# Patent

## Title
Modified peptides having toxin-enhancing effects

## Abstract

This invention relates in part to modifying BtBooster (BtB) peptides, in part to increase their stability in insect midgut digestive juices. Some preferred embodiments of BtB have removed proteinase cleavage sites resulting in increased stability of the modified BtB in the insect gut, while retaining the ability to enhance B.t. proteins for improved insect control. In some preferred embodiments, the protease-stable BtB is used in combination with B.t. spores and/or crystals comprising a Cry protein. Also reported herein is the significant and increased enhancement of Bt toxins against relatively Bt-tolerant insects (Helicoverpa zea, Spodoptera exigua and Agrotis ipsilon), when used with BtBs. We also describe increased toxin enhancement with cadherin fragments that are stabilized against over-digestion by insect midgut proteinases. We also report enhancement of Bt Cry1F toxin by cadherin fragments.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application is a National Stage filing of PCT International Application Serial No. PCT/US2008/072812, filed 11 Aug. 2008, which claims the benefit of U.S. Provisional Application Ser. No. 60/964,249, filed Aug. 10, 2007, U.S. Provisional Application Ser. No. 60/956,618 filed Aug. 17, 2007, and U.S. Provisional Application Ser. No. 61/084,951, filed Jul. 30, 2008, the disclosures each of which are hereby incorporated by reference in their entirety, including all figures, tables and amino acid or nucleic acid sequences.


BACKGROUND OF THE INVENTION
The use of microbial insecticide in agriculture can be part of a larger integrated pest management program. Bacillus thuringiensis (Bt) based biopesticide has been proven after decades of use as a safe alternative to chemical insecticides. The insecticidal crystal proteins produced by Bacillus thuringiensis are broadly used to control insect pests with agricultural importance. Bt proteins can be used in agriculture via microbial pesticides and genetically modified crop plants.
Bt is a spore-forming, Gram-positive bacterium, that can be isolated from many environments (Chaufaux et al., 1997; Martin and Travers, 1989) and new Bt strains have been isolated from soil (Carozzi et al., 1991a; DeLucca et al., 1979; Martin and Travers, 1989; Smith and Couche, 1991), leaves (Kaelin et al., 1994; Smith and Couche, 1991), and insects (Carozzi et al., 1991b) worldwide. Bt produces one or more delta-endotoxins or Cry proteins, which form insoluble inclusions known as insecticidal crystal proteins (ICPs). Although a specific Bt toxin has a narrow spectrum of activity, many different types of Bt toxins have been characterized that have selective toxicity to different orders of insects (Schnepf et al., 1998). Bt is also the main source of genes for transgenic expression in crops to provide pest control with few or no chemical pesticide applications. However, the narrow spectrum of activity for specific Bt toxins also limits efficacy, resulting in additional chemical pesticide applications for adequate pest control.
A generally accepted mode of action for Cry toxins describes the sequential steps of protoxin activation, specific-binding, and cell toxicity (Schnepf et al., 1998). Ingested ICPs are solubilized and activated to a toxic form by the insect's digestive fluids. After crossing the peritrophic matrix, activated toxins bind to specific proteins (i.e. cadherin and aminopeptidase-N) on the midgut microvilli. A recent model (Bravo et al., 2004) proposes that monomeric toxin binds a cad glycosylphosphatidylinositolherin, facilitating further processing necessary for toxin oligomerization. Toxin oligomers have high-affinity to proteins that are attached to the cell membrane by a (GPI) anchor, such as aminopeptidase or alkaline phosphatase. This binding and the localization of GPI-anchored proteins in specific membrane regions called lipid rafts result in toxin oligomer insertion, formation of pores or ion channels, and cell death by osmotic shock. An alternative model proposes the activation of intracellular signaling pathways by toxin monomer binding to cadherin without the need of the toxin oligomerization step to cause cell death (Zhang et al., 2005). Midgut lesions caused by the toxins led to septicemia induced by midgut bacteria that eventually leads to insect death (Broderick et al., 2006).
The cadherin Bt-R1 is a receptor for Bt Cry1A toxins in midgut epithelia of tobacco hornworm (Manduca sexta). We previously identified the Bt-R1 region most proximal to the cell membrane (CR12-MPED) as the essential binding region required for Cry1Ab-mediated cytotoxicity. We also discovered that a peptide containing this region expressed in Escherichia coli functions as an enhancer of Cry1A toxicity against lepidopteran larvae (Chen et al., 2007).
US-2005-0283857-A1, U.S. Pat. No. 7,396,813, and WO 2005/07014A2 relate to the discovery and development of a Bt synergist that enhances Bt toxicity against insects that are agriculturally important pests. More specifically, these patent references relate to fragments of insect cadherins that can be used to enhance the toxicity of insecticidal crystal proteins produced by Bt some of which are commercial microbial biopesticides and some of which are expressed in transgenic plants. For ease of reference, we these peptides can be called “BtBoosters” or “BtB”. BtBooster can be mixed with commercial formulations of Bt to increase the value of the formulations. BtBooster can also be co-expressed with Bt toxin in Bt transgenic plants to offer better pest protection.
United States Patent Applications 2005010188439 (McCutcheon) relates to using a lipase polypeptide having insecticidal activity together with a Bt insecticidal protein.
The use of microbial insecticide in agriculture can be part of a larger integrated pest management program. Bacillus thuringiensis (Bt) based biopesticide has been proven after decades of use as a safe alternative to chemical insecticides. The insecticidal crystal proteins produced by Bacillus thuringiensis are broadly used to control insect pests with agricultural importance. Bt proteins can be used in agriculture via microbial pesticides and genetically modified crop plants.
Bt is a spore-forming, Gram-positive bacterium, that can be isolated from many environments (Chaufaux et al., 1997; Martin and Travers, 1989) and new Bt strains have been isolated from soil (Carozzi et al., 1991a; DeLucca et al., 1979; Martin and Travers, 1989; Smith. and Couche, 1991), leaves (Kaelin et al., 1994; Smith and Couche, 1991), and insects (Carozzi et al., 1991b) worldwide. Bt produces one or more delta-endotoxins or Cry proteins, which form insoluble inclusions known as insecticidal crystal proteins (ICPs). Although a specific Bt toxin has a narrow spectrum of activity, many different types of Bt toxins have been characterized that have selective toxicity to different orders of insects (Schnepf et al., 1998). Bt is also the main source of genes for transgenic expression in crops to provide pest control with few or no chemical pesticide applications. However, the narrow spectrum of activity for specific Bt toxins also limits efficacy, resulting in additional chemical pesticide applications for adequate pest control.
A generally accepted mode of action for Cry toxins describes the sequential steps of protoxin activation, specific-binding, and cell toxicity (Schnepf et al., 1998). Ingested ICPs are solubilized and activated to a toxic form by the insect's digestive fluids. After crossing the peritrophic matrix, activated toxins bind to specific proteins (i.e. cadherin and aminopeptidase-N) on the midgut microvilli. A recent model (Bravo et al., 2004) proposes that monomeric toxin binds a cad glycosylphosphatidylinositolherin, facilitating further processing necessary for toxin oligomerization. Toxin oligomers have high-affinity to proteins that are attached to the cell membrane by a (GPI) anchor, such as aminopeptidase or alkaline phosphatase. This binding and the localization of GPI-anchored proteins in specific membrane regions called lipid rafts result in toxin oligomer insertion, formation of pores or ion channels, and cell death by osmotic shock. An alternative model proposes the activation of intracellular signaling pathways by toxin monomer binding to cadherin without the need of the toxin oligomerization step to cause cell death (Zhang et al., 2005). Midgut lesions caused by the toxins led to septicemia induced by midgut bacteria that eventually leads to insect death (Broderick et al., 2006).
The cadherin Bt-R1 is a receptor for Bt Cry1A toxins in midgut epithelia of tobacco hornworm (Manduca sexta). We previously identified the Bt-R1 region most proximal to the cell membrane (CR12-MPED) as the essential binding region required for Cry1Ab-mediated cytotoxicity. We also discovered that a peptide containing this region expressed in Escherichia coli functions as an enhancer of Cry1A toxicity against lepidopteran larvae (Chen et al., 2007).
US-2005-0283857-A1, U.S. Pat. No. 7,396,813, and WO 2005/07014A2 relate to the discovery and development of a Bt synergist that enhances Bt toxicity against insects that are agriculturally important pests. More specifically, these patent references relate to fragments of insect cadherins that can be used to enhance the toxicity of insecticidal crystal proteins produced by Bt some of which are commercial microbial biopesticides and some of which are expressed in transgenic plants. For ease of reference, these peptides can be called “BtBoosters” or “BtB”. BtBooster can be mixed with commercial formulations of Bt to increase the value of the formulations. BtBooster can also be co-expressed with Bt toxin in Bt transgenic plants to offer better pest protection.
United States Patent Applications 2005010188439 (McCutcheon) relates to using a lipase polypeptide having insecticidal activity together with a Bt insecticidal protein.
WO 03/018810 (by Syngenta) discusses some possibilities for adding Western corn rootworm (WCRW) cathepsin G favored sites (AAPF, AAPM, AVPF, PFLF) to B.t. Cry3A proteins.
BRIEF SUMMARY OF THE INVENTION
This invention relates in part to the modification of BtBoosters (BtB).
The subject invention relates in part to the discovery and demonstration that derivatives of BtBooster (BtB) have enhanced potentiating activity of various Bt products, including the commercial Bt sprayable products “Javelin®,” “Dipel®,” “Xentari®” and “Agree®” in plant-based bioassays and commercial Bt cotton “Bollgard I.”
This invention also relates in part to the modification of BtBooster to increase its stability in insect midgut digestive juices. Some preferred embodiments of BtB have removed proteinase cleavage sites resulting in increased stability of the modified BtB in the insect gut, while retaining the ability to enhance B. t. proteins for improved insect control.
In some preferred embodiments, the protease-stable BtB is used in combination with B.t. spores and/or crystals comprising a Cry protein. In some of these embodiments, a preferred modified BtB is derived from cadherin repeat 12 (CR12) from BT-R1a. Hua et al. (Hua et al., 2004a).
The subject invention also relates in part to the discovery that derivatives of BtB have enhanced potentiating activity of various Bt Cry proteins against important pest species including Helicoverpa zea, Agrotis ipsilon, Spodoptera exigua and Spodoptera frugiperda. 
The subject invention also relates in part that derivatives of BtB have potentiating activity for Cry1Fa protein.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 illustrates the wild-type BtR1 cadherin of M. sexta and the cadherin fragments Ms-CR12-MPED, Ms-CR9-MPED (BtB1), Ms-CR12 (BtB2), the protease-stabilized version Ms-CR12(PS) also called BtB3, Ms-CR10-12 (BtB4) and the proteinase-stabilized Ms-CR10-12(PS) fragment called Bt135 that were tested successfully. FIG. 1 also illustrates the wild type cadherin from Spopdoptera frugiperda and the cadherin fragments Sf-CR10-12 (BtB9) and the protease-stabilized version Sf-CR10-12(PS) (BtB10).
FIG. 2 shows that longer cadherin fragments (Ms-CR7-12, Ms-Cr9-12, and MsCR10-12) enhanced Cry1Ac to a greater extent than did Ms-CR11-12. More specifically, this Figure shows M. sexta cadherin fragments enhanced Cry1Ac in diet overlay bioassay with neonate H. zea. Cry1Ac toxin was mixed with purified inclusion bodies of Ms-CR7-12, Ms-CR9-12, Ms-CR10-12, or Ms-CR11-12 at a fixed toxin to synergist mass ratio of 1:20 and then overlaid on the diet surface.
FIG. 3 demonstrates the increased Cry1Ac toxin enhancing properties of Ms-CR9-MPED and the protease-stabilized Ms-CR12(PS) relative to the Ms-CR12 cadherin fragment. This was a diet surface treatment bioassay with H. zea larvae. More specifically, this Figure shows diet surface contamination using purified Cry1Ac with or without soluble peptides on FL zea neonates. Mass ratio of toxin to BtB was 1:20. Mortality was scored on day 7. BtB1=Ms-CR9-MPED. BtB2=Ms-CR12. BtB3 Ms-CR12(PS).
FIG. 4 shows that the protease-stabilized Ms-CR12(PS) called BtB enhances to preparations of Bt spores and crystals. In FIG. 4A tomato leaves were dipped into suspensions of Bt NRD12 in diluent or Bt NRD12 with BtB1, BtB2 or BtB3. In FIG. 4B tomato leaves were dipped into a formulation of a commercial Bt called Javelin WG® (Certis) without or with BtB1, BtB2 or BtB3. The protease-stabilized Ms-CR12 cadherin fragment called Ms-CR12(PS) or BtB3 enhanced the toxicity of the Bt to H. zea larvae. FIG. 4A shows that tomato leaves were dipped into a suspension of Bt strain NRD12 and Bt plus BtB1, BtB2 or BtB3. Leaves were fed to 2nd instar H. zea larvae. Mortality was scored on day 2. BtB1 Ms-CR9-MPED; BtB2=Ms-CR12; BtB3=Ms-CR12 (PS). FIG. 4B shows that tomato leaves were dipped into a suspension of Bt (Javelin WG) and Bt plus BtB2 or BtB3. Leaves were fed to 2nd instar H. zea larvae. Mortality was scored on day 4. BtB2=Ms-CR12. BtB3=Ms-CR12 (PS).
FIG. 5 illustrates the results of leaves from 4-5 weeks old B. t. cotton that were dipped in BtB inclusion bodies, air dried, and then fed to 2nd instar H. zea larvae. This bioassay was scored on the fourth day. As shown, mortality was significantly increased when the leaves were treated with either BtB2 (Ms-CR12) or BtB3 Ms-CR12(PS)]. Helicoverpa zea larvae were allowed to feed on Bollgard I (BG) leaves or leaves dipped into BtB inclusion body suspensions. Mortality was scored after 4 days. BtB2=Ms-CR12. BtB3=Ms-CR12 (PS).
FIG. 6A shows that a Bt-resistant strain of P. xylostella, based on a diet incorporation bioassay, was about 128-fold more susceptible to Agree WG than to Javelin WG. This was expected since the resistant insects were selected on Bt HD-1, which produces the same Cry toxins present in Javelin WG. Agree WG, however, contain Cry1C which has been shown to have very little cross resistance to Cry1A toxins and is highly active against P. xylostella (Tang et al., 1996). Both BtB2 and BtB3 inclusion bodies were able to significantly enhance Javelin WG® (Certis) (Jav) and Agree WG (Agr) against a Bt-resistant strain of P. xylostella (FIGS. 6B and 6C).
FIG. 7 shows that BtB4 (Ms-CR10-12) and BtB5 [Ms-CR10-12(PS)] enhanced Cry1Ac against H. zea (corn earworms) at low Bt:BtB Mass Ratios. Diet surface treatment bioassays were used to expose neonate H. zea (corn earworm) to increasing mass ratios of BtB to trypsin-activated Cry1Ac. Sample size: 32 larvae/replicate×3 replicates/treatment. The bioassay scored on day 7. BtB4 (CR10-12) and BtB5 were purified using Ni-column and dialysed in 10 mM Tris, 100 mM NaCl at pH 8.0. The results show that both BtB4 and BtB5 enhanced Cry1Ac toxicity to H. zea larvae at low Cry toxin to BtB ratios. Treatments included Cry1Ac toxin alone or Cry1Ac plus the indicated ratios of BtB4 or BtB5. Control was diluent only.
FIG. 8 shows the results of diet surface bioassays against neonate S. exigua. Sample size: 32 larvae/rep×2 rep/treatment. Bioassay was scored on day 7. Purified trypsin-activated Cry1Ab at 2 μg/cm2, Cry1Ac at 4 μg/cm2, and Ni-column purified BtBs at the indicated mass ratios were used in the bioassay. Diet surface treatment bioassay with neonate S. exigua. Sample size: 32 larvae/rep×2 rep/treatment. Bioassay was scored on day 7. Purified trypsin-activated Cry1Ab at 2 μg/cm2, Cry1Ac at 4 μg/cm2, and Ni-column purified BtBs at the indicated mass ratios were used in the bioassay.
FIG. 9 shows that BtB4 (Ms-CR10-12) and BtB5 [Ms-CR10-12(PS)] at low Cry toxin to BtB mass ratios (1:2) lowered the LC50 of Cry1Ac to H. zea (corn earworm, cotton bollworm). Diet surface treatment bioassay with neonate H. zea (corn earworm) to determine LC50 with out and with BtB4 and BtB5. Sample size: 32 larvae/replicate×3 replicates/treatment. Bioassay scored on day 7. BtB4 (CR10-12) and BtB5 were purified using Ni-column and dialysed in 10 mM Tris, 100 mM NaCl at pH 8.0. LC50 for: 1) Cry1Ac alone=0.54 (0.43-0.68) μg/cm2 and 2) Cry1Ac+BtB4=0.07 (0.06-0.09) μg/cm2 estimated (7.7-fold decrease) and 3) Cry1Ac+BtB5=0.04 (0.02-0.07) μg/cm2 estimated (13.5-fold decrease).
FIG. 10A illustrates the results of a cabbage excised-leaf bioassay with 4-day old H. zea showing enhancement of formulated Bt (Javelin WG® Certis) by BtB5 (Ms-CR10-12(PS)] inclusion bodies. Sample size: 30 larvae×3 replicates/treatment. Bioassay was scored on Day 4. Bioassay was done with fixed 1:10 mass ratio of Javelin:BtB. FIG. 10B shows the results of a cabbage excised-leaf bioassay with 4-day old H. zea showing enhancement of formulated Bt (DiPel DF® Valent) by a spray-dried preparation of BtB5 [Ms-CR10-12(PS)]. Sample size: 30 larvae/rep×4 rep/treatment. Bioassay was scored on day 4. Bioassay was done with fixed 1:6 mass ratio of DiPel:BtB. The amount of Ms-CR10-12(PS) in the spray-dried preparation was estimated at 22% of the dry weight by immunological detection (Western blot).
FIG. 11A shows that M. sexta cadherin CR10-12 (BtB4) fragment enhanced Cry1Ac in diet overlay bioassay with neonate A. ipsilon. Cry1Ac toxin was mixed with purified inclusion bodies of Ms-CR10-12 at a fixed toxin to synergist mass ratio of 1:10 and then overlaid on the diet surface. Control treatments with buffer and the synergist alone were not toxic to the larvae. Each data point represents data for the mean±standard errors from three replicate bioassays with 64 larvae per dose. LC50s (μg/cm2, 95% fiducial limits are provided in parentheses where applicable): (i) Cry1Ac alone=19.3 (17.9−20.9); (ii) Cry1Ac+CR10-12= ̃2.0 ( ̃10-fold enhancement). FIG. 11B M. sexta cadherin Ms-CR10-12 fragment (BtB4) enhances Cry1Ab in diet overlay bioassay with neonate S. exigua. Cry1Ab toxin was mixed with purified inclusion bodies of Ms-CR10-12 at a fixed toxin to synergist mass ratio of 1:5 and then overlaid on the diet surface. Control treatments with buffer and the synergist alone were not toxic to the larvae. Each data point represents data for the mean±standard errors from three replicate bioassays with 64 larvae per dose. LC50s (μg/cm2, 95% fiducial limits are provided in parentheses where applicable): (i) Cry1Ab alone= ̃7.0; (ii) Cry1Ab+CR10-12=1.1 (0.9-1.3) ( ̃6-fold enhancement).
FIG. 12 illustrates enhancement of Cry1C toxin by Ms-CR10-12 (BtB4) cadherin fragment in diet overlay bioassay with neonate S. exigua. A fixed amount of Cry1Ca toxin (0.6 μg/cm2) was mixed with purified inclusion bodies of CR10-12 at increasing toxin to synergist mass ratio of 1:1, 1:5, 1:10, 1:50, and 1:100, and then overlaid on the diet surface. Each column represents data for the mean±standard errors from four replicates with 64 larvae per treatment. Treatments: (A) Cry1Ca alone; (B) Cry1Ca+synergist (1:1 ratio); (C) Cry1Ca+synergist (1:5 ratio); (D) Cry1Ca+synergist (1:10 ratio); (E) Cry1Ca+synergist (1:50 ratio); (F) Cry1Ca+synergist (1:100 ratio).
FIG. 13 shows the results of an H. zea diet surface bioassay. Cry1Ab or Cry1Fa toxins were applied alone or with BtB5. BtB5=Ms-CR10-12(PS). This Figure shows that BtB5 substantially shifted the Cry1Ab and Cry1Fa LC50, for H. zea (corn earworm, cotton bollworm). Estimated LC50s Cry1Ab LC50=6.37 (5.03-9.58) μg/cm2; Cry1Ab+BtB5 LC50=0.47 (0.31-0.89) μg/cm2; Cry1Fa LC50=5.53 (4.26-8.74) μg/cm2; Cry1Fa+BtB5 LC50=0.62 (0.52-0.79) μg/cm2.
FIG. 14 shows the results of an S. exigua diet surface bioassay. Cry1Ab or Cry1Fa toxins were applied alone or with BtB5. BtB5=Ms-CR10-12(PS). This Figure shows that BtB5 substantially shifted the Cry1Ab and Cry1Fa LC50s for S. exigua. Diet surface treatment bioassay with neonate S. exigua. Sample size: 32 larvae/rep×2 rep/treatment. Bioassay was scored on Day 5. The addition of BtB5 to Cry1Ab or Cry1Fa significantly shifted the dose response curve to the left. Cry1Ab LC50=not determined. Cry1Ab+BtB5 LC50=2.71 (2.30-3.35) μg/cm2; Cry1Fa LC50=1.82 (1.63-2.04) μg/cm2; Cry1 Fa+ BtB5 LC50=0.66 (0.60-0.72) μg/cm2.
FIG. 15. BtB5 [Ms-CR10-12(PS)] enhanced a mixture of Cry1Ab and Cry1Fa against neonate corn earworms in a diet surface treatment bioassay with neonate H. zea. Sample size: 32 larvae/rep×2 rep/treatment. Bioassay was scored on Day 5. Addition of BtB5 to Cry1Ab or mixture of Cry1Ab and Cry1Fa shifted the dose response curve to the left. Cry1Ab estimated LC50=5.30 (4.59-6.30) μg/cm2; Cry1Fa estimated LC50=5.40 (3.65-11.58) μg/cm2. Cry1Ab+BtB5 estimated LC50=0.48 (0.35-0.78) μg/cm2; Cry1Fa+BtB5 was not tested. Mixture of Cry1Ab+Cry1Fa estimated LC50=1.80 μg/cm2. Cry1Ab Cry1Fa+BtB5 estimated LC50=0.19 μg/cm2.
FIG. 16. A combination of BtBs enhanced a mixture of Cry1Ac and Cry1Fa against neonate corn earworms. Diet surface treatment bioassay with neonate H. zea. Sample size: 32 larvae/rep×2 rep/treatment. Bioassay was scored on Day 7. *Addition of BtB5 and BtB9 to Cry1Ac+Cry1Fa enhanced toxicity significantly. Legend for treatments: (a). 0.025 μg/cm2 Cry1Ac+0.125 μg/cm2 Cry1Fa (1:5 ratio). (b). 0.05 μg/cm2 Cry1Ac+0.25 μg/cm2 Cry1Fa (1:5 ratio). (c). 0.10 mg/cm2 Cry1Ac+0.50 μg/cm2 Cry1Fa (1:5 ratio). (d). 0.025 μg/cm2 Cry1Ac+0.125 μg/cm2 Cry1Fa+0.125 μg/cm2 BtB5+0.125 μg/cm2 BtB9. (e). 0M5 μg/cm2 Cry1Ac+0.25 μg/cm2Cry1Fa+0.25 μg/cm2BtB5+0.125 μg/cm2 BtB9. (f). 0.10 μg/cm2Cry1Ac+0.50 μg/cm2 Cry1Fa+0.50 μm/cm2 BtB5+0.125 μg/cm2 BtB9.
FIGS. 17A and 17B. In diet surface treatment bioassay with neonate S. frugiperda data provide evidence that protease-stabilization made BtB10 [S f-CR10-12(PS)] an improved enhancer relative to BtB9 (Sf10-12) or BtB5 [(Ms-CR10-12(PS)]. A 10-fold mass ratio of BtB10 was the best enhancer ( ̃4-fold enhancement) compared to BtB9 ( ̃2-fold enhancement) and BtB5 (<2-fold enhancement, not significant). Diet surface treatment bioassay with neonate S. frugiperda. Sample size: 32 larvae/rep×2 rep/treatment. Bioassay was scored on Day 5. Total larval weight of survivors per replicate for each treatment was measured and then averaged. FIG. 17A shows the mortality data and FIG. 17B the larval weight data. *Significant enhancement compared to Cry1Fa (treatment A) (P<0.05). BtB10 (stabilized version of SfCR10-12) has better enhancement at 1:10 mass ratio compared to Sf10-12 or BtB5. The arrow in FIG. 17B designates the very significant growth inhibition enhancement by BtB10. Legend: A, 0.1 ug/cm2 Cry1Fa. B, 0.1 μg/cm2 Cry1Fa+0.1 μg/cm2 BtB9 (1:1). C, 0.1 μg/cm2 Cry1Fa+0.5 μg/cm2BtB9 (1:5). D, 0.1 μg/cm2 Cry1Fa+1.0 μg/cm2 BtB9 (1:10). E, 0.1 μg/cm2Cry1Fa+5.0 μg/cm2 BtB9 (1:50). F, 0.1 μg/cm2 Cry1Fa+0.1 μg/cm2 BtB10 (1:1). G, 0.1 μg/cm2 Cry1Fa+0.5 μg/cm2 BtB10 (1:5). H, 0.1 μg/cm2 Cry1Fa+1.0 μg/cm2 BtB10 (1:10). I , 0.1 μg/cm2 Cry1Fa+5.0 μg/cm2 BtB10 (1:50). J, 0.1 μg/cm2 Cry1Fa+0.1 μg/cm2 BtB5 (1:1). K, 0.1 μg/cm2 Cry1Fa+0.5 μg/cm2 BtB5 (1:5). L, 0.1 μg/cm2 Cry1Fa+1.0 μg/cm2 BtB5 (1:1.0). M, 0.1 μg/cm2 Cry1Fa+5.0 μg/cm2BtB5 (1:50). N, 5.0 μg/cm2 BtB9. O, 5.0 μg/cm2BtB10. P, 5.0 μg/cm2 BtB5. Q, dH2O
FIG. 18 shows increased stability of Cry1Ab in S. exigua beet armyworm gut extract in the presence of BtB4 or BtB5. Cry1Ab toxin (10 μg) was preincubated with equal mass of BSA, BtB4, or BtB5 for 30 min at 30° C. (pH 10) before incubation with S. exigua gut extract for 1 h at 30° C. (pH 10). Preincubation with either BtB4 or BtB5 protected the toxin from further proteolytic degradation by the gut extract (lanes 4 and 5) compared to preincubation with BSA (lane 3). Samples were treated with 5.5× protease inhibitor cocktail (Roche Complete) and then heated at 95° C. for 5 min to stop the reaction. Samples were mixed with SDS loading buffer and reheated again prior to loading onto SDS-PAGE, then transferred to a membrane. Cry1Ab was detected with anti-Cry1Ac antibody. The increased stability of toxin in the gut due to BtB presence could be a significant part of the mode of action for BtB.
FIG. 19A shows the mortality of Bt-resistant P. xylostella larvae feeding on leaves of Arabidopsis plant lines containing Cry1Ac protein and Cry1Ac plus BtB I or BIB3. Plants were transformed with a T-DNA vector containing a plant expression cassette directing the expression of a toxic Bt cry1Ac protein. Some Arabidopsis plants were co-transformed with a T-DNA vector with a plant expression cassette for BtB3 (Ms-CR9-MPED) and BtB3 [Ms-CR12(PS)]. The P. xylostella larvae were from a Bt-resistant colony (Benzon Research). Replicates consisted of 20+ cups containing a single Arabidopsis plant (foliage only) with five 4-day onl P. xylostalla larvae. Mortality was determined on day 4. FIG. 19B Cry1Ac levels in assayed Arabidopsis lines were determined from Western blot analysis and normalized to PEP carboxylase.
FIG. 20 shows a protein sequence alignment of the CR12 region from ten different lepidopteran species (Ostrinia nubilalis (European Corn Borer) (SEQ NO:36), Chilo suppressalis (Striped Rice Borer) (SEQ ID NO:35), M. sexta (Tomato Hornworm) (SEQ ID NO:38), P. xylostella (Diamondback Moth) (SEQ ID NO:37), Pectinophora gossypiella (Pink Bollworm) (SEQ ID NO:32), Lymantria dispar (Gypsy Moth) (SEQ ID NO: 31), Bombyx mori (Silkworm) (SEQ ID NO:39), Helicoverpa armigera (Cotton Bollworm) (SEQ ID NO:33), Heliothis virescens (Tobacco Budworm) (SEQ ID NO:34) and S. frugiperda (Fall Armyworm) (SEC) ID NO:40).
SEQ ID NO: 1 is the polynucleotide sequence of Ms-CR9-MPED also called BtB1.
SEQ ID NO:2 is the amino acid sequence of Ms-CR9-MPED (BtB1).
SEQ ID NO:3 is the nucleotide sequence of Ms-CR9-MPED (BtB1).
SEQ ID NO:4 is the polynucleotide sequence that encodes the amino acid sequence of Ms-CR12 also called BtB2—a 12 kDa peptide representing CR12. An N-terminal methionine and C-terminal 6× histidine residues were added for protein expression and purification.
SEQ ID NO:5 is the amino acid sequence of Ms-CR12 (BtB2).
SEQ ID NO:6 is the nucleotide sequence of Ms-CR12 (BtB2).
SEQ ID NO:7 is the polynucleotide sequence of Ms-CR12(PS) also called BtB3—a modified Ms-CR12 with increased protease stability.
SEQ ID NO:8 is the amino acid of Ms-CR12(PS), also called BtB3—a 12 kDa peptide of modified Ms-CR12 with increased protease stability.
SEQ ID NO:9 is the nucleotide sequence of Ms-CR12(PS), also called BtB3.
SEQ ID NO:10 is the polynucleotide that encodes the amino acid sequence ofMs-CR10-12, also called BtB4. Six histidines were added to the C-terminus of the peptide for ease of protein purification.
SEQ ID NO:11 is the amino acid sequence of Ms-CR10-12, also called BtB4.
SEQ ID NO:12 is the nucleotide sequence of Ms-CR10-12, also called BtB4.
SEQ ID NO:13 is the polynucleotide sequence of Ms-CR10-12(PS), also called BtB5—a protease-stabilized version of Ms-CR10-12
SEQ ID NO:14 is the amino acid sequence of Ms-CR10-12(PS), also called BtB5—the protease stabilized version of CR10-12
SEQ ID NO:15 is the nucleotide sequence of Ms-CR10-12(PS), also called BtB5
SEQ ID NO:16 is the polynucleotide sequence of Ms-CR7-12
SEQ ID NO:17 is amino acid sequence of Ms-CR7-12
SEQ ID NO:18 is nucleotide sequence of Ms-CR7-12
SEQ ID NO:19 is the polynucleotide sequence of Ms-CR9-12
SEQ ID NO:20 is amino acid sequence of Ms-CR9-12
SEQ ID NO:21 is nucleotide sequence of Ms-CR9-12
SEQ ID NO:22 is the polynucleotide sequence of Ms-CR11-12
SEQ ID NO:23 is amino acid sequence of Ms-CR11-12
SEQ ID NO:24 is nucleotide sequence of Ms-CR11-12
SEQ ID NO:25 is the polynucleotide sequence that encodes the amino acid sequence of Sf-CR10-12, also called BtB9. Sf-CR10-12 was designed from cadherin repeats 10-12 of S. frugiperda cadherin. Codons were optimized for E. coli expression. Several CG dinucleotides were changed for plant expression. An N-terminal methionine and a lysine residue was added at amino acid position 2.
SEQ ID NO:26 is the amino acid sequence of Sf-CR10-12, also called BtB9
SEQ ID NO:27 is nucleotide sequence of Sf-CR10-12, also called BtB9
SEQ ID NO:28 is the polynucleotide sequence of Sf-CR10-12(PS), also called BtB10. A protease-stabilized version of Sf-CR10-12.
SEQ ID NO:29 is the amino acid sequence of Sf-CR10-12(PS), also called BtB10
SEQ ID NO:30 is nucleotide sequence of Sf-CR10-12(PS), also called BtB10



DETAILED DESCRIPTION OF THE INVENTION
The subject invention relates in part to versions of BtBoosterTM (BtB) that have even better enhancement properties than the originals tested. For example, BtB versions enhanced the Certis USA product Javelin® WG (contains the Bt NRD12 strain) in tomato excised-leaf bioassays against Helicoverpa zea. Additional excised-leaf bioassays using soybean and cabbage consistently demonstrated that BtBoosterTM significantly enhanced Javelin® WG. Bioassays with Javelin® WG plus BtBoosterTM against resistant P. xylostella larvae consistently showed that the addition of BtBoosterTM to the biopesticide significantly enhanced mortality in both excised-leaf and whole plant greenhouse experiments.
The BtB version described in (Chen et al., 2007) is comprised of the CR12-MPED region of M. sexta cadherin BtR1a. Our analysis of diet surface bioassays against H. zea showed that Ms-CR10-12 (BtB4) was significantly better in enhancing trypsin-activated Cry1Ac. We discovered that although BtB4 enhanced Cry toxins, the BIB4 was rapidly degraded by midgut proteases and that a protease-stabilized version, called BtB5 had improved enhancement of BE spores and crystals.
The original BtB is a truncated cadherin peptide derived from Bt-R1a. Three modified versions of BtB were initially generated: i) Ms-CR9-MPED, also called BtB1—a 60 kDa peptide representing cadherin repeat (CR) 9 to the membrane proximal domain, ii) Ms-CR12, also called BtB2—a 12 kDa peptide representing CR12, and iii) Ms-CR12, also called BtB3—a 12 kDa peptide of modified CR12 with increased protease stability. Additional versions of BtB, Ms-CR10-12 (also called BtB4) and the Ms-CR10-12(PS), also called BtB5 (FIG. 1) were also developed and tested successfully.
Our analysis of diet-surface bioassays with Ms-CR9-MPED also called BtB1 (consisted of cadherin repeats (CR) 9-MPED; 28-fold enhancement of Cry1Ac) was significantly better than the original BtBooster (CR12-MPED). Other Manduca cadherin peptides, including Ms-CR7-12, MsCR9-12 and MsCR10-12 had greater enhancement of Cry1Ac toxicity to H. zea than Ms-CR11-12 (FIG. 2) and Ms-CR12 (BtB2). A protease-stabilized Ms-CR12, called Ms-CR12(PS) or BtB3, had improved enhancement with trypsin-activated Bt Cry1Ac toxin against H. zea (FIG. 3). Initial work showed the non-protease stabilized BIB versions such as BtB1 (Ms-CR9-MPED) and BtB2 (Ms-CR12) apparently did not enhance Bt spores and crystals (FIGS. 4A and 4B). Initial work demonstrated that some BtB molecules were unstable under certain conditions, thus reducing their levels of potentiation in those conditions.
BtB3 called Ms-CR12(PS), for the protease-stabilized version of pMs-CR12, was designed to have a reduced number of insect gut protease sensitive sites (specifically trypsin) without unduly increasing stability to human digestive enzymes. This version was tested in tomato excised-leaf bioassays with both a preparation of Bt NRD12 spores and crystals and a formulation of Javelin® (Certis). We found that BtB3 was able to enhance Bt spores and crystals toxicity (FIGS. 4A and 4B). We also found that BtB2 and BtB3 were able to increased mortality of H. zea feeding on Bt cotton (Bollgard I) (FIG. 5). BtB2 and BtB also enhanced Javelin® and Agree® in diet-incorporation bioassays against Bt-resistant Plutella xylostalla larvae (FIGS. 6A and 6B). Since we observed that Ms-CR10-12 had greater Cry toxin enhancing properties than Ms-CRL 2 and MsCR11-12, we designed a protease-stabilized version of Ms-CR10-12 called MsCR10-12(PS) or BtB5. Also, we demonstrated that BtB5 was able to enhance Javelin® against H. zea in cabbage leaf bioassays which. BtB4 did not do (FIGS. 10A and 10B). BtB4 and BtB5 also enhanced other toxins including Cry1Ab and Cry1Fa (these are important toxins because they are in Bt corn) (FIGS. 8, 13, 14, 15, 16). Also, BtB5 enhanced Cry1Fa toxicity to S. frugiperda (Fall armyworm) larvae (FIGS. 17A an 17B). This is a significant development since Fall armyworm is hard to control with Bt, and control of this important pest would improve its Bt's utility as an alternative to chemical control.
Since Fall armyworm is an important lepidopteran pest that is killed by only a few Bt toxins, we reasoned that a BtBooster designed from it's midgut cadherin may have useful and improved toxin-enhancing properties. We designed and expressed Sf-CR10-12 (BtB9) and a protease-stabilized version Sf-CR10-12(PS). Both Sf cadherin peptides enhanced Cry toxicity to Fall armyworm (FIGS. 17A and 17B).
In summary, our results further demonstrate the advantage of applying BtB plus Bt for controlling important agricultural insect pests (i.e. H. zea, S. exigua, S. frugiperda and Bt-resistant P. xylostella).
We also developed a modified BtB (BtB5) that works better than a previous version (BtB3) with a Bt spores and crystals formulation and extends insect control to Spodoptera species and Bt-resistant insects. Performance of BtB5 is being tested in field trials. We also developed other BtBs (BtB9 and BtB10) from S. frugiperda cadherin that have increased enhancing properties for certain Cry1 toxins.
Background on Receptor Bt-R1.
The cadherin protein Bt-R1 from Manduca sexta binds Cry1Aa, Cry1Ab and Cry1Ac toxins on ligand blots (Francis and Bulla, 1997). Purified membranes from COS cells expressing Bt-R1 bound all three Cry1A toxins in binding assays and ligand blots (Keeton and Bulla, 1997). Furthermore, expression of Bt-R1 on the surface of COS7 cells led to toxin-induced cell toxicity as monitored by immunofluorescence microscopy with fixed cells (Dorsch et al., 2002). The Bt-R1 protein has been suggested to induce a conformational change in Cry1Ab that allows the formation of a pre-pore toxin oligomer (Gomez et al., 2002). In Bombyx mori, the cadherin-like protein BtR175 serves as a Cry1Aa receptor (Nagamatsu et al., 1998). Sf9 cells expressing BtR175 swell after exposure to Cry1Aa toxin, presumably due to formation of ion channels in cell membranes (Nagamatsu et al., 1999).
Another cadherin protein was cloned from M. sexta called Bt-R1a (Hua et al., 2004b). Bt-R1a cDNA differs from Bt-R1 by 37 nucleotides that altered two amino acids.
In some examples, BtB is a truncated cadherin peptide derived from Bt-R1a. Three versions of BtB were generated, as discussed above. “BtB1” as referred to herein is a 60 kDa peptide representing cadherin repeat (CR) 9 to the membrane proximal domain. The sequence of BtB1 is provided as SEQ ID NO:1.
Use in Spores and Crystals Formulations.
As discussed in more detail below, it is interesting to note that although BtB1 can enhance purified activated Cry1Ac toxin, it was unable to enhance Bt (NRD12) spores and crystals preparation containing Cry1Aa, Cry1Ab, Cry1Ac, Cry2A and Cry2B crystal proteins when applied to plant surfaces. Since the crystals need to be dissolved and activated in the midgut of target insects, the BtB peptides need to be stabilized from protease degradation so that sufficient BtB remains to react with the activated toxins. Site-directed mutagenesis was done sequentially to remove trypsin and chymotrypsin recognition sequence from BtB2 to create BtB3. BtB2 (SEQ ID NO:4) is a 12 kDa peptide representing CR12, and BtB3 (SEQ ID NO:7) is a 12 kDa peptide of modified CR12 with increased protease stability. Embodiments of modified BtBs according to the subject invention can have at least one trypsin and/or chymotrypsin recognition site removed (by removal or by modification to change the site so that it is no longer recognized by one or more proteases. BtB5 (SEQ ID NO:13) and BtB10 (SEQ ID NO:27) are additional examples of modified BtBs having at least one trypsin and/or chymotrypsin recognition site removed.
Overview of some Experimental Procedures.
BtB was cloned by PCR and inserted into an Escherichia coli expression vector and highly expressed in the cells as inclusion bodies. The inclusion bodies were highly soluble in alkaline buffer (pH 9-12). Both inclusion bodies (FIG. 2) and solubilized BtB (FIG. 3) were successful in enhancing purified Cry1Ac when tested in diet-based bioassays with neonates of H. zea. This is an advantage since BtB did not require further physical or chemical treatment for it to perform as a synergist.
BtB was expressed in E. coli as insoluble inclusion bodies. However, the inclusion bodies were highly soluble in alkaline buffers. This suggests that the inclusion bodies would be soluble in the alkaline environment of the lepidopteran larval midgut. Since the BtB was expressed at very high level in E. coli, the subject invention provides for cost-effective production of BtB for addition to B.t. as an adjuvant or as part of the formulation.
BtB Enhanced Purified Bt Toxin.
In some tests showing the enhancing activity of BtB, purified Cry1Ac toxin that was preactivated with trypsin, and was mixed with BtB inclusion bodies. Toxin to BtB1 mass ratio of 1:20 was maintained through serial dilution with distilled water and applied on the diet surface and allowed to air-dry. Bioassays with H. zea neonates were scored on the seventh day, and the percentage of larval mortality was determined (FIG. 2). The LC50 of the toxin-only treatment and the toxin-BtB1 mixture treatment were calculated by the probit method (SoftTOX ver. 1.1). BtB1 enhanced Cry1Ac by about 28-fold.
In another bioassay, various length BtB peptides were tested for their ability to enhance purified Cry1Ac toxin. BtB inclusion bodies were mixed with purified Cry1Ac toxin and was then applied on the diet surface. Toxin to BtB was maintained at a mass ratio of 1:20. In this bioassay (FIG. 2), Cadherin fragments longer than Ms-CR11-12 enhanced Cry1Ac better than MsCR11-12. This suggests that longer-length cadherin fragments might enhance the synergistic effect. The BtB proteins did not show any toxic effect on the larvae when applied alone.
BtB Enhanced B.t. Spores and Crystals.
Most Bt-based biopesticides contain spores and crystals of the bacteria as the active ingredient. B.t. subsp. kurstaki (NRD12) is the active ingredient of Javelin WG (Certis). The formulation contains several different Cry toxins: Cry1Aa, Cry1Ab, Cry1Ac, Cry2Aa, and Cry2Ab. Spores and crystals suspension from this bacterium was prepared and tested with BtB inclusion bodies in tomato leaf dip bioassay. These leaves were fed to 2nd instar H. zea larvae and the bioassay was scored on the second day. The bioassay results show that only BtB3 the protease-stabilized Ms-CR10-12(PS) significantly enhanced NRD12 (FIG. 4A).
In another leaf dip bioassay, Javelin WG was mixed with either BtB2 or BtB3 and applied on the leaves. These leaves were fed to 2nd instar H. zea larvae, and the bioassay was scored on the fourth day. The results show that only BtB3 significantly enhanced Javelin WG against the larvae (FIG. 4B). This result is significant because it showed that BtB3 was able to enhance a commercial Bt formulation which contains inert materials that might have interfered with the synergist.
BtB Enhanced B.t. Cotton.
Transgenic Cry1Ac B.t. cotton (BollgardTM, Monsanto Co., St. Louis, Mo.) has been available commercially since 1996. Although it is highly efficient in controlling H. virescens and P. gossypiella, supplemental foliar spray is needed to control H. zea (Adamczyk et al., 2001). Leaves from 4-5 weeks old B.t. cotton were dipped in BtB inclusion bodies and air dried. These leaves were fed to 2nd instar H. zea larvae, and the bioassay was scored on the fourth day (FIG. 5). Mortality was significantly increased when the leaves were treated with either BtB2 or BtB3 (P<0.05).
BtBooster Enhanced B.t. Against Bt-Resistant Insect.
As with conventional insecticides, resistance of target insects to B.t. may not be avoidable. Insects have been selected for resistance against Bt in the laboratory settings as well as in the field (reviewed in (Griffitts et al., 2005; McGaughey, 1985)). The diamondback moth, P. xylostella, is an agricultural pest of cruciferous crops including cabbage and canola. Very high resistance against Bt has developed in the field for this pest (Ferre et al., 1991; Liu et al., 1996; Tabashnik et al., 1990).
A laboratory-selected strain of Bt-resistant P. xylostella was tested against Agree WG (a Cry1A and Cry1C producing strain of Bt) and Javelin WG (a Cry1Aa, Cry1Ab, Cry1Ac, Cry2Aa, and Cry2Ab producing strain of Bt). From a diet incorporation bioassay, it was determined that this strain was about 128-fold more susceptible to Agree WG than to Javelin WG (FIG. 6A). This was expected since the resistant insects were selected on Bt HD-1, which produces the same Cry toxins present in Javelin WG. Agree WG, however, contain Cry1C which has been shown to have very little cross resistance to Cry1A toxins and is highly active against P. xylostella (Tang et al., 1996). Both BtB2 and BtB3 inclusion bodies were able to significantly enhance Javelin WG and Agree WG against a Bt-resistant strain of P. xylostella (FIGS. 6B and 6C). These results suggest that the addition of BtB to B.t. formulations can reduce the development of resistance in target insects. This is a very important feature that increases its value in integrated pest management program.
Sequence Alignments.
As BtB2 represents CR12, and BtB3 is a modified CR12 (see FIG. 20), alignment of the CR12 region from ten different lepidopteran species (Ostrinia nubilalis (European Corn Borer), Chilo suppressalis (Striped Rice Borer), M. sexta (Tomato Hornworm), P. xylostella (Diamondback Moth), Pectinophora gossypiella (Pink Bollworm), Lymantria dispar (Gypsy Moth), Bombyx mori (Silkworm), Helicoverpa armigera (Cotton Bollworm), and Heliothis virescens (Tobacco Budworm) and Spodoptera frugiperda) showed more than 50% similarity in sequence (see FIG. 20). This suggests that BIB can work against these insects that share a common protein. All three versions of BtB bound Cry1A toxins strongly in dot-blot assays. This is consistent with a prior report that showed that the CR12 region is required for toxin binding (Hua et al., 2004b).
Further Insights into Mechanism(s) of Action.
Cry toxins bind to BtB and high affinity binding is correlated with toxicity enhancement. For example, as measured by surface plasmon resonance analysis using a BIAcore, the original BtB (Ms-CR12-MPED) bound Cry1Ab at high (9 nM) and low (1 μM) affinity sites. BtB (CR12-MPED)-mediated Cry1A toxicity enhancement was significantly reduced when the high affinity Cry1A-binding epitope (1416GVLTLNIQ1423) within the peptide was altered. The BtB peptide bound brush border membrane vesicles (BBMV) with high affinity (Kd=32 nM) and insect midgut microvilli, but did not alter Cry1Ab or Cry1Ac binding localization in the midgut.
Without being bound by a specific theory, one possible explanation of the observed synergism is that BtB-type peptides bind to the microvilli and attract Cry1A molecules, increasing the probability of toxin interaction with Cry1A receptors such as Bt-R1, GPI-anchored aminopeptidase N and alkaline phosphatase, or sphingoglycolipids (Griffitts et al., 2005). This hypothesis is consistent with the model proposed by Bravo whereby binding of Cry1A monomer to the cadherin Bt-R1 induces structural changes in the toxin that result in further processing and formation of a toxin oligomer. As a Bt-R1 truncation for Cry1A binding, the addition of CR12-MPED might promote the switch of toxin from monomer to oligomer, a form which primarily binds to GPI-anchored receptors resulting in oligomer insertion in the cell membrane.
Since the interaction between BtB and Bt toxins produced a possible novel mode of action for the toxin, the addition of BtB to Bt formulations or Bt transgenic plants might delay resistance.
Several potential advantages can be attributed to BtB: (1) It reduces the amount of Cry protein needed to kill larvae, thus also prolonging pesticidal activity by enhancing the residual activity of Cry protein; (2) It expands host range of Bt biopesticides and (3) It may overcome certain types of acquired resistance to Bt proteins. This discovery suggests that BtB can be used as an additive to Bt to increase its efficacy and potentially increase the usage of Bt biopesticide in agriculture.
We found that low mass ratios (Cry toxin:BtB) were needed for BtB4 and BtB5 to enhance Cry1Ac toxicity to H. zea larvae (FIG. 9). This is significant because H. zea is an important pest of cotton, corn and other crops. The low mass ratios of Cry toxin:BtB are desirable for producing BtB protein for use in biopesticides and transgenic plants.
FIG. 9 shows the shift in LC50 values observed for BtB4 and BtB5 tested at 1:2 (Cry toxin:BtB) mass ratios against H. zea larvae. A lower LC50 value means that with BtB4 or BtB5 the insect larvae at killed at lower Bt toxin concentrations.
We found the BtB4 could enhance Cry1Ac toxicity to A. ipsilon (black cutworm) (FIG. 11A). The black cutworm, A. ipsilon, is very tolerant to most Bt toxins (de Maagd et al., 2003). It is only susceptible to BE toxins at very high doses as demonstrated by diet overlay bioassay. The LC50 for Cry1Ac we determined was 19.3 (17.9-20.9) μg/cm2 (95% fiducial limits in parenthesis). However, the LC50 for Cry1Ac with the addition of 10-fold mass ratio of CR10-12 (BtB4) was reduced to 2 μg/cm2 ( ̃10-fold enhancement). This is important because cutworms are difficult to control crop pests with Bt Cry toxins.
We found that BtB4 and BtB5 could enhance Cry1Ab and Cry1Ac toxicity to S. exigua (FIG. 8). In FIG. 11B, further evidence is presented that that BtB4 could enhance Cry1Ab toxicity to S. exigua (beet armyworm) (FIG. 11B). The diet overlay assays confirmed that the larvae are very tolerant to Cry1Ab. The LC50 for Cry1Ab was estimated at about 7 μg/cm2 (although the fiducial limit could not be determined due to insufficient data). However, the LC50 for Cry1Ab with the addition of 5-fold mass ratio of Ms-CR10-12 (BtB4) was reduced to 1.1 (0.9-1.3) μg/cm2 (about a 6-fold enhancement).
FIG. 12 shows that low ratios of Cry1C:BtB4 (Ms-CR10-12) could enhance Cry1C toxicity to S. exigua. 
BtB5 could also lower the amount of Cry1Ab and Cry1Fa needed to kill H. zea larvae. As shown in FIG. 13 the LC50 values for Cry1Ab and Cry1Fa are substantially lower in the presence of BtB5. This is important because Cry1Ab and Cry1Fa are produced in genetically engineered plants and increased insect control is desirable. Cry1Ab and Cry1Fa may also be used in biopesticides with BtB for enhanced insect control.
In a similar manner BtB5 substantially increased Cry1Ab and Cry1Fa toxicity to S. exigua (beet armyworm) (FIG. 14). BtB5 substantially increased the toxicity of Cry1Ab and Cry1Fa to S. exigua (beet armyworms) The beet armyworm is an important pest that is difficult to control with most Bt Cry proteins. Note in FIG. 14 how without BtB5, Cry1Ab did not cause larval mortality.
Our data presented in FIG. 15 demonstrate that BtB5 could enhance both individual and combinations of Cry toxins. BtB5 enhanced a mixture of Cry1Ab+Cry1Fa toxins when tested in bioassay against H. zea larvae. This is important because Cry1Ab and Cry1Fa toxins are likely to be co-expressed in Bt corn and other crops.
Two BtBs (BtB9 and BtB10 as shown in FIG. 1) were designed with the possibility that they may have increased enhancing properties for Cry toxins active against Spodoptera species. Data presented in FIGS. 17A and 17B are evidence that BtB9 and BtB10 increase Cry1F toxicity to S. frugiperda. Note, BtB5 also increased Cry1F toxicity. These results are because Cry1F is produced in Bt corn and cotton.
Our data presented in FIG. 18 suggest another mechanism that explains the synergism of BtB-type peptides. When BtB was added to Cry1Ab toxin, the toxin was stabilized against digestion by midgut digestive enzymes. This is important because over-digestion of toxin to inactive peptides is a known mechanism of insect resistance to Bt toxins.
Our data presented in FIG. 19 are evidence that BtB cadherin fragments enhance Cry1Ac toxicity to insects when co-expressed in plants. Plant expression constructs for BtB1 (SEQ ID NO: 3) and BtB3 (SEQ ID NO: 4) were co-transformed into Arabidopsis with a synthetic cry1Ac gene coding region using T-DNA vectors in Agrobacterium tumefaciens. Plants were also transformed with only the cry1Ac construct. Plants transgenic for BtB and Cry1ac genes were selected for antibiotic and/or herbicide resistance. Progeny transformed plants expressing the same levels of Cry1Ac protein were identified by quantitative Western blot analysis. The levels of BtB1 and BtB3 were also quantitified by Western blot analysis. FIG. 19 shows the mortality response of Bt-resistant Plutella xylostella larvae feeding on either leaves of Arabidopsis expressing Cry1Ac alone or Cry1Ac plus BtB1 or BtB3. In the left-hand set plant Bt-431 is compared with Bt Cry1Ac plus BtB1 (plant#2) and Bt Cry1Ac plus BtB3 (plant #30). The mortality was greater when a BtB was present with Cry1Ac. In the second set a Cry1Ac producing plant (plant#8) is compared with Cry1Ac plus BtB1 (plant#20) and Cry1Ac plus BtB3 (plant #31). In this plant set, more P. xylostella larvae were killed when a BtB was present.
Various 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.uldhome/Neil_Crickmore/Bt/) for a list of B.t. toxins. These include, but are not limited to, polynucleotides encoding Cry1A toxins such as Cry1Aa, Cry1Ab, Cry1Ac, as well as Cry1B, Cry1C, Cry1F, Cry1E, Cry3A, and the Cry8s, as well as Cry34s+Cry 35s. Cry2 toxins are also preferred for co-administration with peptides of the subject invention.
Various insects can be targeted or otherwise inhibited/controlled by (one or more of) the subject polypeptides/proteins, including:
Agrotis ipsilon, black cutworm
Agrotis orthogonia, pale western cutworm
Anticarsia gemmatalis, velvetbean caterpillar
Chilo partellus, sorghum borer
Diatraea grandiosclla, southwestern corn
Diatraea saccharalis, sugarcane borer
Elasmopalpus lignosellus, lesser cornstalk borer
Feltia subterranea, granulate cutworm
Helicoverpa zea, corn earworm/cotton bollworm
Heliothis virescens, tobacco budworm/cotton boll worm
Homoeosoma electellum, sunflower moth
Ostrinia nubilalis, European corn borer
Pectinophora gossypiella, pink bollworm borer
Plathypena scabra, green cloverworm
Pseudoplusia includens, soybean limper
Pseudaletia unipunctata, army worm
Spodoptera exigua, beet armyworm
Spodoptera frugiperda, fall armyworm
Suleima helianthana, sunflower bud moth
Any genus listed above (and others), generally, can also be targeted as a part of the subject invention. Any additional insects in any of these genera (as targets) are also included within the scope of this invention.
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.
In light of and having the benefit of the subject application, variants of novel BtBs of the subject invention (e.g. those derived from Spodoptera 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.
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
Construction of Booster Peptides Having Increased Stability to Insect Proteases
Several modifications were made to the MS-CR12 peptide to increase its stability in the proteolytic larval gut environment. This was done by replacing amino acids in putative trypsin and cyhmotrypsin cleavage sites on CR12 to remove the cleavage sites. The peptide sequences of the wild type CR12 and a protease stable mutant Ms-CR12(PS)=BtB3 are shown below. The underlined amino acids are the residues that were replaced.







a. MS-CR12 wt (BtB2)


(SEQ ID NO: 5)


MGISTADSIGRELLRLHATQSEGAAITYAIDYDTMVVDPSLEAVRQSAFV


 


LNAQTGVLTLNIQPTATMHGLFKFEVTATDTAGAQDRTDVTVYVVSSQNR


 


VYFVLEHHHHHH


 


b. MS-CRCR12(PS) (BtB3)


(SEQ ID NO: 8)


MGISTADSIGSELLSLHATQSEGAAITYAIDYDTMVVDPSLEAVSQSALV


 


LNAQTGVLTLNIQPTATMHGLINIEVTATDTAGAQDRTDVTVYVVSSQNR


 


VYFVLEHHHHH






    
    
        The Ms-CR10-12 peptide was designed from the CR10-12 region of Bt-R1a (Hua et al., 2004a). Modifications were also made to the pMS-CR10-12 peptide for expression in E. coli and other prokaryotic cells. Modifications included codon optimization for E. coli was performed using online software; see the following worldwide web site: evolvingcode.net/codon/sgd/index.php. We also changed the N-terminus for initiating protein expression and the C-terminus for ease of protein purification using an immobilized nickel column.
    
    









MS-CR10-12(13tB4):


(SEQ ID NO: 11)


MHLERISATDPDGLHAGVVTFQVVGDEESQRYFQVVNDGANLGSLRLLQA


 


VPEEIREFRITIRATDQGTDPGPLSTDMTFRVVFVPTQGEPRFASSEHAV


 


AFIEKSAGMEESHQLPLAQDIKNHLCEDDCHSIYYRIIDGNSEGHFGLDP


 


VRNRLFLKKELIREQSASHTLQVAASNSPDGGIPLPASILTVTVTVREAD


 


PRPVFMRELYTAGISTADSIGRELLRLHATQSEGAAITYAIDYDTMVVDP


 


SLEAVRQSAFVLNAQTGVLTLNIQPTATMHGLFKFEVTATDTAGAQDRTD


 


VTVYVVSSQNRLEHHHHHH






    
    
        Modifications were also made to the Ms-CR10-12 to increase stability in insect midguts. This was accomplished by removing trypsin cleavage sites (R/K-S/A or R-C) in pMS-CR10-12 to yield pMS-CR10-12(PS)=BtB5)(SEQ ID NO:14):
    
    









MHLECISATDPDGLHAGVVTFQVVGDEESQAYFQVVNDGANLGSLSLLQA


 


VPEEIAEFSITICATDQGTDPGPLSTDMTFAVVFVPTQGEPAFASSEHAV


 


AFIEASAGMEESHQLPLAQDIANHLCEDDCHSIYYAIIDGNSEGHFGLDP


 


VANALFLSAELIAEQSASHTLQVAASNSPDGGIPLPASILTVTVTVAEAD


 


PAPVFMAELYTAGISTADSIGCELLALHATQSEGAAITYAIDYDTMVVDP


 


SLEAVCQSAFVLNAQTGVLTLNIQPTATMHGLFNFEVTATDTAGAQDRTD


 


VTVYVVSSQNRLEHHHHHH






The pSf-CR10-12 peptide (SEQ ID NO:26) was designed from the S. frugiperda cadherin sequence and then modified for protease stabilization by the removal of trypsin recognition sites. The approach was as described above for Ms-CR-12(PS) and Ms-CR10-12(PS). Amino acid changes from Sf-CR10-12 to Sf-CR10-12(PS) (SEQ ID NO:29) are indicated the in the alignment of their amino acid sequences below. Changed residues are in bold and designated by 2 in the Prim.cons. sequence.







TABLE 1





Amino acid sequence alignment between Sf-CR10-12 (SEQ ID NO: 26) and Sf-CR10-12(PS) (SEQ ID NO: 29).


Changed residues from Sf-CR10-12 (SEQ ID NO: 26) and Sf-CR10-12(PS) (SEQ ID NO: 29) are shown in bold.


Prim.cons. sequence shows unchanged residues between Sf-CR10-12 (SEQ ID NO: 26) and Sf-CR10-12(PS)


(SEQ ID NO: 29) alone with changed residues designated by 2.




















EXAMPLE 2
Production of BtBooster in E. coli and Formulation of a Dried-Cell Powder BtBooster Product
The BtBooster (MsCR10-12(PS)=BtB5) gene was synthesized with a 5′ NdeI site and a 3′ HindIII restriction site. The BtB5 gene was subcloned into pET30a(+) at the NdeI and HindIII restriction sites. The hexahistidine tag is located at the C-terminus of BtB5 peptide. The start codon (ATG) is located at the NdeI site. The stop codon (TAA) occurs after the his-tag codons, immediately before the HindIII site. The BtB constructs were subcloned into pET30a (Novagen) and expressed in BL21(DE3)pRIL cells. E. coli cells were transformed by a standard electroporation method into E. coli BL21(DE3)pRIL (Novagen). The Ms-CR10-12(PS)peptide was over-expressed in E. coif 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—1.5% 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.
The following conditions illustrate a method for producing BtB for use with Bt formulations in field trials.
Growth Medium:
50 mM (NH4)2SO4, 2 mM MgSO4.7H2O, 40 mM KH2PO4, 55 mM Na2HPO4, 1% (w/v) tryptone, 0.5% (w/v) yeast extract, 30 mM sodium citrate, 1% (v/v) glycerol, 1% (w/v) glucose; Adjust pH to 7.2 with NaOH. Autoclave all components together at 121° C. for 15 min. After autoclaving, add to sterile medium: 10 mM Proline (filter sterilized), and 100 mg/ml Kanamycin (filter sterilized).
Growth and Induction Conditions:
E. coli cells were grown in fermentor (up to 500 L) at 37° C., pH 6.8, and 30% dissolved oxygen. The cells were induced with 1 mM IPTG when the OD600 reached 4 or 5. The cells were grown overnight and harvested the next day (OD600=27-43).
Cell Harvesting and Lysis:
The cells were centrifuged and resuspended in (3 ml per gram pellet ratio) 20 mM NaH2PO4, 0.1% Triton X-100, pH 4.5. The cells were homogenized/broken using a microfluidizer. The lysate was centrifuged and the pellet was resuspended (2 ml per gram pellet ratio) in 20 mM NaH2PO4, 10% (w/v) sucrose, 0.1% Triton X-100, pH 4.5.
Spray Drying Condition:
The lysed cell pellet was spray dried using a Niro Production Minor Spray Dryer (4′ diameter) under standard conditions. Starting weight of material was 62.7 pounds. Material dried without difficulty. At the end of the run, drying chamber brushdown was collected separately and labeled accordingly. Dried samples are stored at 4° C. for later quality control analysis and testing in field trials.
EXAMPLE 3
Preparation of Bt Spores and Crystals-Toxin Preparation
Formulated Bts including Javelin® WG, and Agree® were provided by Certis Corporation. The formulated Bt products XenTari® DiPel® DF were purchased from a local pest control distributor.
Cry1Ac crystal protein was prepared from Bacillus thuringiensis HD-73 essentially as describe previously (Luo et al., 1996). Cry1Ca protein and Cry1Fa were purified from individual Bt strains producing either Cry1Ca or Cry1Fa. The cells were grown in a shaker at 30° C. until cell lysis in a sporulation medium. The spore-crystal preparation was cleaned by a series of sonication and homogenization in detergent (Triton X-100) and high molar salt (1 M NaCl), and finally the spore-crystal preparation was washed and resuspended in distilled water. The Cry1Ac crystal was solubilized in 50 mM Na2CO3 pH 10.5 and activated by trypsin digestion. The toxin was purified by Q-sepharose anion exchange chromatography. Protein concentration was determined using BSA as a standard (Luo et al., 1996). The toxin was aliquoted into 1.5-ml microcentrifuge tubes and stored at −20° C. until needed. Bt NRD12 (the active ingredient in Javelin WG) was also prepared the same way except that the process was stopped after the spore-crystal preparation was resuspended in distilled water. The spore-crystal sample was kept at 4° C. until use.
The cry1Ab gene was over-expressed in Escherichia coli JM103 by using the expression vector pKK223-3 (Lee et al., 1992). Toxin preparation was as described above for preparation of Cry1 toxin derived from Bt crystals.
EXAMPLE 4
Insects and Insect Bioassays
Insects.
All insect eggs were purchased from Benzon Research (Carlisle, Pa.), and emerged larvae were maintained on artificial insect diet (multiple species insect diet, Southland Products, Lake Village, Ark.). The Bt-resistant P. xylostella was selected with Bt kurstaki HD-1 spore-crystal by Benzon Research. The resistant insects were about 800-1000 fold less susceptible to the HD-1 spore-crystal compared to the susceptible (unselected) P. xylostella (data not shown).
Diet Incorporation Bioassay with Bt-Resistant P. xylostella. 
Artificial diet was prepared according to the manufacturer's instruction (Southland Products, Lake Village, Ark.) and cooled to 60° C. before mixing with Javelin WG (Certis, Columbia, Md.) (with or without BtB) or Agree WG (Certis, Columbia, Md.) (with or without BtB) and pipetted into 128-well bioassay trays (C-D International) with a 30-ml plastic syringe. One or three insect neonate was placed in each well. Each treatment contained 16 or 48 larvae with two replicates. Insects were placed in an incubator at 28° C. with a photoperiod of 12:12 h (L:D). Larval mortalities were counted 7 days after treatment.
Diet Surface Overlay Bioassay with IL zea, S. exigua, S. frugiperda and A. ipsilon. 
Artificial diet was prepared according to the manufacturer's instruction (Southland Products, Lake Village, Ark.) and pipetted into 128-well bioassay trays. Using distilled water as diluent, Cry1 toxin (with or without BtB) (50 μl) was applied uniformly on the diet surface diet and allowed to dry. A single neonate was placed in each well. Each treatment contained 32 larvae with two replicates. Insects were placed in an incubator at 28° C. with a photoperiod of 12:12 h (L:D). Larval mortalities were counted 7 days after treatment Diet overlay bioassays for H. zea and S. exigua neonates were scored at 7 days after treatment (DAT) and bioassays for A. ipsilon neonates were scored at 6 DAT.
Tomato Leaf Dip Bioassay with II. zea.
H. zea larvae were grown at 28° C. with a photoperiod of 12:12 h (L:D) for 5 days on artificial diet (Southland Products). The larvae were approximately late second instar at the start of bioassay. Tomato seeds (Better Boy Hybrid—catalog no. 5323-SD) were purchased from Park Seed Wholesale, Greenwood, S.C. The plants were maintained in a growth chamber at 28° C., 14:10 photoperiod, and about 80% relative humidity. When the plants were about 10-12 inches tall, leaves were cut at about 2×3 cm and dipped into treatment solutions. The treated leaves were then air dried and placed individually into lidded plastic cups. Each of the cups also contains a wet Whatman fitter paper. A single larva was added into each cup and mortality was scored on day 2 and 3. Fresh-cut tomato leaves were added on Day 2. Thirty larvae were used per treatment with two replicates. 0.02% Tween 20 (Sigma Aldrich, St. Louis, Mo.) was used as a diluent. Insects were placed in an incubator at 28° C. with a photoperiod of 12:12 h (L:D). Larval mortalities were determined 2 to 4 days after treatment. Larvae were considered dead when no larval movement was detected after being prodded. All insect bioassay was repeated at least once.
Cabbage Excised-Leaf Bioassay.
Copenhagen market early cabbage plants were sprayed in the greenhouse with test solution and allowed to dry. Cabbage leaves were removed and cut into  ̃30×55 mm pieces, placed in 30 ml clear plastic cups with 1 or more larvae (3 or 4 days old) and capped. Typically 3 replicates of 30 larvae for each dose were done. The bioassays were done at 28° C. with a photoperiod of 12:12 h (L:D). Larval mortalities were determined 4 days after treatment. Larvae were considered dead when no larval movement was detected after being prodded. All insect bioassay was repeated at least once.
Statistical Analysis.
The number of surviving and dead larvae in one treatment was compared with another treatment using a 2×2 contingency table by Chi-square analysis. Treatments are not significantly different if P>0.05.
EXAMPLE 5
Results—Longer Cadherin Fragments have Improved Toxin Enhancement Properties
Enhancement of Cry1Ac Toxicity by ‘Longer’ M. sexta Cadherin Fragments.
Previously, a small fragment of a M. sexta cadherin (Ms-CR12-MPED) was demonstrated as a potent enhancer of Cry1Ab and Cry1Ac toxins (Chen et al., 2007b) (Chen et al., 2007a). Mutation of the CR12 region of the CR12-MPED synergist to remove a Cry1Ab-binding region was shown to block the enhancer activity (Chen et al., 2007b), suggesting the importance of CR12 for toxin enhancement activity. Fragments of M. sexta cadherin longer than MS-CR12 were produced to test their synergistic activity with Cry1Ac toxin. Cadherin fragments of various lengths corresponding to CR7-12, CR9-12, CR10-12, and CR11-12, expressed in E. coli, formed insoluble inclusion bodies. The inclusion bodies were washed and resuspended in deionized water for use in bioassays.
Diet overlay bioassays on neonate H. zea were performed using trypsin-activated Cry1Ac with cadherin fragments set at a fixed toxin to cadherin mass ratio of 1:20 (FIG. 2). The LC50 for Cry1Ac alone was estimated to be 1 μg/cm2 (fiducial limits could not be determined due to insufficient data). The LC50 of Cry1Ac in the presence of the cadherin fragments was reduced by 12- to 50-fold. Toxin enhancements by CR7-12, CR9-12, and CR10-12 have overlapping confidence limits, suggesting that the enhancement levels were not significantly different. However, enhancement by CR11-12 was significantly lower compared to the other three longer cadherin fragments. Based on our evaluation of these results, it was decided that CR10-12 will be used for further testing because it was the minimum cadherin length that provided a high enhancement level.
EXAMPLE 6
Cadherin Fragments Increase Cry Toxicity to Difficult to Control Insect Species
Other studies, combining Bt with BtBoosterTM, tested against H. zea, S. exigua, S. frugiperda, Plutella xylostella (Bt-resistant and susceptible populations), and Agrotis ipsilon have also demonstrated significant increases in mortality. It is important to note that BtBoosterTM alone has demonstrated no effects on a wide range of insects, including these species. Artificial diet bioassays, leaf bioassays and whole plant bioassays in the laboratory and greenhouse have provided supportive data. A random selection of different bioassay results are provided below.
Enhancement of Cry1Ac Toxicity Against A. epsilon. 
The black cutworm, A. ipsilon, is very tolerant to most Bt toxins (de Maagd et al., 2003). It is only susceptible to Bt toxins at very high doses as demonstrated by diet overlay bioassay (FIG. 11A). The LC50 for Cry1Ac was determined to be 193 (17.9-20.9) μg/cm2 (95% fiducial limits in parenthesis). However, the LC50 for Cry1Ac with the addition of 10-fold mass ratio of CR10-12 was reduced to approximately 2 μg/cm2 ( ̃10-fold enhancement).
Enhancement of Cry1Ab Toxicity Against S. exigua. 
The beet armyworm, S. exigua, is highly tolerant to Bt toxins, especially Cry1A type toxins (Hernandez-Martinez et al., 2008). Diet overlay bioassay on neonate S. exigua using trypsin-activated Cry1Ab confirmed that the larvae were very tolerant to the toxin (FIG. 11B). The LC50 for Cry1Ab was estimated to be  ̃7 μg/cm2. However, the LC50 for Cry1Ab with the addition of 5-fold mass ratio of CR10-12 was reduced to 1.1 (0.9-1.3) μg/cm2. ( ̃6-fold enhancement).
Enhancement of Cry1Ca and Cry1Ab Toxicity Against S. exigua. 
Spodoptera species are among the more difficult to control lepidopteran pests on cotton, corn, vegetables and cruciferous crops with Bt toxins. In a diet surface overlay bioassay (FIG. 8), the two tested cadherin fragments Ms-CR10-12 and Ms-CR10-12(PS), BtB4 and BtB5 respectively enhanced the toxicity of Cry1Ab toxin to S. exigua in diet surface treatment bioassays. Since Cry1Ca toxin is more toxic than Cry1Ab against S. exigua larvae (de Maagd et al., 2000; Luo et al., 1999) was also tested for BtB enhancement of Cry1Ca toxin. Diet overlay bioassay with neonate S. exigua using trypsin-activated Cry1Ca showed significant enhancement by Ms-CR10-12 over the Cry1Ca alone treatment at varying mass ratios from 1:1 to 1:100 (FIG. 12). The level of enhancement was at a maximum ( ̃5-fold) at 1:1 toxin to Ms-CR10-12 mass ratio
The results of the S. exigua diet overlay assay presented in FIG. 14 are evidence that Ms-CR10-12(PS), BtB5, significantly enhances both Cry1Ab and Cry1Fa toxicity to S. exigua. Note, that Cry1Ab alone, caused no mortality to S. exigua larvae.
Enhancement of Cry1Ab and Cry1Fa Toxicity to S. frugiperda. 
The Fall armyworm, S. frugiperda is an occasional, yet important pest of corn and cotton (Fuxa, 1989). Since Cry1Fa has toxicity to S. frugiperda (Luo et al., 1999) and Cry1Fa is expressed in Bt corn and cotton, we tested ‘improved’ BtBs in bioassays with Cry1Fa against S. frugiperda. Each BtB tested [Ms-CR10-12(PS), Sf-CR10-12 and Sf-CR10-12(PS)] caused increased larval mortality when combined with Cry1Fa toxin (FIG. 17A). The Sf-CR10-12 and Sf-CR10-12(PS) appeared to be better Cry1Fa enhancers than Ms-CR10-12(PS).
EXAMPLE 7
Protease Stabilized BTBs have Improved Enhancement Properties with Bt Spore Crystal Preparations
The use of Bt biopesticides depends in large part on the combined efficacy of Bt spores and crystals. The lack of H. zea larval mortality observed for combinations of Ms-CR9-MPED (BtB1) and Ms-CR12 when mixed with Bt NRD12 (FIGS. 4A and 4B) prompted us to investigate why these BtBs were not effective under these conditions. By examining the stability of BtBs in digestive juice from insect midgut lumen, we concluded that the BtBs were rapidly degraded by digestive proteases. We solved this instability problem by removing trypsin and some chymotrpsin cleavage sites. Cadherin peptides Ms-CR12(PS) (BtB3), Ms-CR10-12(PS) (BtB5), and SfCR10-12(PS) (BtB10) are examples of cadherin peptides that are modified for increased stability in the presence of insect midgut proteases. The results presented in FIGS. 4A and 4B show that Ms-CR12(PS) enhances Bt strain NRD12 toxicity to H. zea larvae in tomato leaf dip bioassays.
Similar results of a protease-stabilized BtB enhancing formulated Bt were observed in cabbage-excised leaf bioassays against 4-day old H. zea larvae. Older larvae were tested because they are more tolerant to Bt. As seen in FIGS. 10A and 10B protease-stabilized Ms-CR10-12(PS) significantly enhanced the toxicity of Javelin® and Dipel® to H. zea larvae. Protease-stabilized BtB fragments demonstrated improved enhancement of Bt formulations applied to plant leaves.
EXAMPLE 8
Protease Stabilized BtBs have Improved Enhancement Properties with Bt Spore Crystal Preparations
A laboratory-selected strain of Bt-resistant P. xylostella (purchased from Benzon Research, Inc.) was tested against Agree® WG (a Cry1Aa, Cry1Ca, and Cry1D producing strain of Bt) and Javelin® WG (a Cry1Aa, Cry1Ab, Cry1Ac, Cry2Aa, and Cry2Ab producing strain of Bt). We determined that this strain was about 128-fold more resistant to Javelin® WG than to Agree® WG (FIG. 6A). This was expected since the resistant strain was selected on Bt HD-1, which produces the same Cry toxins present in Javelin® WG. Agree® WG, however, contains Cry1C, which has been shown to have a unique mechanism of action and thus has very little cross resistance to Cry1A toxins and is highly active against P. xylostella. Both BtB2 and BtB3 inclusion bodies significantly enhanced Javelin® WG and Agree® WG against the Bt-resistant strain of P. xylostella (FIGS. 6B and 6C). These results suggest that the addition of BtB to Bt formulations might overcome resistance in target insects.
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## Claims

1. A composition for inhibiting lepidopteran insects, wherein said composition comprises a Cry 1 protein and at least one polypeptide that enhances inhibitory effects of said Cry 1 protein against said lepidopteran insect said polypeptide comprising a Cry binding domain and wherein said modified fragment resists degradation by a protease and said polypeptide differs from a wild-type fragment of an insect cadherin ectodomain comprising said binding domain by one or more amino acid deletions or substitutions that make said polypeptide more resistant to protease degradation than said fragment, wherein said polypeptide is at least 95% identical with SEQ ID NO:14, and wherein said lepidopteran insects are selected from the group consisting of Heliothis virescens, Helicoverpa zea, Spodoptera fruqiperda, and Spodoptera exiqua.

2. The composition of claim 1 wherein said composition further comprises B.t. spores and/or crystals that comprise said Cry.

3. The composition of claim 1 wherein said polypeptide is modified to remove at least one protease cleavage site present in said wild type fragment, and said cleavage site is a cleavage site for a protease selected from the group consisting of trypsin and chymotrypsin.

4. A method of inhibiting a lepidopteran insect, said method comprising providing a composition of claim 1 to said insect for ingestion, and wherein said lepidopteran insects are selected from the group consisting of Heliothis virescens, Helicoverpa zea, Spodoptera fruqiperda, and Spodoptera exiqua.

5. The method of claim 4, wherein said Cry protein is present in spores and/or crystals.

6. A method of making a composition of claim 1, said method comprises producing said polypeptide, wherein said polypeptide lacks one or more protease recognition sites present in said wild-type fragment.

7. The method of claim 6, wherein said method comprises obtaining a DNA sequence that encodes said wild-type fragment, and modifying said DNA sequence to remove at least one codon for an amino acid that is part of a protease recognition sequence.

8. The composition of claim 1, wherein said Cry1 protein is selected from the group consisting of Cry1Aa, Cry1Ab, and Cry1Ac.

9. The method of claim 4, wherein said Cry is produced by and is present in a transgenic plant, and said method comprises spraying a composition onto said transgenic plant wherein said composition comprises said polypeptide.

10. The method of claim 4 wherein said insect is a Spodoptera frugiperda.

11. The composition of claim 1 wherein said Cry protein is selected from the group consisting of Cry1A, Cry1B, Cry1C, Cry1F, and Cry1E.