme A carboxylase, * dihydropteroate synthase, chloramphenicol acetyl transferase, and B-glucuronidase. The choice of selectab le or screenable marker for plant transformation is not, however, critical to the invention.

The recom binant DNA described above can be introduced into the plant cell in a : number of art-recognized ways. Those skilled in the art will appreciate that the choice of method might deprend on the type of plant targeted for transformation. Suitable methods of transforming plant cells include microinjection (Crossway et al., BioTechniques 4:.320- 334 (1986)), electropo ration (Riggs et al., Proc. Natl. Acad. Sci. USA 83:5602-5606 (1986),

Agrobacterium-mediated transformation (Hinchee et al., Biotechnology 6:915-321 (19 88);

See also, Ishida et al., Nature Biotechnology 14:745-750 (June 1996) for maize transformation), chirect gene transfer (Paszkowski et al., EMBO J. 3:2717-2722 (1984); * Hayashimoto et z!., Plant Physiol. 93:857-863 (1990)(rice)), and ballistic particle acceleration using devices available from Agracetus, Inc., Madison, Wisconsin and D upont,

Inc., Wilmington, Delaware (see, for example, Sanford et al., U.S. Patent 4,945,050; and

McCabe et al., Bf otechnology 6:923-926 (1988)). See also, Weissinger et al., Annual Rev.

Genet. 22:421-4777 (1988); Sanford et al., Particulate Science and Technology 527-37 91987)(onion); Swab et al., Proc. Natl. Acad. Sci. USA 87: 8526-8530 (1990) (tobacco chloroplast); Christou et al., Plant Physiol. 87:671-674 (1988)(soybean); McCabe et al,

Bio/Technology 6:923-926 (1988)(soybean); Klein et al., Proc. Natl. Acad. Sci. USA, 85:4305-4309 (1988)(maize); Klein et al., Bio/Technology 6:559-563 (1988) (maize); Klein et al., Plant Physiol. 91:440-444 (1988) (maize); Fromm et al., Bio/Technology 8:833—839 (1990); and Gorcdon-Kamm et al., Plant Cell 2: 603-618 (1990) (maize); Koziel et al,

Biotechnology 137: 194-200 (1993) (maize); Shimamoto et al., Nature 338: 274-277 ("1989) (rice); Christou e t al., Biotechnology 9: 957-962 (1991) (rice); Datta et al, Bio/Technology

Claims (38)

1. / - J - What is claimed is:

   "1. Anisolated nucleic acid molecule comprising: (a) a nucleotide sequence substantially similar to a nucleotide sequence selected from the group consisting of: nucleotides 569-979 of SEQ ID NO:1, nucleoticles . 1045-2334 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, and SEQ ID NO:14; or (b) a nucleotide sequence isocoding with the nucleotide sequence of (a); wherein expression of said nucleic acid molecule results in at least one toxin that is active against insects.
2. 2. An isolated nucleic acid molecule according to claim 1, wherein said nucleo tide sequence is isocoding with a nucleotide sequence substantially similar to nucleotides 569- 979 of SEQ ID N0:1, nucleotides 1045-2334 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NC6, SEQ ID NO:8, S EQ ID NO:10, SEQ ID NO:12, or SEQ ID NO:14.
3. 3. An isolate d nucleic acid molecule according to claim 1, wherein said nucleotide sequence is substantially similar to nucleotides 569-979 of SEQ ID NO:1, nucleotides 1€45- 2334 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NOQ:12, or SEQ 1D NO:14. .
4. 4. An isolated nucleic acid molecule according to claim 1, wherein said nucleotide sequence enco«des an amino acid sequence selected from the group consisting of SEQID NOs:2,3,5,7,9, 11, 13, and 15.
5. 5. An isolated nucleic acid molecule according to claim 1, wherein said nucle otide sequence comprises nucleotides 569-979 of SEQ ID NO:1, nucleotides 1045-2334 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:1 2, or SEQ ID NO:14.
6. 6. An isolated nucleic acid molecule according to claim 1, wherein said nucleotide sequence is substantially similar to nucleotides 569-979 of SEQ ID NO:1, SEQ ID INO:4, SEQ ID NO:8, or SEQ ID NO:12.

   --
7. 7. An isolated nucleic acid molecule according to claim 1, wherein said nucleotide sequenc e encodes the amino acid sequence set forth in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:9, or SEQ ID NO:13.
8. 8. An isolated nucleic acid molecule according to claim 1, wherein said nucleotide sequence is substantially similar to nucleotides 1045-2334 of SEQ ID NO:1, SEQ ID NIO:6, SEQ ID NO:10, or SEQ ID NO:14.
9. 9. An isolated nucleic acioc molecule according to claim 1, wherein said nucleotide sequence encodes the amino acid sequence set forth in SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, or SEQ ID NO:15.
10. 10. An isolated nucleic acid molecule according to claim 1, wherein said nucleotide sequence comprises the approximately 3.0 kb DNA fragment comprised in pCIBS369 (NRRL B8-21883).
11. 11. An isolated nucleic acid molecule according to claim 1, wherein the toxin is a clive against Plutella xylostella.
12. 12. An isolated nucleic acid molecule comprising a 20 base pair nucleotide poeriion identical in sequence to a consecutive 20 base pair nucleotide portion of a nucleeotide sequence selected from the group consisting of: nucleotides 569-979 of SEQ ID NRO:1, nucleoticdes 1045-2334 of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:1 0, SEQ ID NO:12, and SEQ ID NO:14, wherein expression of said nucleic acid molecule results in at least one toxin that is active against insects.
13. 13. A chimeric gene comprising a heterologous promoter sequence operatively linked to the nucleic acid molecule of claim 1 or claim 12.
14. 14. A recombinant vector comprising the chimeric gene of claim 13.
15. 15. A host cell comprising the chimeric gene of claim 13.
16. "16. A host cell according to claim 15, which Is a bacterial cell.
17. 17. Ahost cell according to claim 15, which is a yeast cell.
18. 18. A host cell according to claim 15, which is a plant cell. :
19. 19. A plant comprising the plant cell of claim 18.
20. 20. A plant according to claim 19, which is maize.
21. 21. A toxin produced by expression of a DNA molecule according to claim 1 or claim 12.
22. 22. A toxin according to claim 21, wherein said toxin is active against Plutella xylostella. ©
23. 23. A toxin according to claim 21, wherein said toxin is produced by the E. coli strain designated as NRRL accession number B-21883. ©
24. 24. A toxin according to claim 21, wherein said toxin comprises an amino acid sequence - selected from the group consisting of: SEQ ID NOs:2, 3, 5,7, 8, 11, 13, and 15.
25. 25. A toxin according to claim 24, wherein said toxin comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs:2, 5, 9, and 13.
26. 26. A toxin according to claim 24, wherein said toxin comprises an amino acid sequence selected from the group consisting of: SEQ ID NOs:3, 7, 11, and 15.
27. 27. A composition comprising an insecticidally effective amount of a toxin according to claim 21.
28. 28. A method of producing a toxin that is active against insects, comprising: (a) obtaining a host cell according to claim 15; and (b) expressing the nucleic acid molecule in said cell, which results in at least one toxin that is active against insects.

    -F-
29. 29. A method of producing an insect-resistant plant, comprising introducing a nucleic acid molecule according to claim 1 or claim 12 into said plant, wherein said nucleic acid molecule is expressible in said plant in an effective amount to control an insect.
30. 30. The method of claim 29, wherein the insect i s Plutella xylostella.
31. 31. A method of controlling an insect comprising delivering to the insect an effective amount of a toxin according to claim 21.
32. 32. The method of claim 31, wherein the insect i s Plutella xylostella
33. 33. The method of claim 32, wherein the toxin is delivered to the insect orally.
34. 34. A method for mutagenizing a nucleic acid molecule according to claim 1 or claim 12, wherein the nucleic acid molecule has been cle=aved into population of double-stranded random fragments of a desired size, comprising: (a) adding to the population of double-s tranded 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 fragm ents; (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 o f a pair to prime replication of the other, thereby forming a mutagenized double-stra_nded polynucleotide; and (d) repeating the second and third steps feor 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.

    2 ad oe - 0H —
35. 35. A method according to claim 28, substantially as herein described and exemplified.
36. 36. A method according to claim 29, substantially as herein described and exemplified.
37. 37. A method according to claim 31, substantially as herein described and exemplified.
38. 38. A method according to claim 34, substantially as herein described and exemplified. AMENDED SHEET