NZ550315A - Genes and polypeptides from Y. entomophaga MH96 associated with insecticidal activity - Google Patents

Abstract

Disclosed are nucleic acid and polypeptide molecules associated with the insecticidal toxin produced by ersina entomophaga MH96. The nucleic acid and polypeptide molecules can be used in the manufacture of a composition suitable for use as a biopesticide.

Description

550315 PATENTS FORM NO. 5 Fee No. 4: $250.00 Our Ref: 43936 / 49 PATENTS ACT 1953 COMPLETE SPECIFICATION After Provisional No: 550315 Dated: 4 October 2006 GENES AND POLYPEPTIDES ASSOCIATED WITH INSECTICIDAL ACTIVITY WE AgResearch Limited, a New Zealand Company of East Street, Ruakura Campus, Hamilton 2020, New Zealand hereby declare the invention for which We pray that a patent may be granted to us, and the method by which it is to be performed to be particularly described in and by the following statement: 1 550315 GENES AND POLYPEPTIDES ASSOCIATED WITH INSECTICIDAL ACTIVITY STATEMENT OF CORRESPONDING APPLICATIONS This application is based on the provisional specification filed in relation to New 5 Zealand Patent Application Number 550315, and United States Patent Application Number 60/849,855 the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD The present invention relates to novel insecticidal genes and polypeptides. The 10 present invention also includes genes and polypeptides which are associated with the folding and/or transport of insecticidal polypeptides. In particular, the present invention relates to the genes and polypeptides associated with the insecticidal toxin produced by Y. entomophaga MH96.

BACKGROUND ART The original organism name Yersinia entomophagous MH-1 has been amended herein to refer to Yersinia entomophaga MH96, to conform to the official nomenclature of this organism. Thus, it should be clear that both names refer to the same organism as originally described in the provisional specification.

A novel insecticidal bacterium was isolated from an infected grass grub field 20 collected from New Zealand soils. The gram-negative bacterium is a new species residing within the genus Yersinia and has been named Yersinia entomophaga MH96. Y. entomophaga MH96 has a broad host range produces a toxin which acts as an insecticide against a range of insects, particularly towards members of the coleopteran and lepidopteran species, amongst others. After infection with the 550315 The infection process appears to be due to a rapid build up in the bacterial population followed by a rapid invasion of the haemocoel leading to the cadaver taking on a deliquescing black appearance.

The continued use of Bacillus thuringiensis (Bt) and derivatives as a biopesticide 5 over many years can lead to an increase in resistant insects. There is, therefore, a need for novel biopesticides to control insects (Moar 2003).

There is also a need for biological control agents such as bioactive compound/s to provide a biopesticide as an alternative to chemical pesticides which can be harmful to the environment.

Recently, a group of toxins have been identified that may provide an alternative to Bt These toxins have been termed tc toxins by Bowen et al. (1988) for toxin complex, as three proteins most easily described as A, B and C, simplistically represented in the S. entomophila system where they reside in the designated gene order sepA, sepB and sepC (Hurst et al., 2000). The components combine 15 to form a complex with insecticidal activity and have been recently reviewed by ffrench-Constant and Waterfield (2006). Studies of Blackburn et al. (2005) have identified that the Photorhabdus luminescens toxin complex a (Tea) is active towards Colorado potato beetle, Leptinotarsa decemlineata, and the sweet potato whitefly, Bemisia tabaci biotype B in parts per million, indicative of a greater level 20 of potency than Bt.

It would also be useful if there could be provided nucleotide and amino acid sequence information associated with the toxin produced by Y. entomophagus MH96 as this would allow for the commercial production of a biopesticide derived from Y. entomophaga MH96. 3 550315 It would also be useful if there could be provided nucleotide and amino acid sequence information relating to a toxin having activity against a broad range of insects, or at least against members of the coleopteran and lepidopteran species.

Furthermore, it would be beneficial in constructing new toxins for controlling 5 insects if individual gene and associated polypeptide sequence and information relating thereto were available to assist in constructing new transgenic organisms with enhanced insect resistance. In particular, the ability to create new transgenic plants which include introduced insecticidal genes, such as those derived from Bacillus thruingiensis has brought to the fore the commercial value and importance 10 of newly identified genes to agriculture.

All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy 15 and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art, in New Zealand or in any other country.

It is acknowledged that the term 'comprise' may, under varying jurisdictions, be 20 attributed with either an exclusive or an inclusive meaning. For the purpose of this specification, and unless otherwise noted, the term 'comprise' shall have an inclusive meaning - i.e. that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non-specified components or elements. This rationale will also be used when the term 'comprised' or 25 'comprising' is used in relation to one or more steps in a method or process. 4 550315 It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice.

Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only.

DISCLOSURE OF INVENTION According to one aspect of the present invention there is provided an isolated nucleic acid molecule having a nucleotide sequence selected from the group consisting of: a) SEQIDNO. 15; b) A functional fragment or variant of the sequence in a); or c) A complement to the sequences in a) or b).

According to a further aspect of the present invention there is provided an isolated nucleic acid molecule having a nucleotide sequence selected from the group consisting of: a) SEQ ID NOs. 2, 4, 6, 8, 10, 12, 14 or a combination thereof; b) A functional fragment or variant of at least one of the sequence(s) in a); or c) A complement to the sequences in a) or b).

According to a further aspect of the present invention there is provided an isolated 20 nucleic acid molecule having a nucleotide sequence selected from the group consisting of: a) SEQ ID NOs. 4, 6, 10, 12, 14 or a combination thereof; 550315 b) A functional fragment or variant of at least one of the sequence(s) in a); or c) A complement to the sequences in a) or b).

According to yet another aspect of the present invention there is provided an 5 isolated nucleic acid molecule which encodes a polypeptide substantially as set forth in SEQ ID NO: 16, or a functional fragment or variant thereof.

According to a further aspect of the present invention there is provided an isolated nucleic acid molecule which encodes a polypeptide selected from the group consisting of: a) SEQ ID NOs. 1, 3, 5,7, 9, 11, 13, or a combination thereof; b) A functional fragment or variant of at least one of the sequence(s) in a); or c) A complement to the sequences in a) or b).

According to a further aspect of the present invention there is provided an isolated 15 nucleic acid molecule which encodes a polypeptide selected from the group consisting of: a) SEQ ID NOs. 3, 5, 9,11, 13, or a combination thereof; b) A functional fragment or variant of at least one of the sequence(s) in a); or c) A complement to the sequences in a) or b).

Preferably, an isolated nucleic acid molecule as substantially described above may be used to produce a toxin. 6 550315 Preferably, there may be an isolated toxin produced from at least one nucleic acid molecule as substantially described above.

Preferably, the toxin may be in the form of a toxic complex. Preferably, the toxin may have an insecticidal activity. Preferably, the toxin may be used as a 5 biopesticide.

Preferably, an isolated nucleic acid molecule as substantially described above may used in the manufacture of a composition suitable as a biopesticide.

According to another aspect of the present invention there is provided there is provided an isolated polypeptide having an amino acid sequence selected from the 10 group consisting of: a) SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13, or a combination thereof; b) A functional fragment or variant of at least one of the sequence(s) in a).

According to a still further aspect of the present invention there is provided an isolated polypeptide having an amino acid sequence substantially as set forth in 15 SEQ ID NO. 16 or a functional fragment or variant thereof.

Preferably, a polypeptide as substantially described above may be in the form of a toxin. Preferably, the toxin may substantially correspond to the polypeptide set forth in SEQ ID NO. 16. Preferably, the isolated toxin may be in the form of at least one polypeptide as substantially described above.

Preferably, the toxin may be in the form of a toxic complex. Preferably, the toxin may have an insecticidal activity. Preferably, the toxin may be used as a biopesticide.

Preferably, an isolated polypeptide as substantially described above may be used in the manufacture of a composition suitable as a biopesticide. 7 550315 According to yet another aspect of the present invention there is provided a construct or vector which includes a nucleotide sequence as selected from the group consisting of: a) SEQ ID NO. 15; b) A functional fragment or variant of the sequence in a); or c) A complement to the sequences in a) or b).

According to yet another aspect of the present invention there is provided a construct or vector which includes a nucleotide sequence as selected from the group consisting of: a) SEQ ID NOs. 4, 6, 10, 12 and 14 or a combination thereof; b) A functional fragment or variant of at least one of the sequence(s) in a); or c) A complement to the sequence(s) in b).

According to a further aspect of the present invention there is provided a construct 15 or vector as substantially described above, wherein the combination of the nucleotide sequences from a) and/or b) and/or c) has each coding region of the subsequent sequence(s) joined to the coding region of the earlier sequence(s) in the combination by a short oligonucleotide segment which encodes a linker peptide.

According to a further aspect of the present invention there is provided a construct or vector substantially described above, wherein the combination of the nucleotide sequences from a) and/or b) and/or c) has each coding region of the subsequent sequence(s) joined to the coding region of the earlier sequence(s) so as to encode a single fusion protein. 8 550315 According to a still further aspect of the present invention there is provided a construct or vector which includes a nucleotide sequence of the present invention substantially as described herein.

According to another aspect of the present invention there is provided a host cell 5 transformed with a construct or vector of the present invention.

According to a further aspect of the present invention there is provided a host cell which includes a non- endogenous a nucleotide sequence as selected from the group consisting of: a) SEQ ID NO. 15; b) A functional fragment or variant of the sequence in a); or c) A complement to the sequences in a) or b).

According to a further aspect of the present invention there is provided a host cell which includes a non-endogenous a nucleotide sequence as selected from the group consisting of: a) SEQ ID NOs. 2, 4, 6, 8, 10, 12, 14 or a combination thereof; b) A functional fragment or variant of at least one of the sequence(s) in a); or c) A complement to the sequence(s) in a) or b).

According to a further aspect of the present invention there is provided a host cell 20 which includes at least two nucleic acid molecules having a nucleotide sequence selected from the group consisting of: a) SEQ ID NOs. 2, 4, 6, 8, 10, 12, 14, or any combination thereof; 9 550315 b) A functional fragment or variant of at least one sequence in a); c) A complement to the sequence(s) in a) or b); wherein the combination of the nucleotide sequences from a) and/or b) and/or c) has each coding region of the subsequent sequence(s) joined to the coding 5 region of the earlier sequence(s) in the combination by a short oligonucleotide segment which encodes a linker peptide.

According to a further aspect of the present invention there is provided a host cell which includes at least two nucleic acid molecules having a nucleotide sequence selected from the group consisting of: a) SEQ ID NOs. 2, 4, 6, 8, 10, 12,14, or any combination thereof; b) A functional fragment or variant of at least one sequence in a); c) A complement to the sequence(s) in a) or b); wherein the combination of the nucleotide sequences from a) and/or b) and/or c) has each coding region of the subsequent sequence(s) joined to 15 the coding region of the earlier sequence(s) so as to encode a single fusion protein.

Preferably, the host cell as substantially described above may include a nucleic acid molecule which has a nucleotide sequences which have been modified to assist with expression of the molecule in a host cell.

Preferably, the host cell as substantially described above may include a nucleotide sequence also has at least one regulatory sequence to assist with expression of the nucleic acid molecule in the host cell. 550315 According to one further aspect of the present invention there is provided a host cell which includes at least two nucleic acid molecules having a nucleotide sequence selected from the group consisting of: a) SEQ ID NOs. 2, 4, 6, 8, 10, 12, 14, or any combination thereof; b) A functional fragment or variant of at least one sequence in a); c) A complement to the sequence(s) in a) or b); together with at least one regulatory sequence to assist with expression of the nucleic acid molecule in the host cell; and wherein the combination of the nucleotide sequences from a) and/or b) and/or 10 c) has each coding region of the subsequent sequence(s) joined to the coding region of the earlier sequence(s) in the combination by a short oligonucleotide segment which encodes a linker peptide.

According to another aspect of the present invention there is provided a host cell which includes at least two nucleic acid molecules having a nucleotide sequence 15 selected from the group consisting of: a) SEQ ID NOs. 2,4, 6, 8,10, 12, 14, or any combination thereof; b) A functional fragment or variant of at least one sequence in a); c) A complement to the sequence(s) in a) or b); together with at least one regulatory sequence to assist with expression of the 20 nucleic acid molecule in the host cell; and wherein the combination of the nucleotide sequences from a) and/or b) and/or c) has each coding region of the subsequent sequence(s) joined to the coding region of the earlier sequence(s) so as to encode a single fusion protein. 11 550315 According to yet a still further aspect of the present invention there is provided a host cell substantially as described above wherein said cell is a plant cell. In other preferred embodiments, a host cell may be any suitable bacteria or algae.

It should be appreciated that other organisms can be host cells, provided the cells 5 can be successfully transformed with a vector or construct of the present invention.

According to one further aspect of the present invention there is provided a plant which includes a host cell substantially as described herein.

According to another aspect of the present invention there is provided a transgenic plant which includes a nucleic acid molecule of the present invention substantially 10 as described herein. Preferably, a seed may be obtained from a transgenic plant.

According to yet a still further aspect of the present invention there is provided a seed comprising a transgenic plant cell substantially as described herein.

Preferably, the host cell as substantially described above may produce a toxin. Preferably, the toxin may be in the form of a toxic complex. Preferably, the toxin 15 may have an insecticidal activity. Preferably, the toxin may be used as a biopesticide.

According to a further aspect of the present invention there is provided a host cell substantially as described above wherein the host cell may be used in the manufacture of a composition suitable as a biopesticide.

According to a further aspect of the present invention there is provided transgenic plant which includes a host cell substantially as described above.

Preferably, the toxin may be in the form of a toxic complex. Preferably, the toxin has an insecticidal activity. Preferably, the toxin may be used as a biopesticide.

According to a still further aspect of the present invention there is provided an 12 550315 isolated toxin produced by at least one nucleic acid molecule of the present invention.

According to yet another aspect of the present invention there is provided an isolated toxic complex produced a host cell transformed with a vector or construct 5 of the present invention.

According to another aspect of the present invention there is provided a toxin which corresponds to the polypeptide set forth in SEQ ID NO. 16.

According to another aspect of the present invention there is provided the use of an isolated toxin substantially as described herein as a biopesticide.

According to one further aspect of the present invention there is provided a plant, seed or plant cell which has been originally derived from a construct or vector of the present invention.

According to a yet further aspect of the present invention there is provided a method of protecting a plant from insects characterised by the step of providing in 15 said plant a toxin or toxin complex derived from one or more of the nucleic acid molecules of the present invention.

According to a further aspect of the present invention there is provided a probe having a nucleotide sequence capable of binding to a nucleic acid molecule of the present invention.

Preferred, plants may include wheat, rice, cotton, corn (maize), barley, canola, and soy beans.

According to another aspect of the present invention there is provided the use of a host cell which includes at least one nucleic acid molecule of the present invention to produce a biopesticide or biopesticide effect. 13 550315 According to a further aspect of the present invention there is provided a composition which includes as an active ingredient an effective amount of a toxin produced as a result of the expression of a nucleic acid molecule having a nucleotide sequence selected from the group consisting of: a) SEQ ID NO. 15; b) A functional fragment or variant of the sequence in a); or c) A complement to the sequences in a) or b).

According to a further aspect of the present invention there is provided a composition which includes as an active ingredient an effective amount of a toxin 10 produced as a result of expression of a nucleic acid molecule having a nucleotide sequence selected from the group consisting of: a) SEQ ID NOs. 2, 4, 6, 8, 10, 12, 14 or a combination thereof; b) A functional fragment or variant of at least one of the sequence(s) in a); or c) A complement to the sequences in a) or b).

According to a further aspect of the present invention there is provided a composition includes as an active ingredient an effective amount of toxin produced as a result of expression of a nucleic acid molecule which encodes a polypeptide substantially as set forth in SEQ ID NO: 16, or a functional fragment or variant 20 thereof.

According to a further aspect of the present invention there is provided a composition that includes a toxin in the form of a polypeptide having an amino acid sequence substantially as set forth in SEQ ID NO. 16 or a functional fragment or variant thereof. 14 550315 According to a further aspect of the present invention there is provided a composition for use as a biopesticide which includes as an active ingredient an effective amount of a polypeptide having an amino acid sequence substantially as set forth in SEQ ID NO. 16.

According to a further aspect of the present invention there is provided a composition for use as a biopesticide which includes as an active ingredient an effective amount of at least one polypeptide substantially as set forth in the sequence listing of the present application.

Preferably, the composition may be formulated with at least one biopolymer 10 compound. Preferably, at least one biopolymer compound is at least one type of gum compound.

Preferably, the composition may be formulated as a gel composition.

In some preferred embodiments, the composition may be formulated with at least one biopolymer compound and at least one desiccating agent. In other preferred 15 embodiments, the composition may be formulated with at least one type of gum compound and the at least one desiccating agent is at least one inert clay compound.

A composition may be formulated as a dough or granular material.

A composition may be formed into a prill or granule shape.

A composition may be mixed with an aqueous liquid and sprayed onto a substrate. Other embodiments, the composition may be coated onto a substrate. Preferably, the substrate may be a seed. 550315 According to a further aspect of the present invention there is provided a composition for use as a biopesticide which may be for the application against insect species listed in Table 13 and/or the larvae thereof.

Compositions of the present invention may be formulated in a variety of different ways without departing from the scope of the present invention. In general the formulation chosen will be dependent on the end application. For example, possible formulations include, but should not be limited to: • Vectors such as the Trojan vector • Matrixes • Soluble powders • Granules • Micro encapsulation • Aqueous suspensions • Non-aqueous suspensions • Emulsions • Pastes • Emulsifiable concentrations • Baits • As an aerosol allowing spray application in powder or wet form It will be appreciated that other suitable formulations and/or methods of preparing the formulations and/or compositions will be known to those skilled in the art. 16 550315 Examples of other such methods to stabiles or prepare a composition include the methods described in patent applications WO 02/15702 or WO 02/15703.

As used herein the term "biopesticide" refers to a biologically derived substance having the ability to kill, or retard the growth or development of, insects and/or the 5 larvae thereof. In particular, a biopesticide derived from the present invention should be capable of retarding growth, or killing, at least one, but preferably all or most of the species listed in the best modes and/or Table 13, and/or the larvae thereof.

The term insecticidal' as used herein refers to the ability to kill or harm insects. 10 Preferably, the term 'insecticidal' refers to the ability to kill or harm the insect species listed in the best modes and/or Table 13, and/or the larvae thereof.

The term 'toxin' as used herein refers to a substance or substances derived from that produced by Y. entomophagus MH96 which is harmful and/or distasteful to insects. In particular, the term 'toxin' should be taken to indicate a toxic complex.

The term 'toxic complex' (Tc) as used herein refers to a toxin which comprises more than one protein encoded by more than one gene. Preferably, the toxin may comprise at least 3 proteins, namely A, B and C components of the Tc. In some cases as with Y. enomophaga the A component is encoded by two separate genes the translated products of which have similarity to either the A amino or the A 20 carboxyl region. That is both ORFs together encode two proteins that have similarity to the A group of proteins. In further preferred embodiments the toxin may comprise at least four or five proteins produced by four or five genes. In some embodiments the Tc may also include one but preferably two Chitinase genes.

It is known that mixing the A components with the B and C component and vice versa derived from the a different organism will yield an active toxin that may differ 17 550315 in its target specificity and or toxicity (Waterfield et al. 2005; Hey et al. 2005; Hey et al. 2006). The use of the Y. enomophaga Tc constituents for such a system is also encompassed in the present invention.

The term 'toxin cluster' as used herein refers to the genes responsible for 5 production of a toxic complex. These genes reside in the virulence region of the V. entomophaga MH96 chromosome and may include one or more Chitinase genes.

The composition of the present invention may preferably include formulations suitable for: • direct application to insect affected areas e.g. drench, spray form; 10 • suspended in a bait matrix • slow release prills for subterranean applications; • hydrophobic matrixes facilitating buoyancy for aquatic surface filter feeders.

Other suitable formulations will be known to those skilled in the art.

The term bait as used herein refers to any foodstuff or other attractant to an insect 15 or larvae thereof which includes an effective amount of toxin, toxin complex; or components thereof.

The term 'effective amount' as used herein refers to a suitable quantity for a biopesticide activity to be exhibited.

It is envisaged numerous delivery systems could be employed to take advantage 20 of the toxin of the present invention.

In other embodiments the toxin of the present invention could be indirectly delivered to insects via a transgenic microorganism which expresses the gene(s) 18 550315 of the present invention and wherein said organisms inhabit the phylloplane, phyilosphere or rhizoplane, rhizosphere of plants of inertest.

In some preferred embodiments the formulated toxin complex components could be applied directly to surfaces where insects may contact such as artificial/cultural 5 surfaces (e.g. milled wood, concrete, and urban dwellings); as well as in agricultural systems such as plant surfaces seed coats or matter of plant origin.

The present invention has application in both terrestrial and aquatic environments and may be applied in both soil and phylloplane systems.

The nucleic acid molecule may be an RNA, cRNA, genomic DNA, cDNA, or PNA 10 molecule, and may be single- or double-stranded. The nucleic acid molecule may also optionally comprise one or more synthetic, non-natural or altered nucleotide bases, or combinations thereof.

For ease of reference only the nucleic acid molecule may generally be referred to as simply being a DNA molecule.

The term 'homology' as used herein in relation to nucleic acid molecules refers to how closely related two or more strands of DNA are to one another based on their respective nucleotide sequences.

The term 'homology' as used herein in relation to polypeptide molecules refers to how closely related two or more polypeptides are to one another based on their 20 respective amino acid sequences.

The term 'heterologous' as used herein means derived from a different Yersinia source. 19 550315 It will therefore be appreciated by those skilled in the art that the term 'homology' as used herein encompasses the term 'percent identity' which refers to the percentage of identical nucleotides or amino acids in each sequence.

The term 'percent positive' as used herein refers to how closely related two or 5 more polypeptides are to one another based upon the similarity of their respective amino acid sequences taking into account not only identical amino acids but also equivalent amino acids which have the same physio-chemical properties.

As can be found in the appendices of a "New England BioLabs catalog" or at the WWW site: http://www.neb.com/nebecomm/tech reference/general data/amino acid structur es.asp and includes groupings of similar amino acids as outlined page 113 Lehninger et al (1993).

The term 'alignment' as used here in refers to the process of lining up two or more sequences to achieve maximal levels of identity (and conservation, in the case of 15 amino acid sequences) for the purpose of assessing the degree of similarity and the possibility of homology.

The term 'similarity' as used herein refers to the extent to which nucleotide or protein sequences are related. The extent of similarity between two sequences can be based on percent sequence identity and/or conservation. In BLAST similarity 20 refers to a positive matrix score.

As used herein the term 'conservation' refers to changes at a specific position of an amino acid or (less commonly, DNA) sequence that preserve the physico-chemical properties of the original residue.

As used herein the term 'identity' refers to the extent to which two (nucleotide or 25 amino acid) sequences are invariant. 550315 As used herein the term 'substitution' refers to the presence of a non-identical amino acid at a given position in an alignment. If the aligned residues have similar physico-chemical properties the substitution is said to be "conservative".

A fragment of a nucleic acid is a portion of the nucleic acid that is less than full 5 length and comprises at least a minimum sequence capable of hybridising specifically with a nucleic acid molecule according to the present invention (or a sequence complementary thereto) under stringent conditions as defined herein.

The term 'exogenous' as used herein, refers to a nucleic acid molecule originating from outside an organism.

A fragment of a polypeptide is a portion of the polypeptide that is less than full length but which still retains a substantially equivalent biological activity to the full length polypeptide. A fragment according to the invention has at least one of the biological activities of a nucleic acid or polypeptide of the invention.

The polypeptides of the invention can be prepared in a variety of ways. For 15 example, they can be produced by isolation from a natural source, by synthesis using any suitable known techniques (such as by stepwise, solid phase, synthesis described by Merryfield (1963), or as preferred, through employing DNA techniques.

The term "variant" as used herein refers to nucleic acid molecule or polypeptide 20 wherein the nucleotide or amino acid sequence exhibits: at least substantially 70%, or at least substantially 75% homology with the nucleotide or amino acid sequences contained in the sequence listing; preferably exhibits at least substantially 80% or 85% homology or 25 greater with said sequences; and 21 550315 most preferably exhibits a homology selected from substantially 90-99% homology to the sequences contained in the sequence listing and which may include at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to said sequences in the 5 sequence listing; as assessed by GAP or BESTFIT (nucleotides and peptides), or BLASTP (peptides) or BLASTN (nucleotides) Altschul et al, (1997), and Schaffer, et al. (2001). The variant may result from modification of the native nucleotide, or amino acid sequence, by such modifications as; insertion, 10 substitution or deletion of one or more nucleotides or amino acids, or it may be a naturally-occurring variant.

Thus, the term variant should be taken to include changes (i.e. conservative substitution) to the nucleotide sequences set forth herein which do not alter the amino acid being coded for, due to the degenerate nature of the genetic code.

The term "variant" also includes homologous sequences which hybridise to the sequences of the invention under standard, but most preferably under stringent conditions.

The term "variant" also encompasses sequences which have been deliberately altered.

In general "stringent conditions" for determining the degree of homology may refer to. a) low salt concentrations (i.e. less than 1M, preferably less than 500mM and most preferably less that 200mM); and b) high hybridization temperatures (i.e. at least 30°C, preferably greater 25 than 37°C and most preferably greater than 50°C). 22 550315 The terms "polypeptide", "peptide" and "protein" are all used interchangeably herein to refer to a molecule comprising a chain or chains of two or more amino acids with amide bond linkages. The terms "polypeptide", "peptide" and "protein" may herein also include modifications of structure in general and more specifically 5 include additions, substitutions, biochemical oxidation or reduction, variations of amino acids including amino acids not commonly found in proteins such as D-amino acids, and prosthetic groups such as may be required for enzymatic activity.

According to one further aspect of the present invention there is provided a transgenic host cell which includes a nucleic acid molecule of the present invention 10 substantially as described herein.

It is envisaged fusion proteins and plants expressing fusion proteins comprising the nucleic acid molecules of the present invention can be made as a matter of mere routine following the teachings of: US 2006/0168683; Hey, T., Bevan, S., et. al. (2005); Hey, T. Cai, C„ (2005).

The term "fusion protein" as used herein refers to a single polypeptide encoded by more than one gene wherein the nucleic acid molecule(s) of the additional gene(s) have been altered so they effectively form a single gene. In general, this is achieved by forming a 'fusion gene' in which the stop codon from the first gene (and any other intermediary genes) is/are removed and the subsequent gene(s) 20 are joined thereto, so they are in frame. The last gene of the 'fusion gene' retains the stop codon. If the genes of the 'fusion gene' each encode proteins linker (or "spacers") nucleotide sequences are added in between to join the genes encoding separate proteins to allow for the proteins to fold independently or be cleaved. However, this definition is not intended to be limiting and is merely illustrative of 25 what is known in the art, such as for example, what is described in US 2006/0168683 and the two separate papers on this subject by Hey et. al. 23 550315 The term 'in frame' refers to the placement of a gene in the fusion gene so that the gene has the correct open reading frame so that the codons of the gene specify the correct amino acids to create the desired protein. In general, the desired protein may be that normally provided by the gene.

The term 'codon' refers to the triplet code created by the nitrogenous bases (ademine (A), guamine (G), cytosine (C) and thymine (T)) of 3 adjacent nucleotides in a nucleic acid molecule which together specify a particular amino acid.

The term 'open reading frame' (or 'ORF') as used herein refers to the sequence of 10 nucleotides in between the start-codon and stop-codon (termination-codon) which contain the Condons that can be translated into a protein.

The term "host cell" refers to a cell, which has been, or is capable of being, transformed by a vector or construct, and can support the replication and/or expression of the gene of interest from the vector or construct. The term "host 15 cell" also encompasses cells derived from an originally transformed cell. Suitable host cells may be prokaryotic cells such as bacteria and viruses, or, eukaryotic cells such as algae, yeast, plant, fungal, insect, amphibian, or mammalian cells.

In some preferred embodiments plant viruses maybe used as a vehicle to express the toxin in plants infected with the virus.

In some further preferred embodiments insect viruses, baculoviruses may be suitable host cells given their ability to act as an effective biopesticide (toxin) delivery system if transformed with genes encoding a toxin or toxin complex derived from the nucleic acid molecule of the present invention.

Understandably, the term "host cell" should also be taken to include a non-human 25 transgenic organism which comprises a host cell. 24 550315 The term "construct" as used herein refers to an artificially assembled or isolated nucleic acid molecule which includes the gene of interest. In general a construct may include the gene or genes of interest and appropriate regulatory sequences. It should be appreciated that the inclusion of regulatory sequences in a construct is 5 optional for example, such sequences may not be required in situations where the regulatory sequences of a host cell are to be used. The term construct includes vectors but should not be seen as being limited thereto.

The term "vector" as used herein encompasses both cloning and expression vectors. Vectors are often recombinant molecules containing nucleic acid 10 molecules from several sources.

A "cloning vector" refers to a nucleic acid molecule originating or derived from a virus, a plasmid or a cell of a higher organism into which another exogenous (foreign) nucleic acid molecule of interest, of appropriate size can be integrated without loss of the vector's capacity for self-replication. Thus vectors can be used 15 to introduce at least one foreign nucleic acid molecule of interest (e.g. gene of interest) into host cells, where the gene can be reproduced in large quantities.

An "expression vector" refers to a cloning vector which also contains the necessary regulatory sequences to allow for transcription and translation of the integrated gene of interest, so that the gene product of the gene can be expressed.

The term "transformation", or grammatical variants thereof, as used herein refers to a process by which the genetic material carried by an individual host cell is altered by incorporation of exogenous DNA into its genome.

The term "expression" as used herein broadly refers to the process by which a nucleic acid molecule is converted by transcription and then translation into a 25 protein. 550315 In relation to plants in order to obtain high expression of heterologous genes preferably the exact nucleotide sequence of a gene may be altered prior to transformation - see for example US 5,380,831.

The term "isolated" as used herein means substantially separated, or purified away from, contaminating sequences in the cell or organism in which the nucleic acid molecule(s) or polypeptide(s) naturally occur(s). The term 'isolated' therefore includes nucleic acids purified by standard purification techniques as well as nucleic acids prepared by recombinant technology, including PCR technology, and those chemically synthesised. Thus, a nucleic acid molecule of the present invention placed in a plant is an isolated nucleic acid molecule.

It is to be clearly understood that the invention also encompasses peptide analogues, which include but are not limited to the following: 1. Compounds in which one or more amino acids are replaced by its corresponding D-amino acid. The skilled person will be aware that retro-inverso amino acid sequences can be synthesised by standard methods; see for example Choreo and Goodman, 1993; 2. Peptidomimetic compounds, in which the peptide bond is replaced by a structure more resistant to metabolic degradation. See for example Olson et al., 1993; and 3. Compounds in which individual amino acids are replaced by analogous structures for example, gem-diaminoalkyl groups or alkylmalonyl groups, with or without modified termini or alkyl, acyl or amine substitutions to modify their charge. 26 550315 The use of such alternative structures can provide significantly longer half-life in the body, since they are more resistant to breakdown under physiological conditions.

Methods for combinatorial synthesis of peptide analogues and for screening of 5 peptides and peptide analogues are well known in the art {see for example Gallop etal., 1994; Hogan, 1997).

For the purposes of this specification, the term "peptide and peptide analogue" includes compounds made up of units which have an amino and carboxy terminus separated in a 1,2, 1,3, 1,4 or larger substitution pattern. This includes the 20 10 naturally-occurring or "common" a-amino acids, in either the L or D configuration, the biosynthetically-available or "uncommon" amino acids not usually found in proteins, such as 4-hydroxyproline, 5-hydroxylysine, citrulline and ornithine; synthetically-derived a-amino acids, such as a-methylalanine, norleucine, norvaline, Co.- and A/-alkylated amino acids, homocysteine, and homoserine; and 15 many others as known in the art.

It also includes compounds that have an amine and carboxyl functional group separated in a 1,3 or larger substitution pattern, such as p-alanine, y-amino butyric acid, Freidinger lactam (Freidinger et al., 1982), the bicyclic dipeptide (BTD) (Freidinger et al., 1982; Nagai and Sato, 1985), amino-methyl benzoic acid 20 (Smythe and von Itzstein, 1994), and others well known in the art. Statine-like isosteres, hydroxyethylene isosteres, reduced amide bond isosteres, thioamide isosteres, urea isosteres, carbamate isosteres, thioether isosteres, vinyl isosteres and other amide bond isosteres known to the art are also useful for the purposes of the invention.

A "common" amino acid is a L-amino acid selected from the group consisting of glycine, leucine, isoleucine, valine, alanine, phenylalanine, tyrosine, tryptophan, 27 550315 aspartate, asparagirie, glutamate, glutamine, cysteine, methionine, arginine, lysine, proline, serine, threonine and histidine. These are referred to herein by their conventional three-letter or one-letter abbreviations.

An "uncommon" amino acid includes, but is not restricted to, one selected from the group consisting of D-amino acids, homo-amino acids, N-alkyl amino acids, dehydroamino acids, aromatic amino acids (other than phenylalanine, tyrosine and tryptophan), ortho-, meta- or para-aminobenzoic acid, ornithine, citrulline, norleucine, a-glutamic acid, aminobutyric acid (Abu), and a-a disubstituted amino acids.

Thus, preferred embodiments of the present invention may have a number of advantages over the prior art which can include: • providing a new biopesticide which has a broad efficacy across a range of insects; • providing a new method for controlling insects; • providing a new biopesticide which has a range of different forms; • providing a non-living biopesticide; • providing genes and polypeptides and sequence information which can be used to produce a biopesticide; • providing recombinant host cells which produce a biopesticide; and • providing new transgenic organisms including plants, and/or ability to produce new transgenic organisms including plants which can produce a novel biopesticide.

BRIEF DESCRIPTION OF SEQUENCE LISTING 28 550315 SEQ ID No. 1: shows the amino acid sequence information for the chil protein.

SEQ ID No. 2: shows the nucleotide sequence information for the chil nucleotide.

SEQ ID No. 3: shows the amino acid sequence information for the yenA1 protein.

SEQ ID No. 4: shows the nucleotide sequence information for the yenA1 nucleotide.

SEQ ID No. 5: shows the amino acid sequence information for the yenA2 10 protein.

SEQ ID No. 6: shows the nucleotide sequence information for the yenA2 nucleotide.

SEQ ID No. 7: shows the amino acid sequence information for the chi2 protein.

SEQ ID No. 8: shows the nucleotide sequence information for the chi2 nucleotide.

SEQ ID No. 9: shows the amino acid sequence information for the yenB protein.

SEQ ID No. 10: shows the nucleotide sequence information for the yenB 20 nucleotide SEQ ID No. 11: shows the amino acid sequence information for the yenCI protein 29 550315 SEQ ID No. 12: SEQ ID No. 13: SEQ ID No. 14: SEQ ID No. 15: SEQ ID No. 16: SEQ ID No. 17: SEQ ID No. 18: BRIEF DESCRIPTION OF DRAWINGS Further aspects of the present invention will become apparent from the following description which is given by way of example only and with reference to the accompanying drawings in which: Figure 1 shows a SDS-PAGE of the cell pellets derived from a 3 ml overnight culture (30°C), of each of the V. entomophaga MH69 mutant derivatives. Lanes: 1, V. entomophaga MH96; 2, Y.ent.i; 3, Y.ent::2; 4, Y.ent::3; 5, Y.ent::4; 6,Y.ent::5, 7 Y.e/?f6; 8, BioRad marker; 9 Y.ent::7; WY.ent::8; 11, Y.ent::9; 12, contaminant; 13,Y.enf::11; 14 Y.entrA2\ 15, Y.ent:: 13 (refer table 1). Marker sizes are listed on the right in kDa, (Lane 16). shows the nucleotide sequence information for the yenCI nucleotide. shows the amino acid sequence information for the yenC2 protein. shows the nucleotide sequence information for the yenC2 nucleotide. shows chil to yenC2 nucleotide sequence information. shows a Chi 1, YenA1, YenA2, Chi2, YenB1, YenCI, YenC2 protein complex encoded by SEQ ID No. 15. shows a YenA1, YenA2, YenB1, YenCI, YenC2 protein complex encoded by SEQ ID No. 18 shows YenA1, YenA2, YenB1, YenCI, YenC2 nucleotide sequence information. 550315 Figure 2 shows a schematic of Y. entomophaga MH69 virulence determinants black bars denote genomic regions flanking the Pathogenicity island. The flanking tRNA sequences that typify the boundary of a pathogenicity island are indicated. The Pacl sites 5 used to clone the virulence encoding region are shown. The Bg!\\ restriction enzyme sites use to construct the Y. entomophaga MH96A3 derivative (V. enfA3) are shown. Sequence related similarities are listed in Table 2; Figure 3 shows a schematic depicting a virulent Tn5 based mutations in Y. 10 entomophaga MH69 showing ORF designations; Figure 4 shows a SDS-PAGE/coomassie stain analysis of Y. entomophaga MH69 grown in LB showing the presence of the A toxin band (denoted by arrow). Note the absence of the toxin band in the Y. entomophaga MH69 A3 derivative (V. entA3) and the restoration of 15 the band when the Y. entomophaga MH69 derived clone pPAC14 is reintroduced to the strain Y. en/A3(PAC14) and wild type Y. entomophaga. Bioassays have shown the Y. e/?£A3(pPAC14) strain to cause virulence indicating the pPAC14 clone likely encodes the main Y. entomophaga virulence determinants. Marker sizes are 20 listed on the right in kDa; Figure 5 shows a V. entomophaga MH69 Tc toxin on SDS-PAGE gel. Lanes; 1, Marker (sizes in KDa) are indicated; 2, Y. entomophaga culture supernatant; 3, V. e/rtA3 culture supernatant; 4, Y. ent ultra centrifuged culture supernatant; 5, Y. entLZ ultra centrifuged culture 25 supernatant, 6; 7, Y. ent ultra centrifuged pellet;8; 9 Y. entAS ultra centrifuged pellet; 10;11; 12 Y. ent, (via glycerol gradient; 13 Y. 31 550315 entAZ (via glycerol gradient);location of the predicted Y. entomophaga insecticidal components are indicated.

Figure 6 Coomassie stained SDS-PAGE of Ultracentrifuged Y.entomophaga supernatant toxins showing the locations of the excised bands 5 labelled 1-9 used for MS-MS analysis (refer to table 8 for results from MS-MS analysis); Figure 7 shows a schematic depicting the pPAC14 Avrll/Pmll (pPAC 14AAvrll/Pmll) and Swal (pPAC14ASwal) self ligations which delete the YenAI-YenA2 and the YenB YenCI and YenC2 regions 10 respectively; Figure 8a shows a SDS-PAGE/silver stain visualisation of Y. ent:: 15 a mutation which resides in the SepB like protein (YenB, Fig. 3) note the absence of the YenB band lanes 10 and 11 (arrow). The Y. enf;:15 mutation which resides in yenB still allows the 15 ultracentrifugation of the complex. In addition the reduced level of the C component seen in lane 10 compared with lane 11, indicates that the C components (YenCI and YenC2) are being expressed, refer to the Y. entomophaga wild type lane 14. Conversely the amount of the YenCI 12 like components in the ultracentrifuged 20 pellet (lane 11) is relatively higher compared to the YenA1 and YenA2 components and Y. entomophaga wild type, lanes 15 and 16. Note the extra products in the Y ent::6 (yenA1) ultracentrifuged pellet (lane 5). Visual analysis of the Y. ent A3(pPAC14AAvrll/Pmll) shows the presence of the Chi2 25 component (lane 12 arrow); 32 550315 Figure 8b shows Y. entAZ (pPAC14ASwal) as expected shows the absence of YenB, YenCI and YenC2 encompassed by the Swal deletion (Table 1; Fig. 7)) this validates that band #4 (Fig. 6) represents the YenCI and YenC2 components. With reference to Fig. 8B it is evident that 5 both the YenB and the YenCI ad YenC2 components are missing a result of the pPAC14ASwal deleting these genes. All of the constructs assessed in Fig. 8 were assessed for affect against grass grub larvae. Only the wild-type Y. entomophaga supernatant was able to give an affect towards grass grub larvae; Assessment of the Y. ent:9 and Y. ent:9 (pACXH) The complementation of the chaperone mutant Y ent::9 showed that when the bacteria are grown at 25°C the supernatant show an absence of YenB (arrow) compared to the Y. ent::9 complementing clone Y. ent: :9 (pACXH) lane 2. Results at growth at 30°C differ to this result (lane 3 and 2). The chaperone mutant Y. ent:9 and its complimenting derivative Y. enf::9(pACXB) show the complementation of the Y. ent:9 to deliver the toxins outside of the cell as can be seen by the comparisons of lanes 15-17. Temperatures are indicated above the figure. Results from Figure 9 suggest the integral role of the A components (YenA1 and YenA2) to the formation of the mature Y. entomophagous complex. This data indicates the possible role of the chaperone cluster being implicated in the secretion of the YenB like component from the cells at 25°C; Figure 10 shows cumulative mortality (%) of third-instar Helicoverpa larvae fed control or Y. entomophaga MH96-treated diet. The sonicated filtrate from a Y. entomophaga MH96 culture was added to the diet Figure 9 33 550315 undiluted or at dilutions of 1:1 (50%), 1:9 (10%) or 1:99 (1%). Values are the mean of four (two for 50%) replicate dishes; and Figure 11 shows a photograph at day 5 of Spodoptera litura in tray with either 7 micro litres of neat toxin applied to a -4mm cube (Toxin) or 7 5 micro litres of LB media.

BEST MODES FOR CARRYING OUT THE INVENTION Identification of the Yersinia entomophaga MH96 virulence determinants Standard suicide mutagenesis was undertaken as described by Delorenzo et al. (1990). A total of 1400 independent mutants were screened for loss of virulence 10 against grass grub larvae. Fifteen mutants were identified that were unable to cause virulence towards 3rd instar grass grub larvae.

Sequence analysis derived off the Tn5 insertion points showed that thirteen of the fifteen a virulent mutations reside in genes belonging to an insecticidal toxin complex (Tc). The nature of insecticidal toxins were initially identified by Bowen et 15 al (1998) and recently reviewed by ffrench-Constant and Waterfield (2006). Toxin complexes consist of three proteins most easily described as A, B and C, simplistically represented in the S. entomophila system, where they reside in the designated gene order sepA sepB and sepC (Hurst et al. 2000). Eleven avirulent mutations reside in the A and B components, two in each of the chitinase like 20 genes (Fig. 2; Fig. 3); One in a methylation gene (Y. ent::4), SDS-PAGE analysis was undertaken for each of the mutants and compared to wildtype (Fig. 1) Cloning the Y. entomophaga MH69 virulence region Tn5 mutagenesis had identified 11 mutations. Using the kanamycin antibiotic selection derived from the Tn5 antibiotic and associated restriction enzyme sites 25 the region flanking each Tn5 insertion point were cloned and the DNA sequenced.. 34 550315 Two large clones (pK3X and pKsal) were identified, from which peripheral sequence analysis showed a high degree of DNA similarity to the genomic DNA of Y. pestis. It was therefore derived that the complete virulence associated region could be sequenced.

From the completed DNA sequence it was evident that the complete Tc cluster resided in the genome of Y. entomophaga and that this cluster was a component of a Pathogenicity Island as defined by Hacker et al. (1997) and is flanked by two tRNA repeats (Figure 2). In addition the Tc cluster could be cloned using the restriction enzyme Pad (Fig. 2).

DNA sequence data identified two Sph\ site in the pKsal clone and that one resided in DNA which exhibited high similarity to the Y. pestis like genome. The pKsal clone was restricted with Hind\\\ and self ligated to form pKsalH3, resulting in the omission of the inner most Sph\ site which resided internal to the predicted Y. entomophaga Pathogenicity Island, the remaining Sph\ site could then be used to 15 insert an antibiotic cassette without affecting virulence, allowing downstream antibiotic selection, facilitating the rapid cloning of the predicted Y. entomophaga Tc MH96 virulence region. A spectinomycin cassette flanked by Sph\ restriction enzyme sites was PCR amplified and inserted into the Sph\ site of pKsalH3. From the resultant construct, the Spectinomycin resistance gene and its flanking region 20 were digested with the restriction enzyme HindlU and inserted into the H/ndlll site of the suicide vector pVIK165 (Kalogeraki and Winans, 1997),. The resultant construct was then conjugated back to Y. entomophaga MH69 and the Spectinomycin resistance gene recombined into the Y. entomophaga MH69 genome to make Y. entomophaga Sm. The strain was assessed by standard 25 bioassay towards grass grub larvae and the strain found to be virulent indicating that the spectimoycin cassette did not affect the virulence of Y. entomophagaSm 550315 Genomic DNA from Y. entomophagaSm was isolated and digested with the restriction enzyme Pacl (Fig. 2). The resultant digest was then ligated into the Pad site of the vector pBRminicospac (Table 1) and the ligation packaged using a Gigapack®IIIXL packaging extract (Stratagene, California, USA). The packaged 5 extract was transformed into the E. coli strain XL1-Blue and plated on LB agar plates containing Ampicillin and Spectinomycin. In total 16 colonies were identified. The restriction enzyme profile of each transformant was then validated by agarose gel electrophoresis. Two constructs designated pPAC12 and pPAC14 were chosen for further analysis as their profiles matched the predicted restriction enzyme 10 profile derived from the generated DNA sequence of the Y. entomophaga Tc related region. The pPAC12 and pPAC14 clones were assessed for their ability to induce virulence in an E. coli background towards grass grub larvae by standard bioassay. Bioassays showed that neither of the pPAC12 and pPAC14 clones were able to induce virulence towards grass grub larvae.

The Y. entomophaga MH69 Tc virulence clone (pPAC14) was also transformed into the Y. entomophaga MH69 related Yersinia, Ewingella americana and S. entomophila. However through bioassay analysis these were found to be avirulent. For this reason a Y. entomophaga MH69 tc deletion derivative was constructed.

Construction of the Y. entomophaga Tc MH96A3 deletion derivative To asses the ability of the pPAC clones to induce virulence in a Yersinia type strain and assess he role of the Y. ent Tc like region towards virulence a Y. ent strain with the Tc like region deleted was constructed The pPAC14 clone was digested with the restriction enzyme Pacl and ligated into the analogous site of a pLAFR3 derivative pLAFRP (Table 1). The resultant 25 construct was digested with the restriction enzyme Bg/ll and a chloramphenicol cassette PCR amplified and inserted. The resultant clone was then electroporated 36 550315 back into Y. entomophaga MH69 and the chloramphenicol based deletion variant allowed to recombine by homologous recombination. Several recombinants were identified and were found by standard bioassay to be non virulent towards grass grub larvae. In addition SDS-PAGE analysis showed the absence of the potential 5 toxin bands (Fig. 4, arrow). One recombinant was stored and termed Y. entomophaga MH69 A3 Complementation of the Y. entomophaga MH69 derivative The Y. entomophaga MH69 toxin complex virulence derived plasmid clone pPAC14 was electroporated into Y. entomophaga MH96A3. Three resultant 10 transformants were chosen and assessed by SDS-PAGE (Fig. 4) and assessed for virulence against grass grub larvae. Results showed that the transformants were virulent to grass grub larvae, demonstrating that the cloned region encodes the main virulence determinants of Y. entomophaga MH96.

Table 2: Similarities of products of putative ORFs to translated amino acid 15 sequences in the database detected using BlastP gene % identity/% similarity Locus tag, protein, species, (a.a size) accession number Yen1 984-4652 86/92 (1-1520) FOG: PAS/PAC domain [Y. frederiksenii ATCC 336411(1522) ail77976874 Yert2 4649-5605 87/93 (1-315) COG3706: Response regulator containing a CheY-like receiver domain and a GGDEF domain [Y. intermedia ATCC 299091 (319) ail77976875 Yen3 5920-6459 91/94 (1-179) nucleoprotein/polynucleotide-associated enzyme [Y. pestis KIM] (179) Yen4 6683-7381 90/93 (1 -232)COG0564: Pseudouridylate synthases, 23S RNA-specific [Y. mollaretii ATCC 439691 (232) ail77962388 Yen5 8767-9600 82/912 (1-276)Signal transduction histidine kinase [Y. mollaretii ATCC 43969] (451) ail77962052 Yen6 9962-10324 55/71 (2-120) hypothetical protein UTI89 C4899 IE. coli UTI891 (122) Yen7 10519-10935 NO MATCHES Chil 55/71 (2-541) unnamed protein product [P. luminescens subsp. Laumondii TT01 ](544) ail36785785 yenA1 55/71 (1-1164) Insecticidal toxin complex protein TccA2 [P. luminescens subsp. Laumondii TT011 (1173) ail36785784 37 550315 yenA2 68/81(1-1363) Insecticidal toxin complex protein TccB2 [P. luminescens subsp. Laumondii TTOI ](1362) CHI36785783 chi2 72/82 (1-627) unnamed protein product [P. luminescens subsp. LaumondiiTT01K622) ail36785782 yenB 59/74 (1-1483) insecticidal toxin complex protein TcaC [P. luminescens] (1485) ail16416891 yenCI 59/73 (8-683) TccC1/XptB1 protein [Xenorhabdus nematophila] (1016) PNF cytotocix ai!42742522 yenC2 70-79 (1-690) putative insecticidal toxin complex [Y. pseudotuberculosis IP 32953] (994) ai!51589839 yenU 28/47(546-729)hypothetical protein Pfl_2644 [Pseudomonas fluorescens PfO-1] (1288) ail77382872 yenV Unidentifierd ORF yenT 45/58 (1-51) Yersinia Heat-stable Enterotoxin Type B \Y. enterocolitis], (71) ail3913874 yenX 72/80 (25-150) hypothetical protein ECA2267 [Erwinia carotovora subsp. Atroseptica SCRI1043] <152) ail49611721 Yen8 64/73 (1-594) amidase [E. carotovora subsp. Atroseptica SCR110431, (600) ail49611595 Yen9 71-82 (1-1207) urea amidolyase [E. carotovora subsp. Atroseptica SCRI1043], (1204) ai!49611596 Yen 10 59/75 (3-229) GntR-family transcriptional regulator [E. carotovora subsp. Atroseptica SCRI10431, (267) ail49611597 Percent identities and similarities were calculated in relation to the deduced gene products A of the sequenced ORF. indicates position of amino-acid similarity (% identity/% similarity over a.a residue - a.a residue) in relation to sequence generated in this study. Refer Fig. 2 5 for schematic of Y. entomophaga MH69 virulence associated region.

Purification of the Y. entomophaga MH69 Tc like toxins (Depicted in Figure 1 and 5) Through growth curve analysis over various temperature increments in conjunction with SDS-PAGE/Coomassie stain and bioassays it has been identified that the Y. 10 entomophaga MH69 Tc-like protein toxins are secreted into the supernatant at temperatures 25 °C and less. Above 30 °C no toxin is present in the supernatant, however the toxin can be identified in the cell pellet, indicating that the toxin is expressed but not secreted at this time point. With reference to Fig. 5 it is evident that the supernatant is relatively clean as assessed by SDS-PAGE Fig. 5 lanes 2-15 5. It was determined that the protein components giving toxicity could be 38 550315 electrophoretically separated by SDS/PAGE and visualised by Coomassie or Silver stain and be easily purified by ultracentrifugation (Fig. 5, lane 7) and further purified by subsequent purification in a 40% glycerol gradient (Fig. 5, lane 12). Bioassays showed that the purified Y. entomophaga MH69 Tc-like toxins are able 5 to cause disease symptoms and mortality, except the Y.enth3 derivatives supernatant fractions of which remain inactive towards grass grub larvae (Table 3). Bioassays of the crude Y. entomophaga MH69 supernatant to Diamond Back Moth, Plutella xylostella (L.) are given in Tables 4-7.

Note the absence of protein bands in the Y.enfA3 strain where the central Y. 10 entomophaga MH69 virulence region has been deleted (Fig. 2; Fig. 5 lanes 3,5,8,11 and13). The predicted location of the Y. entomophaga MH69 virulence associate region toxins are indicated on the right (refer Table 2).

Assessment of Y. entomophaga mutants and Y. ent LZ containing two pPAC14 derived clones pPAC14Aavrll/Pmll and pPAC14Aswal to produce a 15 Tc Each of the Y. entomophaga mutants and two pPAC14 derived deletion clones (pPAC14Aavrll/Pmll and pPAC14Aswal; Table 1) were assessed for the ability to form a complex by ultracentrifugation of culture supernatants grown at 25°C (refer Fig, 8a, and 8b).

MS-MS analysis of the Y. entomophaga bands depicted in figure 6 ESI ion trap tandem MS analysis of 1DE protein bands shown in Figure 6 Table 8 contains some protein identifications of the insecticidal proteins separated by 1DE. These 9 bands were cut out of the gel, destained, alkylated with DTT and iodoacetamide then trypsin digested and extracted from the gel. The peptide 25 mixture from each band was then cleaned up with zip tip (C18 coated pipette tips) 39 550315 to remove any insoluble material or interfering ions and submitted to tandem mass spectrometry analysis (appendix attached for a method).

The resulting data was imported into the database search tool Mascot in an appropriate format and searched against public database NCBI nr species 5 Eubacteria and a small in-house database called "MH96" containing the specific protein sequences of interest. This search tool compares experimentally derived average peptide masses to those obtained via theoretical tryptic cleavage of the protein sequence.

The search parameters were; 1) tryptic cleavages missed - up to 2 2) Modifications - carbamidomethyl, methionine oxidation 3) Protein mass tolerance 1.0 + Da and peptide tolerance 0.6 + Da. The search stringencies for a positive identification using MASCOT include; 1) A total protein Mowse score with P < 0.001 ) 2) A minimum total of two peptides matching the sequence. 3) Peptide MOWSE score P < 0.0001. 4) Where M is underlined it means oxidation These stringencies and reporting standards are based on international proteomic journal reporting guidelines and I used both databases to verify if the result were 20 correct. 40 550315 Where the same peptides match in different databases the peptide amino acid sequence font is in italics. The most predominant protein has the highest protein MOWSE score and is listed in numerical order of abundance.

Table 8: Results of MS/MS analysis of the bands depicted in Figure 6 Band Protein Theoret No.

Total Peptide Sequence and Peptide Peptide number description ical pept protein modifications charge / MASCOT and ides missed database mat MOWSE cleavages MOWSE che score searched MW/pl d Score P<0.05 = expectation 43 166504/ Band 1 gi|78459183 .26 3 244 R EYKVITAK.K 2+/1 56 / 0.024 insecticidal toxin complex R.SSAQFWLDEK.L 2+/0 85 / 3e-005 protein NCBInr [Photorhabdus luminescens .MQDSPEVSITTLSLPK.G 3+/0 17 eubacteria subsp. 1.8e+002 Luminescens] Band 1 V 167795/ 22 21437 R.LIQDFIK.I 2+/0 48/2.3e-06 4.90 R.FWGTEAGK.D 2+/0 49/3.1e-06 MH 1 R.EYQVITAK.G 2+/0 43 / 7e-06 R.ILFSDGFGR.L 2+/0 45/3.3e-06 R.VEAGESWQR.S 2+/0 70 / 8.9e-09 K.IEYWQPVK.Q 2+10 / 4.5e-05 R.GAETCLFYR.S 2+10 49 /1,8e-06 R. SSAQFWLDEK. Q 2+/0 50/ 5.1e-07 R.LDWVITSAGVR.G 2+/0 84/7.6e-10 R.LQGIPLPFSYK.V 2+/0 53 / 8.4e-07 K.TLQAFVGVLDSK.T 2+/0 70/5.4e-09 R SEIFDEITGNR.L 2+/0 4912e-06 R.VESEPDVWVAR.Q 2+/0 65 / 3.4e-08 R.NDGYQYDQER.A 2+/0 45 / 3e-06 R.GDDQAFSPFSPR.F 2+/0 55 / 4.5e-07 R.DVTDEIPVLVNR.L 2+/0 65 / 7e-08 R.FVTEDGYVCALGR.R 2+/0 59 / 6.7e-08 41 550315 R.VYQPYFLDDWR.Y 2+/0 53 / 5.3e-07 R.QIGGDSNAVTYGAMK.A 2+/0 761 2.4e-09 R.VAADPQCSQQWIK.A 2+/0 75 / 3,3e-09 R.YGSPLLSVAINYPR.R 2+/0 65 / 5e-08 R.LQEGATLMDINGDGR.L 2+/0 77 / 9.7e-10 R.WIADNGTLWNALLQAR.F 2+/0 102/ 1.2e-11 R.LFRVESEPOVWVAR.Q 3+/1 38 / 7.8e-06 R.TDLFADTHIYDPLGR.E 3+/0 49/1,2e-06 .MQNSQEMAITTLSLPK.G 2+/0 91 / 3.8e-11 R. DTDGFAQGTVDI PTHPSR.T 3+/0 74/ 1.8e-09 R.LTLDYDLNNSVSTLVAVR.Q 2+ 10 58 / 2.1 e-07 R.LTQEQEYAHGSWDGQER.E /O 45 / 3.5e-06 R.ALPTTDATVPSAWCSTIETR.S /O 62 / 2.8e-08 R.FGQPLTLEGFSQPQTSFNPDR.V 3+/0 39/1,2e-05 R.QVAYETDGSPITQPPLEFDYQR.F /O 34/1,9e-05 Band 1 2. YeriA2 156517/ 4.89 773 R.LNTLFAK.E K.ASLEALQVSK.Q 42 / 2.9e-06 55/1,6e-07 MH96 K.SESLSVSES YR. R R.YGYWAANQQLR.M K.ELYDENISSTEQK.V 75 / 3.5e-09 56 / 5.2e-07 40/ 1.1e-05 Band 2 NCBInr eubacteria gi|14041726 XptD1 protein [Xenorhabdus nematophila] 146332/ 5.29 1 86 K. TEFFFQLESALNQGK. 1 2+/0 86/ 2.2e-00e Band 2 YenA2 156517/ 4.89 242 R.LYISWFEVAEEK.E K. TEFFFQLESALNQGK. L 2+/0 2+/0 52 / 3.1 e-07 59 / 6e-08 MH 1 K.NVDDLYDHLLLDTQVSAK.V R.KDSLSFDAYQMIQSGDLYR.F K.QfiEWEVNSVEQQINLQNMQIK.A 3+/0 3+/1 3+/0 54 / 5.5e-07 37 / 4.3e-05 40 / 5.5e-06 Band 3 No match NCBInr 42 550315 eubacteria Band 3 130232/ 31 27510 R.NAMTIIR.A 2+/0 54/7.1 e-07 Z 4,82 K.EFIFLR.L 2+/0 32 / 6.6e-05 MH 1 R.SVVPLQLR.W 2+/0 48 / 7.3e-07 R.HLNLQYK.I 2+/0 43 / 2.3e-06 R.GENIITYQR.T 2+/0 58 / 3.3e-07 R.EIQSLSNFR.L 2+/0 65! 5.9e-08 K.SDEEMLMEK.I 2+/0 46 /1,4e-06 K.LVAFGDQTGSK.A 2+/0 72 / 6.9e-09 K.SSISPLLAAAAK.I 2+/0 72 / 1e-08 K.MEALSAWLTK.N 2+/0 66 / 3.8e-08 K.LSLDNDALYK.E 2+/0 61 /8.1e-08 K. EGVGEAVMAALK. A 2+/0 57 / 2.2e-07 R.SFEVVSSLYR.L 2+/0 68 / 3.2e-08 K. AVSMANQVLQK. Y 2+/0 71 / 8.6e-09 R.EYIDQSGQADK.A 2+/0 58 / 8.7e-08 K.IEPEITVLSSASK.S 2+/0 65 / 3.5e-08 R.MVPFDNSSPFSR.Q 2+/0 67 / 9.8e-09 R.LTQPNWLGLTQFK.1 2+/0 57 / 2.9e-07 K.GLGASNIELGTVIQR.V 2+/0 64 / 4e-08 K.FIQSVEL DGSNQAR.K 2+/0 65 / 3.4e-08 R.VDPQFSWNTTQEK.Y 2+/0 65 / 3.3e-08 R.LAVVPTNNMVTFFK.G 2+/0 63/1 e-07 R.HDESLLGNGAVIFDK.A 3+/0 52 / 3.4e-07 K.NNLTPAS LALLLGVTR.L 2+/0 66 / 2.6e-08 K.LTFVAEDNPSLGNLDK.G 2+/0 72 / 8.5e-09 K.NEDSIHEYLEFANIK.K 3+/0 38 /1,4e-05 R.SQYSQSLADTLLGLGYR.S 2+/0 104 / 4.1e-12 K.GIANGLSENVCLTTDDFQR.Q 2+/0 93 / 5.4e-11 K.NAQENLLSQTISAEYGVER.S 2+/0 93 / 7.4e-11 43 550315 R.ENVSSETMVSRPFLTMTYR.I 3+/0 / 3.5e-05 K.SNSPLVPQTSSSTDASSESQTNK.L 2+/0 60/ 1.7e-07 Band 3 2. 156517/ 2390 R.LNTLFAK.E 2+/0 47 / 2.2e-06 4.89 YenA2 R.FEPMLNR.S 2+/0 27 / 9.3e-05 K.ASLEALQVSK.Q 2+ / 0 73 / 2.8e-09 R.LSYSTASSLLGR.R 2+/0 73 / 4.5e-09 R.RQEWELQYK.Q 2+/1 39 / 7e-06 K.QLILYPVIINR.D 2+/0 75 /1,6e-09 R.YGYWAANQQLR.M 2+/0 60/1 9e-07 K.NIIDQGDMEYR.K 2+/0 52 /1,3e-06 K.ELYDENISSTEQK.V 2+/0 51 / 7.5e-07 R.FSIDLQQQDIDINK.A 2+/0 43/1 3e-05 Band 4 s gi|14041726 146332/ 2 157 R.LNTLFAK.K 2+/0 62 / 0.0068 .29 K.NLLDQGDMEYR K 2+/0 64 / 0.0037 XptDl NCBInr protein [Xenorhabdu eubacteria s nematophila ] Band 4 a A 130232/ 26 21617 R.NAMTIIR.A 2+/0 57 / 5.2e-07 4.82 R.LQTLSEK.T 2+ / 0 39 / 2.6e-05 MH 1 K.EFIFLR.L 2+/0 32 / 3.4e-05 R.SWPLQLR.W 2+/0 47/1,8e-06 R.HLNLQYK.I 2+/0 46 /1,2e-06 R.GENIITYQR.T 2+/0 58 / 3.4e-07 R.EIQSLSNFR.L 2+/0 64 / 8.2e-08 K.SDEEMLMEK.I 2+/0 40/1.5e-05 K.LVAFGDQTGSK.A 2+/0 72 / 6.9e-09 K.MEALSAWLTK.N 2+10 64 / 5.7e-08 K.LSLDNDALYK.E 2+10 61 / 4.3e-08 R.SFEVV5SLYR.L 2+/0 65 / 5.7e-08 K.AVSMANQVLQK.Y 2+/0 55 / 3.3e-07 K.EGVGEAVMAALK.A 2+/0 56 / 7e-07 R.EYIDQSGQADK.A 44 550315 K.LRGENIITYQR.T R.MVPFDNSSPFSR.Q R. LTQPNWLGLTQPK.L K.GLGASNIELGTVIQR.V K.FIQSVELDGSNQAR.K R.VDPQFSWNTTQEK.Y R.LAVVPTNNMVTFFK.G K.NNLTPASLALLLGVTR.L K.LTFVAEDNPSLGNLDK.G R.SQYSQSLADTLLGLGYR.S K.NAQENLLSQTISAEYGVER.S 2+/0 2+ /1 2+/0 2+/0 2+/0 2+/0 2+/0 2+/0 2+/0 2+/0 2+/0 2+/0 57 / 1.1e-07 51 /1.3e-06 67 / 9.9e-09 56/4.1 e-07 70/ 1.1e-08 63 / 4.8e-08 65 / 3.3e-08 66 / 5.6e-08 65 / 3e-08 81 / 2.1e-09 83 / 2.4e-10 87 / 3.6e-10 Band 4a 2. YenA2 156517/ 17 5959 R.LNTLFAK.E 2+/0 51 / 4.4e-07 4.89 R.CWLNYK.L 2+/0 42 / 6.8e-06 MH 1 R.FEPMLNR. 2+/0 32 / 6.3e-05 K.AVFNFLTK.N 2+/0 65 / 3.3e-08 K.ASLEALQVSK.Q 2+/0 76 / 1.3e-09 K.SESLSVSESYR.R 2+/0 59/1.2e-07 R.LSYSTASSLLGR.R 2+/0 73 / 4.5e-09 R.RQEWELQYK.Q 2+/1 45/1.5e-06 K.QLILYPVIINR.D 2+/0 78 / 8.5e-10 K.ETDSDGNIIVSGR.Y 2+/0 63/1,4e-07 K.NIIDQGDMEYR.K 2+/0 59 / 2.4e-07 R.YGYWAANQQLR.M 2+/0 58 / 3e-07 K.ALLGESWPAELNK.L 2+/0 61 / 8e-08 R.MFPEIYVDPTLR.L 2+/0 49/ 1.4e-06 K.ELYDENISSTEQK.V 52 / 9.9e-07 2+/0 R<LSLVTQSVQQyINRiI 2+/0 55 / 4.6e-07 R.NANSQELYPTAWSEWK.A 2+/0 65 / 1.5e-08 Band 5 NCBInr gi!14041726 XptD1 protein [Xenorhabdus 146332/ 5.29 2 282 R.LNTLFAK.K K.NLLDQGDMEYR.K 2+/ 0 2+/0 62 / 0.0068 67/0.0018 45 550315 eubacteria nematophila] Band 5 1. YenA2 156517/ 24 12222 K.DFTNLR.S 1+/0 /1,6e-05 4.89 R. LNTLFAK. E 2+/0 50 / 5.3e-07 MH1 R.CWLNYK.L 2+/0 44 1 3.6e-06 K.LTDDVAQK.A 2+/0 40 / 5.4e-06 R.FEPMLNR.S 2+/0 31 / 8.3e-05 K.AVFNFLTK.N 2+/0 68/ 1.8e-08 K.NIIYGIWK.E 2+/0 / 4.8e-05 K.ASLEALQVSK.Q 2+/0 73 / 2.7e-09 K.SESLSVSESYR.R 2+/0 60 / 1.1e-07 R.LSYSTASSLLGR.R 2+/0 56 / 2.6e-07 R.RQEWELQYK.Q 2+/1 43 / 2.6e-06 K.QLILYPVIINR.D 2+/0 77 / 9.1e-10 K.ETDSDGNIIVSGR.Y 2+/0 55 / 8.7e-07 K.NIIDQGDMEYR.K 54 / 8.2e-07 2+/0 R.YGYWAANQQLR.M 61 /1,6e-07 2+/0 K.ALLGESWPAELNK.L 2+ 1 0 57 /1.8e-07 R.MFPEIYVDPTLR.L 2+/0 65 / 4.8e-08 K.ELYDENISSTEQK.V 2+/0 54 / 3.6e-07 K.EINMDLYDSSISPR.G 2+/0 51 / 7.4e-07 R.SLDLVLSWETQNSR.L 2+/0 34 / 58e-05 R. LSLVTQSVQQYINR . I 2+/0 52 / 9.8e-07 R.FSIDLQQQDIDINK.A 2+/0 53/1.4e-06 R.NANSQELYPTAWSEWK.A 2+/0 73 / 2.4e-09 K.GYSLTQPSDPDAIAASDPIHYR.K 3+/0 39 / 2e-05 Band 5 2. YenA1 130232/ 22 7230 R.NAMTIIR.A 2+/0 55 / 6.3e-07 4.82 K.EFIFLR.L 2+/0 32 13.5e-05 MH 1 R.SWPLQLR.W 2+/0 31 / 7.4e-05 R.HLNLQYK.I 2+/0 40 / 4.8e-06 R.GENIITYQR.T 2+/0 53 / 1e-06 46 550315 R.EIQSLSNFR.L 2+/0 56 / 4.6e-07 K.SDEEMLMEK.I 2+/0 45/1 7e-06 K.LVAFGDQTGSK. A 2+/0 66 / 2.4e-08 K.SSISPLLAAAAK.I 2+/0 72 / 8.6e-09 K.MEALSAWLTK.N 2+/0 62 / 8.7e-08 K.LSLDNDALYK.E 2+/0 61 / 8.8e-08 K.EGVGEAVMAALK.A 2+/0 41 / 7.8e-06 R.SFEVVSSLYR.L 2+/0 54 / 7.8e-07 K.AVSMANQVLQK. Y 2+/0 50 / 9.8e-07 R. EYIDQSGQADK.A 2+/0 61 / 7.6e-08 K.IEPEITVLSSASK.S 2+/0 44 / 3.6e-06 R.MVPFDNSSPFSR.Q 2+10 56 / 4.2e-07 R.LTQPNWLGLTQPK.L 2+10 50/ 1.3e-06 K.GLGASNIELGTVIQR.V 2+/0 64 / 5.4e-08 K.FIQSVELDGSNQAR. K 2+/0 71 / 7.9e-09 R.VDPQFSWNTTQEK.Y 2+10 56 / 4.7e-07 R. LAW PTNNMVT FFK . G 2+10 60/1.9e-07 Band 6 NCBInr eubacteria gi|36785782 Score: 530 unnamed protein product [Photorhabdus luminescens subsp.

Laumondii TT01] 69106 1 4.94 2 530 R. KEISIACSGVK, T K. LSAYITDWCQYDAR. L 2+/1 2+/0 71 / 0.00076 81 / 6.5e-005 Band 6 8 70009 / 5.03 30540 K.EIYLK.S R • &AGL L GG Xj R. E 1+/0 2+/0 ! 4.7e-05 66 14.5e-08 MH 1 R.&ATADEINK.I R.SAVGGDLTTR.Q R.VFNVIFDGK.V K.EISIACSGVK.A K.GHFISLDTPR.T K.THLGYANYGR.S K.DGENYVLLIK,E R.KEISIACSGVK.A 2+/0 2+/0 2+/0 2+/0 2+/0 2+/0 2+/0 2+/1 67 12.8e-08 65 / 3.4e-08 66 / 2.3e-08 37 / 3.1e-05 54 / 1.9e-07 44 / 5.5e-06 39 / 2e-05 54 / 7.4e-07 47 550315 K.VYTNTYWVER.W 2+/0 61 / 7.9e-08 K.NYMDAEHSLSMGK.N 2+/0 57 / 1,9e-Q7 K.EDGSQGNLSYTATR.V 2+/0 49 / 2e-06 R.VCAPMYNHYVGDK.T 2+/0 53 / 2.2e-07 K.GHIVPLDPYGDLGTAR.N 3+/0 80 / 1.7e-09 K.YTEETYSRPDVNFK.E 2+/0 50 /1 1e-06 K. LSA YITDWCQYDAR. L 2+/0 59 / 5.6e-08 R.GFDLATLMQNPATYDR.L 2+/0 92 / 6.5e-11 R.WQVPGIGSSDGNPHNAWK.F 3+/0 48 / 2.3e-06 K.NGPALGTMENGAPEFFDIVK.N 2+/0 77 / 1e-09 K.LGGVFSWSGDQDCGLLANAAR.E 2+/0 86/ 1.3e-10 K.AMSDISDVIGEPLAAWDSQVGGR.V 3+ / 0 63 / 7e-08 K.IGNPTTADVKPTENIPSPILVEDK.

Y 3+/0 60 / 9.5e-08 3+/0 46 / 2.4e-06 R.EGLGYVADSNQETIDMGPLYN PGK. E 3+/0 45 / 3e-06 R.NVGLPPESADTSIESGTFLPYYQQN R. A Band 6 2. YenA1 130232/ 4.82 1021 R.NAMTIIR.A R.EIQSLSNFR.L 2+/0 2+/0 50 / 1.9e-06 47 / 4.3e-06 MH 1 K.LVAFGDQTGSK.A K.AVSMANQVLQK.Y K.FIQSVELDGSNQAR.K 2+/Q 2+/0 2+/0 57 / 2.2e-07 51 / 8.1 e-07 71 / 8.7e-09 Band 6 YenA2 156517/ 4.89 2 264 K.ASLEALQVSK.Q K.NIIDQGDMEYR.K 2+/0 2+/0 48 / 1.5e-06 52/3.1 e-07 MH 1 Band 7 No match NCBInr Eubacteria 48 550315 Band 7 Chil 60727 / 4.78 9 608 K.LTWTPTR.L R.TNFVEGIK.D 2+/0 2+/0 29/0.00018 47 / 2.2e-06 MH 1 K.VFISLDTPR.S K.GISIASSADPAK.I K.ADYLYSEATK.V K.YSADGNASIAVR.L R.LGVATDPDDAIANHK.G K.NATITTSIPSEEALK.G K.LSHHTNIYRDPSDVYSK.Y 2+/0 2+/0 2+/0 2+/0 2+/0 2+/0 3+/1 71 / 1.9e-08 71 / 1.3e-08 64 / 6,1 e-08 81 / 1.2e-09 55 / 4.6e-07 29 / 5.8e-05 35/ 1.4e-05 Bands heat shock protein 60 [Yersinia enterocolitica] 57785 / 4.87 6 1006 K.LAGGVAVIK.V R.GVNILADAVK.V 2+/0 2+/0 71 / 0.00047 69 / 0.00091 NCBInr K.VGAATEVEMK.E 2+/0 81 / 5.5e-005 Eubacteria K.TTLEDLGQAK.R R.EMLPVLEAVAK.A R. AAVEEGVVAGGGVALIR. A 2+/0 2+/0 2+/0 78 / 0.00011 88 / 9.5e-Q06 100 / 6e-007 Band 8 1. YenA1 130232/ 4.82 3 194 K.LSLDNDALYK.E K.GLGASNIELGTVIQR.V 2+/0 2+ / 0 61 / 7.8e-08 51 / 7.3e-07 MH 1 K.FIQSVELDGSNQAR.K 2+/0 641 3.7e-08 Band 8 2. YenA2 156517/ 4.89 3 167 K.QLILYPVIINR.D R.YGYWAANQQLR.M 2+/0 2+/0 71 / 3.8e-09 46 / 5.6e-06 MH 1 K.ALLGESWPAELNK.L 2+/0 54 / 4.2e-07 Band 9 No Match NCBInr Eubacteria Band 9 2. YenA2 156517/ 4.89 8 499 R.FEPMLNR.S K.ASLEA1QVSK.Q 2+/0 2+/0 /3.1e-05 79 / 5.8e-10 MH 1 R.QEWELQYK.Q K.SESLSVSESYR.R K.ALLGESWPAELNK.L K.ELYDENISSTEQK.V K.EINMDLYDSSISPR.G 2+/0 2+/0 2+/0 2+/0 2+/0 31 / 8.1e-05 59/1.1e-07 59/1 4e-07 62 / 9.5e-08 67/1.1e-08 49 550315 R.FSIDLQQQDIDINK.A 2+/0 62/1,6e-07 Refer to Figure 6 for an explanation of this band Table 3: The effect of Y. entomophaga and Y. ent A3 derived culture supernatants (grown at 25°C), sonicated filtrates and ultracentrifuge derived fractions on the efficacy of the Y. entomophaga toxin complex towards Costelytra zealandica (grass grub) larvae Y. ent toxin in Culture Supernal ant (CS) 25°C, dilut on series 1:10...1:50 grubs discoloured 12 larvae (3 replicates) Grubs dead day 7 day24 day 7 day 24 Y. ent A14hrCS Neat 100% 100% 8% 33% 1:10 dilution 50% 33% 0% 8% 1:20 dilution 50% 33% 8% 8% 1:50 dilution % 50% 0% 0% Y. ent son filtrate Neat (14 hours) 100% 100% 17% 75% SF 1:10 dilution 92% 75% 17% 17% SF 1:20 dilution 50% 0% 0% 0% SF 1:50 dilution 8% 0% 0% 0% control Y. entA3 supernatent 0% 0% 0% 0% Y. ent Toxin in Culture Supernatant (CS); sonicated filtrate (SF) derived from a 15,16 20 and 21 hour culture.

Grubs discoloured grubs dead day 6 day 17 day 6 day 17 CS 15hr Neat 67% 50% 0% 0% CS 21 hr Neat 83% 83% 0% 0% SF 15hr Neat 100% 100% 8% 8% day 6 day 14 day 6 day 14 CS 16hr Neat 83% 67% 8% % CS 20hr Neat 75% 67% % 50% SF16hr Neat 100% 100% 75% 75% Y. ent Toxin Purification by Ultra Centrifugation (UC) 6 grubs / Rep Grubs Amber (denoting gut clearance; or dead (D) Preparation Day3 Day 7 Day 13 Y. ent CS Pre UC Rep1 6 6 2, 4D 50 550315 Rep2 6 6 2, 4D Y. ent UC Pellet. Gradient Load Rep1 6 6 4, 1D Rep2 6 6 3, 2D Y. ent post Gradient UC Rep1 4 Rep2 3 4 , 1D Y. ent A3 CS pre-UC Rep1 0 0 0 Rep2 0 0 0 Y. ent A3 UC Pellet Gradient Load Rep1 0 0 0 Rep2 0 0 0 Grubs Amber Grubs Dead Y. ent Cult Supernatant UC preparation day 6 day 15 day 15 CS Neat 100% 64% 0 UC1 Supernatant Neat % 0% 1 UC2 pellet Neat 100% 54% 0 CS 1:5 dilution 33% 27% 0 UC2 pellet 1:5 dilution 100% 82% 1 CS 1:10 dilution % 0% 0 UC2 pellet 1:10 dilution 92% 54% 0 Y. entA3 (pPAC14ASwal) day 6 day 15 day 15 CS Neat 0% 0% 0% UC2 pellet Neat 0% 0% 0% day 6 day 15 day 15 Y. ent A3 (pPAC14AAvrll/Pmll) 0% 0% 0% CS Neat 0% 0% 0% UC2 pellet Neat 0% 0% 0% UC = ultrcentrifuged; SF = sonicated filtrate; CS = culture supernatant; D= dead; Neat = undiluted.

Pathogenicity of Tc Fractions from the Bacteria Y. entomophaga MH69 to Diamondback Moth, Plutella xylostella (L.) To prepare the Tc fraction cells from a 50 ml broth (OD600,) 14-20 hour culture 5 representing late mid log were harvested by centrifugation at 8 000 x g for 3 min and resuspended in 1.2 ml phosphate buffer saline (10 mM sodium phosphate buffer, pH 7.4; 2.7 mM KCI; 137 mM NaCI). Two 0.7 ml samples were transferred to a 1.7 ml microcentrifuge tube and subjected to three 20-sec rounds of sonication on wet ice using a Sanyo Soniprep 150 Sonicater (18 Q). The sonicated 10 samples were centrifuged at 16 000 x g for 3 min and filter-sterilised through a 0.2 51 550315 |jm Sartorius Minisart® filter to a sterile tube. Toxin was then provided as either neat or at a dilution rate indicated on the bioassay table Laboratory bioassay of Y. entomophaga MH69 toxicity to Diamondback Moth, Plutella xylostella (L.) larvae Determination of active fractions Table 4: Effect of the culture broth fractions of Y. entomophaga MH69 on the mortality of diamond back moth larvae No! larvae No dead Mortality Fraction Rep* tested larvae (%) Mean(%) Live cell broth 1 100.0 2 12 12 100.0 3 12 12 100.0 4 12 12 100.0 100.0 Resuspended live cells 1 100.0 2 12 11 91.7 3 12 12 100.0 4 12 11 91.7 95.8 Heat killed broth 1 2 .0 2 12 0 0.0 3 12 0 0.0 4 12 1 8.3 7.1 Sonicated cell filtrate 1 100.0 2 12 12 100.0 3 12 11 91.7 4 12 11 91.7 95.8 Broth supernatant 1 2 12 0 0 0.0 0.0 3 12 0 0.0 4 12 0 0.0 0.0 Control 1 (PBS) 1 2 .0 2 12 0 0.0 3 12 0 0.0 4 12 0 0.0 .0 Control 2 (LB broth) 1 2 12 1 0 .0 0.0 3 12 0 0.0 4 12 0 0.0 2.5 Sonicated cell filtrate 52 550315 Table 5: Effect of the sonicated cell filtrate concentration of Y. entomophaga MH69 on mortality of diamondback moth larvae No No No larvae dead dead Mortality Mean Concentration Rep tested larvae pupae (%) (%) 100% 1 12 11 0 91.7 2 12 11 0 91.7 3 12 1 91.7 91.7 50% 1 12 6 1 58.3 2 12 11 0 91.7 3 12 11 0 91.7 80.6 % 1 12 9 0 75.0 2 12 9 0 75.0 3 12 7 2 75.0 75.0 % 1 12 0 41.7 2 12 7 0 58.3 3 12 8 1 75.0 58.3 2% 1 12 2 0 16.7 2 12 3 0 .0 3 12 1 50.0 .6 1% 1 12 0 0 0.0 2 12 0 0 0.0 3 12 1 0 8,3 2.8 Control 1 12 0 0 0.0 2 12 0 0 0.0 3 12 0 0 0.0 0.0 Screenings of stability of active fractions Live cell broth Table 6: Effect of ambient temperature and length of storage period on toxicity of Y. entomophaga MH69 live cells to Diamondback Moth, Plutella xylostella (L.) larvae Treatment Rep No larvae tested No dead larvae Mortality (%) Mean (%) 0 d (Fresh culture) 1 12 11 91.7 2 12 83.3 3 12 9 75.0 83.3 1d, 20X 1 12 12 100.0 2 12 83.3 3 12 9 75.0 86.1 7d,20"C 1 12 83.3 2 12 11 91.7 53 550315 3 12 7 58.3 77.8 Id, 4°C 1 12 8 66.7 2 12 7 58.3 3 12 8 66.7 63.9 7d, 4°C 1 12 83.3 2 12 11 91.7 3 12 11 91.7 88.9 Control 1 12 0 0.0 2 12 1 8.3 3 12 1 8.3 .6 Sonicated cell filtrate Table 7: Effect of temperature and length of storage period on toxicity of Y. entomophaga MH69 sonicated cell filtrate to Diamondback Moth, Plutella xylostella (L.) larvae No Treatment Rep No larvae tested dead larvae Mortality (%> Mean (%) 0 d (Fresh culture) 1 12 12 100.0 2 12 9 75.0 3 12 11 91.7 88.9 1d, 20°C 1 12 11 91.7 2 12 9 75.0 3 12 11 91.7 86.1 7d,20°C 1 12 12 100.0 2 12 83.3 3 12 11 91.7 91.7 1d, 4°C 1 12 8 66.7 2 12 9 75.0 3 12 7 58.3 66.7 7d, 4°C 1 12 11 91.7 2 12 8 66.7 3 12 9 75.0 77.8 Control 1 12 1 8.3 2 12 0 0.0 3 12 1 8.3 .6 METHODS Bacterial strains and methods of culture Table 1, lists bacterial strains and plasmids used in this study. Bacteria were grown in LB broth or on LB agar, at 37°C for Escherichia coli and 30°C for Y. 54 550315 entomophaga MH69 and its derivatives. Antibiotic concentrations used (Mg/ml) for V. entomophaga MH69 were chloramphenicol 90, tetracycline 30 and spectinomycin 100, and for E, coli were kanamycin 50, chloramphenicol 30, tetracycline 15, and spectinomycin 100,.

Mutagenesis Transposon insertions were using the mini-Tn5 derivative Tn- Kn1 were undertaken as described by DeLorenzo et al (1990). Insertions were recombined into y. entomophaga MH69 by transforming Y. entomophaga MH69 (Table 1) with the desired pl_AFR3-based construct. After five - ten days of growth in non-10 selective medium, bacteria were selected for resistance to spectinomycin and loss of the pl_AFR3 tetracycline resistance marker. Recombinants were validate by Southern analysis. Alternately suicide mutagenesis was undertaken as described (Kalogeraki and Winans, 1997), DNA isolation and manipulation. Standard DNA techniques were carried out as 15 described by Sambrook et al. (1989). Plasmid DNA was transferred into E. coli by electroporation using a Biorad Gene Pulser (25 |jF, 2.5 kV, and 200 ohms). Plasmid templates for DNA sequencing were prepared using the High Pure Plasmid Isolation Kit (Roche Diagnostics GmbH). Sequences were determined on both strands using custom primers. The DNA was sequenced by using a capillary 20 ABI3730 Genetic Analyzer, from Applied Biosystems Inc (http://awcmee.massev.ac.nz/qenome-service.htm). Sequences were assembled using SEQMAN (DNASTAR Inc., Madison, Wis). Databases at the National Center for Biotechnology Information were searched using BlastN, BlastX and BlastP.

Polymerase chain reaction. PCR reactions were set up in thin-walled 0.5 ml microcentrifuge tubes and performed in a Perkin-Elmer DNA Thermal Cycler 55 550315 (model 480). Each reaction contained the following components: 1x Reddy Mix PCR buffer, 1.5 mM MgCI2, 1.25U Thermoprime Plus DNA polymerase (Abgene; Advanced Biotechnologies Ltd. UK), 0.2 mM each dNTP, 2 |jM each primer and 1 fjl of DNA in a final volume of 25 pi, adjusted with sterile distilled water. The 5 standard cycle times for the PCR reactions were as follows: template DNA was denatured with a preliminary step of 94°C for 2 min, then five PCR cycles of denaturing at 94°C for 15 sec, annealing at 55°C for 30 sec, and elongating at 72°C for 45 sec, followed by 30 cycles at 94°C for 15 sec, 50°C for 30 sec, and 72°C for 45 sec. PCR products were purified using the High Pure PCR Product 10 Purification Kit (Roche Diagnostics GmbH) following the manufacturer's instructions.

Preparation of sterile sonicated filtrates. Cells from a 50 ml broth (OD600, 14-20 hour culture representing late mid log were harvested by centrifugation at 8 000 x g for 3 min and resuspended in 1.2 ml phosphate buffer saline (10 mM sodium 15 phosphate buffer, pH 7.4; 2.7 mM KCI; 137 mM NaCI). Two 0.7 ml samples were transferred to a 1.7 ml microcentrifuge tube and subjected to three 20-sec rounds of sonication on wet ice using a Sanyo Soniprep 150 Sonicater (18 O). The sonicated samples were centrifuged at 16 000 x g for 3 min and filter-sterilised through a 0,2 Mm Sartorius Minisart® filter to a sterile tube.

SDS-PAGE/siiver stain And Western anlaysis. Standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Laemmli (1970), using 4-12% Tris-Glycine gel. Proteins were visualised by silver staining according to Blum et al. (1987).

Induction and purification of Y. entomophaga MH69 toxin From an overnight culture grown at 25°C. Bacterial debris was removed by centrifugation (10 min; 7,500 g; 4°C) and supernatant filtered sterilised through a 56 550315 0.2 |jm Sartorius Minisart® filter to a sterile tube. 4 ml of the supernatent pelleted by ultracentrifugation (2 hours; 80,000 rpm, 4°C) in a sorvall RZ M120EX ultra centrifuge. The pellet was resuspended in 100 pi half-strength LB broth The sample was further centrifuged (10 min; 12000 g; 4°C) and the supernatant 5 applied to the surface of 1.0 ml 5% glycerol to a 3.5 ml of 40% glycerol in half strength LB broth and centrifuged (2 hours; 80000 rpm; 4°C). The pellet resuspended in 0.25 ml TM buffer.

Methodolgy of MS/MS Preparation of proteins for identification by MS.

Gel pieces were destained with 25 mM NH4HC03 and acetonitrile (ACN), (50 %: 50 %). The liquid was discarded and gel pieces dehydrated in ACN, then dried in a vacuum Speed Vac centrifuge at 37°C. The gel plugs were reduced with 25 mM NH4HC03 and 10 mM DTT at 56°C for 10 min then for 20 min at RT and then alkylated with 25 mM NH4HCO3 and 55 mM iodoacetamide (IAA) for 30 min prior to 15 tryptic digestion. The gel plugs were washed and in 25 mM NH4HCO3 then 100 % ACN and dried in a Speed Vac centrifuge at 37°C. Trypsin digests were carried out with 0.1 pg modified, sequencing grade trypsin (Promega, Madison, Wl) at 37 0 C overnight. After digestion the peptides were extracted and protonated with 5 % formic acid (FA) in 25 mM NH4HCO3 15 min x 3 with shaking, then 100 % ACN. 20 The pooled extract solution was collected in a separate clean tube after each step of extraction and dried in a Speed Vac centrifuge at 37°C. The lyophillised peptides were redissolved in 60 p I of 0.1 % (v/v) FA and 2 % ACN and placed in 200 p L auto sampler vials with a glass insert (LC — Packings, Amsterdam, Netherlands).

MS I MS analysis Nano - flow reverse phase LC MS / MS was performed using an Ultimate 3000 nano HPLC system (LC - Packings, Amsterdam, Netherlands) in combination with 57 550315 a LTQ MS / MS (Thermo Electron Corp.). 50 |j I of protein solution was injected by the auto sampler and separated by a Ci8 reverse phase column (75 mm x 15 cm LC-Packings, Amsterdam, Netherlands) at a flow rate of 300 nl / min. The mobile phases consisted of buffer A HPLC grade water with 0.1 % FA and buffer B 98 % 5 ACN and 0.1% FA. Peptides were separated prior to ion trap analysis using a 90 min linear gradient from 2-98 % buffer B.

The sample was injected into the MS (LTQ ion trap; Thermo Electron Corp, USA) through a 30 |j m silica tip (New Objective, Woburn, MA, USA). Data was acquired in information dependent acquisition mode. Each full MS scan was taken in a mass 10 range between 400 - 1600 (m/z) and MS / MS scans with a mass range between (100 - 1600 m/z). The top 6 most abundant peaks in each MS scan were submitted for gas collision dissociation and the identification of peptide amino acid sequence.

Standard Bioassay Healthy, feeding larvae, collected from the field, were individually fed squares of carrot which had been rolled in colonies of putative pathogenic bacteria that had grown overnight on solid media. Twelve second or third instar larvae were used for each treatment. Inoculated larvae were maintained at 15°C, in ice-cube trays. Larvae were left feeding on treated carrot for 3-4 days, then transferred to fresh 20 trays and re-fed with untreated carrot for up to 10-14 days and signs of disease noted.

Bioassay of efficacy of Y. entomophaga MH69 live cells and the toxic proteins on Diamondback Moth, Plutella xylostella (L.) Five fractions of the bacterial culture tested: 1. Live cell broth; 1 ml freshly cultured broth used. 58 550315 2. Concentrated live cells. 10ml of broth centrifuged at 8000 rpm for 8 min, the resulting pellet harvested, and resupended in 1 ml of PB; 3. Resuspended live cells. 1ml broth centrifuged at 8000 rpm for 8 min, the resulting pellet harvested and cells resuspended in equal volume of PB; 4. Heat killed broth. 1ml broth subjected to boiling water for 10 min; broth plated out in LB plate to confirm if any live cells.

. Sterile filtrate broth. 10 ml broth centrifuged at 8000 rpm for 8 min, the resulting pellet harvested, resuspended in 1ml PB, then sonicated and centrifuged at 1300 rpm, 5 min; the supernatant harvested. Supernatant plated out in LB plate to confirm if any live cells.

All fractions mixed with 0.2% Tween 80 as emulsifier. LB broth and PB plus 0.2% Tween 80 used as controls.

Assessment: leaf disc method. 2. The 2nd to 4th instars collected from plants and place in a container supplied with cabbage leaves, if not enough for an experiment, larvae stored in fridge for an extended 2 to 3 day period until further collections. 3. Larvae transferred to clean or sterile Petri dishes containing no cabbage leaves by a sterile fine art brush at least 4 h prior to being exposed to treatments to ensure sufficient uptake of bacteria and the fractions tested. 4. Leaf discs (1.0 cm in diameter) punctured from tender leaves of cabbage seedlings, and stored in a Petri dish containing a small piece of wet tissue . Label clean or sterile 24-well tissue culture plates and their wells, with treatment details plus date on each plate. 550315 6. Using freshly flame sterilized soft tweezers transfer the leaf discs individually into the wells of plates, with the upper surface of the leaves upward. 7. 5jjI of test suspension pipetted onto the upper surface of the leaf disc and 5 spread with a sterile glass rod or homogenizer. 8. Larvae transferred individually onto a leaf disc with alcohol sterilized fine art brushes carefully. All larvae used for a treatment pooled in a plate covered by parafilm to prevent larval escaping from wells. 9. Recode the developmental stage of each larva. 10. Plates sealed in plastic bags and held at 15C under 14:10 (L:D) h photoperiod. 11. Leaf discs renewed daily using the method above. Mortality monitored within 5 d post-inocuiation. 8-12 larvae tested for each treatment, Experiments carried out three replications.

Table 1: Bacterial strains and plasmids Strain/Plasmid genotype Escherichia coli DH10B F~ mcrk Amrr-hsdRMS-mcrBCA80d /acZAM15 AlacX74 endA1 recA1 deoRAara, leu 7697 araD139 ga/U ga/K nupG rps L A".

XL1-BlueMRA AmcrA1&3AmcrCB-hsdSMR-mrr173 endA1 supE44 thi-1 reA1 gyrA96 relA1 Y. entomophaga MH69 (Y. ent) Unique insecticidal bacterium, a new member of the Genus Yersinia Y. ent. MH96 Novel species of insecticidal bacteria Y. ent. MH96Sp Y. ent. MH96Sm with spectiniomycin resistance cassette recombined into the genome of Y. ent. MH96 Y. ent MH96A3 Y. ent MH96 deletion of the central BglII restriction enzyme of the Y. ent MH96 virulence associate region. Cm* 60 550315 Y. entSm Y. ent containing a spectinomycin cassette in an Sphl site flanking the Y ent Tc Pathogenicity Island Ewingella americana Plasmid free avirulent Serratia entomophila Plasmid free avirulent Plasmids pBRminicos2 pBR322 containing pLAFR3 derived Bg/ll cos site inserted into its BamH\ site, ApR pBRminicospac pBRminicos2 with Kanamycin ressitance gene flanked by EcoRI and Pacl flanked restriction enzyme sites inserted into the EcoRI site pK3X Kn Genomic Xba\ clone derived from Y. ent :3 pKsal Kn Genomic Sa/I clone derived from Y. ent: 14 pKsalH3 pKsal digested with Hind\\\ and self ligated, Amk pLAFR3 pRK290 with cos, lacZ and multi-cloning site from pUC8. TcR pLAFR3p pLAFR3with Kan gene flanked by EcoRI and Pacl restriction enzyme sites inserted into the EcoRI site of PLAFR3 TcR pPAC12 Y. ent MH96 Pad virulence clone ArrT, SpK pPAC14 Y. ent MH96 Pad virulence clone Amk, Sp" pSalH 112221 bp Hind\\\-Sal\ fragment of derived from Y. entomophaga MH96 PVIK165 Suicide plasmid. KnK Yersinia entomophaga MH96 bioassay against locusts (Locusta migratoria) Neonate and small locusts were purchased from Biosuppliers Ltd, Auckland, New Zealand. The small locusts were set up in containers (5 per container). Locusts 5 were fed on rolled oats to which either toxin was added and the wet mix allowed to dry, or LB was added and the mix allowed to dry. 0.5 grams of the dried mix was placed in each container to which 5 locusts were added and allowed to feed.

The containers were kept on the lab bench at ambient temperature (approx 22°C) and observed daily. Three control treatments of 3 containers of five locusts and 5 10 containers of 5 locusts containing toxin were set up 10 locust and five separate treatments. 61 550315 Results (Table 9 below) show that the V. entomophaga MH96 toxin has high activity towards Locusta migratoria with mortality occurring by day 4.

Table 9: Bioassays of Yersinia entomophaga toxin supernant against Locusta 5 migratoria Locusts Locusta migratoria day 4 6 8 Control 0/12 0/12 0/12 Control 0/12 0/12 1/12 Control 0/12 0/12 0/12 Toxin 24/25* 24/25* 24/25* n/n -number dead/starting number. * combined total of 5 treatments.

Bioassays of yersinia entomophaga toxin supernatant against the Red headed chockchafer Adoryphorus couloni; Tasmania grass grub 10 Acrossidius tasmaniae; Chafer beetle Odontria sp and Pyronota setosa Bioassays of Yersinia entomophaga toxin supernatant against the Red headed chockchafer Adoryphorus couloni; Tasmania grass grub Acrossidius tasmaniae; Chafer beetle Odontria sp and Pyronota setosa were undertaken as described for grass grub larvae. Breifly 10 micolitres of neat toxin was applied to the surface of 15 a partially air dried (1 hour) 3mm carrot cube and the cube placed in a ice cube tray containing the larvae to be tested. Control is carrot with 10 microlitres of LB added. 62 550315 With reference to Table 10 it is evident that Y. entomophaga MH96 toxin supernatants are effective against all of these species.

Table 10: Bioassays of Y. entomophaga MH96 toxin supernatant against the Red headed chockchafer Adoryphorus couloni; Tasmania grass grub Acrossidius Red headed chockchafer Adoryphorus couloni day 4 6 8 Control 0/12 0/12 0/12 Control 0/12 0/120 0/12 Toxin 0/12 1/12 /12 Toxin 0/12 1/12 12/12 Tasmania grass grub Acrossidius tasmaniae day 4 6 8 Control 0/12 0/12 0/12 Control 0/12 0/120 0/12 Toxin 12/12 12/12 12/12 Toxin 12/12 12/12 12/12 Chafer beetle Odontria sp day 4 6 8 Control 0/12 0/12 0/12 Control 0/12 0/120 0/12 Toxin 11/12 11/12 11/12 Toxin /12 11/12 11/12 Pyronota setosa 63 550315 day 4 6 8 Control 1/12 1/12 1/12 Control 0/12 0/12 0/12 Toxin 11/12 11/12 11/12 Toxin 12/12 12/12 12/12 n/n =number dead/starting number Y. entomophaga MH96 sonicated filtrate against Helicoverpa armigera The previous data has shown sonicated filtrates only contain the V. entomophaga MH96 Tc toxin.

Sonicated cell filtrate. 10 ml broth centrifuged at 8000 rpm for 8 min, the resulting pellet harvested, resuspended in 1mi PB, then sonicated and centrifuged at 1300 10 rpm, 5 min; the supernatant harvested. Supernatant plated out in LB plate to confirm if any live cells.

A rectangle (approximately 20 x 15 x 4 mm) of artificial leaf roller diet (HortResearch, Mt Albert) minus antibiotics was placed in a Petri dish. 100 |jl of Y. entomophaga MH96 sonicated filtrate was pipetted onto the treated pieces of diet. 15 Controls consisted of untreated diet. 18 second-instar Helicoverpa were placed into the Petri dish. Dishes were sealed with parafilm and incubated at 20°C.

With reference to Table 11 below, all larvae fed Y. entomophaga MH96 sonicated filtrate treated diet were died by day 6.

Table 11: Cumulative number of dead Helicoverpa second-instar larvae out of 18 20 larvae per container, respectively.

Days post treatment 1 2 3 6 7 Helicoverpa 64 550315 Control 0 0 0 0 0 Treated 4 12 17 18 18 1Evidence of cannibalism since only head capsules remaining as opposed to complete cadavers.

Effect of Y. entomophaga MH96 sonicated filtrate on Helicoverpa armigera 50 |jl of either Y. entomophaga MH96 sonicated filtrate or dilutions of 1:2, 1:10 or 5 1:100 of the sonicated filtrate was pipetted onto the pieces of diet. Controls consisted of untreated diet. Ten third-instar larvae of Helicoverpa were placed into the Petri dish. There were four replicate dishes per treatment except for the 1:2 dilution where there were only two replicate dishes. Dishes were sealed with parafilm and incubated at 20°C.

With reference to Table 12 after 7 days, all Helicoverpa larvae treated with undiluted sonicated filtrate were dead. For the diet treated with sonicated filtrate diluted 1:2, all larvae were dead in one dish by 6 days but only 3 died in the other dish. Two of the four dishes containing larvae fed the diet treated with 1:100 Y. entomophaga MH96 sonicated filtrate had no mortality, one dish had 2 15 cannibalised larvae and the other dish had six dead larvae. There was one dead larva among the untreated controls (Table 12; Fig 10).

There was a significant effect of undiluted Y. entomophaga MH96 sonicated filtrate on Helicoverpa larvae but little effect of diluted solutions.

Table 12: Cumulative number of dead Helicoverpa third-instar larvae out of 10 20 larvae per container.

Days post treatment Treatment 1 2 3 6 7 Control 1 0 0 0 0 0 Control 2 0 0 0 0 0 Control 3 0 0 0 0 0 Control 4 0 0 0 0 1 65 550315 Undiluted Y. entomophaga MH96 0 0 1 8 10 sonicated filtrate: 1 Undiluted Y. entomophaga MH96 0 0 0 8 10 sonicated filtrate: 2 Undiluted Y. entomophaga MH96 0 0 1 9 10 sonicated filtrate: 3 Undiluted Y. entomophaga MH96 0 0 1 9 10 sonicated filtrate: 4 1:2 Y. entomophaga MH96 sonicated 0 0 11 10 10 filtrate: 1 1:2 Y. entomophaga MH96 sonicated 0 0 0 3 3 filtrate: 2 1Cannibalism.

Y. entomophaga MH96 gave 100% mortality when applied as a sonicated filtrate to diet fed to third instar Helicoverpa larvae. Dilution of the sonicated filtrate reduced the effectiveness of the treatment with all larvae surviving after being fed a diet 5 treated with 1:10 so n i cated filtrate.

Effect of Y. entomophaga MH96 supernatanets on Epiphyas postvittana and Cydia pomonella.

Insects were obtained from Anne Barrington Hort Research Auckland (New 10 Zealand) Insect artificial diet was cut into small 3mm cubes and then air dred in a laminar flow for 1-2 hours with intermittent turning and then applying 7 micro litres of 50% toxin to the dehydrated cube effectively rehydrating it allowing the absorption of toxin to the matrix. Two cubes were then place in a container to which 4-7 15 caterpillar hatchlings were added. Lids were placed and the containers left in a chamber at 18°C and results monitored over a 10 day period. This method was used for on Epiphyas postvittana; Spodoptera litura and Cydia pomonella insects. 66 550315 Table 13: Effect of Y. entomophaga MH96 supernatanets on Epiphyas postvittana; Spodoptera litura and Cydia pomonella.

Day4 Day8 Day9 Epiphyas postvittana expt 1 light brown apple moth T /7 6/7 6/7 Small in size T 6/7 7/7 7/7 Small in size C 0/7 0/7 0/7 C 0/8 0/8 1/8 Epiphyas postvittana expt 2 light brown apple moth T 9/10* 9/10* 9/10* Small in size T 3/12* 4/12* /12* Small in size C 1/14* 1/14* 1/14* C 3/12* 3/12* 3/12* Epiphyas postvittana expt 3 light brown apple moth T 0/5 0/5 0/5 Small in size T 0/6 0/6 0/6 Small in size T 0/5 1/5 215 Small in size T 0/6 0/6 0/6 Small in size C 0/5 0/5 0/5 All food eaten C 0/5 0/5 0/5 half food eaten Spodoptera litura Cluster Caterpillar T 0/5 0/5 0/5 Small in size T 0/5 0/5 1/5 Small in size T 0/5 0/5 0/5 Small in size T 0/5 0/5 0/5 Small in size C 0/5 0/5 0/5 C 0/5 0/5 1/5 67 550315 Cydia pomonella Codling moth expt 1 T /5 /5 /5 T 1/5 /5 /5 T /5 /5 /5 C 1/5 /5 /5 C 1/5 2/5 2/5 c 3/5 3/5 2/5 Cydia pomonella Codling moth expt 2 T /5 /5 /5 T 1/5 /5 /5 T 4/5 /5 /5 T 1/5 /5 /5 T 1/5 1/5 1/5 C 1/5 1/5 2/5 C 2/5 2/5 2/5 c 2/5 2/5 2/5 C= control; T=toxin; H= healthy; D=diseased. * cumulative results of two experiments, n/n =number dead/starting number.

Results (Table 13 above and Figure 11) showed good effect of toxin against the 5 codling moth C. pomonella A lot of control death was identified in experiment 1 and put down to a lack of food, this was rectified in experiment 2 where 2 cubes were provided. Little mortality was observed towards Epiphyas postvittana light brown apple moth. However there was a clear reduction in size indicative or an effect.

Though the insects are not dying there is a significant reduction in growth of Toxin 10 compared to LB (control). In addition there is a greater abundance of grass present in the LB control relative to Toxin treated sample, tt is also noted that Y. 68 550315 entomophaga MH96 has been found ineffective at control of Spodoptera hence the reduction in growth is of interest may show a greater scope of the semi pure toxin. In addition note the difference ion grass levels between control (LB) and toxin.

White butterfly Pieris rapae Y. entomophaga MH96 toxin supernatant with known activity was diluted 50% in Duwett (Elliot chemicals Ltd Christchurch) 50 |jL was then applied to the surface of a 1.0 cm in diameter leaf disc and dispersed using a glass hockey stick. The leaf 10 discs were then air dried for 2 hours allowing the toxin to bind to the leaf. A 3cm diameter piece of Whatman filter paper was then placed into small plastic container. The treated leaf disc then applied and a single caterpillar added. 6 controls were set up containing a leaf treated with only 50 pL Duwett and 12 further treatments were set up of caterpillars to treated with the toxin. In addition 15 three large caterpillars were treated with to leaf discs 2 were given toxin and the third Duwett as a control. In addition three large caterpillars (3-4cm in length) were assessed for susceptibility to the toxin. These caterpillars were supplied with two treated or untreated leaf discs.

Results (Table 14 below) showed a significant level of mortality or a reduction in 20 food eaten as denoted by an absence of leaf disc consumed. A further observation is an aversion effect where the caterpillars in toxin treated containers were often found away from the leaf area. Of the three large caterpillars all of the treated died with one pupating but not emerging while the control pupated and emerged.

Table 14: Effect of Y. entomophaga MH96 toxin supernatent against Pieris rapae 69 550315 Treatment of small caterpillars S=1Q-15mm in length SL=15-20mm in length. L= large - Day3 Day4 Day6 SL Little leaf eaten Little leaf eaten Little leaf eaten SL Little leaf eaten H H SL Little leaf eaten Little leaf eaten H Little leaf eaten SL Little leaf eaten Little leaf eaten H SL H Not feeding 50% H SL Little leaf eaten Little leaf eaten D SL Little leaf eaten H Little leaf eaten S Little leaf eaten Little leaf eaten D S Little leaf eaten D D S Little leaf eaten D D S Little leaf eaten 50%healthy D 15 S Little leaf eaten D D C1 H H* H C2 H l_l* H C3 H H* H C4 H H* H 20 C5 H H* H Treatment of three large caterpillars L H 50%H* D L H H* Pupae and died C6 H H* Pupae and hatched 25 * fresh untreated leaf disc added. H=healthy, D =dead; C = control. EXAMPLES OF OTHER SUSCEPTIBLE INVERTEBRATE SPECIES 70 550315 Table 15 below summaries a list of various other invertebrate species, including the DBM and Wiseana spp tested for susceptibility to whole Yersinia entomophaga MH96 cells, Table 15: Summary of 5 entomophaga MH96 toxin the susceptibility of invertebrates to purfied Yersinia complex.

Insect Lepidoptera Diamondback moth Plutella xylostella Cotton bollworm Helicoverpa amigera Codling moth Cydia pomonella Cluster Caterpillar Spodoptera litura Lightbrown apple moth Epiphyas postvittana Pieris rapae white butterfly Coleoptera New Zealand grass grub Costelytra zealandica Red headed cockchafer Adoryphorus couloni Tasmania grass grub Acrossidius tasmaniae Chafer beetles Odontria sp.

Developmental Class: Family stage Pathogenic? Lepidoptera: Lepidoptera: Lepidoptera: Lepidoptera Lepidoptera: Tortricidae Lepidoptera: ?Pieridae Coleoptera: Scarabaeidae Coleoptera: Scarabaeidae Coleoptera: Scarabaeidae Coleoptera: Scarabaeidae 1 st-4th instar larvae larvae larvae larvae larvae larvae larvae larvae larvae larvae yes yes yes Reduced growth Reduced growth yes yes yes yes yes 71 550315 Developmental Insect Class: Family stage Pathogenic? Orthoptera Locusts Orthoptera: neonates yes Locusta migratoria Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope thereof as defined in the appended claims. 72 550315 REFERENCES Adang et al (1995) Synthetic Insecticidal Crystal Protein Gene US 5,380,831, Jan, 10, 1995 Altschul, S. F., T. L. Madden., A. A. Schaffer., J. Z. Zhang., W. Miller, and D. J., Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl. Acids Res. 25:3389-3402.

Blackburn MB, J M. Domek, D B. Gelman and J S. Hu (2005)The broadly insecticidal Photorhabdus luminescens toxin complex a (Tea): Activity against the Colorado potato beetle, Leptinotarsa decemlineata, and sweet potato whitefly, Bemisia tabaci Journal of Insect Science 5.32 1-11 Blum, H., H. Beier, and H. J. Gross. 1987. Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8, 93-99. de Lorenzo V, Herrero M, Jakubzik U, Timmis KN. (1990) Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol. 172:6568-6572.

Bowen D, Rocheleau TA, Blackburn M, Andreev O, Golubeva E, Bhartia R, ffrench-Constant RH. (1998) Insecticidal toxins from the bacterium Photorhabdus luminescens Science. 1998 280:2129-32 Choreo and Goodman, Acc. Chem. REs., 1993 26 266-273 ffrench-Constant R, Waterfield N (2006) An ABC Guide to the Bacterial Toxin Complexes. Adv Appl Microbiol. 2005;58C: 169-183.

Freidinger, R.M., Perlow, D.S., Veber, D.F., J. Org. Chem. 1982, 59,104-109 _ JUN 20fl9 73 550315 I ffrench-Constant, R, and N. Waterfield. 2005. An ABC Guide to the Bacterial Toxin Complexes. Adv. Appl. Microbiol. 58C: 169-183.

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Hacker J, Blum-Oehler G, Muhldorfer I, Tschape H. (1997) Pathogenicity islands of virulent bacteria: structure, function and impact on microbial evolution. Mol Microbiol. 23:1089-1097.

Hey T, Bevan, S., Schleper, A., Birkhold, P., Burton, S. Meade, T Merlo, D, Sheets, J. Thompson, R and Moon, H., (2005), Mixing And Matching Of Toxin Complex Proteins 38th Annual Meeting Society For Invertebrate Pathology 7-11 August 2005 Anchorage, Alaska Hey, T., Cai, C., Woosley, A., Burton, S, Sheets, J, Waldman, B, Moon, H, Meade, T., and Merlo, D. (2005) Novel Toxin Complex Constructions 38th Annual Meeting Society For Invertebrate Pathology 7-11 August 2005 Anchorage, Alaska Hey et al (2006) Insecticidal toxin complex fusion proteins US 2006/0168683 A1 Jul 27 2006 Hurst, M. R. H., T. R. Glare, T. A. Jackson, and C. W. Ronson. 2000. Plasmid-located pathogenicity determinants of Serratia entomophila, the causal agent of amber disease of grass grub, show similarity to the insecticidal toxins of Photorhabdus luminescens. J. Bacteriol. 182:5127-5138.

Kalogeraki V.S. and Winans, S.C. (1997), Suicide plasmids containing promoterless reporter genes can simultaneously disrupt and create fusions to target genes of diverse bacteria. Gene 188:69-75 74 550315 ) Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.

Lehninger A. L. Nelson D.L. Cox M.M. Principles of Biochemistry (1993) Second edition Wort publishers 33 Irving Place New York, NY 10003, ISBN: 0-87901-500-4 Nagai, U., Sato, K. Tetrahedron Lett. 1985, 26, 647-650.

Merryfield RB (1963) Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide J. Am. Chem. Soc. 85: 2149-2154 Moar, WJ (2003) Breathing new life into insect resistant plants Nature Biotechnology 10:1152-1154.

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Schaffer, A. A., L. Aravind, T. L., Madden, S. Shavirin, J. L. Spouge, Y. I. Wolf, E. V. Koonin, and S. F. Altschul. 2001. Improving the accuracy of PSI-BLAST protein database searches with composition-based statistics and other refinements. Nucleic Acids Res. 29:2994-3005.

Smythe, M.L., von Itzstein, M., J. Am. Chem. Soc. 1994, 116, 2725-2733 Waterfield N, Hares M, Yang G, Dowling A, ffrench-Constant R. (2005) Potentiation and cellular phenotypes of the insecticidal Toxin complexes of Photorhabdus bacteria. Cell Microbiol. 2005 75 550315 As can be found in the appendices of a "New England BioLabs catalog" or at the WWW site: http://www.neb.com/nebecomm/tech reference/general data/amino acid structures, asp and includes groupings of similar amino acids as outlined page 113 Lehninger et al (1993). 76 550315

Claims (65)

WHAT WE CLAIM IS:

1. An isolated nucleic acid molecule having a nucleotide sequence selected from the group consisting of: a) SEQ ID NO. 15; b) A functional fragment or variant of the sequence in a); or c) A complement to the sequences in a) or b).

2. An isolated nucleic acid molecule having a nucleotide sequence selected from the group consisting of: a) SEQ ID NOs. 2, 4, 6, 8, 10, 12, 14 or a combination thereof; b) A functional fragment or variant of at least one of the sequence(s) in a); or c) A complement to the sequences in a) or b).

3. An isolated nucleic acid molecule having a nucleotide sequence selected from the group consisting of: a) SEQ ID NOs. 4, 6,10, 12, 14 or a combination thereof; b) A functional fragment or variant of at least one of the sequence(s) in a); or c) A complement to the sequences in a) or b).

4. An isolated nucleic acid molecule which encodes a polypeptide as set forth in SEQ ID NO: 16, or a functional fragment or variant thereof.

5. An isolated nucleic acid molecule which encodes a polypeptide substantially selected from the group consisting of: 14 a o 6 ?m 550315 7. 8. 9. 10. 11. 12. a) SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13, or a combination thereof; b) A functional fragment or variant of at least one of the sequence(s) in a); or c) A complement to the sequences in a) or b).

An isolated nucleic acid molecule which encodes a polypeptide selected from the group consisting of: a) SEQ ID NOs. 3, 5, 9, 11, 13, or a combination thereof; b) A functional fragment or variant of at least one of the sequence(s) in a); or c) A complement to the sequences in a) or b).

The use of an isolated nucleic acid molecule as claimed in any one of claims 1 - 6 to produce a toxin.

An isolated toxin produced by at least one nucleic acid molecule as claimed in any one of claims 1 to 6.

The toxin as claimed in claim 8 wherein the toxin is in the form of a toxic complex.

The toxin as claimed in any one of claims 8 to 9, wherein the toxin has an insecticidal activity.

The use of an isolated toxin, as claimed in any one of claims 8 to 10 wherein the toxin is used as a biopesticide.

The use of an isolated nucleic acid molecule as claimed in any one of claims 1 - 6, in the manufacture of a composition suitable as a biopesticide.

An isolated polypeptide having an amino acid sequence selected from the group consisting of: 78 550315 15. 16. 17. 18. 19. 20. 21. a) SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13, or a combination thereof; b) A functional fragment or variant of at least one of the sequence(s) in a).

An isolated polypeptide having an amino acid sequence as set forth in SEQ ID NO. 16 or a functional fragment or variant thereof.

The use of an isolated polypeptide as claimed in claim 13 or claim 14 as a toxin or component therefore.

An isolated toxin in the form of at least one polypeptide as claimed in claim 13 or claim 14.

A toxin which substantially corresponds to the polypeptide set forth in SEQ ID NO. 16.

The toxin as claimed in claim 16 or claim 17, wherein the toxin is in the form of a toxic complex.

The toxin as claimed in any one of claims 16 to 18, wherein the toxin has an insecticidal activity.

The use of an isolated toxin, as claimed in any one of claims 16 to 19, wherein the toxin is used as a biopesticide.

The use of an isolated polypeptide as claimed any one of claims 11 to 14, in the manufacture of a composition suitable as a biopesticide.

A construct or vector which includes a nucleotide sequence as selected from the group consisting of: b) A functional fragment or variant of the sequence in a); or a) SEQ ID NO. 15; 79 550315 c) A complement to the sequences in a) or b).

23 A construct or vector which includes a nucleotide sequence as selected from the group consisting of: a) SEQ ID NOs. 4, 6, 10, 12 and 14 or a combination thereof; b) A functional fragment or variant of at least one of the sequence(s) in a); or c) A complement to the sequence(s) in a) or b).

24. A construct or vector as claimed in claim 22 or claim 23, wherein the combination of the nucleotide sequences from a) and/or b) and/or c) has each coding region of the subsequent sequence(s) joined to the coding region of the earlier sequence(s) in the combination by a short oligonucleotide segment which encodes a linker peptide.

25. A construct or vector as claimed in claim 22 or claim 23, wherein the combination of the nucleotide sequences from a) and/or b) and/or c) has each coding region of the subsequent sequence(s) joined to the coding region of the earlier sequence(s) so as to encode a single fusion protein.

26. A non-human host cell which has been transformed with a construct or vector as claimed in any one of claims 22 to 25.

27. A non-human host cell which includes non-endogenous nucleotide sequence as selected from the group consisting of: a) SEQ ID NO. 15; b) A functional fragment or variant of the sequence in a); or c) A complement to the sequences in a) or b). 80 550315

A non-human host cell which includes a non-endogenous nucleotide sequence as selected from the group consisting of: a) SEQ ID NOs. 2, 4, 6, 8, 10, 12,14 or a combination thereof; b) A functional fragment or variant of at least one of the sequence(s) in a); or c) A complement to the sequence(s) in a) or b).

A non-human host cell which includes at least two nucleic acid molecules having a nucleotide sequence selected from the group consisting of: a) SEQ ID NOs. 2, 4, 6, 8, 10, 12, 14, or any combination thereof; b) A functional fragment or variant of at least one sequence in a); c) A complement to the sequence(s) in a) or b); wherein the combination of the nucleotide sequences from a) and/or b) and/or c) has each coding region of the subsequent sequence(s) joined to the coding region of the earlier sequence(s) in the combination by a short oligonucleotide segment which encodes a linker peptide.

A non-human host cell which includes at least two nucleic acid molecules having a nucleotide sequence selected from the group consisting of: a) SEQ ID NOs. 2, 4, 6, 8, 10, 12, 14, or any combination thereof; b) A functional fragment or variant of at least one sequence in a); c) A complement to the sequence(s) in a) or b); wherein the combination of the nucleotide sequences from a) and/or b) and/or c) has each coding region of the subsequent sequence(s) joined to the coding region of the earlier sequence(s) so as to encode a single fusion protein. 81 550315

31.

32.

33.

34.

35.

36.

37.

38.

39.

40. A non-human host cell as claimed in any one of claims 27 to 30, wherein the nucleic acid molecule has a nucleotide sequences which have been modified to assist with expression of the molecule in the host cell. A non-human host cell as claimed in any one of claims 27 to 30, wherein the nucleotide sequence also has at least one regulatory sequence to assist with expression of the nucleic acid molecule in the host cell. A non-human host cell as claimed in any one of claims 26 to 32, wherein the host cell produces a toxin. An isolated toxin produced from a non-human host cell as claimed in any one of claims 26 to 33. A toxin, as claimed in claim 34, wherein the toxin is used as a biopesticide. A toxin as claimed in claim 35, wherein the toxin is in the form of a toxic complex. A non-human host cell as claimed in any one of claims 26 to 33, wherein the host cell is bacteria or algae. A non-human host cell as claimed in any one of claims 26 to 33, wherein the host cell is a plant cell. A non-human host cell as claimed any one of claims 26 to 33, wherein the plant cell is selected from: wheat, rice, cotton, corn (maize), barley, canola, or soy bean. The use of a non-human host cell as claimed in any one of claims 26 to 32, in the manufacture of a composition suitable as a biopesticide. 82 550315

41. A transgenic plant which includes a non-human host cell as claimed in any one of claims 26 to 33 and 37to 40.

42. A transgenic plant which includes a nucleic acid molecule as claimed in any one of claims 1 to 6.

43. A seed obtained from a transgenic plant as claimed in claim 41 or claim 42.

44. A composition which includes as an active ingredient an effective amount of a toxin produced as a result of the expression of a nucleic acid molecule having a nucleotide sequence selected from the group consisting of: a) SEQ ID NO. 15, r ^" b) A functional fragment or variant of the sequence in a); or 1 ' * AtlS ® c) A complement to the sequences in a) or b).

45. A composition which includes as an active ingredient an effective amount of a toxin produced as a result of expression of a nucleic acid molecule having a nucleotide sequence selected from the group consisting of: a) SEQ ID NOs. 2, 4, 6, 8, 10, 12, 14 or a combination thereof; b) A functional fragment or variant of at least one of the sequence(s) in a); or c) A complement to the sequences in a) or b).

46. A composition which includes as an active ingredient an effective amount of a toxin in the form of a polypeptide having an amino acid sequence selected from the group consisting of: a) SEQ ID NOs. 1, 3, 5, 7, 9, 11, 13, or a combination thereof; b) A functional fragment or variant of at least one of the sequence(s) in a). 83

47

48

49

50

51.

52.

53

54

55

56 550315 A composition which includes as an active ingredient an effective amount of toxin produced as a result of expression of a nucleic acid molecule which encodes a polypeptide as set forth in SEQ ID NO: 16, or a functional fragment or variant thereof. A composition that includes a toxin in the form of a polypeptide having an amino acid sequence as set forth in SEQ ID NO. 16 or a functional fragment or variant thereof. A composition as claimed in any one of claims 44 to 48, wherein the composition is suitable for use as a biopesticide. A composition as claimed in any one of claims 44 to 49 wherein the composition is formulated with at least one biopolymer compound. A composition as claimed in claim 50 wherein the at least one biopolymer compound is at least one type of gum compound. A composition as claimed in claim 50 or claim 51 wherein the formulated composition is a gel. A composition as claimed in any one of claims 50 to 52 wherein the composition is formulated with at least one biopolymer compound and at least one desiccating agent. A composition as claimed in claim 53 wherein the desiccating agent is at least one inert clay compound. A composition as claimed in claim 53 or claim 54 wherein the formulated composition is a dough or granular material. 550315

57. A composition as claimed in any one of claims 50 to 54 wherein, on application, the composition is mixed with an aqueous liquid and sprayed onto a substrate.

58. A composition as claimed in any one of claims 50 to 57 wherein the composition is coated onto a substrate.

59. A composition as claimed in claim 58 wherein the substrate is a seed.

60. A method of protecting a plant from insects characterised by the step of expressing in said plant a toxin or toxin complex derived from one or more of the nucleic acid molecules as claimed in any one of claims 1 to 6.

61. A probe capable of binding to a nucleic acid molecules as claimed in any one of claims 1 to 6.

62. A plant, seed or plant cell which has been originally derived from a construct or vector as claimed in any one of claims 22 to 25.

63. A non-human host cell as claimed in any one of claims 26 to 32 wherein the host cell produces a component of the toxin.

64. A plant which has been originally derived from a construct or vector as claimed in any one of claims 22-25 wherein the plant expresses a toxin or a component of the toxin.

65. A method of protecting a plant from insects characterised by the step of expressing at least one nucleic acid molecule as claimed in any of claims 1 to 6. 85