# Patent

## Title
Modified Bacillus thuringiensis cry proteins that inhibit coleopterans

## Abstract

The present invention includes domain I modifications that improve various attributes of various Coleopteran-active Cry proteins. These attributes can include improved target pest spectrum, potency, and insect resistance management. The subject modifications can affect protoxin activation and the efficiency of pore formation, which can lead to enhanced insect intoxication.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application is a continuation of U.S. patent application Ser. No. 12/512,865, filed Jul. 30, 2009, which claims the benefit of Provisional Application Ser. No. 61/084,944, filed Jul. 30, 2008, the disclosures of which are expressly incorporated by reference herein.


BACKGROUND
Billions of dollars are spent each year to control insect pests and additional billions are lost to the damage they inflict. Synthetic organic chemical insecticides have been the primary tools used to control insect pests but biological insecticides, such as the insecticidal proteins derived from Bacillus thuringiensis (Bt), have played an important role in some areas. The ability to produce insect resistant plants through transformation with Bt insecticidal protein genes has revolutionized modern agriculture and heightened the importance and value of insecticidal proteins and their genes.
Western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is an economically important corn pest that causes an estimated $1 billion revenue loss each year in North America due to crop yield loss and expenditures for insect management (Metcalf 1986). WCR management practices include crop rotation with soybeans, chemical insecticides and, more recently, transgenic crops expressing Bt Cry proteins. However, to date only a few examples of Bt Cry proteins provide commercial levels of efficacy against WCR, including Cry34Ab1/Cry35Ab1 (Ellis et al. 2002), modified Cry3Aa1 (Walters et al. 2008) and modified Cry3Bb1 (Vaughn et al 2005). These Bt proteins are highly effective at preventing WCR corn root damage when expressed in the roots of transgenic corn (Moellenbeck et al. 2001, Vaughn et al. 2005, Syngenta U.S. Pat. No. 7,361,813).
Despite the success of WCR-resistant transgenic corn, several factors create the need to develop additional Bt proteins to control WCR. First, although the current Cry proteins expressed in transgenic corn products are robust in preventing WCR root damage and thereby protecting grain yield, some WCR adults emerge in artificial infestation trials, indicating less than complete larval insect control. Second, development of resistant insect populations threatens the long-term durability of Cry proteins; Lepidopteran insects resistant to Cry proteins have developed in the field for Plutella xylostella (Tabashnik, 1994), Trichplplusia ni (Janmaat and Myers 2003), and Helicoverpa zea (Tabashnik et al. 2008). Development of new high potency Cry proteins will provide additional tools for WCR management. Cry proteins with different modes of action can be expressed in combination in transgenic corn to prevent the development WCR insect resistance and protect the long term utility of Bt technology for WCR control.
Bacillus thuringiensis (Bt) is a soil-borne bacterium that produces insecticidal crystal proteins known as delta endotoxins, or Cry proteins (reviewed in Schnepf et al., 1998). Many B. thuringiensis serovars exist in nature that together account for a large number of diverse Cry proteins with various insecticidal properties (see Worldwide Website: lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/intro.html). Cry proteins are oral intoxicants that function by acting on midgut cells of susceptible insects. The active form of many Cry proteins comprises three distinct protein domains. Domain I is a seven α-helix bundle in which six helices surround a central helix. This domain is involved in midgut membrane insertion and pore formation. Domain II is formed by three antiparallel beta sheets packed together in a beta prism. In Cry1A proteins, surface exposed loops at the apices of domain II beta sheets are involved in binding to lepidopteran cadherin receptors; Cry3Aa domain II loops bind a Leptinotarsa decemlineata (Say) (Colorado potato beetle; CPB) membrane-associated a metalloprotease in a similar fashion (Ochoa-Campuzano et al. 2007). Domain III is a beta sandwich structure that interacts with a second class of receptors, examples of which are aminopeptidase and alkaline phosphatase in the case of Cry1A proteins (Piggot and Ellar, 2007). Analogous Cry domain III receptors have yet to be identified in Coleoptera.
One proposed model for Cry protein mode of action is based on pore formation in the midgut membranes of susceptible insects (Knowles and Ellar, 1987). In the most current version of this model (Bravo et al. 2007), binding to both cadherin and aminopeptidase receptors on Lepidopteran midgut membranes are required for Cry protein toxicity. According to the pore formation model, Cry protein intoxication involves several steps: 1) Proteolytic processing of soluble Cry protoxin to an activated core toxin; 2) Cry protein binding to cadherin receptors on the insect midgut; 3) further proteolytic cleavage at the core toxin N-terminus to remove an α-helical region; 4) Cry protein oligomerization to form a pre-pore; 5) pre-pore binding to second site membrane receptors (aminopeptidases and alkaline phosphatases); 6) pre-pore insertion into the membrane and 7) osmotic cell lysis leading to midgut disruption and insect death.
α-helices 4 and 5 of domain I are known to play roles in membrane insertion and pore formation (Walters et al. 1993, Gazit et al. 1998; Nunez-Valdez et al. 2001; FIG. 2), with the other helices proposed to contact the membrane surface like the ribs of an umbrella (Gazit et al. 1998; FIG. 3). Chymostrypsin activation of Cry3Aa1 occurs via cleavage in the loop region between domain I α-helix 3 and α-helix 4 (Carrol et al. 1997). Some α-helix 3 mutants are able to bind receptors but do not form oligomers and are non-toxic to Manduca sexta (reviewed in Jimenez-Juarez et al. 2008). Gazit et al. 1998 showed that α-helix 1 does not bind phospholipid membranes. In Cry1A proteins α-helix 1 is removed following receptor binding and is followed by oligomerization (Gomez et al. (2002). Soberon et. al (2007) have further shown that N-terminal deletion mutants of Cry1Ab and Cry1Ac lacking approximately 60 amino acids encompassing α-helix 1 on the three dimensional Cry structure are capable of assembling ca. 60 kDa monomers into pre-pores. These results contrast with those of Hofte et al. 1986 who reported that deletion of 36 amino acids from the N-terminus of Cry1Ab resulted in loss of insecticidal activity. (Hofte et al., “Structural and functional analysis of a cloned delta endotoxin of Bacillus thuringiensis berliner 1715” Eur. J. Biochem. 161; 273-280 (1986).)
Cry3Aa1 is the best studied three domain Coleopteran-active Bt protein. Cry3Aa1 mode of action follows similar steps as described above for Lepidopteran-active Cry proteins (Bravo et al. 2007). However, there are fundamental differences in the activation steps for Coleopteran-active Cry protoxins. The midgut of coleopteran insects is slightly acidic rather than alkaline as in the case of Lepidopterans (Koller et al 1992) and native Cry3Aa1 is not soluble under acidic conditions. Processing with chymotrypsin resulted in conversion of the full length 67 kDa polypeptide to a 55 kDa derivative that was fully soluble across a broad pH range and retained activity against Colorado potato beetle (Carroll et al. 1997).
BRIEF SUMMARY
The present invention includes domain I modifications that improve various attributes of various Coleopteran-active Cry proteins. These attributes can include improved target pest spectrum, potency, and insect resistance management. The subject modifications can affect protoxin activation and the efficiency of pore formation, which can lead to enhanced insect intoxication.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a ribbon diagram of the crystal structure of Cry3Aa1 (after Li et al. 1991). Alpha helices 1, 2a and 2b are indicated.
FIG. 2 is a schematic representation of a proposed model for the interaction of domain I alpha helices with phospholipid membranes (Gazit et al. 1998).
FIG. 3 is a schematic representation of a Cry protein oligomeric pore (Gazit and Shai 1995).
FIG. 4 is a multiple sequence alignment of Coleopteran-active Cry proteins. Locations of alpha helices are indicated.



BRIEF SUMMARY OF THE SEQUENCES
SEQ ID NOS: 1-109 are Cry3Aa deletions as explained herein.
SEQ ID NOS: 110-220 are Cry3Ba deletions as explained herein.
SEQ ID NOS: 221-326 are Cry7Ab deletions as explained herein.
SEQ ID NOS: 327-436 are Cry8Ba deletions as explained herein.
SEQ ID NO: 437 is the full-length Cry3Aa sequence.
SEQ ID NO: 438 is the full-length Cry3Ba sequence.
SEQ ID NO: 439 is the full-length Cry7Ab sequence.
SEQ ID NO: 440 is the full-length Cry8Ba sequence.
SEQ ID NOs:441 and 442 are DNA and protein sequences for DIG-230
SEQ ID NOs:443-462 are DNA and protein sequences for DIG-230 protease site variants
SEQ ID NO: 463-466 are protein sequences for Cry1Ab, Cry1Ac, Cry1Ah and Cry1Fa respectively.
A further supplement Sequence Listing in txt format is submitted to include these sequences in the originally filed drawing FIG. 4. No new matter is submitted by this amendment.
DETAILED DESCRIPTION
Several Coleopteran-active Cry proteins comprise three distinct domains (FIG. 1) that function in a multi-step process leading to pore formation as described above. The present invention relates to improved Cry proteins designed to have N-terminal deletions in regions with putative secondary structure homology to α-helix one and α-helix 2 in domain I of Cry3Aa. These modified Cry proteins have surprisingly improved activity on Coleopteran pests including (Diabrotica virgifera virgifera), (Diabrotica barberi), and (Diabrotica virgifera zeae).
Specifically, the subject inventors sought to further improve upon Coleopteran activity of Cry3Aa1 (U.S. Pat. No. 4,771,131), Cry3Ba1 (U.S. Pat. No. 4,996,155), Cry7Ab1 (U.S. Pat. No. 5,286,486) and Cry8Ba1 (U.S. Pat. No. 5,554,534). To improve insecticidal properties of these Cry proteins, serial, step-wise deletions are described that remove part of the gene encoding the N-terminus of each respective Cry protein. The deletions remove all of α-helix 1 and all or part of α-helix 2 in domain I, while maintaining the structural integrity of the α-helices 3 through 7.
The range of independent deletions in each series is shown in Table 1 below. (Unless indicated otherwise, all residue numbering discussed herein is with respect to the respective full-length sequences (SEQ ID NOS: 437-440).







TABLE 1







N-terminal deletion series for four Cry proteins.










Cry protein
N-terminal amino acid residue series







Cry3Aa1
F92 through N146



Cry3Ba1
F92 through E147



Cry7Ab1
G77 through Q132



Cry8Ba1
F88 through E144










Deletions were designed as follows. The first sequence in each Cry protein deletion series begins near the N-terminus of α-helix 2a (FIG. 4). Serial, stepwise deletions removing single amino acid residues are then constructed that processively remove α-helix 2a and α-helix 2b. The last deletion in each series begins near the N-terminus of α-helix 3. An initiator methionine codon was added to the beginning of each deleted gene. In addition, a second version of each deleted protein was created where glycine was added at position 2 for protein stability (Varshaysky 1997).
Preferred modified Cry proteins include but are not limited to SEQ ID NOS: 2, 35, 110, 145, 221, 254, 328, 362.
For Cry3Aa the N-terminally deleted variants include the following:

    
    
        SEQ ID NO 2 Deleted residues 1-91, N-terminus at the beginning of α-helix 2a
        SEQ ID NO 35 Deleted residues 1-109, N-terminus at the beginning of α-helix 2b
    
    


For Cry3Ba the N-terminally deleted proteins include:

    
    
        Seq ID NO 110 Deleted residues 1-91, N-terminus at the beginning of α-helix 2a
        Seq ID NO 145 Deleted residues 1-109, N-terminus at the beginning of α-helix 2b
    
    


For Cry7Ab the N-terminally deleted proteins include:

    
    
        Seq ID NO 221 Deleted residues 1-76, N-terminus at the beginning of α-helix 2a
        Seq ID NO 254 Deleted residues 1-94, N-terminus at the beginning of α-helix 2b
    
    


For Cry8Ba the N-terminally deleted proteins include:

    
    
        Seq ID NO 328 Deleted residues residues 1-87, N-terminus at the beginning of α-helix 2a
        Seq ID NO 362 Deleted residues residues 1-105, N-terminus at the beginning of α-helix 2b
    
    


Genes encoding the improved Cry proteins described herein can be made by a variety of methods well-known in the art. For example, synthetic genes and synthetic gene segments can be made by phosphite trimester and phosphoramidite chemistry (Caruthers et al, 1987). Full-length genes can be assembled in a variety of ways including, for example, by ligation of restriction fragments or polymerase chain reaction assembly of overlapping oligonucleotides (Stewart and Burgin, 2005). Further, terminal gene deletions can be made by PCR amplification using site-specific terminal oligonucleotides.
WCR protease recognition sequences can be inserted at specific sites in the Cry protein structure to affect protein processing at desired deletion points within the WCR midgut. Midgut proteases that can be exploited for activation of Cry proteins include by way of example cathepsin B or cathepsin L (Bown et al 2004), chymotrypsin and Asp-N endopeptidase. Further, recognition sites identified empirically by sequencing Cry protein digestion products generated with unfractionated WCR midgut protease preparations can be engineered to effect protein activation. Modified Cry proteins generated either by gene deletion or by introduction of protease cleavage sites have improved activity on Coleopteran pests including (Diabrotica virgifera virgifera), (Diabrotica barberi), and (Diabrotica virgifera zeae).
It is known in the art that many B.t. Cry proteins are about 130 kDa in their full-length form. Proteins exemplified herein represent “core toxins.” B.t. core toxins can be used in conjunction with C-terminal “tails.” The tails can be the natural “tails” or they can be synthetic tails, such as those disclosed in U.S. Pat. Nos. 6,218,188 and 6,673,990.
As stated herein, modified proteins of the subject invention can have surprisingly improved activity against one or more Coleopteran pests including (Diabrotica virgifera virgifera), (Diabrotica barberi), and (Diabrotica virgifera zeae). Various other insects that can also be targeted or otherwise inhibited/controlled by (one or more of) the subject proteins 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
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 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 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.
The subject proteins can be used to protect practically any type of plant from damage by a coleopteran 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.
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.
EXAMPLES
Example 1
Construction of Modified Bt Cry Proteins and Expression in Bacterial Hosts
Standard cloning methods were used in the construction of P. fluorescens expression plasmids. Restriction endonucleases were obtained from New England Biolabs and T4 DNA Ligase (Invitrogen, Cat#15224-025) was used for DNA ligation. Plasmid preparations were performed using the Machery-Nagel Nucleobond Xtra Kit (Cat#740-410) or the Plasmid Midi Kit (Qiagen, Cat#12143), following the instructions of the manufacturers. DNA fragments were purified using the Millipore Ultrafree-DA cartridget (Cat#42600) after agaraose tris-acetate gel isolation. All cloning PCR reactions were performed using Phusion High-Fidelity PCR Master Mix (New England Biolabs, Cat# F-531 S), according to the manufacturers' suggested protocols.
Plasmid constructs carrying full length genes encoding Cry3Aa1, Cry3Ba1, Cry7Ab1 and Cry8Ba1 variants were used as templates to amplify modified genes encoding N-terminal deletions or insertions. Each plasmid DNA was first prepared from the original host using the Nucleobond kit. The toxin coding region was PCR-amplified using the purified plasmid DNA as template. The 5′ forward primer included specific restriction sites to facilitate insertion into expression vectors as well as a Shine-Dalgarno (ribosome binding) sequence with appropriate spacing upstream of the initiating methionine codon. The forward primers bound to the full length templates at positions to create the truncated reading frames. A glycine codon was inserted as the second residue on deletions that had possible destabilizing residues at the second position. If the deletion already had a glycine or valine residue in the second position it was not modified. These guidelines were as described in A. Varshaysky (1997). The reverse primer or 3′ primer bound to the end of the full length coding region and carried a translational stop signal and unique restriction sites to facilitate cloning into expression vectors. Alternatively, protease cleavage sites are introduced at desired deletion locations by gene synthesis or splice overlap PCR (Horton et al. 1989).
The PCR product was then subcloned into pPCR-Script Cam SK(+) from Stratagene (Cat#211192) according to the manufacturer's protocol. The entire PCR fragment was confirmed by sequencing, before restriction digestion with the appropriate enzymes and subsequent cloning into P. fluorescens expression vector pDOW1169.
The basic cloning strategy was to subclone the modified Cry coding sequences (CDS) into pDOW1169 at the SpeI and XhoI restriction sites. A pair of gene-specific forward and reverse primers was designed for amplification of each toxin CDS from the source plasmid via PCR such that appropriate restriction enzymes were introduced at each end of the CDS for cloning into pDOW1169. For ligation to the SpeI site of pDOW1169, the forward primer of each set contained a restriction enzyme recognition site and a ribosome binding site, followed by the translation start codon ATG and a stretch of nucleotides homologous to the 5′ end of the toxin CDS. For ligation to the XhoI site of pDOW1169, the reverse primer contained a stretch of nucleotides homologous to the 3′ end of the toxin CDS, followed by a series of three stop codons for translation termination and a restriction recognition site.
Construction of Expression Strains
The PCR product of each cry gene was digested with the appropriate restriction enzymes and ligated into the SpeI and XhoI sites in pDOW1169 directly downstream of the Ptac promoter. Each of the expression plasmids was transformed into DC454, the standard wild-type P. fluorescens strain, or its derivatives, by electroporation, recovered in SOC-Soy hydrolysate medium and plated on selective medium (M9 glucose agar with additive, if necessary). Colonies were first screened by PCR and positive clones were then analyzed by restriction digestion of miniprep plasmid DNA. Selected clones containing inserts were sequenced.
DNA Sequencing
Clones were analyzed by sequencing using Big Dye Terminator version 3.1 (Applied Biosystems) or sent out for commercial sequencing (MWG Biotech, Inc. High Point, N.C.). Reactions consisted of 3.5 uL 5× sequencing buffer, 1 uL premix, 1 uL primer (6.4 uM), 50 fmol (200 ng) of DNA template, and H2O to adjust volume to 20 uL. Sequencing reactions were purified using Sephadex G-50 (Sigma) and loaded onto the ABI Prism 3100 Genetic Analyzer (Applied Biosystems). Sequence data were assembled and analyzed using the Sequencher software (Gene Codes Corp.).
Growth and Expression Analysis in Shake Flasks
The P. fluorescens strains that carried the expression constructs were analyzed by shake-flask expression. Briefly, seed cultures grown in M9 medium, supplemented with 1% glucose and trace elements, were used to inoculate 50 mL of defined minimal salts medium with 9.5% glycerol as the carbon source at  ̃2% inoculum. Following an initial growth phase at 30° C. with shaking for 24 hours, expression via the Ptac promoter was induced with 0.3 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG). Cultures were sampled at the time of induction and at various times post-induction. Cell density was measured by optical density at 600 nm (OD600).
Cell Fractionation and SDS-PAGE Analysis of Shake Flask Samples
At each sampling time, the cell density of samples was adjusted to OD600=20 and 1 mL aliquots were centrifuged at 14000×g for five minutes and the cell pellets were frozen at −80° C.
Soluble and insoluble fractions from frozen shake flask cell pellet samples were generated using EasyLyse Bacterial Protein Extraction Solution (Epicentre Technologies). Each pellet was resuspended in 1 mL EasyLyse solution and further diluted 1:4 in lysis buffer and incubated with shaking at room temperature for 30 minutes. The lysate was centrifuged at 14,000 rpm for 20 minutes at 4° C. and the supernatant was recovered as the soluble fraction. The pellet (insoluble fraction) was then resuspended in an equal volume of PBS.
Samples were mixed 1:1 with 2× Laemmli sample buffer containing β-mercaptoethanol (Bio-Rad) and boiled for 5 minutes, prior to loading onto Criterion XT Bis-Tris 12% gels. Electrophoresis was performed in the recommended XT MOPS buffer. Gels were stained with Bio-Safe Coomassie Stain according to the manufacturer's protocol and imaged using the Alpha Innotech Imaging system.
Inclusion Body Preparation
Inclusion body preparations were done on P. fluorescens fermentations that showed insoluble Bt insecticidal protein production by SDS-PAGE and MALDI-MS. P. fluorescens fermentation pellets were thawed from the freezer in ambient temperature water bath. The cells were resuspended to 10% w/v in lysis buffer (50 mM Tris, pH7.5, 200 mM NaCl, 5% Glycerol, 2 mM EDTA, 0.5% Triton X-100, and 1 mM—added just prior to use). 25 mL protease inhibitor cocktail (Sigma or AEBSF 1 mM Biosynth AG) was added for every 100 g of cell paste. The slurry was passed two times through a Microfluidics Microfluidizer at 12000+ psi. The lysate was centrifuged in JLA 10.500 rotor, 18,000×g at 4° C. for 30 min. The supernatant was retained. The inclusion pellet was washed in wash buffer (50 mM Tris, pH7.5, 200 mM NaCl, 5% Glycerol, 2 mM EDTA, 1 mM DTT) 2-3 times (or more, until no bacterial odor remains), by gently homogenizing using a spatula or mechanical mixer and centrifuging as described each time. The resultant supernatants and final pellet were retained and stored at −20° C. Small aliquots of each sample were taken and stored in a microcentrifuge tube for PAGE analysis. The inclusion body preparation pellets were then freeze-dried using a Virtis Advantage freeze dryer at ambient shelf temperature and a maximum vacuum drawn for 2 days. The resulting powders were then stored at −20° C.
SDS-PAGE Analysis
Frozen fermentor cell broths (100 μt) were diluted in chilled water by 5 fold and 200 uL sonicated for 10 minutes (Branson Ultrasonics). The lysates were centrifuged at 14000 rpm for 20 minutes (4° C.) and the supernatants removed (soluble fraction). The pellets were then resuspended in 200 uL of phosphate buffered saline (pH 7.2). Further dilutions of both the soluble and insoluble fractions of an equivalent of up to 20 fold of the original fermentation broth were performed in phosphate buffered saline. These fractions were then mixed 1:1 with Laemmli sample buffer with β-mercaptoethanol and boiled for 5 minutes prior to loading 10-20 uL on a Criterion 10% Bis-Tris gel with MOPS buffer. Gels were stained with Simply Blue SafeStain.
SDS-PAGE analysis and quantitation of IB preparations was done by resuspending 25 mg of IB pellet in 1 mL HTS buffer (20 mM Tris, pH 7.5, 50 mM NaCl, 5% v/v glycerol, 10 mM EDTA disodium salt, 0.5% v/v Triton X-100) and sonicated for 1 minute on ice. The resuspended samples were then diluted 1:1 in Laemmli buffer containing 0.2M DTT. The samples were then diluted 10× and 20× with Laemmli buffer lacking DTT and 10 μL was loaded on to a Criterion 18 well 10% Bis-Tris gel run with 1× NuPAGE MES buffer. Gels were run for 5 min at 100V and then 45 min at 200V. Gels were then washed in water for 20 min and stained with Simply Blue Safe Stain. Quantification of target bands was done by comparing densitometric values for the bands against a BSA densitometric standard curve run on the same gel.
Example 2
Insecticidal Activity of Modified Cry Proteins Expressed in Pseudomonas Fluorescens 
Modified Cry proteins listed as SEQ ID NO. 1 through 436 are demonstrated to be active on corn rootworm species including the southern corn rootworm (SCR), Diabrotica undecimpunctata howardi Barber, and the western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte.
Sample Preparation and Bioassays
Inclusion body powders from the relevant P. fluorescens strains are suspended in 100 mM carbonate buffer, pH 11 (approximately 120 mg powder per 3.5 ml buffer). They are incubated overnight at 4° C. on a rocker, and then for about an hour at room temperature, in order to solubilize the toxins. Suspensions are centrifuged at 15,000×g for 20 minutes to pellet insoluble material. To prepare for bioassays, the samples are then transferred to 10 mM CAPS, pH 10 using disposable PD-10 columns (GE Healthcare). All subsequent dilutions of the samples are prepared using 10 mM CAPS, pH 10 and all bioassays contained a treatment of this buffer only which served as a background check.
To quantify toxins in bioassay buffer, samples are prepared for gel electrophoresis by diluting 20-50× into NuPAGE reducing sample buffer (Invitrogen) and heated to 70° C. for 10 minutes. Using BSA to create a standard curve for gel densitometry, varying amounts of toxin sample are loaded on the same gel. The gels are run in MES buffer according to manufacturer's instructions and then stained with a Coomassie Blue based stain. Gels are destained until the background cleared and then are scanned using a BioRad imaging system (Biorad Fluor-S MultiImager with Quantity One software version 4.5.2). BSA standard curves are generated by the software and used to calculate concentrations of toxin in solution.
Bioassays are conducted with first instars on artificial diet in 128-well trays specifically designed for insect bioassays (Bio-Serv, Frenchtown, N.J.). Samples are pipetted into each well so that the surface of the diet is completely covered. Treated trays are placed under a constant airflow at ambient temperature until there is no visible liquid on the diet surface. A single larva is place into each well and the well is sealed using a ventilated lid designed specifically for use with the trays.
Treated trays containing insects are incubated under controlled environmental conditions (28° C.,  ̃40% r.h., 16:8 [L:D]) for 5 days at which point the total number of insects in the treatment, the number of dead insects, and the weight of frozen surviving insects are recorded. Percent mortality and percent growth inhibition are calculated for each treatment. Growth inhibition is calculated as follows:

Growth Inhibition=[1−(Average Weight of Insects in Treatment/Average Weight of Insects in the Background Check)]

Example 3
Expression of Modified Cry Proteins in Plants
One aspect of the subject invention is the transformation of plants with genes encoding the insecticidal protein. The transformed plants are resistant to attack by the target pest.
Genes encoding modified Cry proteins, as disclosed herein, can be inserted into plant cells using a variety of techniques which are well known in the art. For example, a large number of cloning vectors comprising a replication system in E. coli and a marker that permits selection of the transformed cells are available for preparation for the insertion of foreign genes into higher plants. The vectors comprise, for example, pBR322, pUC series, M13mp series, pACYC184, etc. Accordingly, the sequence encoding the modified Cry protein can be inserted into the vector at a suitable restriction site. The resulting plasmid is used for transformation into E. coli. The E. coli cells are cultivated in a suitable nutrient medium, then harvested and lysed. The plasmid is recovered.
Sequence analysis, restriction analysis, electrophoresis, and other biochemical-molecular biological methods are generally carried out as methods of analysis. After each manipulation, the DNA sequence used can be cleaved and joined to the next DNA sequence. Each plasmid sequence can be cloned in the same or other plasmids. Depending on the method of inserting desired genes into the plant, other DNA sequences may be necessary. If, for example, the Ti or Ri plasmid is used for the transformation of the plant cell, then at least the right border, but often the right and the left border of the Ti or Ri plasmid T-DNA, has to be joined as the flanking region of the genes to be inserted.
The use of T-DNA for the transformation of plant cells has been intensively researched and sufficiently described in EP 120 516; Hoekema (1985) In: The Binary Plant Vector System, Offset-durkkerij Kanters B. V., Alblasserdam, Chapter 5; Fraley et al., Crit. Rev. Plant Sci. 4:1-46; and An et al. (1985) EMBO J. 4:277-287.
Once the inserted DNA has been integrated in the plant genome, it is relatively stable. The transformation vector normally contains a selectable marker that confers on the transformed plant cells resistance to a biocide or an antibiotic, such as bialophos, kanamycin, G 418, bleomycin, or hygromycin, inter alia. The individually employed marker should accordingly permit the selection of transformed cells rather than cells that do not contain the inserted DNA.
A large number of techniques are available for inserting DNA into a plant host cell. Those techniques include transformation with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agent, fusion, injection, biolistics (microparticle bombardment), or electroporation as well as other possible methods. If Agrobacteria are used for the transformation, the DNA to be inserted has to be cloned into special plasmids, namely either into an intermediate vector or into a binary vector. The intermediate vectors can be integrated into the Ti or Ri plasmid by homologous recombination owing to sequences that are homologous to sequences in the T-DNA. The Ti or Ri plasmid also comprises the vir region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate themselves in Agrobacteria. The intermediate vector can be transferred into Agrobacterium tumefaciens by means of a helper plasmid (conjugation). Binary vectors can replicate themselves both in E. coli and in Agrobacteria. They comprise a selection marker gene and a linker or polylinker which are framed by the right and left T-DNA border regions. They can be transformed directly into Agrobacteria (Holsters et al. [1978] Mol. Gen. Genet. 163:181-187).
The Agrobacterium used as host cell is to comprise a plasmid carrying a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be contained. The bacterium so transformed is used for the transformation of plant cells. Plant explants can advantageously be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes for the transfer of the DNA into the plant cell. Whole plants can then be regenerated from the infected plant material (for example, pieces of leaf, segments of stalk, roots, but also protoplasts or suspension-cultivated cells) in a suitable medium, which may contain antibiotics or biocides for selection. The plants so obtained can then be tested for the presence of the inserted DNA. No special demands are made of the plasmids in the case of injection and electroporation. It is possible to use ordinary plasmids, such as, for example, pUC derivatives.
The transformed cells grow inside the plants in the usual manner. They can form germ cells and transmit the transformed trait(s) to progeny plants. Such plants can be grown in the normal manner and crossed with plants that have the same transformed hereditary factors or other hereditary factors. The resulting hybrid individuals have the corresponding phenotypic properties.
In a preferred embodiment of the subject invention, plants will be transformed with genes wherein the codon usage has been optimized for plants. See, for example, U.S. Pat. No. 5,380,831, which is hereby incorporated by reference. Methods for creating synthetic B.t. genes for use in plants are known in the art (Stewart 2007).
Agrobacterium Transformation
Standard cloning methods are used in the construction of binary plant expression plasmids. Restriction endonucleases are obtained from New England Biolabs and T4 DNA Ligase (New England Biolabs, Cat# M0202T) was used for DNA ligation. Plasmid preparations are performed using the Nucleospin Plasmid Preparation kit (Machery Nagel, Cat#740 588.250) or the Nucleobond AX Xtra Midi kit (Machery Nagel, Cat#740 410.100), following the instructions of the manufacturers. DNA fragments are purified using the QIAquick PCR Purification Kit (Qiagen, Cat#28104) or the QIAEX II Gel Extraction Kit (Qiagen, Cat#20021) after gel isolation.
DNA fragments are synthesized at DNA2.0 to create the modified Cry genes. Unique restriction sites internal to each gene are identified and a fragment of each gene was synthesized, each containing the specific deletion or insertion. The modified Cry fragments are subcloned into the full length genes at the BbsI restriction site at the start of the gene and a second internal restriction site specific for each gene.
The basic cloning strategy was to subclone full length and the modified Cry coding sequences (CDS) into pDAB8863 at the NcoI and SacI restriction sites. The resulting plasmids are subcloned into the binary plasmid, pDAB3776, utilizing Gateway® technology. LR ClonaseTM (Invitrogen, Cat#11791-019) was used to recombine the full length and modified gene cassettes into the binary expression plasmid.
Electro-competent Agrobacterium tumefaciens (strain Z707S) cells are prepared and transformed using electroporation (Weigel and Glazebrook, 2002). 50 μl of competent agro cells are thawed on ice and 10-25 ng of the desired plasmid was added to the cells. The DNA and cell mix was added to pre-chilled electroporation cuvettes (2 mm). An Eppendorf Electroporator 2510 was used for the transformation with the following conditions, Voltage: 2.4 kV, Pulse length: 5 msec. After electroporation, 1 mL of YEP broth was added to the cuvette and the cell-YEP suspension was transferred to a 15 ml culture tube. The cells are incubated at 28° C. in a water bath with constant agitation for 4 hours. After incubation, the culture was plated on YEP+agar with erythromycin (200 mg/L) and streptomycin (Sigma Chemical Co., St. Louis, Mo.) (250 mg/L). The plates are incubated for 2-4 days at 28° C. Colonies are selected and streaked onto fresh YEP+agar with erythromycin (200 mg/L) and streptomycin (250 mg/L) plates and incubated at 28° C. for 1-3 days.
Colonies are selected for PCR analysis to verify the presence of the gene insert by using vector specific primers. Qiagen Spin Mini Preps, performed per manufacturer's instructions, are used to purify the plasmid DNA from selected Agrobacterium colonies with the following exception: 4 mL aliquots of a 15 mL overnight mini prep culture (liquid YEP+spectinomycin (200 mg/L) and streptomycin (250 mg/L)) are used for the DNA purification. Plasmid DNA from the binary vector used in the Agrobacterium transformation was included as a control. The PCR reaction was completed using Taq DNA polymerase from Invitrogen per manufacture's instructions at 0.5× concentrations. PCR reactions are carried out in a MJ Research Peltier Thermal Cycler programmed with the following conditions; 1) 94° C. for 3 minutes 2) 94° C. for 45 seconds 3) 55° C. for 30 seconds 4) 72° C. for 1 minute per kb of expected product length 5) 29 times to step 2 6) 72° C. for 10 minutes. The reaction was maintained at 4° C. after cycling. The amplification was analyzed by 1% agarose gel electrophoresis and visualized by ethidium bromide staining A colony was selected whose PCR product was identical to the plasmid control.
Arabidopsis Transformation
Arabidopsis thaliana Col-01 is transformed using the floral dip method. The selected colony was used to inoculate one or 15 mL culture of YEP broth containing appropriate antibiotics for selection. The culture is incubated overnight at 280 C with constant agitation at 220 rpm. Each culture is used to inoculate 2 500 ml cultures of YEP broth containing antibiotics for selection and the new cultures are incubated overnight at 280 C with constant agitation. The cells are then pelleted at approx. 8700×g for 10 minutes at room temperature, and the resulting supernatant discarded. The cell pellet is gently resuspended in 500 mL infiltration media containing: 1/2× Murashige and Skoog salts/Gamborg's B5 vitamins, 10% (w/v) sucrose, 0.044 μM benzylamino purine (10 μl/liter of 1 mg/ml stock in DMSO) and 300 μl/liter Silwet L-77. Plants approximately 1 month old are dipped into the media for 15 seconds, being sure to submerge the newest inflorescence. The plants are then laid down on their sides and covered (transparent or opaque) for 24 hours, washed with water, and placed upright. The plants are grown at 220 C, with a 16-hour light/8-hour dark photoperiod. Approximately 4 weeks after dipping, the seeds are harvested.
Arabidopsis Growth and Selection
Freshly harvested seed is allowed to dry for at least 7 days at room temperature in the presence of desiccant. Seed is suspended in a 0.1% Agar (Sigma Chemical Co., St. Louis, Mo.) solution. The suspended seed is stratified at 4° C. for 2 days. Sunshine Mix LP5 (Sun Gro Horticulture Inc., Bellevue, Wash.) is covered with fine vermiculite and sub-irrigated with Hoaglan's solution until wet. The soil mix is allowed to drain for 24 hours. Stratified seed is sown onto the vermiculite and covered with humidity domes (KORD Products, Bramalea, Ontario, Canada) for 7 days. Seeds are germinated and plants are grown in a Conviron (models CMP4030 and CMP3244, Controlled Environments Limited, Winnipeg, Manitoba, Canada) under long day conditions (16 hours light/8 hours dark) at a light intensity of 120-150 μmol/m2 sec under constant temperature (22° C.) and humidity (40-50%). Plants are initially watered with Hoagland's solution and subsequently with DI water to keep the soil moist but not wet.
T1 seed is sown on 10.5′′×21′′ germination trays (T.O. Plastics Inc., Clearwater, Minn.) as described and grown under the conditions outlined. The domes are removed 5-6 days post sowing and plants are sprayed with a 1000× solution of Finale (5.78% glufosinate ammonium, Farnam Companies Inc., Phoenix, Ariz.). Two subsequent sprays are performed at 5-7 day intervals. Survivors (plants actively growing) are identified 7-10 after the final spraying and transplanted into pots prepared with Sunshine mix LP5. Transplanted plants are covered with a humidity dome for 3-4 days and placed in a Conviron with the above mentioned growth conditions.
Example 4
Protein Extraction and Detection
Plant tissue is harvested and immediately frozen in liquid nitrogen or on dry ice and stored at −80° C. in a plastic freezer bag. Upon extraction the sample in the bag is removed from the freezer and placed into liquid nitrogen. The tissue is disrupted by rolling a glass rod across the bag. The tissue is then placed into a tissue homogenizer and homogenization buffer is added (50 mM Tris, buffer, pH 7.8, containing 1 mM DTT, 1 mM EDTA, 1 mM Benzamidine, 0.1% Na Metabisulfate, and 1× plant protease inhibitor cocktail (Sigma)) at a rate of 2 ml buffer/gram of tissue. The tissue is ground well in the homogenizer keeping the unit on wet ice. The sample is placed in a polypropylene centrifuge tube and centrifuged at 14,0000× gravity for 20 minutes at 4° C. The supernatant and pellet are separated and represent both soluble and insoluble fractions respectively. The fractions are then analyzed for recombinant protein as described below.
Proteins are electrotransferred to Nitrocellulose membranes (Amersham Biosciences), which are then blocked with 5% powdered milk in phosphate-buffered saline and 0.05% Tween 20 (Sigma) for 1 h and then incubated with anti-SUP antibody 1:3000 diluted in 3% milk, phosphate-buffered saline, and 0.05% Tween 20. Blots are subsequently incubated with horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit (Amersham Biosciences) and the signal is detected by ECLTM (Amersham Biosciences).
Example 5
Insect Bioassays of Transgenic Arabidopsis 
Transgenic Arabidopsis lines expressing modified Cry proteins listed as SEQ ID NO. 1 through 436 are demonstrated to be active against sensitive rootworm species, including WCR and SCR, in artificial diet overlay assays. Non-transgenic Arabidopsis and/or buffer and water are included in assays as background check treatments.
Protein is extracted from transgenic and non-transgenic Arabidopsis lines using the following procedure. All available rosette leaf tissue from 7-8 week old Arabidopsis T2 plants carrying the modified Cry transgene are harvested into 15 ml conical tubes and frozen on dry ice. Stainless steel balls ( 3/16′′, Small Parts Inc., Miami Lakes, Fla.) are added to each tube and tissue is ground to a fine powder by vortexing. Two ml of extraction buffer (50 mM KPO4, 10 mM dithiothreitol) is added to each tube and the tubes are vortexed for 2 min and kept on ice. Samples are centrifuged at 2750×g and the supernatants are transferred to fresh tubes. Total protein is quantified by Bradford method (REF) and sample volumes are adjusted to normalize protein concentration.
Bioassays are conducted on artificial diet in 128-well trays specifically designed for insect bioassays (Bio-Serv, Frenchtown, N.J.). Samples are pipetted into each well so that the surface of the diet is completely covered. Treated trays are placed under a constant airflow at ambient temperature until there is no visible liquid on the diet surface. A single larva is placed into each well, and the well is sealed using a ventilated lid designed specifically for use with the trays.
Treated trays containing insects are incubated under controlled environmental conditions (28° C.,  ̃40% r.h., 16:8 [L:D]) for 5 days at which point the total number of insects in the treatment, the number of dead insects, and the weight of frozen surviving insects are recorded. Percent mortality and percent growth inhibition are calculated for each treatment. Growth inhibition is calculated as follows:

% Growth Inhibition=[1−(Average Weight of Insects in Treatment/Average Weight of Insects in the Background Check)]

Example 6
Agrobacterium-Mediated Transformation of Maize
Agrobacterium Transformation for Generation of Superbinary Vectors
To prepare for transformation, two different E. coli strains (DH5a containing the pSB11 precursor {either pDAB7702 or pDAB3878 in this case} or pRK2013) are grown at 37° Celsius overnight. The DH5α strain is grown on a petri plate containing LB (5 g Bacto Tryptone, 2.5 g Bacto Yeast Extract, 5 g NaCl, 7.5 g Agar, in 500 ml DI H2O)+Spectinomycin (100 μg/ml) and the pRK2013 strain is grown on a petri plate containing LB+Kanamycin (50 μg/ml). After incubation the plates are placed at 4° Celsius to await the availability of the Agrobacterium strain. Agrobacterium strain LBA4404 containing pSB1 (Japan Tobacco) is grown on AB medium (5 g Glucose, 15 g Agar, in 900 ml DI H2O) with Streptomycin (250 n/ml) and Tetracycline (10 μg/ml) at 28° Celsius for 3 days. After the Agrobacterium is ready, transformation plates are set up by mixing one inoculating loop of each bacteria (pDAB7702 or pDAB3878, pRK2013, and LBA4404+pSB1) on a LB plate with no antibiotics. This plate is incubated at 28° Celsius overnight.
After incubation 1 ml of 0.9% NaCl (4.5 g NaCl in 500 ml DI H2O) solution is added to the mating plate and the cells are mixed into the solution. The mixture is then transferred into a labeled sterile Falcon 2059 (Becton Dickinson and Co. Franklin Lakes, N.J.) tube or equivalent. Another ml of 0.9% NaCl is added to the plate and the remaining cells are mixed into the solution. This mixture is then transferred to the same labeled tube as above. Serial dilutions of the bacterial cells are made ranging from 101-104 by placing 100 μl of the bacterial “stock” culture into labeled Falcon 2059 (Becton Dickinson and Co. Franklin Lakes, N.J.) tubes and then adding 900 μl of 0.9% NaCl. To ensure selection, 100 μl of the dilutions are then plated onto separate AB+Spectinomycin (100 μg/ml)/Sterpromycin (250 μg/ml)/Tetracycline (10 μg/ml) and incubated at 28° Celsius for 4 days.
The colonies are then “patched” onto AB+Spec/Strep/Tet plates as well as lactose medium (0.5 g Yeast Extract, 5 g D-lactose monohydrate, 7.5 g Agar, in 500 ml DI H2O) plates and placed in the incubator at 28° Celsius for 2 days. A Keto-lactose test is performed on the colonies on the lactose media by flooding the plate with Benedict's solution (86.5 g Sodium Citrate monobasic, 50 g Na2CO3, 9 g CuSO4-5H2O, in 500 ml of DI H2O) and allowing the agro colonies to turn yellow.
Any colonies that are yellow (positive for Agrobacterium) are then picked from the patch plate and streaked for single colony isolation on AB+Spec/Strep/Tet plates at 28° Celsius for 2 days. One colony per plate is picked for a second round of single colony isolations on AB+Spec/Strep/Tet media and this is repeated for a total of three rounds of single colony isolations. After the isolations, one colony per plate is picked and used to inoculate separate 3 ml YEP (5 g Yeast Extract, 5 g Peptone, 2.5 g NaCl, in 500 ml DI H2O) liquid cultures containing Spectinomycin (100 μg/ml), Streptomycin (250 μg/ml), and Tetracycline (10 μg/ml). These liquid cultures are then grown overnight at 28° Celsius in a drum incubator at 200 rpm.
Validation cultures are then stated by transferring 2 ml of the inoculation cultures to 250 ml disposable flasks containing 75 ml of YEP+Spec/Strep/Tet. These are then grown overnight at 28° Celsius while shaking at 200 rpm. Following the Qiagen® protocol, Hi-Speed maxi-preps (Qiagen Valencia, Calif.) are then performed on the bacterial cultures to produce plasmid DNA. 500 μl of the eluted DNA is then transferred to 2 clean, labeled 1.5 ml tubes and the Edge BioSystems (Gaithersburg, Md.) Quick-Precip Plus® protocol was followed.
After the precipitation the DNA is resuspended in a total volume of 100 μl TE (10 mM Tris HCl, pH 8.0; 1 mM EDTA). 5 μl of plasmid DNA is added to 50 μl of chemically competent DH5a (Invitrogen; Carlsbad, Calif.) E. coli cells and gently mixed. This mixture is then transferred to chilled and labeled Falcon 2059 (Becton Dickinson and Co. Franklin Lakes, N.J.) tubes. The reaction is incubated on ice for 30 minutes and then heat shocked at 42° Celsius for 45 seconds. The reaction is placed back into the ice for 2 minutes and then 450 μl of SOC medium (Invitrogen; Carlsbad, Calif.) is added to the tubes. The reaction is then incubated at 37° Celsius for 1 hour, shaking at 200 rpm. The cells are then plated onto LB+Spec/Strep/Tet (using 50 μl and 100 μl) and incubated at 37° Celsius overnight.
Three or Four colonies per plate are picked and used to inoculate separate 3 ml LB (5 g Bacto Tryptone, 2.5 g Bacto Yeast Extract, 5 g NaCl, in 500 ml DI H2O) liquid cultures containing Spectinomycin (100 μg/ml), Streptomycin (250 μg/ml), and Tetracycline (10 μg/ml). These liquid cultures are then grown overnight at 37° Celsius in a drum incubator at 200 rpm. Following the Qiagen® protocol, mini-preps (Qiagen Valencia, Calif.) are then performed on the bacterial cultures to produce plasmid DNA. 5 μl of plasmid DNA is then digested in separate reactions using HindIII and SalI enzymes (New England Biolabs Beverly, Mass.) at 37° Celsius for 1 hour before being ran on a 1% agarose (Cambrex Bio science Rockland, Inc. Rockland, Me.) gel. The culture that showed the correct banding pattern is then used to create glycerol stocks by adding 500 μl of culture to 500 μl of sterile glycerol (Sigma Chemical Co.; St. Louis, Mo.) and inverting to mix. The mixture is then frozen on dry ice and stored at −80° Celsius until needed.
Agrobacterium-Mediated Transformation of Maize
Seeds from a High II F1 cross (Armstrong et al., 1991) are planted into 5-gallon-pots containing a mixture of 95% Metro-Mix 360 soilless growing medium (Sun Gro Horticulture, Bellevue, Wash.) and 5% clay/loam soil. The plants are grown in a greenhouse using a combination of high pressure sodium and metal halide lamps with a 16:8 hour photoperiod. For obtaining immature F2 embryos for transformation, controlled sib-pollinations are performed. Immature embryos are isolated at 8-10 days post-pollination when embryos are approximately 1.0 to 2.0 mm in size.
Infection and Cocultivation
Maize ears are surface sterilized by scrubbing with liquid soap, immersing in 70% ethanol for 2 minutes, and then immersing in 20% commercial bleach (0.1% sodium hypochlorite) for 30 minutes before being rinsed with sterile water. The Agrobacterium suspension is prepared by transferring 1-2 loops of bacteria grown on YEP medium (40 g/L peptone, 40 g/L yeast extract, 20 g/L NaCl, 15 g/L Bacto agar) containing 100 mg/L spectinomycin, 10 mg/L tetracycline, and 250 mg/L streptomycin at 28 C for 2-3 days into 5 mL of liquid infection medium (LS Basal Medium (Linsmaier and Skoog, 1965), N6 vitamins (Chu et al, 1965), 1.5 mg/L 2,4-D, 68.5 g/L sucrose, 36.0 g/L glucose, 6 mM L-proline, pH 5.2) containing 100 uM acetosyringone. The solution is vortexed until a uniform suspension is achieved, and the concentration is adjusted to a final density of 200 Klett units, using a Klett-Summerson colorimeter with a purple filter. Immature embryos are isolated directly into a micro centrifuge tube containing 2 mL of the infection medium. The medium is removed and replaced with 1 mL of the Agrobacterium solution with a density of 200 Klett units. The Agrobacterium and embryo solution is incubated for 5 minutes at room temperature and then transferred to co-cultivation medium (LS Basal Medium, N6 vitamins, 1.5 mg/L 2,4-D, 30.0 g/L sucrose, 6 mM L-proline, 0.85 mg/L AgNO3,1, 100 μM acetosyringone, 3.0 g/L Gellan gum, pH 5.8) for 5 days at 25° C. under dark conditions.
After co-cultivation, the embryos are transferred to selective media after which transformed isolates are obtained over the course of approximately 8 weeks. For selection, an LS based medium (LS Basal medium, N6 vitamins, 1.5 mg/L 2,4-D, 0.5 g/L MES, 30.0 g/L sucrose, 6 mM L-proline, 1.0 mg/L AgNO3, 250 mg/L cephotaxime, 2.5 g/L Gellan gum, pH 5.7) is used with Bialaphos. The embryos are transferred to selection media containing 3 mg/liter Bialaphos until embryogenic isolates are obtained. Any recovered isolates are bulked up by transferring to fresh selection medium at 2-week intervals for regeneration and further analysis.
Regeneration and Seed Production
For regeneration, the cultures are transferred to “28” induction medium (MS salts and vitamins, 30 g/L sucrose, 5 mg/L benzylaminopurine, 0.25 mg/L 2, 4-D, 3 mg/liter Bialaphos, 250 mg/L cephotaxime, 2.5 g/L Gellan gum, pH 5.7) for 1 week under low-light conditions (14 μE/m2/s) then 1 week under high-light conditions (approximately 89 μE/m2/s). Tissues are subsequently transferred to “36” regeneration medium (same as induction medium except lacking plant growth regulators). When plantlets grew to 3-5 cm in length, they are transferred to glass culture tubes containing SHGA medium (Schenk and Hildebrandt salts and vitamins (1972), 1.0 g/L myo-inositol, 10 g/L sucrose and 2.0 g/L Gellan gum, pH 5.8) to allow for further growth and development of the shoot and roots. Plants are transplanted to the same soil mixture as described earlier herein and grown to flowering in the greenhouse. Controlled pollinations for seed production are conducted.
Example 7
Bioassay of to Transgenic Maize
WCR eggs are received in soil from Crop Characteristics (Farmington, Minn.). WCR eggs are incubated at 28 C for approximately 10-11 days. Eggs are placed into 0.15% agar solution until the concentration is approximately 75-100 eggs per 0.25 mL aliquot. A hatch plate is set up in a Petri dish with an aliquot of egg suspension on moistened filter paper and sealed with Parafilm. The hatch plate is checked daily for the emergence of WCR larvae. The grade date for the test is 2 weeks after larval hatch.
The soil around the plants is infested with approximately 150-200 WCR eggs, for an estimate of 50-120 larvae per plant during testing. The insects are allowed to feed for 2 weeks, after which a Root Rating is given to each plant. The Node-Injury Scale developed by Oleson, et al. 2005 is utilized for grading, although only scores from 0.01 to 1.0 are given. See Table 2 for a detailed listing of the root ratings and amount of damage caused by the rootworms. The root material is disturbed as little as possible and soil is not washed completely from the plant, so the entire 3-point scale is not used.
Plants which pass the bioassay (with a root rating of 0.5 or less) are transplanted immediately for seed production, and seeds produced by these plants are saved for evaluation as the T1 generation of plants.
Statistical analysis is conducted using JMP software. Protein expression (ng/mL of Cry protein) values are transformed using a natural log transformation.
TABLE 2: Summary of Node-Injury Rating System (Oleson, et al, 2005)


    0.01 No damage or only a few minor feedings
    0.02 Feeding scars evident, but no roots eaten off to within 4 cm of stalk (a root eaten to within 4 cm of the stalk is considered a “pruned root)
    0.10 Many noticeable feeding scars on the outer portion of root, but no root pruning
    0.25 One to three roots pruned, But less than an entire node pruned; outer roots with a moderate amount of feeding damage
    0.50 4-5 roots pruned, considerable feeding damage on the outer portion of the root system
    0.075 6+roots pruned, but with extensive feeding on outer portion of the root system 1.00, 1.25, 1.5, etc.


At least one full node of roots pruned, but fewer than two full nodes pruned 2.00, 2.25, etc.
At least two full nodes of roots pruned, but less than three full nodes pruned

    3.00 Three or more full nodes of roots pruned


Example 8
Construction of Dig-230 Toxin Variants with Protease Sites Engineered within Domain 1
Activity of Cry proteins may be modified by genetic deletion of various structural elements with Domain 1; including Helix 1, Helix 2A and the associated inter-helices loops. Such modifications may impart specific qualities to the engineered toxins which make them desirable over other toxins. Some of these qualities may include activity against Cry-toxin resistant insects, activity against a broader spectrum of insects, and/or increased protein activity, stability or accumulation in transgenic plants. Modification of structural elements within the toxin may be accomplished by means other than by genetic deletion. One alternate method involves the precise mutation of selected amino acid residues within Helix 1, such mutations designed specifically to destabilize the helical structure and increase its processing. Yet another approach by means of which one may modify Domain 1 structural elements is through the inclusion of protease sensitive sites at specific locations intended to enhance processing of the structural elements. In some embodiments it may be desirable to combine these methods.
In this Example, genes encoding variant Cry toxins with improved or broadened activity against certain classes of insects were designed. The variants comprise insertion of sequences encoding protease sensitive sequences into the 5′ end of the DIG-230 coding sequence, with the objective being to affect the removal of Helix1 and Helix2A, and the associated loops, from otherwise wild-type proteins. In one embodiment, protease sites were introduced into the loop region between Helix1 and Helix2A. In a second embodiment, protease sites were introduced into the loop region between Helix1 and Helix2A, and amino acid substitutions were made to destabilize Helix1. In a third embodiment, protease sites were introduced into the loop region between Helix2A and Helix2B. This example discloses such protease site variants of SEQ ID NO: 441, which presents a DNA sequence that encodes a Cry3B-class protein toxin herein referred to as DIG-230. SEQ ID NO: 442 presents the amino acid sequence of the DIG-230 protein encoded by SEQ ID NO: 441. The DIG-230 protein is highly related to the unnamed protein of GenBank Accession No. CAA34983.
Design of Deletion Variants.
We have deduced the likely beginnings and ends of the 3-dimensional structures corresponding to Helix1, Helix2A, and Helix2B, and the locations of the intervening loop regions between them, in Domain 1 of the DIG-230 toxin. These features were predicted by comparing the amino acid sequence of the DIG-230 protein of SEQ ID NO: 442 with the amino acid sequence of a Cry3Bb protein (UniProtKB/Swiss-Prot Accession Number Q06117), for which the crystal structure is known and published (RCSB Protein Structure Database Number 1JI6; and Galitsky et al, (2001)). The homology alignments were conducted using the Jalview 2.3 program (Clamp et al., 2004). The predicted structure locations (Table 3) were used to design the locations of the protease insertion sites of the novel proteins of this invention.







TABLE 3







Amino acid coordinates of projected α-helices of DIG-230 


core toxin Domain 1.













Helix1
spacer
Helix2A
spacer
Helix2B





Residues of
65-80
81-90
91-99
100-104
105-112


SEQ ID NO: 442









One skilled in the field of protein engineering will recognize the dual importance of preserving the accessibility of an engineered protease site and the maintenance of required adjacent protein structural features. The specific protease site sequences employed in this example were derived from corn rootworm gut protease sensitive regions of other Cry toxins, with variations tested to alter charge and structure with the intent of increasing processing. Thus, we tested protease-sensitive sites five or seven residues long, with the object of discovering protease site compositions that would preserve the essential protein structural features of DIG-230 protein Domain 1. In some instances introduction of the engineered protease site was accompanied by the introduction of a helix-destabilizing amino acid at a position distant from the protease insertion site.
Eight protease sensitive sequences were engineered into the loop region between Helix1 and Helix2A by replacing residues 83 to 87 of SEQ ID NO: 442. In addition, amino acid substitutions at two positions were engineered in combination with one of the protease sensitive sequence insertions between Helix1 and Helix2A. Further, two protease sensitive sequences were introduced into the loop region between Helix2A and Helix2B by replacing residues 102-104 of SEQ ID NO: 442. (Deduced co-ordinates of the helices and spacers are presented in Table 3). The insertion positions of the engineered sites and their coordinates in the respective DIG proteins are listed along with the associated SEQ ID Numbers in Table 4. Also disclosed are the amino acid substitutions. All of the introduced sequence changes are intended to increase proteolytic processing within Domain 1 of the engineered protein and to thereby increase insect toxin activity. Processing at the engineered sites may occur within a transgenic plant or within the insect gut. In either or both cases, the effectiveness of the engineered protein toxin will be one of increased activity. Further, such an engineered protein may be inherently toxic to a broader spectrum of insect species than is exhibited by the wild type toxin from which it is derived.







TABLE 4







Protease insertion sites of DIG-230 protease variants. SEQ ID


Numbers associated with each protease variant (both DNA coding


sequence and encoded protein) are listed. Protease Site Residues are


those of the corresponding protein SEQ ID NO. for each DIG protein.













DIG

DIG-230
DIG-230
SEQ ID
SEQ ID
Protease


Num-
Insertion
Insertion
Deleted
NO:
NO:
Site


ber
Location
Positiona
Residues
(DNA)
(Protein)
Residues
















DIG-
Between
82
83-87
443
444
83-89


52
Helix1








and Helix2A







DIG-
Between
82
83-87
445
446
83-89


53
Helix1








and Helix2A







DIG-
Between
82
83-87
447
448
83-89


54
Helix1








and Helix2A







DIG-
Between
82
83-87
449
450
83-89


55
Helix1








and Helix2A







DIG-
Between
82
83-87
451
452
83-89


56
Helix1








and Helix2A







DIG-
Between
82
 83-87b
453
454
83-89


57
Helix1








and Helix2A







DIG-
Between
82
 83-87c
455
456
83-89


58
Helix1








and Helix2A







DIG-
Between
82
 83-87d
457
458
83-89


99
Helix1








and Helix2A







DIG-
Between
101
102-104
459
460
102-106


59
Helix2A








and Helix2B







DIG-
Between
101
102-104
461
462
102-106


60
Helix2A








and Helix2B





aProtease sensitive site was engineered immediately after the listed amino acid position of the DIG-230 sequence disclosed in SEQ ID NO: 442.


bResidue 70 (I) of SEQ ID NO: 442 replaced with D


cResidue 74 (G) of SEQ ID NO: 442 replaced with D


dResidues 70 (I) and 74 (G) of SEQ ID NO: 442 both replaced with D






The coding sequences for proteins DIG-52 through DIG-60, DIG-99, and DIG-230 are inserted into the Pseudomonas expression vector pDOW1169 (WO/2008/151319) and expressed as described below.
Example 9
Construction of Expression Plasmids Encoding Dig-Proteins and Expression in Bacterial Hosts
Construction of Expression Vectors.
Standard cloning methods as taught in Sambrook et al. (1989) and Ausubel et al. (1995) are used in the construction of Pseudomonas fluorescens (Pf) expression plasmids engineered to produce the DIG-52, DIG-53, DIG-54, DIG-55, DIG-56, DIG-57, DIG-58, DIG-59, DIG-60, DIG-99, or DIG-230 proteins. Restriction endonucleases are obtained from NEB and T4 DNA Ligase (Invitrogen) is used for DNA ligation. Plasmid preparations are performed using the NucleoBond® Xtra Kit (Macherey-Nagel Inc, Bethlehem, Pa.) or the Plasmid Midi Kit (Qiagen), following the instructions of the suppliers. DNA fragments are purified using the Millipore Ultrafree®-DA cartridge (Billerica, Mass.) after agarose Tris-acetate gel electrophoresis.
The basic cloning strategy consists of subcloning a DIG coding sequence into pDOW1169 with SpeI and XhoI restriction sites, whereby it is placed under the expression control of the Ptac promoter and the rrnBT1T2 terminator from plasmid pKK223-3 (PL Pharmacia, Milwaukee, Wis.). It is understood, however, that other vectors may be used. pDOW1169 is a medium copy plasmid with the RSF1010 origin of replication, a pyrF gene, and a ribosome binding site preceding the restriction enzyme recognition sites into which DNA fragments containing protein coding regions may be introduced, (US Patent Application No. 20080193974). The expression plasmid is transformed by electroporation into DC454 (a near wild-type P. fluorescens strain having mutations ΔpyrF and lsc::lacIQI), or its derivatives, recovered in SOC-Soy hydrolysate medium, and plated on selective medium (M9 glucose agar lacking uracil, Sambrook et al., supra). Details of the microbiological manipulations are available in Squires et al., (2004), and in US Patent Application No. 20060008877, US Patent Application No. 20080193974, and US Patent Application No. 20080058262, incorporated herein by reference. Colonies are screened by restriction digestion of miniprep plasmid DNA. Plasmid DNA of selected clones containing inserts is sequenced by a commercial sequencing vendor such as Eurofin MWG Operon (Huntsville, Ala.). Sequence data are assembled and analyzed using the SequencherTM software (Gene Codes Corp., Ann Arbor, Mich.).
Growth and Expression Analysis in Shake Flasks.
Production of DIG toxins for characterization and insect bioassay is accomplished by shake-flask-grown P. fluorescens strains harboring expression constructs. Seed cultures grown in M9 medium supplemented with 1% glucose and trace elements are used to inoculate 50 mL of defined minimal medium with 5% glycerol (Teknova Cat. #3D7426, Hollister, Calif.). Expression of the DIG toxin gene via the Ptac promoter is induced by addition of isopropyl-β-D-1-thiogalactopyranoside (IPTG) after an initial incubation of 24 hours at 30° C. with shaking. Cultures are sampled at the time of induction and at various times post-induction. Cell density is measured by optical density at 600 nm (OD600). One skilled in the field of Pseudomonas gene expression will understand that other culture media suitable for growth of Pseudomonas fluorescens may also be utilized, for example, as described in Huang et al., 2007 and US Patent Application No. 20060008877.
Cell Fractionation and SDS-PAGE Analysis of Shake Flask Samples.
At each sampling time, 2 mL aliquots are centrifuged at 14000×g for two minutes. The cell pellets are frozen at −20° C. Soluble and insoluble fractions from frozen shake flask cell pellet samples are generated for analysis. The cell pellet is resuspended in 1 mL of phosphate buffer (Fisher Scientific Catalog #02-686-201A 0.3 mM Phosphate pH7) and transferred to a 15 mL conical screw cap centrifuge tube. The cells are cooled on ice and lysed by sonication with a 2 mm tip and a Branson 250 sonicator using 2 bursts of 45 seconds each at an output of 20 using the constant duty cycle. The lysate is transferred to a 1.5 mL microfuge tube and centrifuged at 14,000 rpm for 10 minutes using a table top microcentrifuge. The supernatant fraction is removed and represents the soluble or supernatant fraction. The pellet is suspended in 0.5 mL of phosphate buffer and represents the pellet or insoluble fraction. The protein concentrations of the fractions are determined using BioRad Protein Dye Assay Reagent. Samples are diluted appropriately and mixed with 4× Laemmli sample buffer containing β-mercaptoethanol (Sambrook et al., supra) and boiled for 5 minutes prior to loading onto Criterion XT Bis-Tris 12% gels (Bio-Rad Inc., Hercules, Calif.) Electrophoresis is performed in the recommended XT MOPS buffer. Gels are stained with Bio-Safe Coomassie Stain according to the manufacturer's (Bio-Rad) protocol and imaged using the Alpha Innotech Imaging system (San Leandro, Calif.).
Inclusion Body Preparation.
Cry protein inclusion body (IB) preparations are prepared from P. fluorescens cells that produce insoluble DIG protein. Cell pellets are thawed in a 37° C. water bath. The cells are adjusted to 25% w/v in lysis buffer (50 mM Tris, pH 7.5, 200 mM NaCl, 20 mM EDTA disodium salt, 1% Triton X-100, and 5 mM Dithiothreitol (DTT), 5 mL/L of bacterial protease inhibitor cocktail (P8465 Sigma Chemicals, St. Louis, Mo.) are added just prior to use). The cells are suspended using a hand-held homogenizer at lowest setting (Tissue Tearor, BioSpec Products, Inc Bartlesville, Okla.). Lysozme is added to the cell suspension (Sigma L7651, from chicken egg white), mixed with a metal spatula, and incubated at room temperature for one hour. After incubation, the suspension is cooled on ice for 15 minutes and sonicated using a Branson Sonifier 250 (two 1-minute sessions, at 50% duty cycle, 30% output), then checked for cell lysis by microscopy. Additional lysozyme is added and the sonication is repeated until cell lysis is confirmed via microscopy. The lysate is centrifuged at 11,500×g for 25 minutes to form the IB pellet. The supernatant is discarded and the IB pellet is resuspended in 100 mL of lysis buffer, homogenized with the hand-held mixer, and centrifuged again. The IB pellet is washed again by resuspension (in 50 mL lysis buffer), homogenization, sonication, and centrifugation until the supernatant becomes colorless and the IB pellet becomes firm and off-white in color. The final pellet is suspended in a sterile solution of 2 mM EDTA and stored at −80° C. in 1 mL aliquots.
Solubilization and Fractionation of IBs.
Two hundred fifty mg of IBs are thawed and centrifuged, the storage buffer is removed, and the pellets are suspended in 1 mL of 0.5M CAPS pH11 and transferred to a 15 mL centrifuge tube. Urea (3.6 gm) is added to the IB suspension and the solution is rocked gently to dissolve the pellet, then 25 mg of DTT, 50 mg of benzamidine and 200 μL of 0.5M EDTA are added. Water is added to adjust the final volume to 10 mL. The tube is kept in a 37° C. incubator and shaken vigorously at 250 rpm for 1 hr, then is left at room temperature overnight. Insoluble material is spun down and the solution is filtered through a 0.45 μM filter and loaded onto a Superose 6 column (GE Healthcare Life Sciences). The column is run at 0.7 mL/min using 25 mM CAPS pH11 containing 0.3M NaCl and 4M urea as the mobile phase. Fractions are collected and pooled on the basis of toxin aggregate size, and the urea in the pooled fractions is removed either by repeated passage through Amicon ultrafiltration units with a 10-30 kDa size cutoff (Millipore) or by dialyzing against 10 mM CAPS pH11.
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## Claims

1. A Cry3A protein having a protease cleavage site inserted between α-Helix2A and α-Helix2B in domain I.

2. The protein of claim 1 wherein said protein is a Cry3Aa protein.

3. A method of inhibiting an insect, said method comprising providing the protein of claim 1 to said insect for ingestion.

4. A polynucleotide encoding the protein of claim 1.

5. A plant cell comprising the polynucleotide of claim 4.

6. A plant comprising a plurality of the plant cell of claim 5.

7. A method of protecting a plant from insect damage, said method comprising providing the protein of claim 1 to said insect for ingestion.

8. The method of claim 7, wherein said protein is produced by and is present in said plant.

9. The protein of claim 1, wherein said protease cleavage site is more susceptible to cleavage by an insect protease than by a plant protease.