# Patent

## Title
Enhancement of Bacillus thuringiensis Cry protein toxicities to coleopterans, and novel insect cadherin fragments

## Abstract

The subject invention relates in part to the discovery that a fragment from a cadherin of the western corn rootworm enhances Cry3 toxicity to larvae of naturally susceptible species. The subject invention also relates in part to the discovery that a cadherin fragment from a beetle enhances Cry3Aa and Cry3Bb toxicity to coleopteran larvae, particularly those in the family Chrysomelidae. Such cadherin fragments are referred to as Bt Boosters (BtBs). The subject invention can be extended to the use of BtBs with other coleopteran-toxic Cry proteins for controlling a wide range of coleopterans.

## Background
This application is a National Stage filing of PCT International Application Serial No. PCT/US2008/72806, filed 11 Aug. 2008, which claims the benefit of U.S. Provisional Application Ser. Nos. 60/964,249, filed 10 Aug. 2007, and 61/084,952 filed 30 Jul. 2008, the disclosures each of which are expressly incorporated herein by reference.


This invention was made in part with government support under USDA award no. 2004-35607-14936. The government has certain rights in this invention.


BACKGROUND OF THE INVENTION
The Cry3 class of Bacillus thuringiensis (Bt) Cry proteins is known for toxicity to coleopteran larvae in the family Chrysomelidae. The Cry3Aa and Cry3Bb insecticidal proteins of Bacillus thuringiensis (Bt) are used in biopesticides and transgenic crops to control larvae of leaf feeding beetles and rootworms. Due to limited efficacy of Cry3-based biopesticides and the success of competing chemical pesticides, these biopesticides have had limited usage and sales (Gelernter, 2004). Cry3Bb is toxic to corn rootworms (Donovan et al., 1992; Johnson et al., 1993), and a modified version is expressed in commercialized MON863 corn hybrids (Vaughn et al., 2005).
Cry3 toxins have a mode-of-action that is similar yet distinct from the action of lepidopteran-active Cry1 toxins. The Cry3A protoxin (73-kDa) lacks the large C-terminal region of the 130-kDa Cry1 protoxins, which is removed by proteases during activation to toxin. Cry3A protoxin is activated to a 55-kDa toxin and then further cleaved within the toxin molecule (Carroll et al., 1997; Loseva et al., 2002). Activated Cry3A toxin binds to brush border membrane vesicles (BBMV) with a Kd  ̃37 nM (Martinez-Ramirez and Real, 1996) and recognizes a 144-kDa binding protein in BBMV prepared from the yellow mealworm Tenebrio molitor (Coleoptera: Tenebrionidae) (Belfiore et al., 1994). Ochoa-Campuzano et al. (Ochoa-Campuzano et al., 2007) identified an ADAM metalloprotease as a receptor for Cry3Aa toxin in CPB larvae.
Structural differences between Cry3Bb and Cry3Aa toxins could account for the unique rootworm activities of Cry3Bb toxin. As noted by Galitsky et al. (Galitsky et al., 2001), differences in toxin solubility, oligomerization, and binding are reported for these Cry3 toxins. Recently, Cry3Aa was modified to have activity against western corn rootworm (WCRW) Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae (Walters et al., 2008). Those authors introduced a chymotrypsin/cathepsin G into domain 1 of Cry3Aa that allowed processing of the 65-kDa form to a 55-kDa toxin that bound rootworm midgut.
Cadherins localized in the midgut epithelium function as receptors for Cry toxins in lepidopteran and dipteran larvae. Sayed et al. (2007) identified a novel cadherin-like gene in WCRW and proposed this protein as a candidate Bt toxin receptor. The cadherin-like gene is highly expressed in midgut tissue of larval stages. The encoded protein is conserved in structure relative to other insect midgut cadherins.
An important Cry1 toxin binding site is localized within the final cadherin repeat (CR) 12 of cadherins from tobacco hornworm Manduca sexta (Lepidoptera: Sphingidae) and tobacco budworm Heliothis virescens (Lepidoptera: Noctiudae) (Hua et al., 2004; Xie et al., 2005). Unexpectedly, a fragment of Bt-R1 cadherin, the Cry1A receptor from M. sexta, not only bound toxin but enhanced Cry1A toxicity against lepidopteran larvae (Chen et al., 2007). If the binding residues within cadherin repeat 12 (CR) were removed, the resulting peptide lost the ability to bind toxin and lost its function as a toxin synergist. A fragment of a cadherin from Anopheles gambiae enhanced the toxicity of the mosquitocidal toxin Cry4Ba to mosquito larvae (Hua et al., 2008).
Terminal cadherin repeats from the Cry1A-binding M. sexta cadherin and the Cry4Ba-binding A. gambiae cadherin were reported to bind toxin and enhance Cry1A toxicity against lepidopteran larvae and Cry4 toxicity against dipteran larvae (Chen et al., 2007; Hua et al., 2008).
BRIEF SUMMARY OF THE INVENTION
The subject invention relates in part to the discovery that a fragment from a cadherin of the western corn rootworm enhances Cry3 toxicity to larvae of naturally susceptible species. The subject invention also relates in part to the discovery that a cadherin fragment from a beetle enhances Cry3Aa and Cry3Bb toxicity to coleopteran larvae, particularly those in the family Chrysomelidae. Such cadherin fragments are referred to herein as Bt Boosters (BtBs).
The subject invention also relates to the extension of these discoveries to the use of BtBs with other coleopteran-toxic Cry proteins for controlling a wide range of coleopterans.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates wild-type insect cadherin and cadherin fragments (i.e. peptides) with synergistic activities, including:
wild-type BtR1 cadherin of M. sexta, the cadherin fragment Ms-CR10-12 (BtB4), and the proteinase-stabilized MsCR10-12(PS) fragment (also called BtB5);
cadherin fragment Tm-CR10-MPED from Tenebrio molitor (Coleoptera: Tenebrionidae); and
cadherin fragments Dv-CR8-10 from Diabrotica virgifera virgifera (Western corn rootworm, WCRW) and the protease stabilized version Dv-CR8-10(PS).
FIG. 2 illustrates enhancement of Bt tenebrionis spore and Cry3Aa crystal toxicity to CPB larvae by the cadherin peptides Ms-CR10-12, Ms-CR10-12(PS) (Panel A), Tm-CR10-12 and Ms-CR10-12(PS) (Panel B), and Ms-CRL0-12 and Tm-CR10-MPED (Panel C). In Panels A and C, Bt suspensions were weighed as a dried powder and mixed in diluent alone or with the cadherin peptides in a 1:10 Bt:Cadherin Peptide mass ratio. In Panel. C, the amount of Cry3Aa in the Btt spore crystal mixture was estimated by SDS-PAGE and scanning densitometry. The indicated amounts were mixed in diluent alone or with the indicated cadherin peptides.
FIG. 3. SDS-PAGE of Cry3Aa, Cry3Bb and Dv-CR8-10 (or CR8-10) peptide (Panel A) and binding saturation of biotinylated Dv-CR8-10 peptide to Cry3 toxins (Panels B and C). Panel A, lanes: Cry3Aa (crystals), Cry3Bb (crystal inclusions), and Dv-CR8-10 (crystal inclusions), solubilized Cry3Aa and Cry3Bb before and after chymotrypsin treatment (designated with XT). In Panels B and C: binding of biotinylated Dv-CR-8-10 to Cry3Aa or Cry3Bb protein was determined using an ELISA-based binding assay. Microtiter plates were coated with chymotrypsin-treated Cry3Aa or Cry3Bb and then incubated with increasing various concentrations of biotinylated Dv-CR8-10 peptide. Bound biotinylated Dv-CR8-10 peptide was detected with a Streptavidin-HRP conjugate and substrate. Non-specific binding was determined in the presence of 1000-fold excess unlabeled CR8-10 peptide. Each data point is the mean of two experiments done in duplicate. Error bars depict standard deviation. Binding affinities (Kd) were calculated based on specifically bound biotinylated Dv-CR8-10 peptide using a one site saturation binding equation.
FIG. 4. Dv-CR8-10 peptide enhances Cry3Aa and Cry3Bb toxicity to CPB larvae. Suspensions of Cry3Aa crystals (Panel A) or Cry3Ba (Panel B) inclusions without or with Dv-CR8-10 inclusions at a toxin:peptide mass ratio of 1:10 were sprayed onto excised potato leaves. In Panel C, treatments consisted of Cry3Aa or Cry3Bb alone, or with varying toxin:peptide mass ratios of Dv-CR8-10 peptide. Buffer and CR8-10 peptide at 400 μg/ml were not toxic to the larvae. Each bioassay consisted of 5 CPB larvae per Petri dish with 10 cups per treatment. Larval mortality was scored on Day 3. Concentrations are expressed as micrograms Cry protein per ml suspension. Each data point represents data for the mean±standard error from a bioassay with 50 larvae per dose.
FIG. 5. Comparison of Cry3Aa and Cry3Bb toxicity to SCRW larvae without and with Dv-CR8-10 peptide. Suspensions of Cry3Aa crystals (Panel A) or Cry3Ba (Panel B) inclusions without or with Dv-CR8-10 inclusions at a toxin:peptide mass ratio of 1:10 were fed to 1st instar SCRW larvae in a surface overlay bioassay. Panel C shows the enhancement effects of increasing ratios of Dv-CR8-10 peptide in Cry3Aa or Cry3Bb suspensions. No toxicity was observed when treated with Dv-CR8-10 peptide alone. Mortality was scored on day 7. Each data point represents data for the mean±standard error from a bioassay with 32 larvae per dose.
FIG. 6. Dv-CR8-10 peptide enhancement of Cry3Bb toxicity to WCRW larvae. Larvae were exposed to diet treated with Cry3Bb crystals alone or Cry3Bb plus Dv-CR8-10 inclusions at fixed 1:10 (Panel A) or varying toxin:peptide ratios (Panel B). In Panel B the Cry3Bb concentration was 1.0 μg/cm2. Mortality was scored on day 6. Each data point represents data for the mean±standard error from a bioassay with 32 larvae per dose. (Panel C) Diet surface bioassay showing enhancement of Cry3Bb (@ 1 μg/cm2 well) with 1:10 and 1:100 ratio of Dv-CR8-10 or Ms-CR10-12(PS) against 1st instar western corn rootworm. Dv-CR8-10 shows better enhancement of toxicity than Ms-CR10-12(PS). No toxicity was observed when treated with Dv-CR8-10 or Ms-CR10-12(PS) alone (@ 100 μg/cm2). Bioassay result was scored on 6th day after treatment.
FIG. 7. Diet surface treatment bioassay with neonate western corn rootworm (WCRW). Sample size: 16 larvae/rep×2 rep/treatment. One μg/cm2 Cry3Bb was applied alone or with 10-fold mass ratio of the different BtBs on diet surface. Soluble BtBs were purified with Ni-column. Cry3Bb was partially solubilized with 20 mM Na2CO3 pH 10.0. Bioassay was scored on Day 6. (Panel A) Mortality data. (Panel B) Larval weight data.



BRIEF DESCRIPTION OF THE SEQUENCES
SEQ ID NO:1 provides the combined polynucleotide/amino acid sequences of Ms-CR10-12 (BtB4).
SEQ ID NO:2 provides the protein sequence from SEQ ID NO:1 (BtB4).
SEQ ID NO:3 provides the combined polynucleotide/amino acid sequences of Ms-CR10-12(PS) proteinase stabilized CR10-12. The protein/peptide/polypeptide of SEQ ID NO:3 is also referred to as BtB5.
SEQ ID NO:4 provides the protein sequence from SEQ ID NO:3 (BtB5).
SEQ ID NO:5 is the Dv-CR8-10/CR8-10 nucleotide sequence from WCRW cadherin with EcoRI, HindIII cloning sites.
SEQ ID NO:6 provides the combined polynucleotide/amino acid sequences of Dv-CR8-10/CR8-10 nucleotide and amino acid sequence from the WCRW cadherin. The polypeptide/protein of SEQ ID NO:6 is also known as BtB7.
SEQ ID NO:7 provides the protein sequence from SEQ ID NO:6 (BtB7).
SEQ ID NO:8 is the Tm-CR10-MPED nucleotide sequence encoding the last cadherin repeat (CR) and membrane proximal extracellular domain (MPED) of Tenebrio molitor cadherin. The ends (underlined) contain Nde1 and Xho1 sites for cloning into a E. coli expression vector pET30A. A 6× his coding sequence was included at the C-terminus.
SEQ ID NO:9 provides the combined nucleotide and amino acid sequences of the Tm-CR10-MPED cadherin fragment from the Tenebrio molitor cadherin. The coding region includes the last cadherin repeat (CR) and membrane proximal extracellular domain (MPED) of the Tenebrio molitor cadherin. The 5′ and 3′ ends contain Nde1 and Xho1 sites for cloning into an E. coli expression vector. A 6× his coding sequence was included at the C-terminus. This region was cloned into pET30a and expressed in. E. coli. 
SEQ ID NO:10 provides the protein sequence from SEQ ID NO:9.
SEQ ID NO:11 provides the combined polynucleotide/amino acid sequences of Dv-CR8-10(PS)—the Proteinase Stabilized Dv-CR8-10 from the WCRW cadherin. The polypeptide of SEQ ID NO:11 is also called BtB8.
SEQ ID NO:12 provides the protein sequence from SEQ ID NO:11 (BtB8).
DETAILED DISCLOSURE OF THE INVENTION
The subject invention relates in part to the discovery that a fragment from a cadherin of the western corn rootworm enhances Cry3 toxicity to larvae of naturally susceptible species. The subject invention also relates in part to the discovery that a cadherin fragment from a beetle enhances Cry3Aa and Cry3Bb toxicity to coleopteran larvae, particularly those in the family Chrysomelidae.
The cadherin repeat (CR) 8-10 of Diabrotica virgifera virgifera was expressed in E. coli as an inclusion body. Larvae ingesting CR10-12 inclusions had increased susceptibility to Cry3Aa or Cry3Bb toxins. The Cry3 toxin-enhancing effect of CR8-10 was demonstrated for Colorado potato beetle (Leptinotarsa decimlineata), southern corn rootworm (D. undecimpunctata howardi) and western corn rootworm (D. virgifera virgifera). The extent of Cry3 toxin enhancement, which ranged from 3- to 8-fold, has practical applications for insect control.
The region spanning CR10-12 of the WCRW cadherin (Siegfried et al. 2007) was cloned and expressed in E. coli. This cadherin fragment significantly enhanced the toxicities of Cry3Aa and Cry3Bb toxins to CPB and rootworms.
The western corn rootworm midgut cadherin (Sayed et al., 2007) was used as a template to design a cadherin fragment that includes a potential toxin binding site. The designed CR8-10 peptide includes the predicted binding site 1311SSLNVTVN1318 which has similarity to Cry1A toxin binding region 2 (TBR 2) of M. sexta cadherin (Sayed et al., 2007). The WCRW cadherin and the CR8-10 cadherin peptide do not contain a clear match to TBR 3 (GVLTLNIQ; residues 1416-1423) of M. sexta cadherin (Chen et al., 2007). This is significant because when Chen et al. (Chen et al., 2007) deleted the critical TBR 3, their cadherin fragment did not bind or enhance Cry1A toxicity. Three cadherin repeats were included in our cadherin peptide, in part because of observations that CR10-12 of M. sexta cadherin has greater Cry1A toxin binding and enhancing properties than CR12 alone.
The CR8-10 cadherin fragment from WCRW increased the potency of Cry3Aa and Cry3Bb proteins between three and about eight-fold against CPB and two rootworm species. This level of potentiation is comparable and in some cases greater than our published reports of cadherin fragment enhancement of Cry1Ab toxicity to M. sexta and Cry4Ba toxicity to A. gambiae (Chen et al., 2007; Hua et al., 2008). In these reports the cadherin fragments consisted of terminal CR and membrane proximal extracellular domains derived from the respective species and both cadherin fragments bound toxin. The toxin-enhancing cadherin fragments of those studies also bound Cry toxin. Soberon et al. (Soberon et al., 2007) provide an explanation for how cadherin fragments are involved in Cry1Ab toxicity. The CR12 fragment induces formation of a pre-pore Cry1Ab-cadherin oligomer, a critical step in intoxication process (Jimenez-Juarez et al., 2008).
There is practical importance of a three to eight-fold level of synergy on field performance of biopesticides. In the case of susceptible CPB larvae and ‘high-dose’ Cry3Aa expression in transgenic Bt Newleaf potato, a 3-fold enhancement of Cry3Aa toxicity can have some impact on inhibition of insects. In addition, where weekly sprays of a microbial biopesticide are required due to short field half-life and tolerance in older larvae (Zhender and Galertner, 1989), a three-fold enhancement of Cry3Aa toxicity may result in highly efficacious control of CPB.
Diabrotica species are major pests of corn in the U.S. significantly impacting corn grown for food and ethanol production, and adult D. undecimpunctata howardi (SCRW or cucumber beetle) is an important insect pest of cucurbits. The LC50 value (1.6 μg/cm2) we determined for SCRW larvae is considerably less than the 61.5 μg/cm2 previously reported (Johnson et al., 1993). The Cry3Bb LC50 (5 μg/cm2) against WCRW is within the 0.7 to 9.2 μg/cm2 values calculated for laboratory and field populations of WCRW (Siegfried et al. 2007). The MON 863 cultivar of Bt maize (also called Yieldguard Rootworm) expresses a modified Cry3Bb, that has 8-fold increased toxicity relative to Cry3Bb, yet some western and southern corn rootworms survive on MON 863 corn. Although factors such as feeding behavior and environmental conditions can impact rootworm survival, a peptide synergist that increases Cry3Bb potency could lead to improved rootworm control.
Based on current data, the BtBs could enhance the following (but not limited to) examples of coleopteran-active Bt toxins (obtained from the online Bt specificity database: world wide web glfc.forestry.ca/bacillus/). The following Bt toxins are known to have toxicity against Colorado potato beetle: Cry1Ba, Cry1Ia, Cry3Aa, Cry3Ba, Cry3Bb, Cry3Ca, Cry7Aa, Cry8Aa, Cry8Bb, and Cry8Bc. The following Bt toxins are known to have toxicity against corn rootworm in general: Cry3Aa, Cry3Bb, Cry8Bb, Cry8Bc, Cry34Aa+Cry35Aa, Cry34Ab+Cry35Ab, Cry34Ac+Cry34Ab, Cry34Ba+Cry35Ba and Cry36Aa. The following Bt toxins are known to have toxicity against Japanese beetle: Cry8Ba, Cry8Da, Cry23Aa, and Cry37Aa. Various other Bacillus thuringiensis Cry proteins can be used with BtB polypeptides of the subject invention. See Crickmore et al. (1998) (world wide web website lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/) for a list of B.t. toxins.
The subject polypeptides and protein toxins can be “applied” or provided to contact the target insects in a variety of ways. For example, transgenic plants (wherein the protein is produced by and present in the plant) can be used and are well-known in the art. Expression of the toxin genes can also be achieved selectively in specific tissues of the plants, such as the roots, leaves, etc. This can be accomplished via the use of tissue-specific promoters, for example. Spray-on applications are another example and are also known in the art. The subject polypeptides/proteins can be appropriately formulated for the desired end use, and then sprayed (or otherwise applied) onto the plant and/or around the plant/to the vicinity of the plant to be protected—before an infestation is discovered, after target insects are discovered, both before and after, and the like. Bait granules, for example, can also be used and are known in the art. Various combinations of approaches are discussed in WO 2005/070214 A2, US-2005-0283857-A1, and U.S. Pat. No. 7,396,813.
The subject proteins can be used to protect practically any type of plant from damage by an insect. Examples of such plants include maize, sunflower, soybean, cotton, canola, rice, sorghum, wheat, barley, vegetables, ornamentals, peppers (including hot peppers), sugar beets, fruit, and turf, to name but a few.
As stated herein, polypeptides (together with a Cry protein) can inhibit one or more Coleopteran pests including the following. (The following can also be used as a source of the insect cadherin ectodomain.) Such insects include:

    Anthonomus grandis, boll weevil
    Bothyrus gibbosus, carrot beetle
    Chaetocnema pulicaria, corn flea beetle
    Colaspis brunnea, grape colaspis
    Cyclocephala borealis, northern masked chafer (white grub)
    Cyclocephala immaculata, southern masked chafer (white grub)
    Diabrotica longicornis barberi, northern corn rootworm
    Diabrotica undecimpunctata howardi, southern corn rootworm
    Diabrotica virgifera virgifera, western corn rootworm
    Eleodes, Conoderus, and Aeolus spp., wireworms
    Epilachna varivestis, Mexican bean beetle
    Hypera punctata, clover leaf weevil
    Lissorhoptrus oryzophilus, rice water weevil
    Melanotus spp., wireworms
    Oulema melanopus, cereal leaf beetle
    Phyllophaga crinita, white grub
    Phylotreta spp., flea beetles including canola flea beetle
    Popillia japonica, Japanese beetle
    Sitophilus oryzae, rice weevil
    Sphenophorus maidis, maize billbug
    Zygogramma exclamationis, sunflower beetle


In light of and having the benefit of the subject application, variants of novel BtBs of the subject invention (e.g. those derived from Diabrotica and Tenebrio cadherins) can be constructed using techniques that are known in the art.
It will be recognized by those skilled in the art that the DNA sequences may vary due to the degeneracy of the genetic code and codon usage. All DNA sequences which code for exemplified and/or suggested peptides (and proteins) are included. The subject invention also includes polynucleotides having codons that are optimized for expression in plants, including any of the specific types of plants referred to herein. Various techniques for creating plant-optimized sequences are know in the art.
Additionally, it will be recognized by those skilled in the art that allelic variations may occur in the DNA sequences which will not significantly change activity of the amino acid sequences of the peptides which the DNA sequences encode. All such equivalent DNA sequences are included within the scope of this invention and the definition of the regulated promoter region. The skilled artisan will understand that exemplified sequences can be used to identify and isolate additional, non-exemplified nucleotide sequences that will encode functional equivalents to the DNA sequences, including those that encode amino acid sequences having at least 85% identity thereto and having equivalent biological activity, those having at least 90% identity, and those having at least 95% identity to a novel BtB polypeptide of the subject invention. Other numeric ranges for variant polynucleotides and amino acid sequences are provided below (e.g., 50-99%). Following the teachings herein and using knowledge and techniques well known in the art, the skilled worker will be able to make a large number of operative embodiments having equivalent DNA sequences to those listed herein without the expense of undue experimentation.
As used herein percent sequence identity of two nucleic acids is determined using the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:402-410. BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences with the desired percent sequence identity. To obtain gapped alignments for comparison purposes, Gapped BLAST is used as described in Altschul et al. (1997) Nucl. Acids. Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (NBLAST and XBLAST) are used. See ncbi.nih.gov website.
Polynucleotides (and the peptides and proteins they encode) can also be defined by their hybridization characteristics (their ability to hybridize to a given probe, such as the complement of a DNA sequence exemplified herein). Various degrees of stringency of hybridization can be employed. The more stringent the conditions, the greater the complementarity that is required for duplex formation. Stringency can be controlled by temperature, probe concentration, probe length, ionic strength, time, and the like. Preferably, hybridization is conducted under moderate to high stringency conditions by techniques well known in the art, as described, for example, in Keller, G. H., M. M. Manak (1987) DNA Probes, Stockton Press, New York, N.Y., pp. 169-170.
As used herein “moderate to high stringency” conditions for hybridization refers to conditions that achieve the same, or about the same, degree of specificity of hybridization as the conditions “as described herein.” Examples of moderate to high stringency conditions are provided herein. Specifically, hybridization of immobilized DNA on Southern blots with 32P-labeled gene-specific probes was performed using standard methods (Maniatis et al.). In general, hybridization and subsequent washes were carried out under moderate to high stringency conditions that allowed for detection of target sequences with homology to sequences exemplified herein. For double-stranded DNA gene probes, hybridization was carried out overnight at 20-25° C. below the melting temperature (Tm) of the DNA hybrid in 6×SSPE, 5× Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature is described by the following formula from Beltz et al. (1983).

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

Washes are typically carried out as follows:

    
    
        (1) Twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS (low stringency wash).
        (2) Once at Tm-20° C. for 15 minutes in 0.2×SSPE, 0.1% SDS (moderate stringency wash).
    
    


For oligonucleotide probes, hybridization was carried out overnight at 10-20° C. below the melting temperature (Tm) of the hybrid in 6×SSPE, 5× Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Tm for oligonucleotide probes was determined by the following formula from Suggs et al. (1981):

Tm (° C.)=2 (number T/A base pairs)+4(number G/C base pairs)

Washes were typically carried out as follows:

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


In general, salt and/or temperature can be altered to change stringency. With a labeled DNA fragment of greater than about 70 or so bases in length, the following can be used:
1 or 2×SSPE, room temperature
1 or 2×SSPE, 42° C.
0.2× or 1×SSPE, 65° C.
0.1×SSPE, 65° C.
Duplex formation and stability depend on substantial complementarity between the two strands of a hybrid, and, as noted above, a certain degree of mismatch can be tolerated. Therefore, polynucleotide sequences of the subject invention include mutations (both single and multiple), deletions, and insertions in the described sequences, and combinations thereof, wherein said mutations, insertions, and deletions permit formation of stable hybrids with a target polynucleotide of interest. Mutations, insertions, and deletions can be produced in a given polynucleotide sequence using standard methods known in the art. Other methods may become known in the future.
The mutational, insertional, and deletional variants of the polynucleotide and amino acid sequences of the invention can be used in the same manner as the exemplified sequences so long as the variants have substantial sequence similarity with the original sequence. As used herein, substantial sequence similarity refers to the extent of nucleotide similarity that is sufficient to enable the variant polynucleotide to function in the same capacity as the original sequence. Preferably, this similarity is greater than 50%; more preferably, this similarity is greater than 75%; and most preferably, this similarity is greater than 90%. The degree of similarity needed for the variant to function in its intended capacity will depend upon the intended use of the sequence. It is well within the skill of a person trained in this art to make mutational, insertional, and deletional mutations that are designed to improve the function of the sequence or otherwise provide a methodological advantage. In some embodiments, the identity and/or similarity can also be 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% as compared to a sequence exemplified herein.
The amino acid identity/similarity and/or homology will be highest in critical regions of the protein that account for biological activity and/or are involved in the determination of three-dimensional configuration that ultimately is responsible for the biological activity. In this regard, certain amino acid substitutions are acceptable and can be expected if these substitutions are in regions that are not critical to activity or are conservative amino acid substitutions which do not affect the three-dimensional configuration of the molecule. For example, amino acids may be placed in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the subject invention so long as the substitution does not materially alter the biological activity of the compound. The following table provides a listing of examples of amino acids belonging to each class.














Class of Amino Acid
Examples of Amino Acids









Nonpolar
Ala, Val, Leu, Ile, Pro, Met, Phe, Trp



Uncharged Polar
Gly, Ser, Thr, Cys, Tyr, Asn, Gln



Acidic
Asp, Glu



Basic
Lys, Arg, His










In some instances, non-conservative substitutions can also be made.

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety to the extent they are not inconsistent with the explicit teachings of this specification. Unless specifically indicated or implied, the terms “a”, “an”, and “the” signify “at least one” as used herein. Following are examples that illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.
EXAMPLE 1
Cloning and Expression of CR8-10 of the Western Corn Rootworm Cadherin-Like Protein
A DNA fragment encoding amino acids 961-1329 (corresponding to CR8-10 sequence) of the D. virgiftra virgifera cadherin-like protein (GI:156144975) was synthesized by GenScript Corp. (Piscataway, N.J.) and the synthetic gene was inserted between the Nde I and HindIII restriction enzyme sites of an expression vector, pET30a(+) (Novagen, Madison, Wis.). The coding sequence and clone orientation were confirmed by sequencing (Molecular Genetics Instrumentation Facility at University of Georgia). The pET construct was transformed into E. coli strain BL21(DE3)/pRIL (Stratagene, La Jolla, Calif.), and positive clones were selected on LB plates containing kanamycin and chloramphenicol. The CR8-10 peptide was overexpressed in E. coli as inclusion bodies. The expression and purification protocol for the truncated cadherin fragment was as described in a previous paper (Chen et al., 2007). The inclusion body form was prepared as a suspension in sterile deionized water. Total protein was measured by Bio-Rad protein assay using bovine serum albumin (BSA) as standard (Bradford, 1976). One microgram of each cadherin peptide was analyzed by sodium dodecyl sulfate-15% polyacrylamide gel electrophoresis (SDS-15% PAGE) with Coomassie brilliant blue R-250 staining. Specific concentration of target protein, such as toxin or the cadherin peptide in total protein was determined from Coomassie-stained gel by gel image analyzer (Alpha Innotech, San Leandro, Calif.) using bovine serum albumin (BSA) as standard.
EXAMPLE 2
Culture of Bt Tenebrionis and Purification of Cry3Aa Crystals
Bt tenebrionis strain was grown in peptone glucose salts medium (Brownbridge and Margalit, 1986) until sporulation and cell lysis. The spore crystal mixture was harvested by centrifugation, suspended in 0.1M NaCl, 2% Triton X-100, 20 mM Bis-Tris, pH6.5 and sonicated. Spores and crystals were pelleted by centrifugation at 12,000 g for 10 min, washed twice with 0.5M NaCl, followed by a single wash in distilled water. Cry3Aa crystals were purified according to (Slaney et al., 1992) and the final Cry3Aa protein concentration was determined based on band density on SDS-15% PAGE, using BSA as standard. Crystals were stored at 4° C. until used for experimentation.
EXAMPLE 3
Cloning and Expression of the Cry3Bb Gene
The Cry3Bb coding region was cloned by PCR using total DNA extracted from the Bt biological insecticide Raven (Ecogen, Inc. Langhorne, Pa.) as template with primers Cry3Bb/FWD (CAGGTCTAGAGTTATGTATTATGATAAGAATGGG) and Cry3Bb/REV (TAAACTCGAGTTACAATTGTACTGGGATAAATTC). The PCR amplified cry3Bb gene was cloned into pGEM-T Easy vector (Promega, Madison, Wis.), and then subcloned between the Xba I and XhoI sites of the pET30a(+). The clone called Cry3Bb/pET was transformed into E. coli strain BL21-CodonPlus (DE3)/pRIL (Stratagene, LaJolla, Calif.). The coding sequence and clone orientation were confirmed by sequencing. The expression and purification protocol for the Cry3Bb protein inclusion bodies was as described above for CR8-10 peptide.
EXAMPLE 4
Insect Bioassays
Adult CPB were obtained from the New Jersey Department of Agriculture (Trenton, N.J.). A CPB colony was maintained on containerized potato plants in an environmental chamber at 25° C. and 70% relative humidity with a photoperiod of 16 hrs light: 8 hrs dark. Neonate CPB larvae were fed fresh potato leaves for 5 hrs before bioassay. Suspensions (30 ml) of Cry3 crystals or crystals plus CR8-10 inclusions prepared in diluent [0.12% v/v Kinetic (Helena Chemical Co, Collierville, Tenn.)] plus 0.12% v/v commercial-grade polyethylene and octyl phenol polyethoxy ethanol spreader-sticker (Southern Agricultural Insecticides, Inc., Boon, N.C.) in tap water], were sprayed on potato leaves and the leaves were air-dried in a fume hood for 15 min. 10 larvae were applied to treated leaves in a petri dish. Each bioassay was conducted with 50 larvae per dose.
Eggs of southern corn rootworm (SCRW) and western corn rootworm (WCRW) were obtained from French Agricultural Research, Inc. (Lamberton, Minn.) and incubated at 25° C. for 8 days and 16 days until hatching, respectively. Molten SCRW artificial diet (Bio-Serv, Frenchtown, N.J.) was adjusted to pH 7.0 (according to the manufacturer) for SCRW bioassay and to pH 9.0 for WCRW bioassay with KOH and then applied onto 128-well bioassay tray (C-D International, Pitman, N.J.). Cry3 crystals or crystals with CR8-10 inclusions were serially diluted with sterile deionized water and then overlaid onto the diet surface and air-dried. One newly hatched larva was transferred into each well; the trays were sealed with perforated lids (C-D International, Pitman, N.J.) and then covered with brown paper to provide a dark environment. Each bioassay was conducted with 16 larvae per replicate and two replicates per dose. The trays were incubated at 28° C. for 6 days for WCRW or 7 days for SCRW before mortality counts were scored. Bioassays were repeated at least twice.
EXAMPLE 5
Results
In this study, the CR8-10 region of WCRW cadherin (Sayed et al., 2007) was over-expressed in E. coli and tested for the ability to enhance Cry3Aa and Cry3Bb toxicity to CPB and rootworm larvae. The CR8-10 region is homologous to toxin-enhancing regions from M. sexta and A. gambiae cadherins and contains a predicted toxin binding region (Sayed et al., 2007). The native WCRW coding sequence was optimized for E. coli expression, and most of the CpG sequences in the native cadherin sequence were removed to decrease potential DNA methylation in planta. The synthetic CR8-10 peptide of 377 amino acid residues has an initiation methionine, a C-terminal 6 histidine tag, and a molecular size of 42,814 Da. Inclusion bodies isolated from recombinant E. coli were composed of the expected 45-kDa protein, plus lesser amounts of two about 30-kDa peptides (FIG. 3A).
Cry3Aa crystals were prepared from Bt tenebrionis, and Cry3Bb inclusions were isolated from recombinant E. coli for testing in insect bioassays with CR8-10 inclusions. The Cry3Aa crystals and Cry3Bb crystals were composed of the expected 73- and 75-kDa protoxin-sized proteins (FIG. 3A). Leaflets of potato were sprayed with suspensions of Cry3 crystals alone or crystals with CR8-10 inclusions, air dried and fed to first instar potato beetle larvae. The calculated LC50 mortality values were 4.4 μg Cry3Aa/ml and 6.5 μg Cry3Bb/ml (Table 1; FIGS. 4A and B). The addition of CR8-10 inclusions at a 1:10 mass ratio of Cry3:CR8-10 to the crystal suspensions reduced the Cry3Aa and Cry3Bb LC50 values 3.3-fold and 6-fold, respectively. To determine the extent that CR8-10 could enhance a low dose of toxin, we added increasing amounts of CR8-10 inclusions to a Cry3 crystal concentration predicted to cause about 20% larval mortality. As seen in FIG. 4C, the enhancement effect reached a plateau at a 1:10 (Cry protein:CR8-10) mass ratio. The CR8-10 peptide enhanced the low Cry3Bb dose to a greater extent than Cry3Aa.
The different and expected insecticidal properties of Cry3Aa and Cry3Bb were detected in our bioassays against SCRW. Whereas Cry3Bb is toxic to southern corn rootworms (SCRW) Diabrotica undecimpactata howardi (Coleoptera: Chrysomelidae), Cry3Aa has lower toxicity to this pest (FIG. 5A); c.f. (Donovan et al., 1992; Slaney et al., 1992). The ability of CR8-10 peptide to enhance Cry3Aa and Cry3Bb toxicity to SCRW was tested using a 1:10 (Cry3:CR8-10) mass ratio. The addition of CR8-10 to Cry3Aa reduced the LC50 value about 1.7-fold. As seen in FIG. 5A, the highest dose of Cry3Aa and Cry3Aa plus CR8-10 killed less than 100% of the larvae. Cry3Bb was about 100-fold more toxic than Cry3Aa to SCRW larvae in diet overlay bioassays (Table 1, FIG. 5B). The addition of Cry8-10 inclusions increased Cry3Aa and Cry3Bb toxicity to SCRW larvae (Table 1) and the mass ratio 1:10 (Cry3:CR8-10) was optimal for toxicity enhancement.
Since Cry3Bb, but not Cry3Aa, is active against WCRW, we tested the ability of CR8-10 to enhance Cry3Bb toxicity. As seen in FIG. 5A, CR8-10 enhanced Cry3Bb toxicity at each toxin dosage tested. The LC50 for Cry3Bb was 5.2 μg/cm2 and the LC50 for Cry3Bb plus a 1:10 toxin to cadherin fragment ratio was 0.7 μg/cm2 representing a 7.9-fold-fold enhancement by CR 8-10 peptide. Maximal enhancement of Cry3Bb toxicity WCRW larvae occurred with a 1:10 Cry3Bb:CR8-10 mass ratio (FIG. 6B).
EXAMPLE 6
Further Results
In this study additional BtBoosters were tested in combination with beetle-active Bt (B. thuringiensis subsp. tenebrionis), which produces Cry3Aa toxins against Colorado Potato Beetle (CPB) larvae. After initial dose response bioassays, a Bt dose that caused about 40% mortality (12.5 μg/ml) was chosen for further bioassays using various BtBoosterTM constructs (TmCad, a T. mollitor-derived cadherin peptide; BtB5 and BtB6, M. sexta-derived cadherin peptides). Several preliminary bioassays were performed, and the results are shown in FIG. 2. The data show that addition of BtB5, BtB6, or TmCad increased the toxicity of Bt tenebrionis against CPB. Statistical analysis by pair-wise Chi-square analysis showed that the enhancements were significant (P<0.05).
These results are very interesting because it shows that a lepidoptera-derived BtBoosterTM (i.e. BtB5 and BtB6) and a coleopteran-derived BtBooster (i.e. TmCad) can be combined with a beetle-active Bt to elicit a synergistic effect.
EXAMPLE 7
Still Further Results
The ability of BtB5 to enhance Cry3Bb toxicity to WCRW larvae was demonstrated in a diet surface bioassay against 1st instar WCRW larvae. The CR8-10 from WCRW was included for comparison. FIG. 2 shows the results of a diet surface bioassay showing enhancement of Cry3Bb (at 1 μg per well) with 1:10 and 1:100 ratios of CR8-10 or BtB5 against 1st instar WCR. CR8-10 shows better enhancement of toxicity than BtB5. No toxicity was observed when treated with WCR8-10 or BtB5 alone (at 100 ug per well). Bioassay result was scored on 6th day after treatment.
EXAMPLE 8
Control of Additional Coleopteran Insects with Additional Cry Proteins
The subject invention can also be extended to other Cry proteins. For example, BtBs of the subject invention can be used in combination with Cry34/35 proteins to enhance control of corn rootworms. In some specific embodiments of these embodiments, BtB5, BtB7, and BtB8 enhance Cry34/Cry35 for inhibiting corn rootworms. In addition, in some preferred embodiments, BtB5, BtB7, and BtB8 can be used with Cry8 proteins for controlling grubs, particularly white grubs.







TABLE 1







Effect of CR8-10 peptide on Cry3Aa and Cry3Bb


toxicity to CPB, SCRW and WCRW larvaea
















Control
CR8-10





Relative
Mortality
Mortality


Treatment
Insect
LC50
Toxicity
(% ± SE)
(% ± SE)
















Cry3Aab
CPB
3.21
(2.17-4.44)
—
3.0 ± 3.0



Cry3Aa + CR8-10d
CPB
0.96
(0.79-1.12)
3.3
3.0 ± 3.0
4.0 ± 2.0


Cry3Bbc
CPB
6.54
(5.54-7.86)
—
6.0 ± 3.5


Cry3Bb + CR8-10
CPB
1.09
(0.88-1.35)
6  
6.0 ± 3.5
2.0 ± 2.0


Cry3Aa
SCRW
91.81
(61.95-134.12)
—
12.5 ± 4.0 


Cry3Aa + CR8-10
SCRW
56.79
(43.34-72.31)
1.6
12.5 ± 4.0 
12.5 ± 3.3 


Cry3Bb
SCRW
1.18
(0.80-1.71)
—
12.5 ± 4.0 


Cry3Bb + CR8-10
SCRW
0.26
(0.14-0.42)
4.5
12.5 ± 3.3 
12.5 ± 4.0 


Cry3Bb
WCRW
5.18
(2.90-10.04)
—
9.3 ± 3.1


Cry3Bb + CR8-10
WCRW
0.65
(0.24-1.21)
7.9
9.3 ± 3.1
9.3 ± 3.1





aResults are 50% lethal doses (LC50) (with 95% confidence intervals) and are expressed as micrograms of Cry protein per ml for CPB bioassays and micrograms of Cry protein per square centimeter of diet surface for SCRW and WCRW bioassays.


bCry3Aa crystals were purified from a sporulated Bt tenebrionis culture.


cCry3Bb inclusions were prepared from recombinant E. coli.


dCry8-10 inclusions were isolated from E. coli expressing the CR8-10 region of WCRW cadherin.






REFERENCES


    Belfiore, C. J., et al., 1994. A specific binding protein from Tenebrio molitor for the insecticidal toxin of Bacillus thuringiensis subsp. tenebrionis. Biochem Biophys Res Commun. 200, 359-64.
    Bradford, M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.
    Brownbridge, M., Margalit, J., 1986. New Bacillus thuringiensis strains isolated in Israel are highly toxic to mosquito larvae. J Invertebr Pathol. 48, 216-22.
    Carroll, J., et al., 1997. Intramolecular proteolytic cleavage of Bacillus thuringiensis Cry3A delta-endotoxin may facilitate its coleopteran toxicity. Journal of Invertebrate Pathology. 70, 41-49.
    Chen, J., et al., 2007. Synergism of Bacillus thuringiensis toxins by a fragment of a toxin-binding cadherin. Proc Natl Acad Sci USA. 104, 13901-6.
    Donovan, W. P., et al., 1992. Characterization of two genes encoding Bacillus thuringiensis insecticidal crystal proteins toxic to Coleroptera species. Appl. Environ. Microbiol. 58, 3921-3927.
    Galitsky, N., et al., 2001. Structure of the insecticidal bacterial delta-endotoxin Cry3Bb1 of Bacillus thuringiensis. Acta Crystallographica Section D-Biological Crystallography. 57, 1101-1109.
    Gelernter, W., 2004. The rise and fall of Bacillus thuringiensis tenebrionis. Phytoparasitica. 32, 321324.
    Hua, G., et al., 2004. Bt-R1a extracellular cadherin repeat 12 mediates Bacillus thuringiensis Cry1Ab binding and toxicity. J Biol. Chem. 279, 28051-28056.
    Hua, G., et al., 2008. Anopheles gambiae cadherin AgCad1 binds the Cry4Ba toxin of Bacillus thuringiensis israelensis and a fragment of AgCad1 synergizes toxicity. Biochemistry. 47, 5101-10.
    Jimenez-Juarez, N., et al., 2008. The pre-pore from Bacillus thuringiensis Cry1Ab toxin is necessary to induce insect death in Manduca sexta. Peptides. 29, 318-23.
    Johnson, T. B., et al., 1993. Insecticidal Activity of Eg4961, a Novel Strain of Bacillus-Thuringiensis Toxic to Larvae and Adults of Southern Corn-Rootworm (Coleoptera, Chrysomelidae) and Colorado Potato Beetle (Coleoptera, Chrysomelidae). Journal of Economic Entomology. 86, 330-333.
    Loseva, O., et al., 2002. Changes in protease activity and Cry3Aa toxin binding in the Colorado potato beetleL implications for insect resistance to Bacillus thuringiensis toxins. Insect Biochem. Mol. Biol. 32, 566-577.
    Martinez-Ramirez, A. C., Real, M. D., 1996. Proteolytic processing of Bacillus thuringiensis CryIIIA toxin and specific binding to brush-border membrane vesicles of Leptinotarsa decemlineata (Colorado Potato Beetle). Pesticide Biochemistry and Physiology. 54, 115-122.
    Ochoa-Campuzano, C., et al., 2007. An ADAM metalloprotease is a Cry3Aa Bacillus thuringiensis toxin receptor. Biochem Biophys Res Commun. 362, 437-42.
    Sayed, A., et al., 2007. A novel cadherin-like gene from western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), larval midgut tissue. Insect Mol. Biol. 16, 591-600.
    Slaney, A. C., et al., 1992. Mode of action of Bacillus thuringiensis toxin CryIIIA: An analysis of toxicity in Leptinotarsa decemlineata (Say) and Diabrotica undecimpunctata howardi barber. Insect Biochem. Mol. Biol. 22, 9-18.
    Soberon, M., et al., 2007. Engineering modified Bt toxins to counter insect resistance. Science. 318, 1640-2.
    Vaughn, T., et al., 2005. A method of controlling corn rootworm feeding using a Bacillus thuringiensis protein expressed in maize. Crop Science. 45, 931-938.
    Walters, F. S., et al., 2008. An engineered chymotrypsin/cathepsin G site in domain I renders Bacillus thuringiensis Cry3A active against Western corn rootworm larvae. Appl Environ Microbiol. 74, 367-74.
    Xie, R., et al., 2005. Single amino acid mutations in the cadherin receptor from Heliothis virescens affect its toxin binding ability to Cry1A toxins J. Biol. Chem. 280, 8416-8425.

## Claims

1. A method of inhibiting a rootworm, said method comprising providing said rootworm with a Bacillus thuringiensis Cry protein selected from the group consisting of a Cry 3 protein, a Cry34 protein together with a Cry 35 protein, a Cry 8 protein, and a Cry 36 protein and a polypeptide comprising a segment with at least 85% sequence identity with an coleopteran midgut cadherin ectodomain that binds said Cry protein.

2. The method of claim 1, wherein said polypeptide is selected from the group consisting of SEQ ID NOS:7, 12, and 10.

3. The method of claim 1 wherein said insect is of the genus Diabrotica.

4. The method of claim 1 wherein said insect is of the genus Tenebrio.

5. The method of claim 3 wherein said insect is Diabrotica virgifera.

6. The method of claim 5 wherein said ectodomain is the CR10-12 region.