Polynucleotides, pesticidal proteins, and novel methods of using them Number:7,129,212 from the United States Patent and Trademark Office (PTO) owispatent Disclosed and claimed are novel Bacillus thuringiensis isolates, pesticidal toxins, and genes. The subject invention also provides novel methods of controlling diamond back moths.<H Polynucleotides, pesticidal, Polynucleotides, 7129212.html, Patent, pto, 7129212

Title: Polynucleotides, pesticidal proteins, and novel methods of using them

Abstract: Disclosed and claimed are novel Bacillus thuringiensis isolates, pesticidal toxins, and genes. The subject invention also provides novel methods of controlling diamond back moths.

Patent Number: 7,129,212 Issued on 10/31/2006 to Narva, et al.

Inventors: Narva; Kenneth E. (Carlsbad, CA), Merlo; Donald J. (Carmel, IN)
Assignee: Mycogen Corporation (Indianapolis, IN)

Appl. No.: 10/698,096

Filed: October 31, 2003

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
09850351	May., 2001	6656908
09307106	May., 1999	6603063
09073898	May., 1998	6242669
08960780	Oct., 1997	6204435
60029848	Oct., 1996

Current U.S. Class: 514/12 ; 530/350

Current International Class: A61K 38/16 (20060101); C07K 14/32 (20060101)

References Cited [Referenced By]

U.S. Patent Documents


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Foreign Patent Documents


0 359 472	Mar., 1990	EP
WO 94/04684	Mar., 1994	WO
WO 94/05771	Mar., 1994	WO
WO 94/21795	Sep., 1994	WO
WO 94/24264	Oct., 1994	WO
WO 96/05314	Feb., 1996	WO
WO 96/10083	Apr., 1996	WO
WO 98/18932	May., 1998	WO

Other References

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Primary Examiner: Wax; Robert A.
Attorney, Agent or Firm: Saliwanchik, Lloyd & Saliwanchik

Parent Case Text

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 09/307,106 (filed May 7, 1999, now U.S. Pat. No. 6,603,063), and a continuation-in-part of U.S. Ser. No. 09/850,351 (filed May 7, 2001, now U.S. Pat. No. 6,656,908), the latter of which is a continuation of application U.S. Ser. No. 08/960,780 (filed Oct. 30, 1997, now U.S. Pat. No. 6,204,435); which claims the benefit of provisional application U.S. Ser. No. 60/029,848 (filed Oct. 30, 1996). U.S. Ser. No. 09/307,106 is also a continuation of application U.S. Ser. No. 09/073,898.

Claims

The invention claimed is:

1. An isolated protein that has toxin activity against a lepidopteran pest, wherein said protein comprises SEQ ID No.: 17.

2. A method for controlling a lepidopteran pest wherein said method comprises administering to said pest a protein according to claim 1.

3. The method of claim 2 wherein said lepidopteran pest is a diamond back moth (Plutella xylostella).

4. The method of claim 3 wherein said diamond back moth is resistant to another Bacillus thuringiensis toxin.

5. The method of claim 3 wherein said protein is produced by and is present in a plant.

6. The method of claim 3 wherein said plant produces another Bacillus thuringiensis toxin.

7. The method of claim 5 wherein said plant is selected from the group consisting of cabbage, broccoli, collards, kale, cauliflower, and Brussels sprouts.

8. The method of claim 3 wherein said protein is used as part of a strategy to prevent or control the development of resistant diamond back moths.

9. A truncated or chimeric toxin comprising a segment consisting of residues 200 to the C terminus of SEQ ID No.: 17.

10. The toxin of claim 9 wherein said segment comprises residues 191 to the C terminus of SEQ ID No.: 17.

11. The toxin of claim 9 wherein said segment comprises residues 200 to 412 of SEQ ID No.: 17.

12. The toxin of claim 11 wherein said segment comprises residues 200 to 455 of SEQ ID No.: 17.

13. The chimeric toxin comprising a segment consisting of residues 412 to the C terminus of SEQ ID NO.: 17.

Description

BACKGROUND OF THE INVENTION

Insects and other pests cost farmers billions of dollars annually in crop losses and in the expense of keeping these pests under control. The losses caused by insect pests in agricultural production environments include decrease in crop yield, reduced crop quality, and increased harvesting costs.

The soil microbe Bacillus thuringiensis (B.t.) is a Gram-positive, spore-forming bacterium characterized by parasporal crystalline protein inclusions. These inclusions often appear microscopically as distinctively shaped crystals. The proteins can be highly toxic to pests and specific in their toxic activity. Certain B.t. toxin genes have been isolated and sequenced, and recombinant DNA-based B.t. products have been produced and approved for use. In addition, with the use of genetic engineering techniques, new approaches for delivering these B.t. endotoxins to agricultural environments are under development, including the use of plants genetically engineered with endotoxin genes for insect resistance and the use of stabilized intact microbial cells as B.t. endotoxin delivery vehicles (Gaertner, F. H., L. Kim [1988] TIBTECH 6:S4 S7). Thus, isolated B.t. endotoxin genes are becoming commercially valuable.

Hofte and Whiteley classified B.t. crystal protein genes into four major classes (Hofte, H., H. R. Whiteley [1989] Microbiological Reviews 52(2):242 255). The classes were CryI (Lepidoptera-specific), CryII (Lepidoptera- and Diptera-specific), CryIII (Coleoptera-specific), and CryIV (Diptera-specific). The discovery of strains specifically toxic to other pests has been reported (Feitelson, J. S., J. Payne, L. Kim [1992] Bio/Technology 10:271 275). CryV has been proposed to designate a class of toxin genes that are nematode-specific. Lambert et al. (Lambert, B., L. Buysse, C. Decock, S. Jansens, C. Piens, B. Saey, J. Seurinck, K. van Audenhove, J. Van Rie, A. Van Vliet, M. Peferoen [1996] Appl. Environ. Microbiol. 62(1):80 86) describe the characterization of a Cry9 toxin active against lepidopterans. Published PCT applications WO 94/05771 and WO 94/24264 also describe B.t. isolates active against lepidopteran pests. Gleave et al. ([1991] JGM 138:55 62), Shevelev et al. ([1993] FEBS Lett. 336:79 82; and Smulevitch et al. ([1991] FEBS Lett. 293:25 26) also describe B.t. toxins. Many other classes of B.t. genes have now been identified.

WO 94/21795, WO 96/10083, related U.S. patents, and Estruch, J. J. et al. (1996) PNAS 93:5389 5394 describe toxins obtained from Bacillus microbes, wherein the toxins were purportedly produced during vegetative cell growth. These toxins were thus termed vegetative insecticidal proteins (VIP). These toxins were reported to be distinct from crystal-forming .delta.-endotoxins. These applications make specific reference to toxins designated Vip1A(a), Vip1A(b), Vip2A(a), Vip2A(b), Vip3A(a), and Vip3A(b). See also Lee et al., AEM vol. 69, no. 8 (August 2003), pages 4648 4657, for a discussion of Vip3 mechanism of action and truncation. There are no known reports of Vip3 proteins having activity against diamondback moths (Plutella xylostella).

Diamondback moths are known to develop resistance to various chemical pesticides, as well as some B.t. Cry toxins such as Cry1Ab, Cry1Ac, and Cry1C. See, e.g., Syed, A. R. (1992), Insecticide resistance in diamondback moth in Malaysia, pp. 437 442, in N. S. Talekar (ed.) Management of Diamondback Moth and Other Pests: Proceedings of the 2.sup.nd International Workshop, AVRDC, Taiwan; Shelton, A. M., et al. (1993), Resistance of diamondback moth to Bacillus thuringiensis subspecies in the field, J. Econ. Entomol. 86:697 705; Tabashnik, B. E., et al. (1990), Field development of resistance to Bacillus thuringiensis in diamondback moth, J. Econ. Entomol. 83:1671 1676; Tabashnik, B. E., et al. (1993), Increasing efficiency of bioassays: evaluating resistance to Bacillus thuringiensis in diamondback moth, J. Econ. Entomol. 86:635 644; Tanada, H. (1992), Occurrence of resistance to Bacillus thuringiensis in diamondback moth, and results of trials for integrated control in a watercress greenhouse, pp. 165 173, in N. S. Talekar (ed.) Management of Diamondback Moth and Other Crucifer Pests: Proceedings of the 2.sup.nd International Workshop, AVRDC, Taiwan; Zhao, J. Z., et al. (1993), On-farm insecticide resistance monitoring methods for diamondback moth, Acta Agriculturae Sinica 1(1):(in press); Zhu, G. R., et al. (1991), Insecticide resistance and management of diamondback moth and imported cabbage worm in P. R. China, Resistant Pest Management Newsletter 3(2):25 26; Tabashnik, B. E., (1994), Evolution of resistance to Bacillus thuringiensis, Annual Review of Entomology 39:47 49; Metz, T. D., et al. (1995), Transgenic broccoli expressing a Bacillus thuringiensis insecticidal crystal protein: Implications for pest resistance management strategies, Molecular Breeding 1:309 317; Perez, C. J., et al. (1995), Effect of application technology and Bacillus thuringiensis subspecies on management of B. thuringiensis subsp. kurstaki-resistant diamondback moth (Lepidoptera: Plutellidae), J. Econ. Entomol. 88:1113 1119; Shelton, A. M., Jr., et al. (1993), Resistance of diamondback moth (Lepidoptera: Plutellidae) to Bacillus thuringiensis subspecies in the field, J. Econ. Entomol. 86:697 705; Tang, J. D., et al. (1996), Toxicity of Bacillus thuringiensis spore and crystal protein to resistant diamondback moth (Plutella xylostella), Appl. Environ. Microbiol. 62:564 569; Zhao, J. Z., et al. (2001), Different cross-resistance patterns in the diamondback moth (Lepidoptera: Plutellidae) resistant to Bacillus thuringiensis toxin Cry1C, Journal of Economic Entomology 94(6):1547 1552; Cao, J., et al. (1999), Transgenic broccoli with high levels of Bacillus thuringiensis Cry1C protein control diamondback moth resistant to Cry1A or Cry1C, Molecular Breeding, 5(2):131 141.

New classes of toxins and genes are described in WO 98/18932. They are distinct from those disclosed in WO 94/21795, WO 96/10083, WO 98/44137, and Estruch et al.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns materials and methods useful in the control of non-mammalian pests and, particularly, plant pests. In one embodiment, the subject invention provides novel B.t. isolates having advantageous activity against non-mammalian pests. In a further embodiment, the subject invention provides new toxins useful for the control of non-mammalian pests. In a preferred embodiment, these pests are lepidopterans. The toxins of the subject invention are preferably soluble toxins that can be obtained from the supernatant of Bacillus cultures.

The subject invention further provides nucleotide sequences that encode toxins of the subject invention. The nucleotide sequences of the subject invention encode toxins that are distinct from previously described toxins. In a specific embodiment, the subject invention provides new toxins having advantageous pesticidal activities.

A preferred class of toxins of the subject invention includes SUP-1 toxins. These toxins, and the genes that encode them, can be characterized in terms of, for example, the size of the toxin or gene, the DNA or amino acid sequence, pesticidal activity, and/or antibody reactivity. In a preferred embodiment, toxins of the subject invention have advantageous and surprising activity against diamond back moths (DBM; Plutella xylostella). This is advantageous in part because the subject invention provides a new alternative for controlling DBMs, which are known to develop resistance to some B.t. and other pesticides. Thus, the subject invention includes using a toxin of the subject invention in methods of controlling or inhibiting DBMs that have developed resistance (DBM.sup.R) to at least one other type of toxin.

The subject invention includes plants cells transformed with at least one polynucleotide sequence of the subject invention such that the transformed plant cells express pesticidal toxins in tissues consumed by target pests. Toxins of the subject invention can be used in combination with other toxins. Transformation of plants with the genetic constructs disclosed herein can be accomplished using techniques well known to those skilled in the art and would typically involve modification of the gene to optimize expression of the toxin in plants. One such preferred sequence is disclosed herein.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 shows correlation of SEQ ID NO:26 (plant-optimized KB59A4-6) expression and toxicity to tobacco budworm in Arabidopsis T1 lines.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO. 1 is a forward primer, designated "the 339 forward primer," used according to the subject invention.

SEQ ID NO. 2 is a reverse primer, designated "the 339 reverse primer," used according to the subject invention.

SEQ ID NO. 3 is a nucleotide sequence encoding a toxin from B.t. strain PS36A.

SEQ ID NO. 4 is an amino acid sequence for the 36A toxin.

SEQ ID NO. 5 is a nucleotide sequence encoding a toxin from B.t. strain PS81F.

SEQ ID NO. 6 is an amino acid sequence for the 81F toxin.

SEQ ID NO. 7 is a nucleotide sequence encoding a toxin from B.t. strain Javelin 1990.

SEQ ID NO. 8 is an amino acid sequence for the Javelin 1990 toxin.

SEQ ID NO. 9 is a forward primer, designated "158C2 PRIMER A," used according to the subject invention.

SEQ ID NO. 10 is a nucleotide sequence encoding a portion of a soluble toxin from B.t. PS158C2.

SEQ ID NO. 11 is a forward primer, designated "49C PRIMER A," used according to the subject invention.

SEQ ID NO. 12 is a nucleotide sequence of a portion of a toxin gene from B.t. strain PS49C.

SEQ ID NO. 13 is a forward primer, designated "49C PRIMER B," used according to the subject invention.

SEQ ID NO. 14 is a reverse primer, designated "49C PRIMER C," used according to the subject invention.

SEQ ID NO. 15 is an additional nucleotide sequence of a portion of a toxin gene from PS49C.

SEQ ID NO. 16 is the nucleotide sequence of the SUP toxin gene from B.t. strain PS49C.

SEQ ID NO. 17 is the amino acid sequence of the SUP toxin gene from B.t. strain PS49C.

SEQ ID NO. 18 is the nucleotide sequence of the SUP toxin gene from B.t. strain PS158C2.

SEQ ID NO. 19 is the amino acid sequence of the SUP toxin gene from B.t. strain PS158C2.

SEQ ID NO. 20 is a forward primer, designated "SUP-1A," used according to the subject invention.

SEQ ID NO. 21 is a reverse primer, designated "SUP-1B," used according to the subject invention.

SEQ ID NO:22 is a SUP primer for use according to the subject invention.

SEQ ID NO:23 is a SUP primer for use according to the subject invention.

SEQ ID NO:24 is a nucleotide sequence for a SUP gene from KB59A4-6.

SEQ ID NO:25 is an amino acid sequence for a SUP toxin from KB59A4-6.

SEQ ID NO:26 is a plant-optimized polynucleotide that encodes a KB59A4-6 SUP toxin.

SEQ ID NO:27 is a protein encoded by SEQ ID NO:26.

DETAILED DISCLOSURE OF THE INVENTION

The subject invention concerns materials and methods for the control of non-mammalian pests. In specific embodiments, the subject invention pertains to new Bacillus thuringiensis isolates and toxins that preferably have activity against lepidopterans. The subject invention further concerns novel genes which encode pesticidal toxins and novel methods for identifying and characterizing Bacillus genes which encode toxins with useful properties. The subject invention concerns not only the polynucleotide sequences which encode these toxins, but also the use of these polynucleotide sequences to produce recombinant hosts which express the toxins. The proteins of the subject invention are distinct from protein toxins which have previously been isolated from Bacillus thuringiensis (B.t.).

B.t. isolates useful according to the subject invention have been deposited in the permanent collection of the Agricultural Research Service Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street; Peoria, Ill. 61604, USA. The culture repository numbers of the B.t. strains are as follows:

TABLE-US-00001 TABLE 1 Culture Repository No. Deposit Date Patent No. B.t. PS11B (MT274) NRRL B-21556 Apr. 18, 1996 B.t. PS31G1 (MT278) NRRL B-21560 Apr. 18, 1996 B.t. PS36A NRRL B-18929 Dec. 27, 1991 B.t. PS49C NRRL B-21532 Mar. 14, 1996 B.t. PS81A2 NRRL B-18484 Apr. 19, 1989 5,164,180 B.t. PS81F NRRL B-18424 Oct. 7, 1988 5,045,469 B.t. PS81GG NRRL B-18425 Oct. 11, 1988 5,169,629 B.t. PS81I NRRL B-18484 Apr. 19, 1989 5,126,133 B.t. PS85A1 NRRL B-18426 Oct. 11, 1988 B.t. PS86BB1 (MT275) NRRL B-21557 Apr. 18, 1996 B.t. PS86V1 (MT276) NRRL B-21558 Apr. 18, 1996 B.t. PS86W1 (MT277) NRRL B-21559 Apr. 18, 1996 B.t. PS89J3 (MT279) NRRL B-21561 Apr. 18, 1996 B.t. PS91C2 NRRL B-18931 Feb. 6, 1991 B.t. PS158C2 NRRL B-18872 Aug. 27, 1991 5,268,172 B.t. PS185U2 (MT280) NRRL B-21562 Apr. 18, 1996 B.t. PS192M4 NRRL B-18932 Dec. 27, 1991 5,273,746 B.t. PS244A2 NRRL B-21541 Mar. 14, 1996 PS94R1 NRRL B-21801 Jul. 1, 1997 PS101DD NRRL B-21802 Jul. 1, 1997 PS202S NRRL B-21803 Jul. 1, 1997 PS213E5 NRRL B-21804 Jul. 1, 1997 PS218G2 NRRL B-21805 Jul. 1, 1997

Cultures which have been deposited for the purposes of this patent application were deposited under conditions that assure that access to the cultures is available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122. The deposits will be available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

Further, the subject culture deposits will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., they will be stored with all the care necessary to keep them viable and uncontaminated for a period of at least five years after the most recent request for the firnishing of a sample of the deposit, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the culture(s). The depositor acknowledges the duty to replace the deposit(s) should the depository be unable to furnish a sample when requested, due to the condition of a deposit. All restrictions on the availability to the public of the subject culture deposits will be irrevocably removed upon the granting of a patent disclosing them.

Many of the strains useful according to the subject invention are readily available by virtue of the issuance of patents disclosing these strains or by their deposit in public collections or by their inclusion in commercial products. For example, the B.t. strain used in the commercial product, Javelin, is publicly available. The "HD" isolates are publicly available from the Howard Dulmage culture collection.

Mutants of the isolates referred to herein can be made by procedures well known in the art. For example, an asporogenous mutant can be obtained through ethylmethane sulfonate (EMS) mutagenesis of an isolate. The mutants can be made using ultraviolet light and nitrosoguanidine by procedures well known in the art.

In one embodiment, the subject invention concerns materials and methods including nucleotide primers and probes for isolating, characterizing, and identifying Bacillus genes encoding protein toxins that are active against non-mammalian pests. The nucleotide sequences described herein can also be used to identify new pesticidal Bacillus isolates. The invention further concerns the genes, isolates, and toxins identified using the methods and materials disclosed herein.

The new toxins and polynucleotide sequences provided here are defined according to several parameters. One characteristic of the toxins described herein is pesticidal activity. In a specific embodiment, these toxins have activity against coleopteran and/or lepidopteran pests. The toxins and genes of the subject invention can be further defined by their amino acid and nucleotide sequences. The sequences of the molecules can be defined in terms of homology to certain exemplified sequences as well as in terms of the ability to hybridize with, or be amplified by, certain exemplified probes and primers. The toxins provided herein can also be identified based on their immunoreactivity with certain antibodies.

An important aspect of the subject invention is the identification and characterization of new families of Bacillus toxins, and genes which encode these toxins. Members of a preferred family have been designated "SUP" toxins. Toxins within this family, as well as genes encoding toxins within this family, can readily be identified as described herein by, for example, size, amino acid or DNA sequence, and antibody reactivity. Amino acid and DNA sequence characteristics include homology with exemplified sequences, ability to hybridize with DNA probes, and ability to be amplified with specific primers.

SUP toxins of the subject invention are soluble and can be obtained from the supernatant of Bacillus cultures as described herein. In a preferred embodiment, the SUP toxins are active against lepidopteran pests. The SUP toxins typically have a size of about 70 100 kDa and, preferably, about 80 kDa. The SUP family is exemplified herein by toxins from isolates PS49C and PS158C2. The subject invention provides probes and primers useful for the identification of toxins and genes in the SUP family

These toxins can be used alone or in combination with other toxins to control pestsThese toxins may be used, for example, with .delta.-endotoxins which are obtained from Bacillus isolates.

Table 2 provides a summary of SUP toxins and genes of the subject invention, which can be obtained from particular B.t. isolates as shown in Table 2. Genes encoding toxins in each of these families can be identified by a variety of highly specific parameters, including the ability to hybridize with the particular probes set forth in Table 2. Sequence identity in excess of about 80% with the probes set forth in Table 2 can also be used to identify the genes of the various families. Also exemplified are particular primer pairs which can be used to amplify the genes of the subject invention. A portion of a gene within the indicated family would typically be amplifiable with at least one of the enumerated primer pairs. In a preferred embodiment, the amplified portion would be of approximately the indicated fragment size. Primers shown in Table 2 consist of polynucleotide sequences which encode peptides as shown in the sequence listing attached hereto. Additional primers and probes can readily be constructed by those skilled in the art such that alternate polynucleotide sequences encoding the same amino acid sequences can be used to identify and/or characterize additional genes encoding pesticidal toxins. In a preferred embodiment, these additional toxins, and their genes, could be obtained from Bacillus isolates.

TABLE-US-00002 TABLE 2 Probes (SEQ Primer Pairs Fragment Family Isolates ID NO.) (SEQ ID NOS.) size (nt) SUP PS49C, PS158C2 10, 12, 15 53 and 54 370

With the teachings provided herein, one skilled in the art could readily produce and use the various toxins and polynucleotide sequences described herein.

Genes and toxins. The genes and toxins useful according to the subject invention include not only the full length sequences but also fragments of these sequences, variants, mutants, and fusion proteins which retain the characteristic pesticidal activity of the toxins specifically exemplified herein. Chimeric genes and toxins, produced by combining portions from more than one Bacillus toxin or gene, may also be utilized according to the teachings of the subject invention. As used herein, the terms "variants" or "variations" of genes refer to nucleotide sequences which encode the same toxins or which encode equivalent toxins having pesticidal activity. As used herein, the term "equivalent toxins" refers to toxins having the same or essentially the same biological activity against the target pests as the exemplified toxins.

It is apparent to a person skilled in this art that genes encoding active toxins can be identified and obtained through several means. The specific genes exemplified herein may be obtained from the isolates deposited at a culture depository as described above. These genes, or portions or variants thereof, may also be constructed synthetically, for example, by use of a gene synthesizer. Variations of genes may be readily constructed using standard techniques for making point mutations. Also, fragments of these genes can be made using commercially available exonucleases or endonucleases according to standard procedures. For example, enzymes such as Bal31 or site-directed mutagenesis can be used to systematically cut off nucleotides from the ends of these genes. Also, genes which encode active fragments may be obtained using a variety of restriction enzymes. Proteases may be used to directly obtain active fragments of these toxins.

Equivalent toxins and/or genes encoding these equivalent toxins can be derived from Bacillus isolates and/or DNA libraries using the teachings provided herein. There are a number of methods for obtaining the pesticidal toxins of the instant invention. For example, antibodies to the pesticidal toxins disclosed and claimed herein can be used to identify and isolate toxins from a mixture of proteins. Specifically, antibodies may be raised to the portions of the toxins which are most constant and most distinct from other Bacillus toxins. These antibodies can then be used to specifically identify equivalent toxins with the characteristic activity by immunoprecipitation, enzyme linked immunosorbent assay (ELISA), or Western blotting. Antibodies to the toxins disclosed herein, or to equivalent toxins, or fragments of these toxins, can readily be prepared using standard procedures in this art. The genes which encode these toxins can then be obtained from the microorganism.

Fragments and equivalents which retain the pesticidal activity of the exemplified toxins are within the scope of the subject invention. Also, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the amino acid sequences disclosed herein. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same, or essentially the same, toxins. These variant DNA sequences are within the scope of the subject invention. As used herein, reference to "essentially the same" sequence refers to sequences which have amino acid substitutions, deletions, additions, or insertions which do not materially affect pesticidal activity. Fragments retaining pesticidal activity are also included in this definition.

A further method for identifying the toxins and genes of the subject invention is through the use of oligonucleotide probes. These probes are detectable nucleotide sequences. Probes provide a rapid method for identifying toxin-encoding genes of the subject invention. The nucleotide segments which are used as probes according to the invention can be synthesized using a DNA synthesizer and standard procedures.

Certain toxins of the subject invention have been specifically exemplified herein. Since these toxins are merely exemplary of the toxins of the subject invention, it should be readily apparent that the subject invention comprises variant or equivalent toxins (and nucleotide sequences coding for equivalent toxins) having the same or similar pesticidal activity of the exemplified toxin. Equivalent toxins will have amino acid homology with an exemplified toxin. This amino acid identity will typically be 60% or greater, preferably 75% or greater, more preferably 80% or greater, more preferably 90% or greater, and can be 95% or greater. These identities are as determined using standard alignment techniques. The amino acid homology will be highest in critical regions of the toxin which account for biological activity or are involved in the determination of three-dimensional configuration which ultimately is responsible for the biological activity. In this regard, certain amino acid substitutions are acceptable and can be expected if these substitutions are in regions which are not critical to activity or are conservative amino acid substitutions which do not affect the three-dimensional configuration of the molecule. For example, amino acids may be placed in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the subject invention so long as the substitution does not materially alter the biological activity of the compound. Table 3 provides a listing of examples of amino acids belonging to each class.

TABLE-US-00003 TABLE 3 Class of Amino Acid Examples of Amino Acids Nonpolar Ala, Val, Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr, Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His

In some instances, non-conservative substitutions can also be made. The critical factor is that these substitutions must not significantly detract from the biological activity of the toxin.

The .delta.-endotoxins of the subject invention can also be characterized in terms of the shape and location of toxin inclusions, which are described above.

As used herein, reference to "isolated" polynucleotides and/or "purified" toxins refers to these molecules when they are not associated with the other molecules with which they would be found in nature. Thus, reference to "isolated and purified" signifies the involvement of the "hand of man" as described herein. Chimeric toxins and genes also involve the "hand of man."

As mentioned above, the subject invention includes truncated toxins and chimeric toxins (derived using SEQ ID NOS:17, 19, and 25, for example). As described in U.S. Pat. No. 6,137,033 for example (see also Lee et al. discussed above in the Background section), Vip3 proteins are proteolytically truncated from about 88 kDa to about 66 kDa. The 66 kDa protein comprises amino acid residues 200 789. The 66 kDa protein appears to be further truncated by proteases to yield a 33 kDa toxic core (the C terminus of the 66 kDa protein, corresponding to residues 200 455 of the full-length) and a 45 kDa protein (corresponding to residues 412 789 of the full-length protein).

In light of the diamond back moth (DBM) toxicity exhibited by the 49C and KB59A-46 SUP toxins, very interesting results can be obtained by aligning the sequences of the SUP toxins of the subject invention (SEQ ID NOS:17, 19, and 25, for example) with, for example, those for the proteins of SEQ ID NO:6 (the 81F toxin) and 8, and/or the Vip3 sequences. As can be determined by such alignments (which are within the skill in the art--PLOT SIMILARITY can be used, for example), most of the sequence divergence between 81F vs. 49C and KB59A4-6 occurs in about the last 200 amino acid residues of the protein. This would correspond to about the last two-thirds of the 45 kDa band discussed in the '033 patent.

Thus, it appears that the last 200 or so residues of the SUP proteins, or other regions where there is sequence divergence, could be involved with the mechanism of action accounting for insect specificity. In light of this and other teachings discussed herein and in the art in general, the subject invention includes chimeric toxins comprising certain fragments of the subject SUP toxins. Residues 412 to the C terminus (of SEQ ID NOS: 17, 19, and 25) are preferred for such uses, as are residues .about.600 to the C terminus. In other embodiments, residues .about.200 455 (of SEQ ID NOS: 17, 19, and 25) can be used for constructing chimerics. Alternatively or in combination with other chimeric approaches, the first 200 or so amino acids of SEQ ID NOS: 17, 19, and 25 can be omitted/removed (in vitro); this would yield truncated toxins or truncated chimeric toxins.

The various segments identified above can be swapped amongst themselves, or they can be used in conjunction with, for example, other sequences disclosed herein or with Vip3 sequences. For example, one of the C terminal segments discussed above (residues 412 to the C terminus of SEQ ID NO:17 and 25, for example [786 and 787, respectively]) can be used with residues 0 412 or 455 of SEQ ID NO:6 (81F) for example. Residues 200 412 or 455 of SEQ ID NO:17 or 25, for example, could be used with the C terminal segment of 81F, for example. Alternatively, Vip3 sequences could be used in place of the 81F segments (together with the 49C or KB59A4-6 segments) discussed above. Thus, if SUP and Vip3 toxins are considered to have three main domains or regions as discussed above, chimerics of the subject invention include those that would comply with the following, where each letter depicts a domain, the subscript number indicates the domains in the order discussed above (N terminal to C terminal), and different letters depict different source SUP or Vip3 proteins: A.sub.1A.sub.2B.sub.3, A.sub.1B.sub.2A.sub.3, A.sub.1B.sub.2B.sub.3, A.sub.1B.sub.2C.sub.3, and the like. Preferred embodiments of such chimerics are designed to have toxin activity against diamond back moths. The relative location/sequence/order (1-2-3) of the domains does not necessarily have to be maintained, and it should also be clear that the subject invention includes truncated chimerics, including those where a domain is wholly or partly removed. Examples include A.sub.2B.sub.3.

Recombinant hosts. The toxin-encoding genes of the subject invention can be introduced into a wide variety of microbial or plant hosts. Expression of the toxin gene results, directly or indirectly, in the production and maintenance of the pesticide. With suitable microbial hosts, e.g., Pseudomonas, the microbes can be applied to the situs of the pest, where they will proliferate and be ingested. The result is a control of the pest. Alternatively, the microbe hosting the toxin gene can be killed and treated under conditions that prolong the activity of the toxin and stabilize the cell. The treated cell, which retains the toxic activity, then can be applied to the environment of the target pest.

A wide variety of ways are available for introducing a Bacillus gene encoding a toxin into a microorganism host under conditions which allow for stable maintenance and expression of the gene. These methods are well known to those skilled in the art and are described, for example, in U.S. Pat. No. 5,135,867, which is incorporated herein by reference.

Synthetic genes which are functionally equivalent to the toxins of the subject invention can also be used to transform hosts. Methods for the production of synthetic genes can be found in, for example, U.S. Pat. No. 5,380,831.

Treatment of cells. As mentioned above, Bacillus or recombinant cells expressing a Bacillus toxin can be treated to prolong the toxin activity and stabilize the cell. The pesticide microcapsule that is formed comprises the Bacillus toxin within a cellular structure that has been stabilized and will protect the toxin when the microcapsule is applied to the environment of the target pest. Suitable host cells may include either prokaryotes or eukaryotes. As hosts, of particular interest will be the prokaryotes and the lower eukaryotes, such as fungi. The cell will usually be intact and be substantially in the proliferative form when treated, rather than in a spore form.

Treatment of the microbial cell, e.g., a microbe containing the Bacillus toxin gene, can be by chemical or physical means, or by a combination of chemical and/or physical means, so long as the technique does not deleteriously affect the properties of the toxin, nor diminish the cellular capability of protecting the toxin. Methods for treatment of microbial cells are disclosed in U.S. Pat. Nos. 4,695,455 and 4,695,462, which are incorporated herein by reference.

Methods and formulations for control of pests. Control of pests using the isolates, toxins, and genes of the subject invention can be accomplished by a variety of methods known to those skilled in the art. These methods include, for example, the application of Bacillus isolates to the pests (or their location), the application of recombinant microbes to the pests (or their locations), and the transformation of plants with genes which encode the pesticidal toxins of the subject invention. Transformations can be made by those skilled in the art using standard techniques. Materials necessary for these transformations are disclosed herein or are otherwise readily available to the skilled artisan.

Formulated bait granules containing an attractant and the toxins of the Bacillus isolates, or recombinant microbes comprising the genes obtainable from the Bacillus isolates disclosed herein, can be applied to the soil. Formulated product can also be applied as a seed-coating or root treatment or total plant treatment at later stages of the crop cycle. Plant and soil treatments of Bacillus cells may be employed as wettable powders, granules or dusts, by mixing with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical materials (powdered corncobs, rice hulls, walnut shells, and the like). The formulations may include spreader-sticker adjuvants, stabilizing agents, other pesticidal additives, or surfactants. Liquid formulations may be aqueous-based or non-aqueous and employed as foams, gels, suspensions, emulsifiable concentrates, or the like. The ingredients may include rheological agents, surfactants, emulsifiers, dispersants, or polymers.

As would be appreciated by a person skilled in the art, the pesticidal concentration will vary widely depending upon the nature of the particular formulation, particularly whether it is a concentrate or to be used directly. The pesticide will be present in at least 1% by weight and may be 100% by weight. The dry formulations will have from about 1 95% by weight of the pesticide while the liquid formulations will generally be from about 1 60% by weight of the solids in the liquid phase. The formulations that contain cells will generally have from about 10.sup.2 to about 10.sup.4 cells/mg. These formulations will be administered at about 50 mg (liquid or dry) to 1 kg or more per hectare.

The formulations can be applied to the environment of the pest, e.g., soil and foliage, by spraying, dusting, sprinkling, or the like.

Diamondback moths (DBMs) are a particularly troublesome pest in Asia, including Southeast Asia. Thus, the subject invention advantageously includes the transgenic plants and seeds of the subject invention, and the use thereof, in Asia, especially for controlling the development of resistant DBMs.

Polynucleotide probes. It is well known that DNA possesses a fundamental property called base complementarity. In nature, DNA ordinarily exists in the form of pairs of anti-parallel strands, the bases on each strand projecting from that strand toward the opposite strand. The base adenine (A) on one strand will always be opposed to the base thymine (T) on the other strand, and the base guanine (G) will be opposed to the base cytosine (C). The bases are held in apposition by their ability to hydrogen bond in this specific way. Though each individual bond is relatively weak, the net effect of many adjacent hydrogen bonded bases, together with base stacking effects, is a stable joining of the two complementary strands. These bonds can be broken by treatments such as high pH or high temperature, and these conditions result in the dissociation, or "denaturation," of the two strands. If the DNA is then placed in conditions which make hydrogen bonding of the bases thermodynamically favorable, the DNA strands will anneal, or "hybridize," and reform the original double stranded DNA. If carried out under appropriate conditions, this hybridization can be highly specific. That is, only strands with a high degree of base complementarity will be able to form stable double stranded structures. The relationship of the specificity of hybridization to reaction conditions is well known. Thus, hybridization may be used to test whether two pieces of DNA are complementary in their base sequences. It is this hybridization mechanism which facilitates the use of probes of the subject invention to readily detect and characterize DNA sequences of interest.

The probes may be RNA, DNA, or PNA (peptide nucleic acid). The probe will normally have at least about 10 bases, more usually at least about 17 bases, and may have up to about 100 bases or more. Longer probes can readily be utilized, and such probes can be, for example, several kilobases in length. The probe sequence is designed to be at least substantially complementary to a portion of a gene encoding a toxin of interest. The probe need not have perfect complementarity to the sequence to which it hybridizes. The probes may be labelled utilizing techniques which are well known to those skilled in this art.

One approach for the use of the subject invention as probes entails first identifying by Southern blot analysis of a gene bank of the Bacillus isolate all DNA segments homologous with the disclosed nucleotide sequences. Thus, it is possible, without the aid of biological analysis, to know in advance the probable activity of many new Bacillus isolates, and of the individual gene products expressed by a given Bacillus isolate. Such a probe analysis provides a rapid method for identifying potentially commercially valuable insecticidal toxin genes within the multifarious subspecies of B.t.

One hybridization procedure useful according to the subject invention typically includes the initial steps of isolating the DNA sample of interest and purifying it chemically. Either lysed bacteria or total fractionated nucleic acid isolated from bacteria can be used. Cells can be treated using known techniques to liberate their DNA (and/or RNA). The DNA sample can be cut into pieces with an appropriate restriction enzyme. The pieces can be separated by size through electrophoresis in a gel, usually agarose or acrylamide. The pieces of interest can be transferred to an immobilizing membrane.

The particular hybridization technique is not essential to the subject invention. As improvements are made in hybridization techniques, they can be readily applied.

The probe and sample can then be combined in a hybridization buffer solution and held at an appropriate temperature until annealing occurs. Thereafter, the membrane is washed free of extraneous materials, leaving the sample and bound probe molecules typically detected and quantified by autoradiography and/or liquid scintillation counting. As is well known in the art, if the probe molecule and nucleic acid sample hybridize by forming a strong non-covalent bond between the two molecules, it can be reasonably assumed that the probe and sample are essentially identical. The probe's detectable label provides a means for determining in a known manner whether hybridization has occurred.

In the use of the nucleotide segments as probes, the particular probe is labeled with any suitable label known to those skilled in the art, including radioactive and non-radioactive labels. Typical radioactive labels include .sup.32P, .sup.35S, or the like. Non-radioactive labels include, for example, ligands such as biotin or thyroxine, as well as enzymes such as hydrolases or perixodases, or the various chemiluminescers such as luciferin, or fluorescent compounds like fluorescein and its derivatives. The probes may be made inherently fluorescent as described in International Application No. WO 93/16094.

Various degrees of stringency of hybridization can be employed. The more severe the conditions, the greater the complementarity that is required for duplex formation. Severity can be controlled by temperature, probe concentration, probe length, ionic strength, time, and the like. Preferably, hybridization is conducted under moderate to high stringency conditions by techniques well known in the art, as described, for example, in Keller, G. H., M. M. Manak (1987) DNA Probes, Stockton Press, New York, N.Y., pp. 169 170.

As used herein "moderate to high stringency" conditions for hybridization refers to conditions which achieve the same, or about the same, degree of specificity of hybridization as the conditions employed by the current applicants. Examples of moderate and high stringency conditions are provided herein. Specifically, hybridization of immobilized DNA on Southern blots with 32P-labeled gene-specific probes was performed by standard methods (Maniatis et al.). In general, hybridization and subsequent washes were carried out under moderate to high stringency conditions that allowed for detection of target sequences with homology to the exemplified toxin genes. For double-stranded DNA gene probes, hybridization was carried out overnight at 20 25.degree. C. below the melting temperature (Tm) of the DNA hybrid in 6.times.SSPE, 5.times. Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature is described by the following formula (Beltz, G. A., K. A. Jacobs, T. H. Eickbush, P. T. Cherbas, and F. C. Kafatos [1983] Methods of Enzymology, R. Wu, L. Grossman and K. Moldave [eds.] Academic Press, New York 100:266 285).

Tm=81.5.degree. C.+16.6 Log [Na+]+0.41(% G+C)-0.61(% formamide)-600/length of duplex in base pairs.

Washes are typically carried out as follows:

(1) Twice at room temperature for 15 minutes in 1.times.SSPE, 0.1% SDS (low stringency wash).

(2) Once at Tm-20.degree. C. for 15 minutes in 0.2.times.SSPE, 0.1% SDS (moderate stringency wash).

For oligonucleotide probes, hybridization was carried out overnight at 10 20.degree. C. below the melting temperature (Tm) of the hybrid in 6.times.SSPE, 5.times. Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Tm for oligonucleotide probes was determined by the following formula:

Tm (.degree. C.)=2(number T/A base pairs)+4(number G/C base pairs) (Suggs, S. V., T. Miyake, E. H. Kawashime, M. J. Johnson, K. Itakura, and R. B. Wallace [1981] ICN-UCLA Symp. Dev. Biol. Using Purified Genes, D. D. Brown [ed.], Academic Press, New York, 23:683 693).

Washes were typically carried out as follows:

(1) Twice at room temperature for 15 minutes 1.times.SSPE, 0.1% SDS (low stringency wash).

(2) Once at the hybridization temperature for 15 minutes in 1.times.SSPE, 0.1% SDS (moderate stringency wash).

In general, salt and/or temperature can be altered to change stringency. With a labeled DNA fragment >70 or so bases in length, the following conditions can be used:

Low: 1 or 2.times.SSPE, room temperature

Low: 1 or 2.times.SSPE, 42.degree. C.

Moderate: 0.2.times. or 1.times.SSPE, 65.degree. C.

High: 0.1.times.SSPE, 65.degree. C.

Duplex formation and stability depend on substantial complementarity between the two strands of a hybrid, and, as noted above, a certain degree of mismatch can be tolerated. Therefore, the probe sequences of the subject invention include mutations (both single and multiple), deletions, insertions of the described sequences, and combinations thereof, wherein said mutations, insertions and deletions permit formation of stable hybrids with the target polynucleotide of interest. Mutations, insertions, and deletions can be produced in a given polynucleotide sequence in many ways, and these methods are known to an ordinarily skilled artisan. Other methods may become known in the future.

Thus, mutational, insertional, and deletional variants of the disclosed nucleotide sequences can be readily prepared by methods which are well known to