Title:
PROTEIN MIXTURES FOR MAIZE INSECT CONTROL
Kind Code:
A1


Abstract:
Embodiments of the present invention relate to insecticidal Bacillus thuringiensis Cry1 and Cry2 polypeptides. Methods for using the polypeptides and nucleic acids of embodiments of the invention to synergistically enhance resistance of plants to insect predation are encompassed in embodiments of the present invention.



Inventors:
Flexner, John Lindsey (Landenberg, PA, US)
Kapka-kitzman, Deirdre (Ankeny, IA, US)
Procyk, Lisa (Ankeny, IA, US)
Stanley, Bruce H. (Wilmington, DE, US)
Zhao, Jianzhou (Johnston, IA, US)
Application Number:
14/254793
Publication Date:
08/07/2014
Filing Date:
04/16/2014
Assignee:
E I DU PONT DE NEMOURS AND COMPANY (Wilmington, DE, US)
PIONEER HI BRED INTERNATIONAL INC (Johnston, IA, US)
Primary Class:
Other Classes:
424/115, 504/116.1, 514/143, 514/229.2, 514/355, 514/359, 514/406, 514/408, 514/478, 514/531, 514/634, 800/302
International Classes:
C12N15/82
View Patent Images:



Other References:
Tounsi et al (J. Appl. MicrobioL 95:23-28; 2003)
de Maagd et al (Appl Environ. Microbiol 65:4369-4374, 1999)
Aronson et al (FEMS Microbiol. Lett. 2001, 195:1-8)
Friedberg (Brief. Bioinformatics (2006) 7: 225-242)
Castle et al (Current Opinion in Biotechnology 2006. 17:105-112)
Tang et al (Mol. Breeding, 2006, 18:1-10)
Primary Examiner:
VISONE, LEE A
Attorney, Agent or Firm:
PIONEER HI-BRED INTERNATIONAL, INC. (7250 NW 62ND AVENUE PO BOX 552, JOHNSTON, IA, 50131-0552, US)
Claims:
What is claimed is:

1. A method of reducing pest damage in a transgenic plant comprising: planting a first transgenic plant seed, wherein the first transgenic plant seed comprises a first transgene and a second transgene, wherein the first transgene causes expression of a Cry1 protein in a plant, wherein the Cry1 protein is selected from the group consisting of (a) SEQ ID NO:1, (b) SEQ ID NO:4, and (c) a polypeptide that is at least 90% identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:4; and the second transgene causes expression of a Cry2 protein in a plant, wherein the Cry2 protein is selected from the group consisting of (a) SEQ ID NO:2 and (b) a polypeptide that is at least 90% identical to the amino acid sequence of SEQ ID NO:2; thereby reducing damage caused by a first target pest to a plant grown from the first transgenic plant seed.

2. The method of claim 1 wherein the transgenic plant is maize.

3. The method of claim 1 wherein the first target pest is a member of order Lepidoptera.

4. The method of claim 3 wherein the first target pest is selected from the group consisting of European corn borer and corn earworm.

5. The method of claim 1 further comprising treating the first transgenic plant seed with a pesticidal agent.

6. The method of claim 5 wherein the pesticidal agent is selected from the group consisting of: an insecticide, an acaricide, a nematicide, a fungicide, a bactericide, a herbicide, or a combination thereof.

7. The method of claim 6 wherein the pesticidal agent is an insecticide.

8. The method of claim 7 wherein the insecticide is selected from the group consisting of: a pyrethrin, a synthetic pyrethrin, an oxadizine, a chloronicotinyl, a nitroguanidine, a triazole, an organophosphate, a pyrrol, a pyrazole, a phenol pyrazole, a diacylhydrazine, a biological/fermentation product, a carbamate, or a combination thereof.

9. The method of claim 1 wherein the first transgenic plant seed further comprises a herbicide resistance gene.

10. The method of claim 9 wherein the herbicide resistance gene is selected from the group consisting of: glyphosate N-acetyltransferase (GAT), 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), phosphinothricin N-acetyltransferase (PAT) or a combination thereof.

11. A method for providing synergistic insecticidal activity against at least one pest comprising: providing a first transgenic plant, wherein the first transgenic plant expresses a Cry1-derived insecticidal polypeptide and a Cry2-derived insecticidal polypeptide, wherein the Cry1-derived insecticidal polypeptide comprises a polypeptide selected from the group consisting of: (a) SEQ ID NO:1 and (b) SEQ ID NO:4, and (c) a polypeptide that is at least 90% identical to the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:4; and wherein the Cry2-derived insecticidal polypeptide comprises a polypeptide selected from the group consisting of (a) SEQ ID NO:2 and (b) a polypeptide that is at least 90% identical to the amino acid sequence of SEQ ID NO:2; thereby resulting in synergistic insect resistance against a first target pest.

12. The method of claim 11 wherein the transgenic plant is maize.

13. The method of claim 11 wherein the first target pest is a member of order Lepidoptera.

14. The method of claim 13 wherein the first target pest is selected from the group consisting of European corn borer and corn earworm.

15. The method of claim 11 further comprising treating the first transgenic plant seed with a pesticidal agent.

16. The method of claim 15 wherein the pesticidal agent is selected from the group consisting of: an insecticide, an acaricide, a nematicide, a fungicide, a bactericide, a herbicide, or a combination thereof.

17. The method of claim 16 wherein the pesticidal agent is an insecticide.

18. The method of claim 17 wherein the insecticide is selected from the group consisting of: a pyrethrin, a synthetic pyrethrin, an oxadizine, a chloronicotinyl, a nitroguanidine, a triazole, an organophosphate, a pyrrol, a pyrazole, a phenol pyrazole, a diacylhydrazine, a biological/fermentation product, a carbamate, or a combination thereof.

19. The method of claim 11 wherein the first transgenic plant further comprises a herbicide resistance gene.

20. The method of claim 19 wherein the herbicide resistance gene is selected from the group consisting of: glyphosate N-acetyltransferase (GAT), 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), phosphinothricin N-acetyltransferase (PAT) or a combination thereof.

21. A method of reducing pest damage in a transgenic plant comprising: planting a first transgenic plant seed, wherein the first transgenic plant seed comprises a first transgene and a second transgene, wherein the first transgene causes expression of a Cry1 protein in a plant and the second transgene causes expression of a Cry2 protein in a plant, the Cry1 protein selected from the group consisting of the polypeptide of SEQ ID NO:1 and the polypeptide of SEQ ID NO:4; and the Cry2 protein comprising the polypeptide of SEQ ID NO: 2, thereby reducing damage caused by a first target pest to a plant grown from the first transgenic plant seed.

22. The method of claim 21 wherein the transgenic plant is maize.

23. The method of claim 21 wherein the first target pest is a member of order Lepidoptera.

24. The method of claim 23 wherein the first target pest is selected from the group consisting of European corn borer and corn earworm.

25. A transgenic plant comprising a first transgene and a second transgene, wherein the first transgene causes expression of a Cry1 protein in a plant and the second transgene causes expression of a Cry2 protein in a plant.

26. The transgenic plant of claim 25, wherein the Cry1 protein selected from the group consisting of the polypeptide of SEQ ID NO:1 and the polypeptide of SEQ ID NO:4; and wherein the Cry2 protein comprises the polypeptide of SEQ ID NO: 2.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119 to provisional application Ser. No. 61/146,875 filed Jan. 23, 2009, herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

Embodiments of the present invention relate generally to the field of pest control, providing insecticidal polypeptides related to combinations of Bacillus thuringiensis (B. t.) Cry1 and Cry2 polypeptides and the polynucleotides that encode them. Embodiments of the present invention also relate to methods and compositions for improved resistance of plants to insect predation, including, but not limited to, transgenic plant production. The Cry1 and Cry2 polypeptide mixtures provide improved insecticidal activity and synergism against key plant pests, including maize pests.

BACKGROUND OF THE INVENTION

Numerous commercially valuable plants, including common agricultural crops, are susceptible to attack by insect and nematode pests, causing substantial reductions in crop yield and quality. For example, growers of maize (Zea mays), commonly referred to as corn in the United States, face a major problem with combating pest infestations. Insects, nematodes, and related arthropods annually destroy an estimated 15% of agricultural crops in the United States and an even greater percentage in developing countries. In addition, competition with weeds and parasitic and saprophytic plants account for even more potential yield losses. Yearly, such pests cause over $100 billion in crop damage in the United States alone.

In an effort to combat pest infestations, various methods have been employed in order to reduce or eliminate pests in a particular plot. These efforts include rotating corn with other crops that are not a host for a particular pest and applying pesticides to the above-ground portion of the crop, applying pesticides to the soil in and around the root systems of the affected crop. Traditionally, farmers have relied heavily on chemical pesticides to combat pest damage. However, the use of chemical pesticides is costly, as farmers apply billions of gallons of synthetic pesticides to combat these pests each growing season, costing nearly $8 billion. In addition, such pesticides are inconvenient for farmers, result in the emergence of insecticide-resistant pests, and they raise significant environmental and health concerns.

Because of concern about the impact of pesticides on public health and the health of the environment, significant efforts have been made to find ways to reduce the amount of chemical pesticides that are used. Recently, much of this effort has focused on the development of transgenic crops that are engineered to express insect toxicants derived from microorganisms. For example, U.S. Pat. No. 5,877,012 to Estruch et al. discloses the cloning and expression of proteins from such organisms as Bacillus, Pseudomonas, Clavibacter and Rhizobium into plants to obtain transgenic plants with resistance to such pests as black cutworms, armyworms, several borers and other insect pests. Publication WO/EP97/07089 by Privalle et al. teaches the transformation of monocotyledons, such as corn, with a recombinant DNA sequence encoding peroxidase for the protection of the plant from feeding by corn borers, earworms and cutworms. Jansens et al., Crop Sci., 37(5):1616-1624 (1997), reported the production of transgenic corn containing a gene encoding a crystalline protein from Bt that controlled both generations of Eastern Corn Borer (ECB). U.S. Pat. Nos. 5,625,136 and 5,859,336 to Koziel et al. reported that the transformation of corn with a gene from Bt that encoded for a δ-endotoxin provided the transgenic corn with improved resistance to ECB. Additionally, a comprehensive report of field trials of transgenic corn that expresses an insecticidal protein from Bt has been provided by Armstrong et al., Crop Science, 35(2):550-557 (1995).

For these and other reasons, there is a demand for alternative insecticidal agents for agricultural crops. For example, maize plants incorporating transgenic genes which cause the maize plant to produce insecticidal proteins providing protection from the target pest(s) is a more environmentally friendly approach to controlling pests. The use of pesticidal crystal proteins derived from the soil bacterium Bt commonly referred to as “Cry proteins” have been utilized. Cry proteins are globular protein molecules which accumulate as protoxins in crystalline form during late stage of the sporulation of Bt. After ingestion by the pest, the crystals are solubilized to release protoxins in the alkaline midgut environment of the larvae. Protoxins (˜130 kDa) are converted into mature toxic fragments (˜66 kDa N terminal region) by gut proteases. Many of these proteins are quite toxic to specific target insects, but harmless to plants and other non-targeted organisms. Some Cry proteins have been recombinantly expressed in crop plants to provide pest-resistant transgenic plants. Among those, Bt-transgenic cotton and corn have been widely cultivated.

A large number of Cry proteins have been isolated, characterized and classified based on amino acid sequence homology. See Crickmore et al., Microbiol. Mol. Biol. Rev., 62:807-813 (1998). This classification scheme provides a systematic mechanism for naming and categorizing newly discovered Cry proteins. Bt toxins have traditionally been categorized by their specific toxicity towards specific insect categories. For example, the Cry1 group of toxins is toxic to Lepidoptera, and includes, but is not limited to, Cry1Aa, Cry1Ab and Cry1Ac. See Hofte et al., Microbiol. Rev., 53:242-255 (1989). The Cry1 classification is the best known and contains the highest number of cry genes, currently totals over 130. Cry1 and Cry2 proteins share a minimal amount of sequence homology. See, e.g., Crickmore et al. (1998) indicating that Cry1A and Cry2A classes are among the most divergent.

It has generally been found that individual Cry proteins possess relatively narrow activity spectra. For example, Cry1Ac was the first toxin to be deployed in transgenic cotton for control of H. virescens and H. zea insect pests. This toxin is known for its high level toxicity to H. virescens. However, it is slightly deficient in its ability to control H. zea and has almost no activity on Spodoptera species. Additionally, Cry1Ab toxin has slightly less activity on H. zea than Cry1Ac but has far superior activity against S. exigua.

Cry2A is an exception as it is unusual in that this subset of Cry proteins possesses a broader effective range that includes toxicity to both the Lepidoptera and Diptera orders of insects. The Cry2A protein was discovered to be a toxin showing a dual activity against Trichoplusia ni (cabbage looper) and Aedes taeniorhynchus (mosquito) (Yamamoto & McLaughlin, Biochem. Biophys. Res. Comm., 130:414-421 (1982)). The nucleic acid molecule encoding the Cry2A protein (termed Cry2Aa) was cloned and expressed in B. megaterium and found to be active against both Lepidoptera and Diptera insects (Donovan et al., J. Bacteriol., 170:4732-4738 (1988)). An additional coding sequence homologous to Cry2Aa was cloned (termed Cry2Ab) and was found to be active only against Lepidoptera larvae (Widner & Whiteley, J. Bacteriol., 171(2):965-974 (1989)).

Second generation transgenic crops could be more resistant to insects if they are able to express multiple, novel and/or synergistic Bt genes.

Accordingly, it is an objective of embodiments of the present invention to provide synergistic resistance to plant insects.

Another objective of embodiments of the invention includes methods for incorporating multiple Cry proteins into transgenic plants, namely maize.

SUMMARY OF THE INVENTION

Embodiments of the present invention relate to Cry polypeptides derived from Bt Cry1 and Cry2 polypeptides and provides a novel means for improved and synergistic insecticidal resistance against key crop pests. Embodiments of the present invention also relate to transgenic plants expressing such a nucleic acid and/or polypeptide. The transgenic plants can express the transgene in any way known in the art, including, but not limited to, constitutive expression, developmentally regulated expression, tissue specific expression, etc. Additionally, seed obtained from a transgenic plant of the invention is also encompassed.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the sequences for the IP1-88 and IP2-127 Cry proteins, as well as the DNA and amino acid sequence for a Cry1 (h) protein relevant to the disclosure.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of this invention belong. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, processes and examples described in the description are illustrative only and not intended to be limiting to the scope of the claims in any manner. Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosure is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

As used herein, “pesticidal agent” or “pesticide” includes any organism, organic substance, or inorganic substance that has pesticidal activity. As used herein, the term “pesticidal activity” refers to activity of an organism or a substance (such as, for example, a protein) that can be measured by, but is not limited to, pest mortality, pest weight loss, pest repellency, and other behavioral and physical changes of a pest after feeding and exposure for an appropriate length of time. Thus, an organism or substance having pesticidal activity adversely impacts at least one measurable parameter of pest fitness. Preferably, pesticidal activity results in reduced damage to a plant with such a pesticidal agent as compared with plants lacking such pesticidal agent. If the pesticidal agent's target pest is an insect, it is referred to as an “insecticide.” If the pesticidal agent's target pest is a mite, it is referred to as an “acaricide.” If the pesticidal agent's target pest is a mite, it is referred to as a “nematicide”. If the pesticidal agent's target pest is a fungus, it is referred to as a “fungicide.” If the pesticidal agent's target pest is a bacterium, it is referred to as a “bactericide.” If the pesticidal agent's target pest is a plant, it is referred to as a “herbicide.”

Combinations of pesticidal agents can have one of three effects on pesticidal activity: antagonistic, additive, or synergistic. If the observed pesticidal activity of the two pesticidal agents together is approximately the expected pesticidal activity of the combination, the combination is said to be “additive.” If the observed pesticidal activity of the two pesticidal agents together is less than the expected pesticidal activity of the combination, the combination is said to be “antagonistic.” If the observed pesticidal activity of the two pesticidal agents together is greater than the expected pesticidal activity of the combination, the combination is said to be “synergistic.” The expected pesticidal activity for a given combination of pesticidal agents is determined by the following method. If X is the observed level of pesticidal activity of pesticidal agent A alone and Y is the observed level of pesticidal activity of pesticidal agent B alone, the expected pesticidal activity of pesticidal agents A and B in combination (assuming the level of pesticidal activity is measured on a scale from 0 to 100) is X+Y−(X*Y)/100.

Embodiments of the invention may show an increase of pesticidal activity of a given combination of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% or greater against the insect target as compared to the expected pesticidal activity of the combination.

As used herein, “nucleic acid” includes reference to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues (e.g., peptide nucleic acids) having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to that of naturally occurring nucleotides.

As used herein, the terms “encoding” or “encoded” when used in the context of a specified nucleic acid mean that the nucleic acid comprises the requisite information to direct translation of the nucleotide sequence into a specified protein. The information by which a protein is encoded is specified by the use of codons. A nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid or may lack such intervening non-translated sequences (e.g., as in cDNA).

The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

The terms “residue” or “amino acid residue” or “amino acid” are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively “protein”). The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass known analogues of natural amino acids that can function in a similar manner as naturally occurring amino acids.

Polypeptides of the embodiments can be produced either from a nucleic acid disclosed herein, or by the use of standard molecular biology techniques. For example, a protein of the embodiments can be produced by expression of a recombinant nucleic acid of the embodiments in an appropriate host cell, or alternatively by a combination of ex vivo procedures.

The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) “reference sequence,” (b) “comparison window,” (c) “sequence identity,” (d) “percentage of sequence identity,” and (e) “substantial identity.”

    • (a) As used herein, “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
    • (b) As used herein, “comparison window” makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; the search-for-local alignment method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 872264, as modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif., USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins et al. (1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153; Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331. The ALIGN program is based on the algorithm of Myers and Miller (1988) supra. A PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences. The BLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990) supra. BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleotide sequence encoding a protein of the embodiments. BLAST protein searches can be performed with the BLASTX program, score=50, wordlength=3, to obtain amino acid sequences homologous to a protein or polypeptide of the embodiments. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used, as described on the National Center for Biotechnology Information website. Alignment may also be performed manually by inspection.

Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. The term “equivalent program” as used herein refers to any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) supra, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the GCG Wisconsin Genetics Software Package for protein sequences are 8 and 2, respectively. For nucleotide sequences the default gap creation penalty is 50 while the default gap extension penalty is 3. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 200. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the GCG Wisconsin Genetics Software Package is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

    • (c) As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity”. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).
    • (d) As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
    • (e)(i) The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70%, 80%, 90%, or 95% or more sequence identity when compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes generally means sequence identity of at least 60%, 70%, 80%, 90%, or 95% or more sequence identity.

Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. Generally, stringent conditions are selected to be about 5° C. lower than the Tm for the specific sequence at a defined ionic strength and pH. However, stringent conditions encompass temperatures in the range of about 1° C. to about 20° C. lower than the Tm, depending upon the desired degree of stringency as otherwise qualified herein. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.

(e)(ii) The term “substantial identity” in the context of a peptide indicates that a peptide comprises a sequence with at least 70%, 80%, 85%, 90%, 95%, or more sequence identity to a reference sequence over a specified comparison window. Optimal alignment for these purposes can be conducted using the global alignment algorithm of Needleman and Wunsch (1970) supra. An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution. Peptides that are “substantially similar” share sequences as noted above except that residue positions that are not identical may differ by conservative amino acid changes.

The term “toxin” as used herein refers to a polypeptide showing pesticidal activity or insecticidal activity or improved pesticidal activity or improved insecticidal activity. “Bt” or “Bacillus thuringiensis” toxin is intended to include the broader class of Cry toxins found in various strains of Bt, which includes such toxins as, for example, Cry1s, Cry2s, or Cry3s.

The terms “proteolytic site” or “cleavage site” refer to an amino acid sequence which confers sensitivity to a class of proteases or a particular protease such that a polypeptide containing the amino acid sequence is digested by the class of proteases or particular protease. A proteolytic site is said to be “sensitive” to the protease(s) that recognize that site. It is appreciated in the art that the efficiency of digestion will vary, and that a decrease in efficiency of digestion can lead to an increase in stability or longevity of the polypeptide in an insect gut. Thus, a proteolytic site may confer sensitivity to more than one protease or class of proteases, but the efficiency of digestion at that site by various proteases may vary. Proteolytic sites include, for example, trypsin sites, chymotrypsin sites, and elastase sites.

Research has shown that the insect gut proteases of Lepidopterans include trypsins, chymotrypsins, and elastases. See, e.g., Lenz et al. (1991) Arch. Insect Biochem. Physiol. 16: 201-212; and Hedegus et al. (2003) Arch. Insect Biochem. Physiol. 53: 30-47. For example, about 18 different trypsins have been found in the midgut of Helicoverpa armigera larvae (see Gatehouse et al. (1997) Insect Biochem. Mol. Biol. 27: 929-944). The preferred proteolytic substrate sites of these proteases have been investigated. See, e.g., Peterson et al. (1995) Insect Biochem. Mol. Biol. 25: 765-774.

Efforts have been made to understand the mechanism of action of Bt toxins and to engineer toxins with improved properties. It has been shown that insect gut proteases can affect the impact of Bt Cry proteins on the insect. Some proteases activate the Cry proteins by processing them from a “protoxin” form into a toxic form, or “toxin.” See Oppert (1999) Arch. Insect Biochem. Phys. 42: 1-12; Carroll et al. (1997) J. Invertebrate Pathology 70: 41-49. This activation of the toxin can include the removal of the N- and C-terminal peptides from the protein and can also include internal cleavage of the protein. Other proteases can degrade the Cry proteins. See Oppert, ibid.

A comparison of the amino acid sequences of Cry toxins of different specificities reveals five highly-conserved sequence blocks. Structurally, the toxins comprise three distinct domains which are, from the N- to C-terminus: a cluster of seven alpha-helices implicated in pore formation (referred to as “domain 1”), three anti-parallel beta sheets implicated in cell binding (referred to as “domain 2”), and a beta sandwich (referred to as “domain 3”). The location and properties of these domains are known to those of skill in the art. See, e.g., Li et al. (1991) Nature, 305:815-821; Morse et al. (2001) Structure, 9:409-417. When reference is made to a particular domain, such as domain 1, it is understood that the exact endpoints of the domain with regard to a particular sequence are not critical so long as the sequence or portion thereof includes sequence that provides at least some function attributed to the particular domain. Thus, for example, when referring to “domain 1,” it is intended that a particular sequence includes a cluster of seven alpha-helices, but the exact endpoints of the sequence used or referred to with regard to that cluster are not critical. One of skill in the art is familiar with the determination of such endpoints and the evaluation of such functions.

In an effort to better characterize and improve Bt toxins, strains of the bacterium Bt have been studied. An effort was undertaken to identify the nucleotide sequences encoding the crystal proteins from the selected strains, and the wild-type (i.e., naturally occurring) nucleic acids of the embodiments were isolated from these bacterial strains, cloned into an expression vector, and transformed into E coli. Depending upon the characteristics of a given preparation, it was recognized that the demonstration of pesticidal activity sometimes required trypsin pretreatment to activate the pesticidal proteins. Thus, it is understood that some pesticidal proteins require protease digestion (e.g., by trypsin, chymotrypsin, and the like) for activation, while other proteins are biologically active (e.g., pesticidal) in the absence of activation.

Such molecules may be altered by means described, for example, in U.S. application Ser. No. 10/606,320, filed Jun. 25, 2003, and Ser. No. 10/746,914, filed Dec. 24, 2003. In addition, nucleic acid sequences may be engineered to encode polypeptides that contain additional mutations that confer improved or altered pesticidal activity relative to the pesticidal activity of the naturally occurring polypeptide. The nucleotide sequences of such engineered nucleic acids comprise mutations not found in the wild type sequences.

The mutant polypeptides of the embodiments are generally prepared by a process that involves the steps of: obtaining a nucleic acid sequence encoding a Cry family polypeptide; analyzing the structure of the polypeptide to identify particular “target” sites for mutagenesis of the underlying gene sequence based on a consideration of the proposed function of the target domain in the mode of action of the toxin; introducing one or more mutations into the nucleic acid sequence to produce a desired change in one or more amino acid residues of the encoded polypeptide sequence; and assaying the polypeptide produced for pesticidal activity.

Many of the Bt insecticidal toxins are related to various degrees by similarities in their amino acid sequences and tertiary structure and means for obtaining the crystal structures of Bt toxins are well known. Exemplary high-resolution crystal structure solution of both the Cry3A and Cry3B polypeptides are available in the literature. The solved structure of the Cry3A gene (Li et al. (1991) Nature 353:815-821) provides insight into the relationship between structure and function of the toxin. A combined consideration of the published structural analyses of Bt toxins and the reported function associated with particular structures, motifs, and the like indicates that specific regions of the toxin are correlated with particular functions and discrete steps of the mode of action of the protein. For example, many toxins isolated from Bt are generally described as comprising three domains: a seven-helix bundle that is involved in pore formation, a three-sheet domain that has been implicated in receptor binding, and a beta-sandwich motif (Li et al. (1991) Nature 305: 815-821).

As reported in U.S. Pat. No. 7,105,332, and pending U.S. application Ser. No. 10/746,914, filed Dec. 24, 2003, the toxicity of Cry proteins can be improved by targeting the region located between alpha helices 3 and 4 of domain 1 of the toxin. This theory was premised on a body of knowledge concerning insecticidal toxins, including: 1) that alpha helices 4 and 5 of domain 1 of Cry3A toxins had been reported to insert into the lipid bilayer of cells lining the midgut of susceptible insects (Gazit et al. (1998) Proc. Natl. Acad. Sci. USA 95: 12289-12294); 2) the inventors' knowledge of the location of trypsin and chymotrypsin cleavage sites within the amino acid sequence of the wild-type protein; 3) the observation that the wild-type protein was more active against certain insects following in vitro activation by trypsin or chymotrypsin treatment; and 4) reports that digestion of toxins from the 3′ end resulted in decreased toxicity to insects.

A series of mutations may be created and placed in a variety of background sequences to create novel polypeptides having enhanced or altered pesticidal activity. See, e.g., U.S. application Ser. No. 10/606,320, filed Jun. 25, 2003, now abandoned, and Ser. No. 10/746,914, filed Dec. 24, 2003. These mutants include, but are not limited to: the addition of at least one more protease-sensitive site (e.g., trypsin cleavage site) in the region located between helices 3 and 4 of domain 1; the replacement of an original protease-sensitive site in the wild-type sequence with a different protease-sensitive site; the addition of multiple protease-sensitive sites in a particular location; the addition of amino acid residues near protease-sensitive site(s) to alter folding of the polypeptide and thus enhance digestion of the polypeptide at the protease-sensitive site(s); and adding mutations to protect the polypeptide from degradative digestion that reduces toxicity (e.g., making a series of mutations wherein the wild-type amino acid is replaced by valine to protect the polypeptide from digestion). Mutations may be used singly or in any combination to provide polypeptides of the embodiments.

In this manner, the embodiments provide sequences comprising a variety of mutations, such as, for example, a mutation that comprises an additional, or an alternative, protease-sensitive site located between alpha-helices 3 and 4 of domain 1 of the encoded polypeptide. A mutation which is an additional or alternative protease-sensitive site may be sensitive to several classes of proteases such as serine proteases, which include trypsin and chymotrypsin, or enzymes such as elastase. Thus, a mutation which is an additional or alternative protease-sensitive site may be designed so that the site is readily recognized and/or cleaved by a category of proteases, such as mammalian proteases or insect proteases. A protease-sensitive site may also be designed to be cleaved by a particular class of enzymes or a particular enzyme known to be produced in an organism, such as, for example, a chymotrypsin produced by the corn earworm Heliothis zea (Lenz et al. (1991) Arch. Insect Biochem. Physiol. 16: 201-212). Mutations may also confer resistance to proteolytic digestion, for example, to digestion by chymotrypsin at the C-terminus of the peptide.

The presence of an additional and/or alternative protease-sensitive site in the amino acid sequence of the encoded polypeptide can improve the pesticidal activity and/or specificity of the polypeptide encoded by the nucleic acids of the embodiments. Accordingly, the nucleotide sequences of the embodiments can be recombinantly engineered or manipulated to produce polypeptides having improved or altered insecticidal activity and/or specificity compared to that of an unmodified wild-type toxin. In addition, mutations may be placed in or used in conjunction with other nucleotide sequences to provide improved properties. For example, a protease-sensitive site that is readily cleaved by insect chymotrypsin, e.g., a chymotrypsin found in the bertha armyworm or the corn earworm (Hegedus et al. (2003) Arch. Insect Biochem. Physiol. 53: 30-47; and Lenz et al. (1991) Arch. Insect Biochem. Physiol. 16: 201-212), may be placed in a Cry background sequence to provide improved toxicity to that sequence. In this manner, the embodiments provide toxic polypeptides with improved properties.

For example, a mutagenized Cry nucleotide sequence can comprise additional mutants that comprise additional codons that introduce a second trypsin-sensitive amino acid sequence (in addition to the naturally occurring trypsin site) into the encoded polypeptide. An alternative addition mutant of the embodiments comprises additional codons designed to introduce at least one additional different protease-sensitive site into the polypeptide, for example, a chymotrypsin-sensitive site located immediately 5′ or 3′ of the naturally occurring trypsin site. Alternatively, substitution mutants may be created in which at least one codon of the nucleic acid that encodes the naturally occurring protease-sensitive site is destroyed and alternative codons are introduced into the nucleic acid sequence in order to provide a different (e.g., substitute) protease-sensitive site. A replacement mutant may also be added to a Cry sequence in which the naturally-occurring trypsin cleavage site present in the encoded polypeptide is destroyed and a chymotrypsin or elastase cleavage site is introduced in its place.

It is recognized that any nucleotide sequence encoding the amino acid sequences that are proteolytic sites or putative proteolytic sites (for example, sequences such as NGSR, RR, or LKM) can be used and that the exact identity of the codons used to introduce any of these cleavage sites into a variant polypeptide may vary depending on the use, i.e., expression in a particular plant species. It is also recognized that any of the disclosed mutations can be introduced into any polynucleotide sequence of the embodiments that comprises the codons for amino acid residues that provide the native trypsin cleavage site that is targeted for modification. Accordingly, variants of either full-length toxins or fragments thereof can be modified to contain additional or alternative cleavage sites, and these embodiments are intended to be encompassed by the scope of the embodiments disclosed herein.

It will be appreciated by those of skill in the art that any useful mutation may be added to the sequences of the embodiments so long as the encoded polypeptides retain pesticidal activity. Thus, sequences may also be mutated so that the encoded polypeptides are resistant to proteolytic digestion by chymotrypsin. More than one recognition site can be added in a particular location in any combination, and multiple recognition sites can be added to or removed from the toxin. Thus, additional mutations can comprise three, four, or more recognition sites. It is to be recognized that multiple mutations can be engineered in any suitable polynucleotide sequence; accordingly, either full-length sequences or fragments thereof can be modified to contain additional or alternative cleavage sites as well as to be resistant to proteolytic digestion. In this manner, the embodiments provide Cry toxins containing mutations that improve pesticidal activity as well as improved compositions and methods for impacting pests using other Bt toxins.

Mutations may protect the polypeptide from protease degradation, for example by removing putative proteolytic sites such as putative serine protease sites and elastase recognition sites from different areas. Some or all of such putative sites may be removed or altered so that proteolysis at the location of the original site is decreased. Changes in proteolysis may be assessed by comparing a mutant polypeptide with wild-type toxins or by comparing mutant toxins which differ in their amino acid sequence. Putative proteolytic sites and proteolytic sites include, but are not limited to, the following sequences: RR, a trypsin cleavage site; LKM, a chymotrypsin site; and NGSR, a trypsin site. These sites may be altered by the addition or deletion of any number and kind of amino acid residues, so long as the pesticidal activity of the polypeptide is increased. Thus, polypeptides encoded by nucleotide sequences comprising mutations will comprise at least one amino acid change or addition relative to the native or background sequence, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 35, 38, 40, 45, 47, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, or 280 or more amino acid changes or additions. Pesticidal activity of a polypeptide may also be improved by truncation of the native or full-length sequence, as is known in the art.

Embodiments of the present invention provide insecticidal polypeptides related to Bt Cry1 and Cry2 polypeptides. Nucleic acid molecules encoding the polypeptides are also provided. Methods for using the polypeptides and nucleic acids to enhance resistance of plants to insect predation are encompassed.

The combination of a Cry1 and Cry2 protein yields a synergistic effect against a plurality of target pests, providing greater than expected mortality and/or resistance to a plurality of target pests. Prior art has in fact taught away from the disclosed embodiments, indicating that synergism is not an expected outcome for insecticidal activity. Others skilled in the art have indicated that such combinations result in the antagonism, rather than synergism, effect on Helicoverpa armigera (Liao, C. et al., Toxicity of B. thuringiensis insecticidal proteins of Helicoverpa armigera and H. punctigera, major pests of cotton, J. Invertebrate Pathology 80:55-63 (2002)). Others have found reported tests using Cry1 and Cry2 proteins with reported synergism effects (Ding et al., Expression and synergism of two cry insecticidal protein genes in P. fluorescens, Chinese J. of Microbiol., 40:573-578 (2000)).

Methods of Enhancing Insect Resistance in Plants

Embodiments of the present invention provide methods of enhancing plant resistance to insect pests including, but not limited to, members of order Lepidoptera, the Helicoverpa ssp. (e.g., Helicoverpa Zea and Heliothis virescens), and/or Spodoptera ssp. (e.g., Spodoptera exigua, Spodoptera frugiperda) through the use of Cry1-derived insecticidal polypeptides combined with Cry2-derived insecticidal polypeptides to produce a synergistic effect. Any method known in the art can be used to cause the insect pests to ingest one or more polypeptides during the course of feeding on the plant. As such, the insect pest will ingest insecticidal amounts of the one or more polypeptides of embodiments of the invention and may discontinue feeding on the plant. In some embodiments, the insect pest is killed by ingestion of the one or more polypeptides. In other embodiments, the insect pests are inhibited or discouraged from feeding on the plant without being killed.

In one embodiment, transgenic plants can be made to express one or more polypeptides. The transgenic plant may express the one or more polypeptides in all tissues (e.g., global expression). Alternatively, the one or more polypeptides may be expressed in only a subset of tissues (e.g., tissue specific expression), preferably those tissues consumed by the insect pest. Polypeptides that are embodiments of the invention can be expressed constitutively in the plant or be under the control of an inducible promoter. Polypeptides that are embodiments of the invention may be expressed in the plant cytosol or in the plant chloroplast either by protein targeting or by transformation of the chloroplast genome.

In another embodiment, a composition comprising one or more polypeptides of embodiments of the invention can be applied externally to a plant susceptible to the insect pests. External application of the composition includes direct application to the plant, either in whole or in part, and/or indirect application, e.g., to the environment surrounding the plant such as the soil. The composition can be applied by any method known in the art including, but not limited to, spraying, dusting, sprinkling, or the like. In general, the composition can be applied at any time during plant growth. One skilled in the art can use methods known in the art to determine empirically the optimal time for administration of the composition. Factors that affect optimal administration time include, but are not limited to, the type of susceptible plant, the type of insect pest, which one or more polypeptides are administered in the composition.

The composition comprising one or more polypeptides may be substantially purified polypeptides, a cell suspension, a cell pellet, a cell supernatant, a cell extract, or a spore-crystal complex of Bt cells. The composition comprising one or more polypeptides embodying the invention may be in the form of a solution, an emulsion, a suspension, or a powder. Liquid formulations may be aqueous or non-aqueous based and may be provided as foams, gels, suspensions, emulsifiable concentrates, or the like. The formulations may include agents in addition to the one or more polypeptides embodying the invention. For example, compositions may further comprise spreader-sticker adjuvants, stabilizing agents, other insecticidal additives, diluents, agents that optimize the rheological properties or stability of the composition, such as, for example, surfactants, emulsifiers, dispersants, or polymers.

In another embodiment, recombinant hosts that express one or more polypeptides that are embodiments of the invention are applied on or near a plant susceptible to attack by an insect pest. The recombinant hosts include, but are not limited to, microbial hosts and insect viruses that have been transformed with and express one or more nucleic acid molecules (and thus polypeptides) of embodiments of the invention. In some embodiments, the recombinant host secretes the polypeptide into its surrounding environment so as to contact an insect pest. In other embodiments, the recombinant hosts colonize one or more plant tissues susceptible to insect infestation.

The nucleotide sequences of the embodiments can also be used to isolate corresponding sequences from other organisms, particularly other bacteria, and more particularly other Bacillus strains. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences set forth herein. Sequences that are selected based on their sequence identity to the entire sequences set forth herein or to fragments thereof are encompassed by the embodiments. Such sequences include sequences that are orthologs of the disclosed sequences. The term “orthologs” refers to genes derived from a common ancestral gene and which are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences share substantial identity as defined elsewhere herein. Functions of orthologs are often highly conserved among species.

In a PCR approach, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any organism of interest. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.), hereinafter “Sambrook”. See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially-mismatched primers, and the like.

In hybridization techniques, all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as 32P or any other detectable marker. Thus, for example, probes for hybridization can be made by labeling synthetic oligonucleotides based on the sequences of the embodiments. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook.

For example, an entire sequence disclosed herein, or one or more portions thereof, may be used as a probe capable of specifically hybridizing to corresponding sequences and messenger RNAs. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique to the sequences of the embodiments and are generally at least about 10 or 20 nucleotides in length. Such probes may be used to amplify corresponding Cry sequences from a chosen organism by PCR. This technique may be used to isolate additional coding sequences from a desired organism or as a diagnostic assay to determine the presence of coding sequences in an organism. Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook).

Hybridization of such sequences may be carried out under stringent conditions. The term “stringent conditions” or “stringent hybridization conditions” as used herein refers to conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold, 5-fold, or 10-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 or 500 nucleotides in length.

Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a final wash in 0.1×SSC at 60 to 65° C. for at least about 20 minutes. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. The duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours.

Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm (thermal melting point) can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: Tm=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, “% form” is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Washes are typically performed at least until equilibrium is reached and a low background level of hybridization is achieved, such as for 2 hours, 1 hour, or 30 minutes.

Tm is reduced by about 1° C. for each 1% of mismatching; thus, Tm, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the Tm can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the Tm for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than the Tm; moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the Tm; low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the Tm.

Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45° C. (aqueous solution) or 32° C. (formamide solution), the SSC concentration can be increased so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See also Sambrook. Thus, isolated sequences that encode a Cry protein of the embodiments and hybridize under stringent conditions to the Cry sequences disclosed herein, or to fragments thereof, are encompassed by the embodiments.

Preferably, a Cry1 and Cry2 polypeptide are produced by a transgenic plant, thereby making the plant resistant to attack from a target pest and providing synergistic resistance to at least one target pest. A discussion of production of such transgenic plants is provided below.

Production of Transgenic Plants

Any method known in the art can be used for transforming a plant or plant cell with a nucleic acid molecule of an embodiment of the present invention. Nucleic acid molecules can be incorporated into plant DNA (e.g., genomic DNA or chloroplast DNA) or be maintained without insertion into the plant DNA (e.g., through the use of artificial chromosomes). Suitable methods of introducing nucleic acid molecules into plant cells include microinjection (Crossway et al., Biotechniques 4:320-334 (1986)); electroporation (Riggs et al., Proc. Natl. Acad. Sci., 83:5602-5606 (1986); D'Halluin et al., Plant Cell, 4:1495-1505 (1992)); Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and 5,981,840; Osjoda et al., Nature Biotechnology, 14:745-750 (1996); Horsch et al., Science, 233:496-498 (1984); Fraley et al., Proc. Natl. Acad. Sci., 80:4803 (1983); Fütterer et al., Gene transfer to plants, 213-263 (Potrykus 1995); direct gene transfer (Paszkowski et al., EMBO J. 3:2717-2722 (1984)); ballistic particle acceleration (U.S. Pat. Nos. 4,945,050, 5,879,918, 5,886,244, and 5,932,782; Tomes et al., Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment, Plant Cell, Tissue, and Organ Culture: Fundamental Methods (Gamborg & Phillips 1995); and McCabe et al., Biotechnology, 6:923-926 (1988)); virus-mediated transformation (U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367 and 5,316,931); pollen transformation (De Wet et al., Experimental Manipulation of Ovule Tissues, 197-209 (Chapman et al. 1985)); Lec 1 transformation (U.S. patent application Ser. No. 09/435,054; International Publication No. WO 00/28058); whisker-mediated transformation (Kaeppler et al., Plant Cell Reports, 9:415-418 (1990); Kaeppler et al., Theor. Appl. Genet., 84:560-566 (1992)); and chloroplast transformation technology (Bogorad, Trends in Biotechnology, 18:257-263 (2000); Ramesh et al., Methods Mol Biol., 274:301-7 (2004); Hou et al., Transgenic Res., 12:111-4 (2003); Kindle et al., Proc. Natl. Acad. Sci., 88:1721-5 (1991); Bateman & Purton, Mol Gen Genet., 263:404-10 (2000); Sidorov et al., Plant J., 19:209-216 (1999)).

The choice of transformation protocols used for generating transgenic plants and plant cells can vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Examples of transformation protocols particularly suited for a particular plant type include those for: potato (Tu et al., Plant Molecular Biology, 37:829-838 (1998); Chong et al., Transgenic Research, 9:71-78 (2000)); soybean (Christou et al., Plant Physiol., 87:671-674 (1988); McCabe et al., BioTechnology, 6:923-926 (1988); Finer & McMullen, In Vitro Cell Dev. Biol., 27P:175-182 (1991); Singh et al., Theor. Appl. Genet., 96:319-324 (1998)); maize (Klein et al., Proc. Natl. Acad. Sci., 85:4305-4309 (1988); Klein et al., Biotechnology, 6:559-563 (1988); Klein et al., Plant Physiol., 91:440-444 (1988); Fromm et al., Biotechnology, 8:833-839 (1990); Tomes et al., Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment, Plant Cell, Tissue, and Organ Culture: Fundamental Methods (Gamborg & Phillips 1995)); and cereals (Hooykaas-Van Slogteren et al., Nature 311:763-764 (1984); U.S. Pat. No. 5,736,369).

In some embodiments, more than one construct is used for transformation in the generation of transgenic plants and plant cells. Multiple constructs may be included in cis or trans positions. In preferred embodiments, each construct has a promoter and other regulatory sequences. Embodiments of the invention relate to combinations of different Cry1 and Cry2 proteins resulting in a synergistic effect against target pests such as those disclosed herein. By way of example, the Cry1 protein may be the polypeptide disclosed in SEQ ID NO:1 or 4, or a polypeptide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.75% identical to the polypeptide of SEQ ID NO:1 or 4. By way of further example, the Cry2 protein may be the polypeptide disclosed in SEQ ID NO:2, or a polypeptide that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.75% identical to the polypeptide of SEQ ID NO:2. As a result, a nucleic acid encoding such a Cry1 (such as, for example, the nucleic acid disclosed in SEQ ID NO:3) or Cry2 protein may be used in such a construct.

Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype and thus the desired phenotype. Such regeneration techniques rely on manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker that has been introduced together with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in the art (e.g., Evans et al., Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, 124-176 (MacMillilan Publishing Co. 1983); and Binding, Regeneration of Plants, Plant Protoplasts, 21-73 (CRC Press, 1985). Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are also described in the art (e.g., Klee et al., Ann. Rev. of Plant Phys., 38:467-486 (1987)).

The term “plant” includes whole plants, shoot vegetative organs/structures (e.g., leaves, stems and tubers), roots, flowers and floral organs/structures (e.g., bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g., vascular tissue, ground tissue, and the like) and cells (e.g. guard cells, egg cells, trichomes and the like), and progeny of same. The class of plants that can be used in embodiments of the present invention includes the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae. Plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid and hemizygous plants are also included.

Embodiments of the invention may use nucleic acid molecules to confer desired traits on essentially any plant. Thus, embodiments of the invention have use over a broad range of plants, including species from the genera Allium, Ananas, Anacardium, Apium, Arachis, Asparagus, Athamantha, Atropa, Avena, Bambusa, Beta, Brassica, Bromus, Browallia, Camellia, Cannabis, Carica, Ceratonia, Cicer, Chenopodium, Chicorium, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Coix, Cucumis, Cucurbita, Cynodon, Dactylis, Datura, Daucus, Dianthus, Digitalis, Dioscorea, Elaeis, Eliusine, Euphorbia, Festuca, Ficus, Fragaria, Geranium, Glycine, Graminae, Gossypium, Helianthus, Heterocallis, Hevea, Hibiscus, Hordeum, Hyoscyamus, Ipomoea, Lactuca, Lathyrus, Lens, Lilium, Linum, Lolium, Lotus, Lupinus, Lycopersicon, Macadamia, Macrophylla, Malus, Mangifera, Manihot, Majorana, Medicago, Musa, Narcissus, Nemesia, Nicotiana, Onobrychis, Olea, Olyreae, Oryza, Panicum, Panieum, Pannisetum, Petunia, Pelargonium, Persea, Pharoideae, Phaseolus, Phleum, Picea, Poa, Pinus, Pistachia, Pisum, Populus, Pseudotsuga, Pyrus, Prunus, Pseutotsuga, Psidium, Quercus, Ranunculus, Raphanus, Ribes, Ricinus, Rhododendron, Rosa, Saccharum, Salpiglossis, Secale, Senecio, Setaria, Sequoia, Sinapis, Solanum, Sorghum, Stenotaphrum, Theobromus, Trigonella, Trifolium, Triticum, Tsuga, Tulipa, Vicia, Vitis, Vigna, and Zea.

In specific embodiments, transgenic plants are maize, potato, rice, soybean, alfalfa, sunflower, canola, or cotton plants.

Transgenic plants may be grown and pollinated with either the same transformed strain or different strains. Two or more generations of the plants may be grown to ensure that expression of the desired nucleic acid molecule, polypeptide and/or phenotypic characteristic is stably maintained and inherited. One of ordinary skill in the art will recognize that after the nucleic acid molecule of embodiments of the present invention is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.

In certain embodiments the polynucleotides of the embodiments can be stacked with any combination of polynucleotide sequences of interest in order to create plants with a desired trait. For example, the polynucleotides of the embodiments may be stacked with any other polynucleotides encoding polypeptides having pesticidal and/or insecticidal activity, such as other Bt toxic proteins (described in, for example, U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; and Geiser et al., Gene, 48: 109 (1986)), lectins (Van Damme et al., Plant Mol. Biol., 24: 825 (1994)), pentin (described in U.S. Pat. No. 5,981,722), and the like. The combinations generated can also include multiple copies of any one of the polynucleotides of interest. The polynucleotides of the embodiments can also be stacked with any other gene or combination of genes to produce plants with a variety of desired trait combinations including, but not limited to, traits desirable for animal feed such as high oil genes (e.g., U.S. Pat. No. 6,232,529); balanced amino acids (e.g., hordothionins (U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802; and 5,703,409); barley high lysine (Williamson et al., Eur. J. Biochem., 165: 99-106 (1987); and WO 98/20122) and high methionine proteins (Pedersen et al., J. Biol. Chem., 261: 6279 (1986); Kirihara et al., Gene, 71: 359 (1988); and Musumura et al., Plant Mol. Biol., 12: 123 (1989)); increased digestibility (e.g., modified storage proteins (U.S. Pat. No. 6,858,778); and thioredoxins (U.S. Pat. No. 7,009,087)); the disclosures of which are herein incorporated by reference in their entirety.

The polynucleotides of the embodiments can also be stacked with traits desirable for disease or herbicide resistance (e.g., fumonisin detoxification genes (U.S. Pat. No. 5,792,931); avirulence and disease resistance genes (Jones et al., Science, 266: 789 (1994); Martin et al., Science, 262: 1432 (1993); Mindrinos et al., Cell, 78:1089 (1994)); acetolactate synthase (ALS) mutants that lead to herbicide resistance such as the S4 and/or Hra mutations; genes encoding resistance to inhibitors of glutamine synthase such as phosphinothricin or basta (e.g., bar or PAT genes); and glyphosate resistance (EPSPS and GAT (glyphosate acetyl transferase) genes (Castle et al., Science, 304:1151 (2004))); and traits desirable for processing or process products such as high oil (e.g., U.S. Pat. No. 6,232,529); modified oils (e.g., fatty acid desaturase genes (U.S. Pat. No. 5,952,544; WO 94/11516)); modified starches (e.g., ADPG pyrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE), and starch debranching enzymes (SDBE)); and polymers or bioplastics (e.g., U.S. Pat. No. 5,602,321; beta-ketothiolase, polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert et al., J. Bacteriol., 170:5837-5847 (1988)) facilitate expression of polyhydroxyalkanoates (PHAs)); the disclosures of which are herein incorporated by reference. One could also combine the polynucleotides of the embodiments with polynucleotides providing agronomic traits such as male sterility (see, e.g., U.S. Pat. No. 5,583,210), stalk strength, flowering time, or transformation technology traits such as cell cycle regulation or gene targeting (e.g., WO 99/61619, WO 00/17364, and WO 99/25821); the disclosures of which are herein incorporated by reference.

These stacked combinations can be created by any method including, but not limited to, cross-breeding plants by any conventional or TopCross methodology, or genetic transformation. If the sequences are stacked by genetically transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order. For example, a transgenic plant comprising one or more desired traits can be used as the target to introduce further traits by subsequent transformation. The traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences will be introduced, the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a transformation cassette that will suppress the expression of the polynucleotide of interest. This may be combined with any combination of other suppression cassettes or over expression cassettes to generate the desired combination of traits in the plant. It is further recognized that polynucleotide sequences can be stacked at a desired genomic location using a site-specific recombination system. See, e.g., WO 99/25821, WO 99/25854, WO 99/25840, WO 99/25855, and WO 99/25853, all of which are herein incorporated by reference.

Determination of Expression in Transgenic Plants

Any method known in the art can be used for determining the level of expression in a plant of a nucleic acid molecule of embodiments of the invention or polypeptide encoded therefrom. For example, the expression level in a plant of a polypeptide encoded by a nucleic acid molecule of embodiments of the invention can be determined by immunoassay, quantitative gel electrophoresis, etc. Expression of nucleic acid molecules of embodiments of the invention can be measured directly by reverse transcription quantitative PCR (qRT-PCR) of isolated RNA from the plant. Additionally, the expression level in a plant of a polypeptide encoded by a nucleic acid molecule of embodiments of the invention can be determined by the degree to which the plant phenotype is altered. In one embodiment, enhanced insect resistance is the phenotype to be assayed.

As used herein, “enhanced insect resistance” refers to increased resistance of a transgenic plant expressing a polypeptide of an embodiment of the invention to consumption and/or infestation by an insect pest as compared to a plant not expressing a polypeptide of an embodiment of the invention. Enhanced resistance can be measured in a number of ways. In one embodiment, enhanced resistance is measured by decreased damage to a plant expressing a polypeptide of an embodiment of the invention as compared to a plant not expressing a polypeptide of an embodiment of the invention after the same period of insect incubation. Insect damage can be assessed visually. For example in cotton plants, damage after infestation can be measured by looking directly at cotton plant bolls for signs of consumption by insects. In another embodiment, enhanced resistance is measured by increased crop yield from a plant expressing a polypeptide of an embodiment of the invention as compared to a plant not expressing a polypeptide of an embodiment of the invention after the same period of insect incubation.

Insect pests include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Lepidoptera.

Larvae of the order Lepidoptera include, but are not limited to, armyworms, cutworms, loopers, and heliothines in the family Noctuidae Spodoptera frugiperda JE Smith (fall armyworm); S. exigua Hübner (beet armyworm); S. litura Fabricius (tobacco cutworm, cluster caterpillar); Mamestra configurata Walker (bertha armyworm); M. brassicae Linnaeus (cabbage moth); Agrotis ipsilon Hufnagel (black cutworm); A. orthogonia Morrison (western cutworm); A. subterranea Fabricius (granulate cutworm); Alabama argillacea Hübner (cotton leaf worm); Trichoplusia ni Hübner (cabbage looper); Pseudoplusia includens Walker (soybean looper); Anticarsia gemmatalis Hübner (velvetbean caterpillar); Hypena scabs Fabricius (green cloverworm); Heliothis virescens Fabricius (tobacco budworm); Pseudaletia unipuncta Haworth (armyworm); Athetis mindara Barnes and Mcdunnough (rough skinned cutworm); Euxoa messoria Harris (darksided cutworm); Earias insulana Boisduval (spiny bollworm); E. vittella Fabricius (spotted bollworm); Helicoverpa armigera Hübner (American bollworm); H. zea Boddie (corn earworm or cotton bollworm); Melanchra picta Harris (zebra caterpillar); Egira (Xylomyges) curialis Grote (citrus cutworm); borers, casebearers, webworms, coneworms, and skeletonizers from the family Pyralidae Ostrinia nubilalis Hübner (European corn borer); Amyelois transitella Walker (naval orangeworm); Anagasta kuehniella Zeller (Mediterranean flour moth); Cadra cautella Walker (almond moth); Chilo suppressalis Walker (rice stem borer); C. partellus, (sorghum borer); Corcyra cephalonica Stainton (rice moth); Crambus caliginosellus Clemens (corn root webworm); C. teterrellus Zincken (bluegrass webworm); Cnaphalocrocis medinalis Guenee (rice leaf roller); Desmia funeralis Hübner (grape leaffolder); Diaphania hyalinata Linnaeus (melon worm); D. nitidalis Stoll (pickleworm); Diatraea grandiosella Dyar (southwestern corn borer), D. saccharalis Fabricius (surgarcane borer); Eoreuma loftini Dyar (Mexican rice borer); Ephestia elutella Hübner (tobacco (cacao) moth); Galleria mellonella Linnaeus (greater wax moth); Herpetogramma licarsisalis Walker (sod webworm); Homoeosoma electellum Hulst (sunflower moth); Elasmopalpus lignosellus Zeller (lesser cornstalk borer); Achroia grisella Fabricius (lesser wax moth); Loxostege sticticalis Linnaeus (beet webworm); Orthaga thyrisalis Walker (tea tree web moth); Maruca testulalis Geyer (bean pod borer); Plodia interpunctella Hübner (Indian meal moth); Scirpophaga incertulas Walker (yellow stem borer); Udea rubigalis Guenee (celery leaftier); and leafrollers, budworms, seed worms, and fruit worms in the family Tortricidae Acleris gloverana Walsingham (Western blackheaded budworm); A. variana Fernald (Eastern blackheaded budworm); Archips argyrospila Walker (fruit tree leaf roller); A. rosana Linnaeus (European leaf roller); and other Archips species, Adoxophyes orana Fischer von Rösslerstamm (summer fruit tortrix moth); Cochylis hospes Walsingham (banded sunflower moth); Cydia latiferreana Walsingham (filbertworm); C. pomonella Linnaeus (coding moth); Platynota flavedana Clemens (variegated leafroller); P. stultana Walsingham (omnivorous leafroller); Lobesia botrana Denis & Schiffermüller (European grape vine moth); Spilonota ocellana Denis & Schiffermüller (eyespotted bud moth); Endopiza viteana Clemens (grape berry moth); Eupoecilia ambiguella Hübner (vine moth); Bonagota salubricola Meyrick (Brazilian apple leafroller); Grapholita molesta Busck (oriental fruit moth); Suleima helianthana Riley (sunflower bud moth); Argyrotaenia spp.; Choristoneura spp.

Selected other agronomic pests in the order Lepidoptera include, but are not limited to, Alsophila pometaria Harris (fall cankerworm); Anarsia lineatella Zeller (peach twig borer); Anisota senatoria J. E. Smith (orange striped oakworm); Antheraea pernyi Guerin-Méneville (Chinese Oak Silkmoth); Bombyx mori Linnaeus (Silkworm); Bucculatrix thurberiella Busck (cotton leaf perforator); Colias eurytheme Boisduval (alfalfa caterpillar); Datana integerrima Grote & Robinson (walnut caterpillar); Dendrolimus sibiricus Tschetwerikov (Siberian silk moth), Ennomos subsignaria Hübner (elm spanworm); Erannis tiliaria Harris (linden looper); Euproctis chrysorrhoea Linnaeus (browntail moth); Harrisina americana Guérin-Méneville (grapeleaf skeletonizer); Hemileuca oliviae Cockrell (range caterpillar); Hyphantria cunea Drury (fall webworm); Keiferia lycopersicella Walsingham (tomato pinworm); Lambdina fiscellaria fiscellaria Hulst (Eastern hemlock looper); L. fiscellaria lugubrosa Hulst (Western hemlock looper); Leucoma salicis Linnaeus (satin moth); Lymantria dispar Linnaeus (gypsy moth); Manduca quinquemaculata Haworth (five spotted hawk moth, tomato hornworm); M. sexta Haworth (tomato hornworm, tobacco hornworm); Operophtera brumata Linnaeus (winter moth); Paleacrita vernata Peck (spring cankerworm); Papilio cresphontes Cramer (giant swallowtail, orange dog); Phryganidia californica Packard (California oakworm); Phyllocnistis citrella Stainton (citrus leafminer); Phyllonorycter blancardella Fabricius (spotted tentiform leafminer); Pieris brassicae Linnaeus (large white butterfly); P. rapae Linnaeus (small white butterfly); P. napi Linnaeus (green veined white butterfly); Platyptilia carduidactyla Riley (artichoke plume moth); Plutella xylostella Linnaeus (diamondback moth); Pectinophora gossypiella Saunders (pink bollworm); Pontia protodice Boisduval & Leconte (Southern cabbageworm); Sabulodes aegrotata Guenée (omnivorous looper); Schizura concinna J. E. Smith (red humped caterpillar); Sitotroga cerealella Olivier (Angoumois grain moth); Thaumetopoea pityocampa Schiffermuller (pine processionary caterpillar); Tineola bisselliella Hummel (webbing clothesmoth); Tuta absoluta Meyrick (tomato leafminer); Yponomeuta padella Linnaeus (ermine moth); Heliothis subflexa Guenée; Malacosoma spp. and Orgyia spp.

In particular embodiments, the insect pests are from the order of Lepidopteran insects including European corn borer, e.g., Ostrinia nubilalis; corn earworm, e.g., Helicoverpa zea; common stalk borer, e.g., Papiapema nebris; armyworm, e.g., Pseudaletia unipuncta; Southwestern corn borer, e.g., Diatraea grandiosella; black cutworm, e.g., Agrotis ipsilon; fall armyworm, e.g., Spodoptera frugiperda; beet armyworm, e.g., Spodoptera exigua; and diamond-back moth, e.g., Plutella xylostella. In specific embodiments, the insect pests are European corn borer, Ostrinia nubilalis, and corn earworm Helicoverpa zea.

Determinations can be made using whole plants, tissues thereof, or plant cell culture.

All publications and patent applications in this specification are indicative of the level of ordinary skill in the art to which embodiments of this invention pertain. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated by reference. The disclosure of each reference set forth herein is incorporated herein by reference in its entirety.

EXAMPLES

Embodiments of this invention can be better understood by reference to the following examples. The foregoing and following description of embodiments of the present invention and the various embodiments are not intended to limit the claims, but rather are illustrative thereof. Therefore, it will be understood that the claims are not limited to the specific details of these examples. It will be appreciated by those skilled in the art that other embodiments of the invention may be practiced without departing from the spirit and the scope of the disclosure, the scope of which is defined by the appended claims.

Example 1

The evaluation of potential synergism between Bt proteins IP1-88 (a Cry1) and IP2-127 (a Cry2) using the bioassay method according to Colby, S. R., Calculating synergistic and antagonistic responses of herbicide combinations, Weeds, 15(1):20-22 (1967). Bt proteins IP1-88 (a Cry1) and IP2-127 (a Cry2) were utilized as test substances to qualitatively confirm the presence and resultant effect of the Bt proteins. Evaluation of the interactive effects between the two insecticidal compounds was required to understand the effectiveness of the two toxins stacked in transgenic plants. Resultant synergy was examined using neonate larvae of European corn borer (ECB), Ostrinia nubilalis.

Standardized Lepidoptera LC50 diet incorporation bioassays were utilized to evaluate the effects of insecticidal proteins Cry1 and Cry2 on Lepidoptera larvae (referring to the immature stage of an insect between the egg and pupal stage of an insect with complete metamorphosis). All synergism experiments were conducted in completely randomized designs with 5 replications. Each replicate consisted of eight wells in a 96-well bioassay plate. There were two basic doses/treatments for each protein based on preliminary data, one close to but <LC50s based on mortality (M) data (as M1×), and the other close to but <IC50s based on response (R) data (as R1×, mortality+severe stunted or <0.1 mg/larva). Apart from the basic 1×:1× ratio, higher doses using 2× dose for one of the two proteins might be also used (1×:2× or 2×:1×). The insecticidal proteins were combined with a Lepidoptera specific artificial diet to create the bioassay diet. Ingredients including boiling water, agar (SeaPlaque), agar (NuSeive), cooling water and Southland Premix were used for the diet. The diet was dispensed and combined with the proteins in 96-well plates and one neonate larva was placed in each well.

All plates in bioassays were placed in a growth chamber with a target temperature of 27±1° C. and relative humidity of >60%. Approximately 4 days after initiation of each bioassay, mortality and severe stunted (ss, <0.1 mg/larva) counts were scored. Data analysis was based on the following: X=Observed result from Compound A at dose p; Y=Observed result from Compound B at dose q; E=Expected result for mixture of A and B at dose (p+q) if there is no synergy or antagonism (assuming responses range from 0 to 100), whereby E=X+(100˜X)(Y/100)=X+Y−(X*Y)/100; if observed value is greater than expected result (Obs>E): synergism; if observed value is similar as expected result (Obs=E): additive; and if observed value is less than expected result (Obs<E): antagonism. Results provided in Table 1 below showed synergism on ECB in the mixture of IP1-88 and IP2-127.

TABLE 1
Mortality of ECB caused by IP1-88 or IP2-127 alone and
in mixture (n = 40, 4 d at 27° C.)
Protein inExpected
single orObserved% mortality,
combinationDose% mortality*Colby's equation
IP 1-88M1x32.5
M2x23.1
IP2-127M1x7.7
M2x20.5
IP1-88: IP2-127M1x: 1x63.237.7
M1x: 2x81.646.4
M2x: 1x66.729.0
*Corrected data based on Abbott equation with CK mortality < 2.5%.

Example 2

Bt proteins IP1-88 (a Cry1) and IP2-127 (a Cry2), as well as additional proteins were utilized as test substances to further confirm the presence and resultant effect of the Bt proteins, according to the same methods of Example 1. Both European corn borers (ECB) and corn earworms (CEW), Helicoverpa zea, were used in the bioassays. The observed response (mortality+ss) of both ECB and CEW in two of three mixture treatments was evidently higher than expected mortality based on Colby's equation (results provided in Table 2), also indicative of synergism between IP1-88 and IP2-127 for both ECB and CEW.

TABLE 2
Response (mortality + ss) of ECB and CEW caused by IP1-88 or IP2-
127 alone and in mixture (n = 40, 4 d at 27° C.)
Protein inObserved/
single orexpected
combinationDose% responseECBCEW
IP 1-88R1xObserved31.619.4
R2xObserved94.527.8
IP2-127R1xObserved7.721.5
R2xObserved20.583.8
IP1-88: IP2-127R1x: 1xObserved94.786.8
Expected36.936.7
R1x: 2xObserved76.994.7
Expected45.686.9
R2x: 1xObserved66.768.4
Expected94.936.7

Example 3

Bt proteins Cry1Ah and IP2-127 (a Cry2), as well as additional proteins were utilized as test substances to further confirm the presence and resultant effect of the Bt proteins, according to the same methods of Example 1. Both European corn borers (ECB) and corn earworms (CEW), Helicoverpa zea, were used in the bioassays. The observed mortality and response (mortality+ss) of both ECB and CEW in the mixture treatments was higher than expected based on Colby's equation (results provided in Table 3), also indicative of synergism between Cry1Ah and IP2-127.

TABLE 3
Mortality (mort) and response (resp; mortality + ss) of ECB and CEW caused by Cry1Ah
or IP2-127 alone and in mixture. (Trial 1: n = 40, trial 2: n = 32; both 4 d at 27° C.)
Protein in singleObserved/expectedECB - trial 1CEW - trial 1ECB - trial 2CEW - trial 2
or combinationDose% response% mort% resp% mort% resp% mort% resp% mort% resp
Cry1AhR1xObserved59.550.628.223.730.262.545.140.9
R2xObserved77.292.266.744.730.275.070.970.4
IP2-127R1xObserved28.86.921.77.959.455.311.934.9
R2xObserved72.633.326.334.267.773.342.462.7
Cry1Ah:1P2-127R1x:1xObserved74.065.469.969.577.092.962.872.9
Expected71.254.043.829.771.683.251.661.5
R1x:2xObserved92.282.074.065.893.4100.079.796.6
Expected88.967.147.149.877.590.068.478.0
R2x:1xObserved92.275.481.462.254.189.786.990.2
Expected83.892.773.949.171.688.874.480.8