USOO8304605B2

(12) United States Patent (10) Patent No.: US 8,304,605 B2 Lira et al. (45) Date of Patent: Nov. 6, 2012

(54) DIG-11 INSECTICIDAL CRY TOXINS C07K 14/325 (2006.01) AOIN57/8 (2006.01) (75) Inventors: Justin M. Lira, Fishers,IN (US); (52) U.S. Cl...... 800/279: 800/302:530/350; 514/4.5; Kenneth Narva, Zionsville, IN (US); 435/320.1 N RGAS in (58) Field of Classification Search ...... None (US); Timothy D. Hey Zionsville, IN See application file for complete search history. (US) (56) References Cited (73) Assignee: Dow AgroSciences, LLC., Indianapolis, IN (US) U.S. PATENT DOCUMENTS 2007/0044178 A1 2/2007 Carozzi et al...... 800,279 (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 OTHER PUBLICATIONS U.S.C. 154(b) by 193 days. de Maagdet al (Trends in Genetics, 2001, 17(4), 193-199).* (21) Appl. No.: 12/814,813 * cited by examiner (22) Filed: Jun. 14, 2010 Primary Examiner — Anne Kubelik (65) Prior Publication Data Assistant Examiner — Lee A Visone (74) Attorney, Agent, or Firm — Ronald Maciak, Faegre US 2010/0319093 A1 Dec. 16, 2010 Baker Daniels LLP

Related U.S. Application Data (57) ABSTRACT (60) Provisional application No. 61/187.460, filed on Jun. 16, 2009. DIG-1 1 Cry toxins, polynucleotides encoding such toxins, s use of Such toxins to control pests, and transgenic plants that (51) Int. Cl. produce Such toxins are disclosed. AOIH 5/00 (2006.01) CI2N 15/32 (2006.01) 19 Claims, No Drawings US 8,304,605 B2 1. 2 DG-11 INSECTICIDAL CRY TOXINS be produced in combination in transgenic corn to prevent the development WCR resistance and protect the long term CROSS-REFERENCE TO RELATED utility of B.t. technology for rootworm control. APPLICATIONS BRIEF SUMMARY OF THE INVENTION This application claims the benefit of U.S. Provisional Application 61/187.460 filed on Jun. 16, 2009, which is The present invention provides insecticidal Cry toxins, expressly incorporated by reference herein. including the protein toxin designated herein as DIG-11 as well as variants of DIG-11, nucleic acids encoding these FIELD OF THE INVENTION 10 toxins, methods of controlling pests using the toxins, methods of producing the toxins in transgenic host cells, and trans This invention concerns new insecticidal Cry toxins and genic plants that express the toxins. The predicted amino acid their use to control . sequence of the wild type DIG-11 protein is given in SEQID NO:2. BACKGROUND OF THE INVENTION 15 The present invention provides easily administered, func Bacillus thuringiensis (B.t.) is a soil-borne bacterium that tional proteins. The present invention also provides a method produces pesticidal crystal proteins known as delta endotox for delivering insect toxins that are functionally active and ins or Cry proteins. Cry proteins are oral intoxicants that effective against many orders of insects, preferably function by acting on midgut cells of Susceptible insects. coleopteran insects. By “functional activity” (or “active Some Cry toxins have been shown to have activity against against) it is meant herein that the protein toxins function as nematodes. An extensive list of delta endotoxins is main orally active insect control agents (alone or in combination tained and regularly updated at http://www.lifesci. Sussex. with other proteins), that the proteins have a toxic effect ac.uk/home/Neil Crickmore/Bt/intro.html. (alone or in combination with other proteins), or are able to Western corn rootworm (WCR), virgifera vir 25 disrupt or deter insect growth and/or feeding which may or gifera LeConte, is an economically important corn pest that may not cause death of the insect. When an insect comes into causes an estimated S1 billion revenue loss each year in North contact with an effective amount of a “toxin' of the subject America due to crop yield loss and expenditures for insect invention delivered via transgenic plant expression, formu management (Metcalf, 1986). WCR management practices lated protein composition(s), sprayable protein include crop rotation with soybeans, chemical insecticides 30 composition(s), a bait matrix or other delivery system, the and, more recently, transgenic crops expressing B.t. Cry pro results are typically death of the insect, inhibition of the teins. However, to date only a few examples of Bt. Cry growth and/or proliferation of the insect, and/or prevention of proteins provide commercial levels of efficacy against WCR, the insects from feeding upon the source (preferably a trans including Cry34Ab1/Cry35Ab1 (Ellis et al., 2002), modified genic plant) that makes the toxins available to the insects. Cry3Aa1 (Walters et al., 2008) and modified Cry3Bb1 35 (Vaughn et al., 2005). These B.t. proteins are highly effective Functional proteins of the Subject invention can also work at preventing WCR corn root damage when produced in the together or alone to enhance or improve the activity of one or roots of transgenic corn (Moellenbecket al., 2001, Vaughnet more other toxin proteins. The terms “toxic,” “toxicity,” or al., 2005, U.S. Pat. No. 7,361,813). “toxin' as used herein are meant to convey that the subject Despite the success of WCR-resistant transgenic corn, sev 40 “toxins have “functional activity” as defined herein. eral factors create the need to discover and develop new Cry Complete lethality to feeding insects is preferred but is not proteins to control WCR. First, although production of the required to achieve functional activity. If an insect avoids the currently-deployed Cry proteins in transgenic corn plants toxin or ceases feeding, that avoidance will be useful in some provides robust protection against WCR root damage, applications, even if the effects are sublethal or lethality is thereby protecting grain yield. Some WCR adults emerge in 45 delayed or indirect. For example, if insect resistant transgenic artificial infestation trials, indicating less than complete larval plants are desired, the reluctance of insects to feed on the insect control. Second, development of resistant insect popu plants is as useful as lethal toxicity to the insects because the lations threatens the long-term durability of Cry proteins in ultimate objective is avoiding insect-induced plant damage. rootworm control. Lepidopteran insects resistant to Cry pro As described in Example 1, a nucleic acid encoding the teins have developed in the field for Plutella xylostella 50 DIG-11 protein was isolated from a B.t. strain internally (Tabashnik, 1994), Trichoplusia ni (Janmaat and Myers, designated by Dow AgroSciences LLC as PS184M1. The 2003, 2005), and Helicoverpa zeae (Tabashnik et al., 2008). nucleic acid sequence for the full length coding region was Insect resistance to B.t. Cry proteins can develop through determined, and the full length protein sequence was deduced several mechanisms (Heckel et al., (2007), Pigott and Ellar, from the nucleic acid sequence. The DIG-11 protein has some 2007). Multiple receptor protein classes for Cry proteins have 55 similarity tO Cry7Ab3 (Genbank Accession been identified within insects, and multiple examples exist No.ABX24522.1) and other B. thuringiensis Cry7-type pro within each receptor class. Resistance to a particular Cry teins (http://www.lifesci.sussex.ac.uk/home/Neil Crick protein may develop, for example, by means of a mutation more/Bt/intro.html). within the toxin-binding portion of a cadherin domain of a Insect active variants of the DIG-11 toxin are also receptor protein. A further means of resistance may be medi 60 described herein, and are referred to collectively as DIG-11 ated through a protoxin-processing protease. Resistance to insect toxins. Individual variants of DIG-11 insect toxin may Cry toxins in species of has a complex genetic be identified by specific DIG-nomenclature. The toxins can basis, with at least four distinct, major resistance genes. Simi be used alone or in combination with other Cry toxins, such as larly, multiple genes are predicted to control resistance to Cry Cry34Ab1/Cry35Ab1 (DAS-59122-7), Cry3Bb1 toxins in species of Coleoptera. Development of new high 65 (MON88017), Cry3A (MIR604), chimeric Cry1Ab/Cry3Aa potency Cry proteins will provide additional tools for WCR (FR8A, WO 2008/121633 A1), CryET33 and CryET34, management. Cry proteins with different modes of action can Vip1A, Cry1 Ia, CryET84, CryET80, CryET76, CryET71, US 8,304,605 B2 3 4 CryET69, CryET75, CryET39, CryET79, and CryET74 to In another embodiment the invention provides a plant com control development of resistant Coleopteran insect popula prising a DIG-11 insect toxin. tions. In another embodiment the invention provides a method DIG-11 insect toxins may also be used in combination with for controlling a pest population comprising contacting said RNAi methodologies for control of other insect pests. For example, DIG-11 insect toxin can be used in transgenic plants population with a pesticidally effective amount of a DIG-11 in combination with a dsRNA for suppression of an essential insect toxin gene in corn rootworm or an essential gene in an insect pest. In another embodiment the invention provides an isolated Such target genes include, for example, vacuolar ATPase, nucleic acid that encodes a DIG-11 toxin. ARF-1, Act42A, CHD3, EF-1C, and TFIIB. An example of a In another embodiment the invention provides a DNA con Suitable target gene is vacuolar ATPase, as disclosed in 10 struct comprising a nucleotide sequence that encodes a DIG WO2007/035650. 11 insect toxin operably linked to a promoter that is not In one embodiment the invention provides an isolated derived from Bacillus thuringiensis and is capable of driving DIG-11 insect toxin polypeptide comprising a core toxin expressionina plant. The invention also provides a transgenic segment selected from the group consisting of plant that comprises the DNA construct stably incorporated (a) a polypeptide comprising the amino acid sequence of 15 residues 142 to 664 of SEQID NO:2: into its genome and a method for protecting a plant from a (b) a polypeptide comprising an amino acid sequence hav pest comprising introducing the construct into said plant. ing at least 90% sequence identity to the amino acid sequence of residues 142 to 664 of SEQID NO:2: BRIEF DESCRIPTION OF THE SEQUENCES (c) a polypeptide comprising an amino acid sequence of residues 142 to 664 of SEQID NO:2 with up to 20 amino SEQ ID NO:1 DNA sequence encoding full-length DIG acid substitutions, deletions, or modifications that do not 11 insect toxin; 3492nt. adversely affect expression or activity of the toxin SEQID NO:2 Full-length DIG-11 protein sequence; 1164 encoded by SEQID NO:2: aa. or an insecticidally active fragment thereof. 25 SEQID NO:3 Maize-optimized DNA sequence encoding In another embodiment the invention provides an isolated DIG-84, a DIG-11 core toxin: 1992 int. DIG-11 insect toxin polypeptide comprising a DIG-11 core SEQID NO:4 Cry1Ab protoxin segment; 545 aa. toxin segment selected from the group consisting of SEQIDNO:5 Chimeric toxin: DIG-84 core toxin segment/ (a) a polypeptide comprising the amino acid sequence of Cry1Ab protoxin segment; 1209 aa. residues 1 to 664 of SEQID NO:2: 30 SEQ ID NO:6 Dicot-optimized DNA sequence encoding (b) a polypeptide comprising an amino acid sequence hav the Cry1Ab protoxin segment; 1635 nt ing at least 90% sequence identity to the amino acid SEQID NO:7 Maize-optimized DNA sequence encoding sequence of residues 1 to 664 of SEQID NO:2: the Cry1Ab protoxin segment; 1635 nt (c) a polypeptide comprising an amino acid sequence of residues 1 to 664 of SEQID NO:2 with up to 20 amino 35 DETAILED DESCRIPTION OF THE INVENTION acid substitutions, deletions, or modifications that do not adversely affect expression or activity of the toxin DIG-11 insect toxins, and insect active variants. In addition encoded by SEQID NO:2: to the full length DIG-11 insect toxin of SEQ ID NO:2, the or an insecticidally active fragment thereof. invention encompasses insect active variants. By the term In another embodiment the invention provides an isolated 40 “variant', applicants intend to include fragments, certain DIG-11 insect toxin polypeptide comprising a DIG-11 core deletion and insertion mutants, and certain fusion proteins. toxin segment selected from the group consisting of The DIG-11 protein is a classic three-domain Cry toxin. As a (a) a polypeptide comprising the amino acid sequence of preface to describing variants of the DIG-11 insect toxin that residues 142 to 1164 of SEQID NO:2: are included in the invention, it will be useful to briefly review (b) a polypeptide comprising an amino acid sequence hav 45 the architecture of three-domain Cry toxins in general and of ing at least 90% sequence identity to the amino acid the DIG-11 insect toxin in particular. sequence of residues 142 to 1164 of SEQID NO:2: A majority of Bacillus thuringiensis delta-endotoxin crys (c) a polypeptide comprising an amino acid sequence of tal protein molecules are composed of two functional seg residues 142 to 1164 of SEQ ID NO:2 with up to 20 ments. The protease-resistant core toxin is the first segment amino acid Substitutions, deletions, or modifications 50 and corresponds to about the first half of the protein molecule. that do not adversely affect expression or activity of the The full -130 kDa protoxin molecule is rapidly processed to toxin encoded by SEQID NO:2: the resistant core segment by proteases in the insect gut. The or an insecticidally active fragment thereof. segment that is deleted by this processing will be referred to In another embodiment the invention provides an isolated herein as the “protoxin segment. The protoxin segment is DIG-11 insect toxin polypeptide comprising a DIG-11 core 55 believed to participate intoxin crystal formation (Arvidson et toxin segment selected from the group consisting of al., (1989). The protoxin segment may thus convey a partial (a) a polypeptide comprising the amino acid sequence of insect specificity for the toxin by limiting the accessibility of residues 1 to 1164 of SEQID NO:2: the core to the insect by reducing the protease processing of (b) a polypeptide comprising an amino acid sequence hav the toxin molecule (Haider et al., (1986) or by reducing toxin ing at least 90% sequence identity to the amino acid 60 solubility (Aronson et al., (1991). B.t. toxins, even within a sequence of residues 1 to 1164 of SEQID NO:2: certain class, vary to some extent in length and in the precise (c) a polypeptide comprising an amino acid sequence of location of the transition from the core toxin portion to pro residues 1 to 1164 of SEQID NO:2 with up to 20 amino toxin portion. The transition from core toxin portion to pro acid substitutions, deletions, or modifications that do not toxin portion will typically occur at between about 50% to adversely affect expression or activity of the toxin 65 about 60% of the full length toxin. SEQID NO:2 discloses the encoded by SEQID NO:2: 1164 amino acid sequence of the full-length DIG-11 polypep or an insecticidally active fragment thereof. tide, of which the N-terminal 664 amino acids comprise a US 8,304,605 B2 5 6 DIG-84 core toxin segment of the DIG-11 protein. The 5'-ter- mers of molecular weight about 60 kDa into pre-pores in the minal 1992 nucleotides of SEQID NO:1 are a coding region absence of cadherin binding. These N-terminal deletion for a DIG-84 core toxin segment. mutants were reported to be active on Cry-resistant insect Three dimensional crystal structures have been determined larvae. Furthermore, Diaz-Mendoza et al., (2007) described for Cry1Aa1, Cry2Aa1, Cry3Aa1, Cry3Bb1, Cry4Aa, 5 Cry1Ab fragments of 43 kDa and 46 kDa that retained activ Cry4Ba and Cry8Ea1. These structures for the core toxins are ity on Mediterranean corn borer (Sesamia nonagrioides). remarkably similar and are comprised of three distinct These fragments were demonstrated to include amino acid domains with the features described below (reviewed in de residues 116 to 423; however the precise amino acid Maagdet al., 2003). sequences were not elucidated and the mechanism of activity Domain I is abundle of seven alpha helices where helix five 10 of these proteolytic fragments is unknown. The results of is surrounded by six amphipathic helices. This domain has Gomez et al., (2002), Soberonet al., 2007 and Diaz-Mendoza been implicated in pore formation and shares homology with et al., (2007) contrast with those of Hofte et al., (1986), who other pore forming proteins including hemolysins and reported that deletion of 36 amino acids from the N-terminus colicins. Domain I of the DIG-11 protein comprises amino of Cry1Ab resulted in loss of insecticidal activity. acid residues 86 to 306 of SEQID NO:2. 15 We have deduced the beginning and end of helices 1, 2A, Domain II is formed by three anti-parallel beta sheets 2B, and 3, and the location of the spacer regions between packed togetherina beta prism. The loops of this domain play them in Domain I of the DIG-11 toxin by comparing the important roles in binding insect midgut receptors. In Cry1A DIG-11 protein sequence with the protein sequence for proteins, Surface exposed loops at the apices of domain II beta Cry8Ea1, for which the structure is known. These locations sheets are involved in binding to Lepidopteran cadherin are described in Table 1. TABLE 1 Amino acid coordinates of projected C-helices of DIG-11 protein. Helix1 spacer Helix2A spacer Helix2B spacer Helix3 spacer Helix4 Residues of 82-99 100-102 103-117 118-126 127-136 137-141 142-171 172-175 176-196 SEQ ID NO: 2 receptors. Cry3Aa domain II loops bind a membrane-associ- " Amino terminal deletion variants of DIG-11 In one of its ated metalloprotease of Leptinotarsa decemlineata (Say) aspects the invention provides DIG-11 insect toxin variants in (Colorado potato ) in a similar fashion (Ochoa-Campu- which all or part of helices 1, 2A, and 2B are deleted to Zano et al., 2007). Domain II shares homology with certain improve insect activity and avoid development of resistance carbohydrate-binding proteins including Vitelline and jaca- is by insects. These modifications are made to provide DIG-11 line. Domain II of the DIG-11 protein comprises amino acid variants with improved attributes, such as improved target residues 311 to 508 of SEQID NO:2. pest spectrum, potency, and insect resistance management. In Domain III is a beta sandwich of two anti-parallel beta Some embodiments of the Subject invention, the Subject modi sheets. Structurally this domain is related to carbohydrate- fications may affect the efficiency of protoxin activation and binding domains of proteins such as glucanases, galactose 40 pore formation, leading to insect intoxication. More specifi oxidase, sialidase and others. Domain III binds certain classes cally, to provide DIG-11 insect toxin variants with improved of receptor proteins and perhaps participates in insertion of an attributes, step-wise deletions are described that remove part oligomeric toxin pre-pore that interacts with a second class of of the gene encoding the N-terminus. The deletions remove receptors, examples of which are aminopeptidase and alka- all of C-helix 1 and all or part of C-helix 2 in Domain I, while line phosphatase in the case of Cry1A proteins (Honée et al., 45 maintaining the structural integrity of the C-helices 3 through (1991), Pigott and Ellar, 2007)). Analogous Cry Domain III 7. The subject invention therefore relates in part to improve receptors have yet to be identified in Coleoptera. Conserved ments to Cry protein efficacy made by engineering the C-he B.t. Sequence blocks 2 and 3 map near the N-terminus and lical components of Domain I for more efficient pore forma C-terminus of Domain 2, respectively. Hence, these con- tion. More specifically, the subject invention relates in part to served sequence blocks 2 and 3 are approximate boundary 50 improved DIG-11 insect toxins designed to have N-terminal regions between the three functional domains. These regions deletions in regions with putative secondary structure homol of conserved DNA and proteinhomology have been exploited ogy to C-helices 1 and 2 in Domain I of Cry 1 proteins. for engineering recombinant B.t. toxins (U.S. Pat. No. 6,090, Deletions to improve the insecticidal properties of the 931, WO 91/01087, WO 95/06730, WO 1998.022595). DIG-11 insect toxins may initiate before the predicted C-he Domain III of the DIG-11 protein comprises amino acid 55 lix 2A start, and may terminate after the C-helix 2B end, but residues 518 to 662 of SEQID NO:2. preferably do not extend into C-helix 3 It has been reported that C-helix 1 of domain I is removed In designing coding sequences for the N-terminal deletion following receptor binding. Aronson et al. (1999) demon- variants, an ATG start codon, encoding methionine, is strated that Cry1Ac bound to BBMV was protected from inserted at the 5' end of the nucleotide sequence designed to proteinase K cleavage beginning at residue 59, just after C-he- 60 express the deletion variant. For sequences designed for use lix 1; similar results were cited for Cry1Ab. Gomez et al., in transgenic plants, it may be of benefit to adhere to the (2002) found that Cry1Ab oligomers formed upon BBMV “N-end rule of Varshaysky (1997). It is taught that some receptor binding lacked the C-helix 1 portion of domain I. amino acids may contribute to protein instability and degra Also, Soberon et al., (2007) have shown that N-terminal dation in eukaryotic cells when displayed as the N-terminal deletion mutants of Cry1Aband Cry1Ac which lack approxi- 65 residue of a protein. For example, data collected from obser mately 60 amino acids encompassing C-helix 1 on the three Vations in yeast and mammalian cells indicate that the N-ter dimensional Cry structure are capable of assembling mono- minal destabilizing amino acids are F, L, W.Y. R. K. H., I, N, US 8,304,605 B2 7 8 Q, D, E and possibly P. While the specifics of protein degra In another embodiment of the invention, protease cleavage dation mechanisms may differ somewhat between organisms, sites may be engineered at desired locations to affect protein the conservation of identity of N-terminal destabilizing processing within the midgut of susceptible larvae of certain amino acids seen above Suggests that similar mechanisms insect pests. These protease cleavage sites may be introduced may function in plant cells. For instance, Worley et al., (1998) by methods such as chemical gene synthesis or splice overlap found that in plants, the N-end rule includes basic and aro PCR (Horton et al., 1989). Serine protease recognition matic residues. It is a possibility that proteolytic cleavage by sequences, for example, can optionally be inserted at specific plant proteases near the start of C-helix 3 of subject B.t. sites in the Cry proteinstructure to effect protein processing at insecticidal proteins may expose a destabilizing N-terminal desired deletion points within the midgut of susceptible lar 10 vae. Serine proteases that can be exploited in Such fashion amino acid. Such processing may target the cleaved proteins include Lepidopteran midgut serine proteases such as trypsin for rapid decay and limit the accumulation of the B.t. insec or trypsin-like enzymes, chymotrypsin, elastase, etc. (Chris ticidal proteins to levels insufficient for effective insect con teller et al., 1992). Further, deletion sites identified empiri trol. Accordingly, for N-terminal deletion variants that begin cally by sequencing Cry protein digestion products generated with one of the destabilizing amino acids, applicants prefer to 15 with unfractionated larval midgut protease preparations or by add a codon that specifies a G (glycine) amino acid between binding to brush border membrane vesicles can be engineered the translational initiation methionine and the destabilizing to effect protein activation. Modified Cry proteins generated amino acid. either by gene deletion or by introduction of protease cleav Example 2 gives specific examples of amino-terminal dele age sites have improved activity on Lepidopteran pests Such tion variants of DIG-11 insect toxins in accordance with the as Ostrinia nubilalis, Diatraea grandiosella, Helicoverpa invention. zea, Agrotis ipsilon, Spodoptera frugiperda, Spodoptera Chimeric Toxins Chimeric proteins utilizing the core toxin exigua, Diatraea saccharalis, Loxagrotis albicosta, domain of one Cry toxin fused to the protoxin segment of Coleopteran pests such as western corn rootworm, Southern another Cry toxin have previously been reported. DIG-11 corn root worn, northern corn rootworm (i.e. Diabrotica variants include insect toxins comprising an N-terminal core 25 spp.), and other target pests. toxin segment of a DIG-11 insect toxin (which may be full Coleopteran serine proteases such as trypsin, chymot length or have the N-terminal deletions described above) rypsin and cathepsin G-like protease, Coleopteran cysteine fused to a heterologous protoxin segment at Some point past proteases such as cathepsins (B-like, L-like, O-like, and the end of the core toxin portion. The transition to the heter K-like proteases) (Koiwa et al., (2000) and Bown et al., ologous protoxin segment can occur at approximately the 30 (2004), Coleopteran metalloproteases such as ADAM10 core toxin/protoxinjunction or, in the alternative, a portion of (Ochoa-Campuzano et al., (2007)), and Coleopteran aspartic the native protoxin (extending past the core toxin portion) can acid proteases such as cathepsins D-like and E-like, pepsin, be retained with the transition to the heterologous protoxin plasmepsin, and chymosin may further be exploited by engi occurring downstream. As an example, a chimeric toxin of the neering appropriate recognition sequences at desired process subject invention has a full core toxin segment of DIG-11 (i.e. 35 ing sites to affect Cry protein processing within the midgut of DIG-84: amino acids 1-664 of DIG-11) and a heterologous Susceptible larvae of certain insect pests. protoxin (amino acids 665 to the C-terminus). In a preferred A preferred location for the introduction of such protease embodiment, the heterologous portion of the protoxin is cleavage sites may be within the “spacer” region between derived from a Cry1Ab delta-endotoxin, as illustrated in SEQ C.-helix2B and C.-helix3, for example within amino acids 137 ID NO:5. 40 to 141 of the full length DIG-11 protein (SEQ ID NO:2 and SEQID NO:4 discloses the 545 amino acid sequence of a Table 1). A second preferred location for the introduction of Cry1Ab protoxin segment useful in DIG-11 insect toxin vari protease cleavage sites may be within the spacer region ants of the invention. Attention is drawn to the last about 100 between C.-helix3 and C-helix4 (Table 1), for example within to 150 amino acids of this protoxin segment, which it is most amino acids 172 to 175 of the full length DIG-11 protein of critical to include in the chimeric toxin of the subject inven 45 SEQ ID NO:2. Modified Cry proteins generated either by tion. gene deletion or by introduction of protease cleavage sites Protease sensitivity variants Insect gut proteases typically have improved activity on insect pests including but not lim function in aiding the insect in obtaining needed amino acids ited to western corn rootworm, Southern corn root worn, from dietary protein. The best understood insect digestive northern corn rootworm, and the like. proteases are serine proteases, which appear to be the most 50 Various technologies exist to enable determination of the common type (Englemann and Geraerts, (1980), particularly sequence of the amino acids which comprise the N-terminal in Lepidopteran species. Coleopteran insects have guts that or C-terminal residues of polypeptides. For example, auto are more neutral to acidic than are Lepidopteran guts. The mated Edman degradation methodology can be used in majority of Coleopteran larvae and adults, for example Colo sequential fashion to determine the N-terminal amino acid rado potato beetle, have slightly acidic midguts, and cysteine 55 sequence of up to 30 amino acid residues with 98% accuracy proteases provide the major proteolytic activity (Wolfson and per residue. Further, determination of the sequence of the Murdock, (1990). More precisely, Thie and Houseman amino acids comprising the carboxy end of polypeptides is (1990) identified and characterized the cysteine proteases, also possible (Bailey et al., (1992); U.S. Pat. No. 6,046,053). cathepsin B-like and cathepsin H-like, and the aspartyl pro Thus, in some embodiments, B.t. Cry proteins which have tease, cathepsin D-like, in . Gillikinet 60 been activated by means of proteolytic processing, for al., (1992) characterized the proteolytic activity in the guts of example, by proteases prepared from the gut of an insect, may western corn rootworm larvae and found primarily cysteine be characterized and the N-terminal or C-terminal amino proteases. U.S. Pat. No. 7,230,167 disclosed that the serine acids of the activated toxin fragment identified. DIG-11 insect protease, cathepsin G, exists in western corn rootworm. The toxinvariants produced by introduction or elimination of pro diversity and different activity levels of the insect gut pro 65 tease processing sites at appropriate positions in the coding teases may influence an insect's sensitivity to a particular B.t. sequence to allow, or eliminate, proteolytic cleavage of a toxin. larger variant protein by insect, plant or microorganism pro US 8,304,605 B2 10 teases are within the scope of the invention. The end result of rially alter the biological activity of the variant. Table 2 pro Such manipulation is understood to be the generation of toxin vides a listing of examples of amino acids belonging to each fragment molecules having the same or better activity as the class. intact (full length) toxin protein. Domains of the DIG-11 insect toxin The separate domains 5 TABLE 2 of the DIG-11 insect toxin, (and variants that are 90, 95, or 97% identical to such domains) are expected to be useful in Class of Amino Acid Examples of Amino Acids forming combinations with domains from other Cry toxins to Nonpolar Side Chains Ala, Val, Leu, Ile, Pro, Met, Phe, Trp provide new toxins with increased spectrum of pest toxicity, Uncharged Polar Side Chains Gly, Ser, Thr, Cys, Tyr, ASn, Gln improved potency, or increased protein stability. Domain I of 10 Acidic Side Chains Asp, Glu Basic Side Chains LyS, Arg, His the DIG-11 protein comprises amino acid residues 86 to 306 Beta-branched Side Chains Thr, Val, Ile of SEQID NO:2. Domain II of the DIG-11 protein comprises Aromatic Side Chains Tyr, Phe, Trp, His amino acid residues 311 to 508 of SEQID NO:2. Domain III of the DIG-11 protein comprises amino acid residues 518 to 662 of SEQ ID NO:2. Domain swapping or shuffling is 15 In some instances, non-conservative Substitutions can also another mechanism for generating altered delta-endotoxin be made. The critical factor is that these substitutions must not proteins. Domains II and III may be swapped between delta significantly detract from the biological activity of the toxin. endotoxin proteins, resulting in hybrid or chimeric toxins Variants include polypeptides that differ in amino acid with improved pesticidal activity or target spectrum. Domain sequence due to mutagenesis. Variant proteins encompassed II is involved in receptor binding, and Domain III binds cer by the present invention are biologically active, that is they tain classes of receptor proteins and perhaps participates in continue to possess the desired biological activity of the insertion of an oligomeric toxin pre-pore. Some Domain III native protein, that is, retaining pesticidal activity. substitutions in other toxins have been shown to produce Variant proteins can also be designed that differ at the Superior toxicity against Spodoptera exigua (de Maagdet al., sequence level but that retain the same or similar overall (1996) and guidance exists on the design of the Cry toxin 25 essential three-dimensional structure, Surface charge distri domain Swaps (Knight et al., (2004). bution, and the like. See e.g. U.S. Pat. No. 7,058,515; Larson Methods for generating recombinant proteins and testing et al., (2002); Stemmer (1994a, 1994b, 1995); and Crameriet them for pesticidal activity are well known in the art (see, for al., (1996a, 1996b, 1997). example, Naimov et al., (2001), de Maagdet al., (1996), Geet Nucleic Acids Isolated nucleic acids encoding DIG-11 al., (1991), Schnepf et al., (1990), Rang et al., (1999)). 30 insect toxins are one aspect of the present invention. This Domain I from Cry1A and Cry3A proteins has been studied includes nucleic acids encoding SEQ ID NO:2 and SEQ ID for the ability to insert and form pores in membranes. C-he NO:5, and complements thereof, as well as other nucleic lices 4 and 5 of domain I play key roles in membrane insertion acids that encode insect active variants of SEQID NO:2. By and pore formation (Walters et al., 1993, Gazit et al., 1998: "isolated' applicants mean that the nucleic acid molecules Nunez-Valdez et al., 2001), with the other helices proposed to 35 have been removed from their native environment and have contact the membrane surface like the ribs of an umbrella been placed in a different environment by the hand of man. (Bravo et al., (2007); Gazit et al., (1998)). Because of the redundancy of the genetic code, a variety of DIG-11 insect toxin variants created by making a limited different DNA sequences can encode the amino acid number of amino acid deletions, Substitutions, or additions sequences disclosed herein. It is well within the skill of a Amino acid deletions, Substitutions, and additions to the 40 person trained in the art to create these alternative DNA amino acid sequence of SEQID NO:2 can readily be made in sequences encoding the same, or essentially the same, toxins. a sequential manner and the effects of Such variations on Gene synthesis Genes encoding the improved Cry proteins insecticidal activity can be tested by bioassay. Provided the described herein can be made by a variety of methods well number of changes is limited in number, Such testing does not known in the art. For example, synthetic gene segments and involve unreasonable experimentation. The invention 45 synthetic genes can be made by phosphite tri-ester and phos includes insecticidally active variants of the core toxin phoramidite chemistry (Caruthers et al., 1987), and commer (amino acids 1-664 of SEQID NO:2, or amino acids 142-664 cial vendors are available to perform gene synthesis on of SEQ ID NO:2) in which up to 10, up to 15, or up to 20 demand. Full-length genes can be assembled in a variety of amino acid additions, deletions, or Substitutions have been ways including, for example, by ligation of restriction frag made. 50 ments or polymerase chain reaction assembly of overlapping The invention includes DIG-11 insect toxinvariants having oligonucleotides (Stewart and Burgin, 2005). Further, termi a core toxin segment that is 90%. 95% or 97% identical to nal gene deletions can be made by PCR amplification using amino acids 1-664 of SEQID NO:2 or amino acids 142-664 site-specific terminal oligonucleotides. of SEQID NO:2. Nucleic acids encoding DIG-11 insect toxins can be made Variants may be made by making random mutations or the 55 for example, by synthetic construction by methods currently variants may be designed. In the case of designed mutants, practiced by any of several commercial Suppliers. (See for there is a high probability of generating variants with similar example, U.S. Pat. No. 7,482.119 B2). These genes, or por activity to the native toxin when amino acid identity is main tions or variants thereof, may also be constructed syntheti tained in critical regions of the toxin which account for bio cally, for example, by use of a gene synthesizer and the design logical activity or are involved in the determination of three 60 methods of, for example, U.S. Pat. No. 5,380,831. Alterna dimensional configuration which ultimately is responsible tively, variations of synthetic or naturally occurring genes for the biological activity. A high probability of retaining may be readily constructed using standard molecular biologi activity will also occur if substitutions are conservative. cal techniques for making point mutations. Fragments of Amino acids may be placed in the following classes: non these genes can also be made using commercially available polar, uncharged polar, basic, and acidic. Conservative Sub 65 exonucleases or endonucleases according to standard proce stitutions whereby an amino acid of one class is replaced with dures. For example, enzymes such as Bal31 or site-directed another amino acid of the same type are least likely to mate mutagenesis can be used to systematically cut off nucleotides US 8,304,605 B2 11 12 from the ends of these genes. Also, gene fragments which http://emboss.sourceforge.net/). wSTRETCHER calculates encode active toxin fragments may be obtained using a vari an optimal global alignment of two sequences using a modi ety of restriction enzymes. fication of the classic dynamic programming algorithm which Given the amino acid sequence for a DIG-11 insect toxin, uses linear space. The Substitution matrix, gap insertion pen a coding sequence can be designed by reverse translating the alty and gap extension penalties used to calculate the align coding sequence using codons preferred by the intended host, ment may be specified. When utilizing the wSTRETCHER and then refining the sequence using alternative codons to program for comparing nucleotide sequences, a Gap open remove sequences that might cause problems and provide penalty of 16 and a Gap extend penalty of 4 can be used with periodic stop codons to eliminate long open coding sequences the scoring matrix file EDNAFULL. When used for compar in the non-coding reading frames. 10 ingamino acid sequences, a Gap open penalty of 12 and a Gap Quantifying Sequence Identity To determine the percent extend penalty of 2 can be used with the EBLOSUM62 scor identity of two amino acid sequences or of two nucleic acid ing matrix file. sequences, the sequences are aligned for optimal comparison A further non-limiting example of a mathematical algo purposes. The percent identity between the two sequences is rithm utilized for the comparison of sequences is that of a function of the number of identical positions shared by the 15 Needleman and Wunsch (1970), which is incorporated in the sequences (i.e. percent identity number of identical posi sequence alignment Software packages GAPVersion 10 and tions/total number of positions (e.g. overlapping positions)x wNEEDLE (http://emboss.sourceforge.net/). GAP Version 100). In one embodiment, the two sequences are the same 10 may be used to determine sequence identity or similarity length. The percent identity between two sequences can be using the following parameters: for a nucleotide sequence, 96 determined using techniques similar to those described identity and % similarity are found using GAP Weight of 50 below, with or without allowing gaps. In calculating percent and Length Weight of 3, and the nwsgapdna. cmp scoring identity, typically exact matches are counted. matrix. Foramino acid sequence comparison, % identity or 96 The determination of percent identity between two similarity are determined using GAP weight of 8 and length sequences can be accomplished using a mathematical algo weight of 2, and the BLOSUM62 scoring program. rithm. A nonlimiting example of Such an algorithm is that of 25 wNEEDLE reads two input sequences, finds the optimum Altschuletal. (1990), and Karlin and Altschul (1990), modi alignment (including gaps) along their entire length, and fied as in Karlin and Altschul (1993), and incorporated into writes their optimal global sequence alignment to file. The the BLASTN and BLASTX programs. BLAST searches may algorithm explores all possible alignments and chooses the be conveniently used to identify sequences homologous best, using a scoring matrix that contains values for every (similar) to a query sequence in nucleic or protein databases. 30 possible residue or nucleotide match. wNEEDLE finds the BLASTN searches can be performed, (score=100, word alignment with the maximum possible score, where the score length=12) to identify nucleotide sequences having homol of an alignment is equal to the sum of the matches taken from ogy to claimed nucleic acid molecules of the invention. the scoring matrix, minus penalties arising from opening and BLASTX searches can be performed (score=50, word extending gaps in the aligned sequences. The Substitution length 3) to identify amino acid sequences having homology 35 matrix and gap opening and extension penalties are user to claimed insecticidal protein molecules of the invention. specified. When amino acid sequences are compared, a Gapped BLAST Altschulet al., (1997) can be utilized to default Gap open penalty of 10, a Gap extend penalty of 0.5, obtain gapped alignments for comparison purposes. Alterna and the EBLOSUM62 comparison matrix are used. When tively, PSI-Blast can be used to performan iterated search that DNA sequences are compared using wNEEDLE, a Gap open detects distant relationships between molecules Altschulet 40 penalty of 10, a Gap extend penalty of 0.5, and the al., (1997). When utilizing BLAST, Gapped BLAST, and EDNAFULL comparison matrix are used. PSI-Blast programs, the default parameters of the respective Equivalent programs may also be used. By “equivalent programs can be used. See www.ncbi.nlm.nih.gov. program' is intended any sequence comparison program that, A non-limiting example of a mathematical algorithm uti for any two sequences in question, generates an alignment lized for the comparison of sequences is the ClustalW algo 45 having identical nucleotide or amino acid residue matches rithm (Thompson et al., (1994). ClustalW compares and an identical percent sequence identity when compared to sequences and aligns the entirety of the amino acid or DNA the corresponding alignment generated by ALIGNX, sequence, and thus can provide data about the sequence con wNEEDLE, or wSTRETCHER. The % identity is the per servation of the entire amino acid sequence or nucleotide centage of identical matches between the two sequences over sequence. The ClustalW algorithm is used in several com 50 the reported aligned region (including any gaps in the length) mercially available DNA/amino acid analysis software pack and the 96 similarity is the percentage of matches between the ages, such as the ALIGNX module of the Vector NTI Program two sequences over the reported aligned region (including Suite (Invitrogen, Inc., Carlsbad, Calif.). When aligning any gaps in the length). amino acid sequences with ALIGNX, one may conveniently Alignment may also be performed manually by inspection. use the default settings with a Gap open penalty of 10, a Gap 55 Recombinant hosts The insect toxin-encoding genes of the extend penalty of 0.1 and the blosumó3mt2 comparison subject invention can be introduced into a wide variety of matrix to assess the percentamino acid similarity (consensus) microbial or plant hosts. Expression of the insect toxin gene or identity between the two sequences. When aligning DNA results, directly or indirectly, in the intracellular production sequences with ALIGNX, one may conveniently use the and maintenance of the pesticidal protein. With suitable default settings with a Gap open penalty of 15, a Gap extend 60 microbial hosts, e.g. Pseudomonas, the microbes can be penalty of 6.6 and the Swgapdnamt comparison matrix to applied to the environment of the pest, where they will pro assess the percent identity between the two sequences. liferate and be ingested. The result is a control of the pest. Another non-limiting example of a mathematical algo Alternatively, the microbe hosting the insect toxin gene can rithm utilized for the comparison of sequences is that of be treated under conditions that prolong the activity of the Myers and Miller (1988). Such an algorithm is incorporated 65 toxin and stabilize the cell. The treated cell, which retains the into the wSTRETCHER program, which is part of the wBM toxic activity, then can be applied to the environment of the BOSS sequence alignment Software package (available at target pest. US 8,304,605 B2 13 14 Where the B.t. toxin gene is introduced via a suitable vector name but a few. Methods for transforming plants are well into a microbial host, and said host is applied to the environ known in the art, and illustrative transformation methods are ment in a living State, it is essential that certain host microbes described in the Examples. be used. Microorganism hosts are selected which are known A preferred embodiment of the subject invention is the to occupy the “phytosphere' (phylloplane, phyllosphere, 5 transformation of plants with genes encoding the Subject rhizosphere, and/or rhizoplane) of one or more crops of inter insecticidal protein or its variants. The transformed plants are est. These microorganisms are selected so as to be capable of resistant to attack by an insect target pest by virtue of the Successfully competing in the particular environment (crop presence of controlling amounts of the Subject insecticidal and other insect habitats) with the wild-type indigenous protein or its variants in the cells of the transformed plant. By microorganisms, provide for stable maintenance and expres 10 incorporating genetic material that encodes the insecticidal sion of the gene expressing the polypeptide pesticide, and, properties of the B. t. insecticidal toxins into the genome of a desirably, provide for improved protection of the pesticide plant eaten by a particular insect pest, the adult or larvae from environmental degradation and inactivation. would die after consuming the food plant. Numerous mem A large number of microorganisms are known to inhabit bers of the monocotyledonous and dicotyledonous classifica the phylloplane (the surface of the plant leaves) and/or the 15 tions have been transformed. Transgenic agronomic crops as rhizosphere (the soil Surrounding plant roots) of a wide vari well as fruits and vegetables are of commercial interest. Such ety of important crops. These microorganisms include bacte crops include but are not limited to maize, rice, soybeans, ria, algae, and fungi. Of particular interest are microorgan canola, Sunflower, alfalfa, Sorghum, wheat, cotton, peanuts, isms, such as bacteria, e.g. genera Pseudomonas, Erwinia, tomatoes, potatoes, and the like. Several techniques exist for Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizo introducing foreign genetic material into plant cells, and for bium, Sinorhizobium, Rhodopseudomonas, Methylophilius, obtaining plants that stably maintain and express the intro Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter; duced gene. Such techniques include acceleration of genetic Azotobacter, Leuconostoc, and Alcaligenes; fungi, particu material coated onto microparticles directly into cells (U.S. larly yeast, e.g. genera Saccharomyces, Cryptococcus, Pat. No. 4,945,050 and U.S. Pat. No. 5,141,131). Plants may Kluyveromyces, Sporobolomyces, Rhodotorula, and Aure 25 be transformed using Agrobacterium technology, see U.S. Obasidium. Of particular interest are such phytosphere bac Pat. No. 5,177,010, U.S. Pat. No. 5,104,310, European Patent terial species as Pseudomonas syringae, Pseudomonas fluo Application No. 0131624B1, European Patent Application rescens, Serratia marcescens, Acetobacter xylinum, No. 120516, European Patent Application No. 159418B1, Agrobacterium tumefaciens, Agrobacterium radiobacter; European Patent Application No. 176112, U.S. Pat. No. Rhodopseudomonas spheroides, Xanthomonas campestris, 30 5,149,645, U.S. Pat. No. 5,469,976, U.S. Pat. No. 5,464,763, Sinorhizobium meliloti (formerly Rhizobium meliloti), U.S. Pat. No. 4,940,838, U.S. Pat. No. 4,693,976, European Alcaligenes eutrophus, and Azotobacter vinelandii; and Patent Application No. 116718, European Patent Application phytosphere yeast species such as Rhodotorula rubra, R. No. 290799, European Patent Application No. 320500, Euro glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. pean Patent Application No. 604662, European Patent Appli difluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, 35 cation No. 627752, European Patent Application No. S. cerevisiae, Sporobolomyces roseus, S. odorus, Kluyvero 0267159, European Patent Application No. 0292435, U.S. myces veronae, and Aureobasidium pollulans. Of particular Pat. No. 5,231,019, U.S. Pat. No. 5,463,174, U.S. Pat. No. interest are the pigmented microorganisms. 4,762,785, U.S. Pat. No. 5,004,863, and U.S. Pat. No. 5,159, Methods of Controlling Insect Pests 135. Other transformation technology includes WHIS When an insect comes into contact with an effective 40 KERSTM technology, see U.S. Pat. No. 5,302,523 and U.S. amount of toxin delivered via transgenic plant expression, Pat. No. 5,464,765. Electroporation technology has also been formulated protein compositions(s), sprayable protein com used to transform plants, see WO 87/06614, U.S. Pat. No. position(s), a bait matrix or other delivery system, the results 5,472,869, U.S. Pat. No. 5,384,253, WO9209696, and WO are typically death of the insect, or the insects do not feed 932 1335. All of these transformation patents and publications upon the source which makes the toxins available to the 45 are incorporated by reference. In addition to numerous tech insects. nologies for transforming plants, the type of tissue which is The subject protein toxins can be “applied' or provided to contacted with the foreign genes may vary as well. Such contact the target insects in a variety of ways. For example, tissue would include but would not be limited to embryogenic transgenic plants (wherein the protein is produced by and tissue, callus tissue type I and II, hypocotyl, meristem, and the present in the plant) can be used and are well-known in the art. 50 like. Almost all plant tissues may be transformed during dedi Expression of the toxin genes can also beachieved selectively fferentiation using appropriate techniques within the skill of in specific tissues of the plants, such as the roots, leaves, etc. an artisan. This can be accomplished via the use of tissue-specific pro Genes encoding DIG-11 insect toxins can be inserted into moters, for example. Spray-on applications are another plant cells using a variety oftechniques which are well known example and are also known in the art. The Subject proteins 55 in the art as disclosed above. For example, a large number of can be appropriately formulated for the desired end use, and cloning vectors comprising a marker that permits selection of then sprayed (or otherwise applied) onto the plant and/or the transformed microbial cells and a replication system func around the plant/to the vicinity of the plant to be protected— tional in Escherichia coli are available for preparation and before an infestation is discovered, after target insects are modification of foreign genes for insertion into higher plants. discovered, both before and after, and the like. Bait granules, 60 Such manipulations may include, for example, the insertion for example, can also be used and are known in the art. of mutations, truncations, additions, or Substitutions as Transgenic Plants desired for the intended use. The vectors comprise, for The Subject proteins can be used to protect practically any example, pFBR322, puC series, M13 mp series, p ACYC184, type of plant from damage by an insect pest. Examples of such etc. Accordingly, the sequence encoding the Cry protein or plants include maize, Sunflower, soybean, cotton, canola, 65 variants can be inserted into the vectorata suitable restriction rice, Sorghum, wheat, barley, vegetables, ornamentals, pep site. The resulting plasmid is used for transformation of E. pers (including hot peppers), Sugar beets, fruit, and turf, to coli, the cells of which are cultivated in a suitable nutrient US 8,304,605 B2 15 16 medium, then harvested and lysed so that workable quantities Protein)), and these promoters may also be used. Promoters of the plasmid are recovered. Sequence analysis, restriction may also be used that are active during a certain stage of the fragment analysis, electrophoresis, and other biochemical plants’ development as well as active in specific plant tissues molecular biological methods are generally carried out as and organs. Examples of Such promoters include but are not methods of analysis. After each manipulation, the DNA limited to promoters that are root specific, pollen-specific, sequence used can be cleaved and joined to the next DNA embryo specific, corn silk specific, cotton fiber specific, seed sequence. Each manipulated DNA sequence can be cloned in endosperm specific, phloem specific, and the like. the same or other plasmids. Under certain circumstances it may be desirable to use an The use of T-DNA-containing vectors for the transforma inducible promoter. An inducible promoter is responsible for tion of plant cells has been intensively researched and suffi 10 expression of genes in response to a specific signal. Such as: ciently described in European Patent Application No. physical stimulus (e.g. heat shock genes); light (e.g. RUBP 120516; Lee and Gelvin (2008), Fraley et al., (1986), and An carboxylase); hormone (e.g. glucocorticoid); antibiotic (e.g. et al., (1985), and is well established in the field. tetracycline); metabolites; and stress (e.g. drought). Other Once the inserted DNA has been integrated into the plant desirable transcription and translation elements that function genome, it is relatively stable throughout Subsequent genera 15 in plants may be used. Such as 5' untranslated leader tions. The vector used to transform the plant cell normally sequences, RNA transcription termination sequences and contains a selectable marker gene encoding a protein that poly-adenylate addition signal sequences. Numerous plant confers on the transformed plant cells resistance to a herbi specific gene transfer vectors are known to the art. cide or an antibiotic, Such as bialaphos, kanamycin, G418, Transgenic crops containing insect resistance (IR) traits bleomycin, or hygromycin, inter alia. The individually are prevalent in corn and cotton plants throughout North employed selectable marker gene should accordingly permit America, and usage of these traits is expanding globally. the selection of transformed cells while the growth of cells Commercial transgenic crops combining IR and herbicide that do not contain the inserted DNA is suppressed by the tolerance (HT) traits have been developed by multiple seed selective compound. companies. These include combinations of IR traits conferred A large number of techniques are available for inserting 25 by B. t. insecticidal proteins and HT traits such as tolerance to DNA into a host plant cell. Those techniques include trans Acetolactate Synthase (ALS) inhibitors such as sulfony formation with T-DNA delivered by Agrobacterium tumefa lureas, imidazolinones, triazolopyrimidine, Sulfonanilides, ciens or Agrobacterium rhizogenes as the transformation and the like, Glutamine Synthetase (GS) inhibitors such as agent. Additionally, fusion of plant protoplasts with lipo bialaphos, glufosinate, and the like, 4-HydroxyPhenylPyru somes containing the DNA to be delivered, direct injection of 30 vate Dioxygenase (HPPD) inhibitors such as mesotrione, the DNA, biolistics transformation (microparticle bombard isoxaflutole, and the like, 5-EnolPyruvylShikimate-3-Phos ment), or electroporation, as well as other possible methods, phate Synthase (EPSPS) inhibitors such as glyphosate and the may be employed. like, and Acetyl-Coenzyme A Carboxylase (ACCase) inhibi In a preferred embodiment of the subject invention, plants tors such as haloxyfop, quizalofop, diclofop, and the like. will be transformed with genes wherein the codon usage of 35 Other examples are known in which transgenically provided the protein coding region has been optimized for plants. See, proteins provide plant tolerance to herbicide chemical classes for example, U.S. Pat. No. 5,380,831, which is hereby incor Such as phenoxy acids herbicides and pyridyloxyacetates porated by reference. Also, advantageously, plants encoding a auxin herbicides (see WO 2007/053482 A2), or phenoxy truncated toxin will be used. The truncated toxin typically acids herbicides and aryloxyphenoxypropionates herbicides will encode about 55% to about 80% of the full length toxin. 40 (see WO 2005107437 A2, A3). The ability to control multiple Methods for creating synthetic B.t. genes for use in plants are pest problems through IR traits is a valuable commercial known in the art (Stewart 2007). product concept, and the convenience of this product concept Regardless of transformation technique, the gene is pref is enhanced if insect control traits and weed control traits are erably incorporated into a gene transfer vector adapted to combined in the same plant. Further, improved value may be express the B. t. insecticidal toxin genes and variants in the 45 obtained via single plant combinations of IR traits conferred plant cell by including in the vector a plant promoter. In by a B.t. insecticidal protein such as that of the subject inven addition to plant promoters, promoters from a variety of tion with one or more additional HT traits such as those Sources can be used efficiently in plant cells to express foreign mentioned above, plus one or more additional input traits genes. For example, promoters of bacterial origin, such as the (e.g. other insect resistance conferred by B.t.-derived or other octopine synthase promoter, the nopaline synthase promoter, 50 insecticidal proteins, insect resistance conferred by mecha the mannopine synthase promoter, promoters of viral origin, nisms such as RNAi and the like, disease resistance, stress such as the 35S and 19S promoters of cauliflower mosaic tolerance, improved nitrogen utilization, and the like), or virus, and the like may be used. Plant promoters include, but output traits (e.g. high oils content, healthy oil composition, are not limited to ribulose-1,6-bisphosphate (RUBP) car nutritional improvement, and the like). Such combinations boxylase Small Subunit (SSu), beta-conglycinin promoter, 55 may be obtained either through conventional breeding phaseolin promoter, ADH (alcohol dehydrogenase) pro (breeding Stack) or jointly as a novel transformation event moter, heat-shock promoters, ADF (actin depolymerization involving the simultaneous introduction of multiple genes factor) promoter, and tissue specific promoters. Promoters (molecular stack). Benefits include the ability to manage may also contain certain enhancer sequence elements that insect pests and improved weed control in a crop plant that may improve the transcription efficiency. Typical enhancers 60 provides secondary benefits to the producer and/or the con include but are not limited to ADH1-intron 1 and ADH1 Sumer. Thus, the Subject invention can be used in combination intron 6. Constitutive promoters may be used. Constitutive with other traits to provide a complete agronomic package of promoters direct continuous gene expression in nearly all improved crop quality with the ability to flexibly and cost cells types and at nearly all times (e.g., actin, ubiquitin, effectively control any number of agronomic issues. CaMV 35S). Tissue specific promoters are responsible for 65 Target Pests gene expression in specific cell or tissue types, such as the The DIG-11 insect toxins of the invention are particularly leaves or seeds (e.g., Zein, oleosin, napin, ACP (Acyl Carrier Suitable for use in control of insects pests. Coleopterans are US 8,304,605 B2 17 18 one important group of agricultural, horticultural, and house Maruca testulalis, Melanchra picta, Operophtera brumata, hold pests which cause a very large amount of damage each Orgvia sp., Ostrinia nubilalis (European corn borer), Paleac year. This insect order encompasses foliar- and root-feeding rita vernata, Papiapena nebris (common stalk borer), larvae and adults, including: weevils from the families Papilio Cresphontes, Pectinophora gossypiella, Phryganidia Anthribidae, Bruchidae, and Curculionidae e.g. boll weevil californica, Phyllonorycter blancardella, Pieris mapi, Pieris (Anthonomus grandis Boheman), rice water weevil (Lissor rapae, Plathylpena scabra, Platynota flouendana, Platynota hoptrus Oryzophilus Kuschel), granary weevil (Sitophilus Stultana, Platyptilia carduidactyla, Plodia interpunctella, grananus Linnaeus), rice weevil (Sitophilus Oryzae Lin Plutella xylostella (diamondback ), Pontia protodice, naeus), clover leaf weevil (Hypera punctata Fabricius), and Pseudaletia unipuncta (armyworm), Pseudoplasia maize billbug (Sphenophorus maidis Chittenden); flea 10 , cucumber beetles, rootworms, leaf beetles, potato includens, aegrotata, concinna, beetles, and leaf miners in the family Chrysomelidae e.g. Sitotroga cerealella, Spilonta ocellana, Spodoptera fru Colorado potato beetle (Leptinotarsa decemlineata Say), giperda (fall armyworm), Spodoptera exigua (beet army western corn rootworm (Diabrotica virgifera virgifera worm), Thaurnstopoea pity'Ocampa, Ensola bisselliella, Tri LeConte), northern corn rootworm (Diabrotica barben Smith 15 choplusia hi, Udea rubigalis, Xylomyges curiails, and & Lawrence); Southern corn rootworm (Diabrotica undeci padella. impunctata howardi Barber), corn flea beetle (Chaetocnema Use of DIG-11 insect toxins to control Coleopteran pests of pulicara Melsheimer), crucifer flea beetle (Phyllotreta cru crop plants is contemplated. In some embodiments, Cry pro ciferae Goeze), grape colaspis (Colaspis brunnea Fabricius), teins may be economically deployed for control of insect cereal (Oulema melanopus Linnaeus), and Sun pests that include but are not limited to, for example, root flower beetle ( exclamationis Fabricius); worms such as Diabrotica undecimpunctata howardi (South beetles from the family Coccinellidae e.g. Mexican bean ern corn rootworm), Diabrotica longicornis barberi (north beetle (Epilachna varivestis Mulsant); chafers and other ern corn rootworm), and Diabrotica virgifera (western corn beetles from the family (e.g. Japanese beetle rootworm), and grubs such as the larvae of (Popillia japonica Newman), northern masked chafer (white 25 borealis (northern masked chafer), Cyclocephala immaculate grub, Cyclocephala borealis Arrow), Southern masked chafer (Southern masked chafer), and Popillia japonica (Japanese (white grub, Cyclocephala immaculata Olivier), European beetle). chafer (Rhizotrogus maialis Razoumowsky), white grub Use of the DIG-11 insect toxins to control parasitic nema (Phyllophaga Crinita Burmeister), and carrot beetle (Ligyrus todes including, but not limited to, root knot nematode (Me gibbosus De Geer); carpet beetles from the family Dermes 30 loidogyne icognita) and Soybean cyst nematode (Heterodera tidae; wireworms from the family Elateridae e.g. Melanotus glycines) is also contemplated. spp., Conoderus spp., Limonius spp., Agriotes spp., Cten Antibody Detection of DIG-11 Insect Toxins icera spp., Aeolus spp.); bark beetles from the family Sco Anti-toxin antibodies. Antibodies to the insect toxins dis lytidae, and beetles from the family Tenebrionidae (e.g. Ele closed herein, or to equivalent toxins, or fragments of these Odes spp). Any genus listed above (and others), generally, can 35 toxins, can readily be prepared using standard procedures in also be targeted as a part of the Subject invention. Any addi this art. Such antibodies are useful to detect the presence of tional insects in any of these genera (as targets) are also the DIG-11 insect toxins. included within the scope of this invention. Once the B. t. insecticidal toxin has been isolated, antibod Lepidopterans are another important group of agricultural, ies specific for the toxin may be raised by conventional meth horticultural, and household pests which cause a very large 40 ods that are well known in the art. Repeated injections into a amount of damage each year. This insect order encompasses host of choice over a period of weeks or months will elicit an foliar- and root-feeding larvae and adults. Lepidopteran immune response and result in significant anti-B. t. toxin insect pests include, but are not limited to: Achoroia grisella, serum titers. Preferred hosts are mammalian species and more gloverana, Acleris variana, Adoxophyes Orana, Agro highly preferred species are rabbits, goats, sheep and mice. tis Ipsilon (black cutworm), Alabama argillacea, Alsophila 45 Blood drawn from Such immunized may be pro pometaria, Amyelois transitella, Anagasta kuehniella, Anar cessed by established methods to obtain antiserum (poly sia lineatella, Anisota Senatoria, Antheraea pernyi, Anticar clonal antibodies) reactive with the B.t. insecticidal toxin. sia gemmatalis, Archips sp., Argyrotaenia sp., Athetis mind The antiserum may then be affinity purified by adsorption to ara, Bombyx mori, Bucculatrix thurberiella, Cadra cautella, the toxin according to techniques known in the art. Affinity Choristoneura sp., Cochylls hospes, Colias eurytheme, Cor 50 purified antiserum may be further purified by isolating the cyra cephalonica, Cydia latiferreanus, Cydia pomonella, immunoglobulin fraction within the antiserum using proce Datana integerrima, Dendrolimus Sibericus, Desmia fenera dures known in the art. The resulting material will be a het lis, Diaphania hyalinata, Diaphania initidalis, Diatraea erogeneous population of immunoglobulins reactive with the grandiosella (Southwestern corn borer), Diatraea sacchara B.t. insecticidal toxin. lis, Ennomos subsignaria, Eoreuma loftini, Esphestia elu 55 Anti-B.t. toxin antibodies may also be generated by pre tella, Erannis tillaria, Estigmene acrea, Eulia salubricola, paring a semi-synthetic immunogen consisting of a synthetic Eupocoelia ambiguella, Eupoecilia ambiguella, Euproctis peptide fragment of the B.t. insecticidal toxin conjugated to chrysorrhoea, messoria, Galleria mellonella, Gra an immunogenic carrier. Numerous schemes and instruments pholita molesta, Harrisina americana, Helicoverpa subflexia, useful for making peptide fragments are well known in the art. Helicoverpa zea (corn earworm), Heliothis virescens, 60 Many Suitable immunogenic carriers such as bovine serum Hemileuca Oliviae, Homoeosoma electellum, Hyphantia albumin or keyhole limpet hemocyanin are also well known cunea, Keiferia lycopersicella, Lambdina fiscellaria fiscel in the art, as are techniques for coupling the immunogen and laria, Lambdina fiscellaria lugubrosa, Leucoma Salicis, carrier proteins. Once the semi-synthetic immunogen has Lobesia botrana, Loxagrotis albicosta (western bean cut been constructed, the procedure for making antibodies spe worm), Loxostege Sticticalis, Lymantria dispar; Macala thy 65 cific for the B. t. insecticidal toxin fragment is identical to risalis, Malacosoma sp., Mamestra brassicae, Mamestra those used for making antibodies reactive with natural B.t. configurata, Manduca quinquemaculata, Manduca Sexta, toxin. US 8,304,605 B2 19 20 Anti-B. t. toxin monoclonal antibodies (MAbs) are readily Detection Using Probes prepared using purified B. t. insecticidal toxin. Methods for A further method for identifying the toxins and genes of the producing MAbs have been practiced for over 15 years and Subject invention is through the use of oligonucleotide are well known to those of ordinary skill in the art. Repeated probes. These probes are detectable nucleotide sequences. intraperitoneal or Subcutaneous injections of purified B.t. These sequences may be rendered detectable by virtue of an insecticidal toxin in adjuvant will elicit an immune response appropriate radioactive label or may be made inherently fluo in most animals. Hyperimmunized B-lymphocytes are rescent as described in U.S. Pat. No. 6,268,132. As is well removed from the and fused with a suitable fusion known in the art, if the probe molecule and nucleic acid partner cell line capable of being cultured indefinitely. Pre sample hybridize by forming strong base-pairing bonds ferred animals whose B-lymphocytes may be hyperimmu 10 between the two molecules, it can be reasonably assumed that nized and used in the production of MAbs are mammals. the probe and sample have Substantial sequence homology. More preferred animals are rats and mice and most preferred Preferably, hybridization is conducted under stringent condi is the BALB/c mouse strain. tions by techniques well-known in the art, as described, for Numerous mammalian cell lines are Suitable fusion part example, in Keller and Manak, (1993). Detection of the probe ners for the production of hybridomas. Many such lines are 15 provides a means for determining in a known manner whether available from the AmericanType Culture Collection (ATCC, hybridization has occurred. Such a probe analysis provides a Manassas, Va.) and commercial suppliers. Preferred fusion rapid method for identifying toxin-encoding genes of the partner cell lines are derived from mouse myelomas and the Subject invention. The nucleotide segments which are used as HL-1(R) Friendly myeloma-653 cell line (Ventrex, Portland, probes according to the invention can be synthesized using a Me.) is most preferred. Once fused, the resulting hybridomas DNA synthesizer and standard procedures. These nucleotide are cultured in a selective growth medium for one to two sequences can also be used as PCR primers to amplify genes weeks. Two well known selection systems are available for of the subject invention. eliminating unfused myeloma cells, or fusions between Hybridization myeloma cells, from the mixed hybridoma culture. The As is well known to those skilled in molecular biology, choice of selection system depends on the strain of mouse 25 similarity of two nucleic acids can be characterized by their immunized and myeloma fusion partner used. The AAT tendency to hybridize. As used herein the terms “stringent selection system, described by Taggart and Samloff, (1983), conditions” or “stringent hybridization conditions are may be used; however, the HAT (hypoxanthine, aminopterin, intended to refer to conditions under which a probe will thymidine) selection system, described by Littlefield, (1964), hybridize (anneal) to its target sequence to a detectably is preferred because of its compatibility with the preferred 30 greater degree than to other sequences (e.g. at least 2-fold mouse strain and fusion partner mentioned above. Spent over background). Stringent conditions are sequence-depen growth medium is then screened for immunospecific MAb dent and will be different in different circumstances. By con secretion. Enzyme linked immunosorbent assay (ELISA) trolling the Stringency of the hybridization and/or washing procedures are best Suited for this purpose; though, radioim conditions, target sequences that are 100% complementary to munoassays adapted for large Volume screening are also 35 the probe can be identified (homologous probing). Alterna acceptable. Multiple screens designed to consecutively pare tively, Stringency conditions can be adjusted to allow some down the considerable number of irrelevant or less desired mismatching in sequences so that lower degrees of similarity cultures may be performed. Cultures that secrete MAbs reac are detected (heterologous probing). Generally, a probe is less tive with the B.t. insecticidal toxin may be screened for cross than about 1000 nucleotides in length, preferably less than reactivity with known B. t. insecticidal toxins. MAbs that 40 500 nucleotides in length. preferentially bind to the preferred B.t. insecticidal toxin may Typically, stringent conditions will be those in which the be isotyped using commercially available assays. Preferred salt concentration is less than about 1.5 M Naion, typically MAbs are of the IgG class, and more highly preferred MAbs about 0.01 to 1.0 MNaion concentration (or other salts) at pH are of the IgG and IgG, Subisotypes. 7.0 to pH 8.3 and the temperature is at least about 30°C. for Hybridoma cultures that secrete the preferred MAbs may 45 short probes (e.g. 10 to 50 nucleotides) and at least about 60° be sub-cloned several times to establish monoclonality and C. for long probes (e.g. greater than 50 nucleotides). Stringent stability. Well known methods for sub-cloning eukaryotic, conditions may also be achieved with the addition of desta non-adherent cell cultures include limiting dilution, soft aga bilizing agents such as formamide. Exemplary low stringency rose and fluorescence activated cell sorting techniques. After conditions include hybridization with a buffer solution of each subcloning, the resultant cultures preferably are be re 50 30% to 35% formamide, 1 MNaCl, 1% SDS (sodium dodecyl assayed for antibody secretion and isotype to ensure that a sulfate) at 37° C. and a wash in 1x to 2xSSC (20xSSC=3.0M stable preferred MAb-secreting culture has been established. NaC1/0.3 M trisodium citrate) at 50° C. to 55° C. Exemplary The anti-B.t. toxin antibodies are useful invarious methods moderate stringency conditions include hybridization in 40% of detecting the claimed B. t. insecticidal toxin of the instant to 45% formamide, 1.0 MNaCl, 1% SDS at 37°C. and awash invention, and variants or fragments thereof. It is well known 55 in 0.5x to 1xSSC at 55° C. to 60° C. Exemplary high strin that antibodies labeled with a reporting group can be used to gency conditions include hybridization in 50% formamide, 1 identify the presence of antigens in a variety of milieus. M. NaCl, 1% SDS at 37° C. and a wash in 0.1XSSC at 60° C. Antibodies labeled with radioisotopes have been used for to 65°C. Optionally, wash buffers may comprise about 0.1% decades in radioimmunoassays to identify, with great preci to about 1% SDS. Duration of hybridization is generally less sion and sensitivity, the presence of antigens in a variety of 60 than about 24 hours, usually about 4 to about 12 hours. biological fluids. More recently, enzyme labeled antibodies Specificity is typically the function of post-hybridization have been used as a substitute for radiolabeled antibodies in washes, the critical factors being the ionic strength and tem the ELISA assay. Further, antibodies immunoreactive to the perature of the final wash solution. For DNA/DNA hybrids, B.t. insecticidal toxin of the present invention can be bound to the thermal melting point (T,) is the temperature (under an immobilizing Substance such as a polystyrene well or 65 defined ionic strength and pH) at which 50% of a comple particle and used in immunoassays to determine whether the mentary target sequence hybridizes to a perfectly matched B.t. toxin is present in a test sample. probe. T is reduced by about 1° C. for each 1% of mismatch US 8,304,605 B2 21 22 ing; thus, T., hybridization conditions, and/or wash condi Once at T-20° C. for 15 minutes in 0.2xSSPE, 0.1% SDS tions can be adjusted to facilitate annealing of sequences of (moderate stringency wash). the desired identity. For example, if sequences with >90% For oligonucleotide probes, hybridization may be carried identity are sought, the T can be decreased 10°C. Generally, out overnight at 10° C. to 20° C. below the T of the hybrid in stringent conditions are selected to be about 5° C. lower than 6xSSPE, 5xDenhardt’s solution, 0.1% SDS, 0.1 mg/mL the T for the specific sequence and its complement at a denatured DNA.T., for oligonucleotide probes may be deter defined ionic strength and pH. However, highly stringent mined by the following formula (Suggs et al., 1981). conditions can utilize a hybridization and/or wash at 1°C.. 2 C., 3° C., or 4°C. lower than the T: moderately stringent T(C.)=2(number of TWA base pairs)+4(number of conditions can utilize a hybridization and/or wash at 6°C., 7° 10 G/C base pairs) C., 8°C., 9° C., or 10° C. lower than the T, and low strin Washes may typically be carried out as follows: gency conditions can utilize a hybridization and/or wash at Twice at room temperature for 15 minutes 1 xSSPE, 0.1% 11° C., 12°C., 13°C., 14°C., 15° C., or 20° C. lower than the SDS (low stringency wash). Tn. Once at the hybridization temperature for 15 minutes in T (in C.) may be experimentally determined or may be 15 1xSSPE, 0.1% SDS (moderate stringency wash). approximated by calculation. For DNA-DNA hybrids, the T. Probe molecules for hybridization and hybrid molecules can be approximated from the equation of Meinkoth and Wahl formed between probe and target molecules may be rendered (1984): detectable by means other than radioactive labeling. Such T(C.)=81.5°C.+16.6(log M)+0.41 (% GC)-0.61(% alternate methods are intended to be within the scope of this formamide)-500/L: invention. where M is the molarity of monovalent cations, 9% GC is the All patents, patent applications, provisional applications, percentage of guanosine and cytosine nucleotides in the and publications referred to or cited herein are incorporated DNA, 96 formamide is the percentage of formamide in the by reference in their entirety to the extent they are not incon hybridization solution, and L is the length of the hybrid in sistent with the explicit teachings of this specification. base pairs 25 By the use of the term “genetic material herein, it is meant Alternatively, the T is described by the following formula to include all genes, nucleic acid, DNA and RNA. The term “dsRNA refers to double-stranded RNA. For designations of (Beltz et al., 1983). nucleotide residues of polynucleotides, DNA, RNA, oligo T(C.)=81.5°C.+16.6(log Na+I)+0.41 (% GC)- nucleotides, and primers, and for designations of amino acid 0.61 (% formamide)-600/L 30 residues of proteins, standard IUPAC abbreviations are where Na+ is the molarity of sodium ions, '% GC is the employed throughout this document. Nucleic acid sequences percentage of guanosine and cytosine nucleotides in the are presented in the standard 5' to 3’ direction, and protein DNA, 96 formamide is the percentage of formamide in the sequences are presented in the standard amino (N) terminal to hybridization solution, and L is the length of the hybrid in carboxy (C) terminal direction. base pairs 35 It should be understood that the examples and embodi Using the equations, hybridization and wash compositions, ments described herein are for illustrative purposes only and and desired T, those of ordinary skill will understand that that various modifications or changes in light thereof will be variations in the stringency of hybridization and/or wash Suggested to persons skilled in the art and are to be included solutions are inherently described. If the desired degree of within the spirit and purview of this application and the scope mismatching results in a T of less than 45° C. (aqueous 40 of the appended claims. These examples should not be con solution) or 32° C. (formamide solution), it is preferred to Strued as limiting. increase the SSC concentration so that a higher temperature Unless specifically indicated or implied, the terms “a”, can be used. An extensive guide to the hybridization of “an’, and “the signify "at least one' as used herein. nucleic acids is found in Tijssen (1993) and Ausubel et al., All percentages are by weight and all solvent mixture pro 1995) Also see Sambrook et al., (1989). 45 portions are by Volume unless otherwise noted. All tempera Hybridization of immobilized DNA on Southern blots with tures are in degrees Celsius. radioactively labeled gene-specific probes may be performed by standard methods Sambrook et al., Supra.). Radioactive EXAMPLE 1. isotopes used for labeling polynucleotide probes may include 32P 33P 14C, or 3H. Incorporation of radioactive isotopes 50 Isolation of a Gene Encoding DIG-11 Toxin into polynucleotide probe molecules may be done by any of several methods well known to those skilled in the field of Unless otherwise indicated, molecular biological and bio molecular biology. (See, e.g. Sambrook et al., Supra.) Ingen chemical manipulations described in this and Subsequent eral, hybridization and Subsequent washes may be carried out Examples were performed by standard methodologies as dis under stringent conditions that allow for detection of target 55 closed in, for example, Ausubel et al. (1995), and Sambrook sequences with homology to the claimed toxin encoding et al. (1989), and updates thereof. Nucleic acid encoding the genes. For double-stranded DNA gene probes, hybridization insecticidal Cry protein designated herein as DIG-11 insect may be carried out overnight at 20°C. to 25°C. below the T. toxin was isolated from B.t. Strain PS184M1. Degenerate of the DNA hybrid in 6xSSPE, 5xDenhardt’s Solution, 0.1% primers to be used as Forward and Reverse primers in PCR SDS, 0.1 mg/mL denatured DNA 20xSSPE is 3M NaCl, 0.2 60 reactions using PS184M1 genomic DNA as template were MNaHPO, and 0.02M EDTA (ethylenediamine tetra-acetic designed based on multiple sequence alignments of each acid sodium salt); 100xDenhardt’s Solution is 20 gm/L Poly class of B.t. insecticidal toxin. The Forward Primer corre vinylpyrollidone, 20 gm/L Ficoll type 400 and 20 gm/L sponds to bases 841 to 865 of SEQID NO:1, and the Reverse Bovine Serum Albumin (fraction V). Primer corresponds to the complement of bases 2227 to 2250 Washes may typically be carried out as follows: 65 of SEQID NO:1. This pair of primers was used to amplify a Twice at room temperature for 15 minutes in 1xSSPE, fragment of 1410 bp, corresponding to nucleotides 841 to 0.1% SDS (low stringency wash). 2250 of SEQID NO:1. This sequence was used as the anchor US 8,304,605 B2 23 24 point to begin genome walking using methods adapted from coding sequence comprising an open reading frame of 3195 the GenomeWalkerTM Universal Kit (Clontech, Palo Alto, bases which encodes a deleted variant DIG-11 protein com Calif.). The nucleic acid sequence of a fragment spanning the prising 1065 amino acids (i.e. methionine plus amino acids DIG-11 coding region was determined. SEQID NO:1 is the 101 to 1164 of the full-length DIG-11 protein). Serial, step 3492 bp nucleotide sequence encoding the full length DIG-11 wise deletions that remove additional codons for a single protein. SEQID NO:2 is the amino acid sequence of the full amino acid corresponding to residues 101 through 141 of the length DIG-11 protein deduced from SEQID NO:1. full-length DIG-11 protein of SEQID NO:2 provide variants missing part or all of C-helix 2A and C-helix 2B. Thus a EXAMPLE 2 second designed deleted variant coding sequence requires 10 elimination of bases 1 to 303 of SEQ ID NO:1, thereby Deletion of Domain I C.-Helices from DIG-11 removing the coding sequence foramino acids 1 through 101. Restoration of a functional open reading frame is again To improve the insect active properties of the DIG-11 accomplished by reintroduction of a translation initiation insect toxin, serial, step-wise deletions are made, each of methionine codon at the beginning of the remaining coding which removes part of the N-terminus of the DIG-11 protein. 15 sequence, thus providing for a second deleted variant coding The deletions remove part or all of C-helix 1 and part or all of sequence having an open reading frame of 3192 bases encod C.-helix 2 in Domain I, while maintaining the structural integ ing a deleted variant DIG-11 protein comprising 1064 amino rity of C-helix 3 through C.-helix 7. acids (i.e. methionine plus amino acids 102 through 1164 of Deletions are designed as follows. This example utilizes the full-length DIG-11 protein). The last designed deleted the full length chimeric DNA sequence encoding the full variant coding sequence requires removal of bases 1 through length DIG-11 protein e.g. SEQID NO:1 and SEQID NO:2, 423 of SEQID NO:1, thus eliminating the coding sequence respectively) to illustrate the design principles with 73 spe for amino acids 1 through 141, and, after reintroduction of a cific variants. It utilizes the chimeric sequence of SEQ ID translation initiation methionine codon, providing a deletion NO:5 (DNA encoding the DIG-84 core toxin segment fused variant coding sequence having an open reading frame of to Cry1Ab protoxin segment) to provide an additional 73 25 3069 bases which encodes a deletion variant DIG-11 protein specific variants. One skilled in the art will realize that other of 1023 amino acids (i.e. methionine plus amino acids 142 DNA sequences encoding all or an N-terminal portion of the through 1164 of the full-length DIG-11 protein). As exempli DIG-11 protein may be similarly manipulated to achieve the fied, after elimination of the deletion sequence, an initiator desired result. To devise the first deleted variant coding methionine codon is added to the beginning of the remaining sequence, all of the bases that encode C.-helix 1 including the 30 coding sequence to restore a functional open reading frame. codon for the Leucine residue near the beginning of C-helix Also as described, an additional glycine codon is to be added 2A (i.e. L100 for the full length DIG-11 protein of SEQID between the methionine codon and the codon for the instabil NO:2), are removed. Thus, elimination of bases 1 through 300 ity-determining amino acid in the instance that removal of the of SEQ ID NO:1 removes the coding sequence for amino deleted sequence leaves exposed at the N-terminus of the acids 1 through 100 of SEQ ID NO:2. Reintroduction of a 35 remaining portion of the full-length protein one of the insta translation initiating ATG (methionine) codon at the begin bility-determining amino acids as provided above. ning (i.e. in front of the codon corresponding to amino acid Table 3 describes specific variants designed in accordance 101 of the full length protein) provides for the deleted variant with the strategy described above. TABLE 3 Deletion variant protein sequences of the full-length DIG-11 protein of SEQID NO. 2 and the fusion protein sequence of SEQ ID NO. 5. DIG-11 Residues DIG-11 Residues Deletion added at Residues of Deletion added at Residues of Variant NH, terminus SEQID NO: 2 Variant NH, terminus SEQID NO: 5 M O1-1164 74 M O1-1209 M O2-1164 75 M O2-1209 M O3-1164 76 M O3-1209 M O4-1164 77 M O4-1209 MG O4-1164 78 MG O4-1209 M OS-1164 79 M OS-1209 MG OS-1164 8O MG OS-1209 M O6-1164 81 M O6-1209 M O7-1164 82 M O7-1209 1 M O8-1164 83 M O8-1209 MG O8-1164 84 MG O8-1209 M O9-1164 85 M O9-1209 MG O9-1164 86 MG O9-1209 M 1O-1164 87 M 10-1209 M 11-1164 88 M 11-1209 M 12-1164 89 M 12-1209 M 13-1164 90 M 13-1209 MG 13-1164 91 MG 13-1209 M 14-1164 92 M 14-1209 MG 14-1164 93 MG 14-1209 M 15-1164 94 M 15-1209 MG 15-1164 95 MG 15-1209 M 16-1164 96 M 16-1209 MG 16-1164 97 MG 16-1209 M 17-1164 98 M 17-1209 US 8,304,605 B2 25 26 TABLE 3-continued Deletion variant protein sequences of the full-length DIG-11 protein of SEQ ID NO. 2 and the fusion protein sequence of SEQID NO. 5. DIG-11 Residues DIG-11 Residues Deletion added at Residues of Deletion added at Residues of Variant NH2 terminus SEQID NO: 2 Variant NH2 terminus SEQID NO: 5 26 MG 17-1164 99 MG 17 209 27 M 18-1164 OO M 18 209 28 MG 18-1164 O1 MG 18 209 29 M 19-1164 O2 M 19 209 30 M 2O-1164 O3 M 20 209 31 MG 2O-1164 O4 MG 20 209 32 M 21-1164 05 M 21 209 33 M 22-1164 O6 M 22 209 34 MG 22-1164 O7 MG 22 209 35 M 23-1164 O8 M 23 209 36 MG 23-1164 09 MG 23 209 37 M 24-1164 10 M 24 209 38 MG 24-1164 11 MG 24 209 39 M 25-1164 12 M 25 209 40 MG 25-1164 13 MG 25 209 41 M 26-1164 14 M 26 209 42 MG 26-1164 15 MG 26 209 43 M 27-1164 16 M 27 209 44 MG 27-1164 17 MG 27 209 45 M 28-1164 18 M 28 209 46 MG 28-1164 19 MG 28 209 47 M 29-1164 2O M 29 209 48 MG 29-1164 21 MG 29 209 49 M 3O-1164 22 M 30 209 50 MG 3O-1164 23 MG 30 209 51 M 31-1164 24 M 31 209 52 MG 31-1164 25 MG 31 209 53 M 32-1164 26 M 32 209 S4 M 33-1164 27 M 33 209 55 MG 33-1164 28 MG 33 209 56 M 34-1164 29 M 34 209 57 MG 34-1164 30 MG 34 209 58 M 35-1164 31 M 35 209 59 MG 35-1164 32 MG 35 209 60 M 36-1164 33 M 36 209 61 MG 36-1164 34 MG 36 209 62 M 37-1164 35 M 37 209 63 MG 37-1164 36 MG 37 209 64 M 38-1164 37 M 38 209 65 MG 38-1164 38 MG 38 209 66 M 39-1164 39 M 39 209 67 MG 39-1164 40 MG 39 209 68 M 40-1164 41 M 40 209 69 MG 40-1164 42 MG 40 209 70 M 41-1164 43 M 41 209 71 MG 41-1164 44 MG 41 209 72 M 42-1164 45 M 42 209 73 MG 42-1164 46 MG 42 209

Nucleic acids encoding the toxins described in Table 3 are tum, 197 CDs), and soybean (Glycine max; ca. 1000 CDs) designed in accordance with the general principles for Syn 50 were downloaded from data at the website http://www.ka thetic genes intended for expression in plants, as discussed Zusa.or.jp/codon?. Biased codon sets that comprise highly above. used codons in maize CDS, and merged dicot CDS datasets, in appropriate rescaled relative amounts, were calculated EXAMPLE 3 after omitting any synonymous codon used less than about 55 Design of Plant-Optimized Versions of Coding 10% of total codon uses for that amino acid in either plant Sequences for DIG-84 and Cry1Ab Protoxin type. To derive a maize optimized sequence encoding the Proteins DIG-84 protein, synonomous codon substitutions to the experimentally determined DIG-11 DNA sequence were DNA sequences having plant codon biases were designed 60 made such that the resulting DNA sequence had the overall and synthesized to produce the DIG-84 protein in transgenic codon composition of the maize codon bias table, while pre monocots and the Cry1Ab protoxin segment in monocot or serving the encoded amino acid sequence. Further refine dicot plants. A codon usage table for maize (Zea mays L.) was ments of the sequence were made to eliminate undesirable calculated from 706 protein coding sequences (CDs) restriction enzyme recognition sites, potential plant intron obtained from sequences deposited in GenBank. Codon 65 splice sites, long runs of A/T or C/G residues, and other motifs usage tables for tobacco (Nicotiana tabacum, 1268 CDs), that might interfere with RNA stability, transcription, or canola (Brassica napus, 530 CDs), cotton (Gossypium hirsu translation of the coding region in plant cells. Other changes US 8,304,605 B2 27 28 were made to introduce desired restriction enzyme recogni translation of the bases comprising the XhoI restriction tion sites, and to eliminate long internal Open Reading enzyme recognition site used to terminate the DIG-84CDS in Frames (frames other than +1). These changes were all made pDAB102007. within the constraints of retaining the maize-biased codon Growth and Expression Analysis in Shake Flasks Produc composition. Synthesis of the designed sequence was per tion of DIG-84 protein for characterization and insect bioas formed by a commercial vendor (DNA2.0, Menlo Park, say was accomplished by shake-flask-grown Pfluorescens Calif.). strain DPfl3591 harboring expression constructs (e.g. plas Additional guidance regarding the production of synthetic mid pl)AB102007). Seed cultures grown in M9 medium genes can be found in, for example, WO 97/13402 and U.S. Supplemented with 1% glucose and trace elements were used 10 to inoculate 50 mL of defined minimal medium with 5% Pat. No. 5,380,831. glycerol (Teknova Cat. #3D7426, Hollister, Calif.). Expres A maize-optimized DNA sequence encoding the DIG-84 sion of the DIG-84 toxin gene via the Ptac promoter was core toxin segment, comprising amino acids 1 to 664 of the induced by addition of isopropyl-B-D-1-thiogalactopyrano full-length DIG-11 protein of SEQID NO:2, is given in SEQ side (IPTG) after an initial incubation of 24 hours at 30° with ID NO:3. Analogous methods were used to design a dicot 15 shaking. Cultures were sampled at the time of induction and optimized DNA sequence encoding the Cry1Ab protoxin seg at various times post-induction. Cell density was measured by ment as disclosed as SEQID NO:6, and a maize-optimized optical density at 600 nm (ODoo). Other culture media suit DNA sequence encoding the Cry1Ab protoxin segment, as able for growth of Pseudomonas fluorescens may also be disclosed as SEQID NO:7. utilized, for example, as described in Huang et al., 2007 and US Patent Application No. 20060008877. EXAMPLE 4 Cell Fractionation and SDS-PAGE Analysis of Shake Flask Samples At each sampling time, the cell density of Construction of Expression Plasmids Encoding samples was adjusted to ODoo 20 and 1 mL aliquots were DIG-84 Insect Toxin and Expression in Bacterial centrifuged at 14000xg for five minutes. The cell pellets were Hosts 25 frozen at -20°. Soluble and insoluble fractions from frozen shake flask cell pellets were generated following re-suspen Standard cloning methods were used in the construction of sion of the pellets in 0.5 mL Butterfield's potassium phos Pseudomonas fluorescens (Pf) expression plasmids engi phate buffer pH7.2 (Thermo-Fisher Scientific, Rockford, neered to produce DIG-84 protein encoded by a maize-opti Ill.). The samples were sonicated twice for 45 seconds at a mized coding region. Restriction endonucleases were 30 constant output of 20, using a 2 mm diameter probe and a obtained from New England BioLabs (NEB; Ipswich, Mass.) Branson Sonifier 250 (Danbury, Conn.), with icing between and T4 DNA Ligase (NEB) was used for DNA ligation. bursts. The lysate was centrifuged at 14,000 rpm for 20 min Plasmid preparations were performed using the Nucleospin(R) utes at 4 and the Supernatant was recovered as the soluble Plasmid Kit (Macherey-Nagel Inc. Bethlehem, Pa.) following fraction. The pellet (insoluble fraction) was then resuspended the instructions of the supplier. DNA fragments were purified 35 in an equal volume of Butterfields's phosphate buffer 0. using the QIAquick Gel Extraction Kit (Qiagen, Valencia, Samples were mixed 1:1 with 2x Laemmlisample buffer Calif.) after agarose Tris-acetate gel electrophoresis. The lin containing B-mercaptoethanol (Sambrook et al., Supra.) and earized vector was phosphatased with NEB Antarctic Phos boiled for 5 minutes prior to loading onto Criterion XT Bis phatase to enhance formation of recombinant molecules. Tris 12% gels (Bio-Rad Inc., Hercules, Calif.) Electrophore The basic cloning strategy entailed Subcloning a DNA 40 sis was performed in the recommended XT MOPS buffer. fragment having the DIG-84 insect toxin coding sequence Gels were stained with Bio-Safe Coomassie Stain according (CDS) as provided by SEQID NO:3 into pIDOW1169 at, for to the manufacturers (Bio-Rad) protocol and imaged using example, Spel and XhoI restriction sites, whereby it was the Alpha Innotech Imaging system (San Leandro, Calif.). placed under the expression control of the Ptac promoter and DIG-84 Insect Toxin Preparation DIG-84 insect toxin was therrnBT1T2 terminator from plasmid pKK223-3 (PL Phar 45 enriched from 45.5 grams of recombinant Pseudomonas cell macia, Milwaukee, Wis.). pl.)OW 1169 is a medium copy paste resuspended in 400 mL of lysis buffer (100 mM CAPS, plasmid with the RSF1010 origin of replication, a pyrF gene, 5 mM EDTA, 5 mMTCEP (Tris(2-carboxyethyl)-phosphine and a ribosome binding site preceding the restriction enzyme hydrochloride)) pH11). The suspension was passed two times recognition sites into which DNA fragments containing pro through an M-11OY Microfluidizer(R) (Microfluidics Inc., tein coding regions may be introduced, (US Patent Applica 50 Newton, Mass.). This device was equipped with two cham tion No. 20080193974). The expression plasmid bers: the H30Z Auxiliary Processing Module (APM), which pDAB102007 was transformed by electroporation into has a nominal passage size of 200 microns, and the H1OZ DC454 (a near wild-type Pfluorescens strain having muta Interaction Chamber (IXC), which has a nominal passage size tions ApyrF and lsc:lacI), or its derivatives, recovered in of 100 microns. The APM was placed downstream from the SOC-Soy hydrolysate medium, and plated on selective 55 IXC as recommended by the manufacturer. Cells were dis medium (M9 glucose agar lacking uracil, Sambrook et al., rupted between 11,000 and 15,000 psi and clarified by cen Supra). Details of the microbiological manipulations are trifugation (SLC1500 rotor, 12,000 rpm, for 20 minutes). The available in Squires et al., (2004), US Patent Application No. Supernatant was decanted and filtered (0.8 um) prior to anion 20060008877, US Patent Application No. 20080193974, and exchange chromatography. US Patent Application No. 2008.0058262, incorporated 60 The Pseudomonas cell lysate was split in half and pro herein by reference. Recombinant colonies were identified by cessed in two batches. DIG-84 protein was enriched by pas restriction enzyme digestion of miniprep plasmid DNA and sage of the lysate through five 5 mL High Trap CaptotMQ one (DPfl3591) was selected for further work. The DIG-84 columns (Amersham BioSciences, Piscataway, N.J.) linked in protein, as produced from the pl)AB102007 expression vec series end-to-end. Lysate was injected through the five col tor, comprises amino acids 1 to 664 of the DIG-11 protein 65 umn series at 5 mL/min. Non-binding proteins were eluted disclosed in SEQ ID NO:2, with an N-terminal addition of with Buffer A (50 mM Bis Tris Propane, 5 mM EDTA, 5 mM two amino acids (Leucine and Glutamine) contributed by DTT, pH9) until the absorbance at 280 nm reached near US 8,304,605 B2 29 30 baseline. Elution was continued with Buffer A containing for gel densitometry, which was measured using a BioRad 0.15 M NaCl to remove additional contaminants. With the imaging system (Fluor-S MultiImager with Quantity One first half of the lysate, the NaCl concentration was increased software version 4.5.2). Proteins in the gel matrix were to 0.2 M for continued elution of contaminants, then bound stained with Coomassie Blue-based stain and destained proteins were eluted with a linear gradient to 0.5 MNaCl over 5 before reading. 240 mL while collecting 10 mL fractions. With the second Purified proteins were tested for insect activity in bioassays half of the lysate, after the first elution with Buffer A, con conducted with neonate insect larvae on artificial insect diet. taminants were removed by elution with 0.15 MNaCl (the 0.2 Larvae of DBM and rDBM were hatched from eggs obtained MNaCl elution step was eliminated), then the bound proteins from a colony maintained by a commercial insectary (BenZon were eluted with a NaCl gradient to 0.5 M was described 10 above. Research Inc., Carlisle, Pa.). WCR eggs were obtained from Pooled fractions were concentrated with an Amicon Ultra Crop Characteristics, Inc. (Farmington, Minn.). 15 regenerated cellulose centrifugal filter device (50,000 The bioassays were conducted in 128-well plastic trays Molecular Weight Cutoff: Millipore) then injected into Slide specifically designed for insect bioassays (C-D International, A-Lyzer R cassettes (10,000 Molecular Weight Cutoff: 15 Pitman, N.J.). Each well contained 1.0 mL of Multi-species Thermo Fisher Scientific) and dialyzed overnight at 4 Lepidoptera diet (Southland Products, Lake Village, Ark.) or against two 4 Liter volumes of dialysis buffer (10 mM CAPS a proprietary diet designed for growth of Coleopteran insects (3-(cyclohexamino)1-propanesulfonic acid), pH10). Total (Dow AgroSciences LLC, Indianapolis, Ind.). A 40 uL or 60 protein concentrations were Subsequently determined by uLaliquot of protein sample was delivered by pipette onto the Bradford total protein assay. 1.5 cm diet surface of each well (27 uL/cm or 40 uL/cm). Gel electrophoresis The concentrated extract was prepared Diet concentrations were calculated as the amount (ng) of for electrophoresis by diluting 1:50 in NuPAGE(R) LDS DIG-84 protein per square centimeter (cm) of surface area in sample buffer (Invitrogen) containing 5 mM dithiothreitol as the well. The treated trays were held in a fume hood until the a reducing agent and heated at 95° for 4 minutes. The sample liquid on the diet surface evaporated or was absorbed into the was loaded in duplicate lanes of a 4-12% NuPAGE(R) gel 25 diet. alongside five BSA standards ranging from 0.2 to 2 ug/lane Within a few hours of eclosion, individual larvae were (for standard curve generation). Voltage was applied at 200V picked up with a moistened camel hairbrush and deposited on using MOPS SDS running buffer (Invitrogen) until the track the treated diet, one or two larvae per well. The infested wells ing dye reached the bottom of the gel. The gel was stained were then sealed with adhesive sheets of clear plastic, vented with 0.2% Coomassie Blue G-250 in 45% methanol, 10% 30 to allow gas exchange (C-D International, Pitman, N.J.). Bio acetic acid, and destained, first briefly with 45% methanol, 10% acetic acid, and then at length with 7% acetic acid, 5% assay trays were held under controlled environmental condi methanol until the background clears. Following destaining, tions (28°, -40% Relative Humidity, 16:8 Light:Dark) for 5 the gel was scanned with a BioRad Fluor-S MultiImager. The days, after which the total number of insects exposed to each instrument's Quantity One V.4.5.2 Software was used to 35 protein sample, the number of dead insects, and the weight of obtain background-Subtracted Volumes of the stained protein Surviving insects were recorded. Percent mortality, average bands and to generate the BSA standard curve that was used live weights, and growth inhibition were calculated for each to calculate the concentration of DIG-84 protein in the stock treatment. Stunting was defined as a decrease in average live Solution. weights. Growth inhibition (GI) was calculated as follows: 40 EXAMPLE 5 where TWIT is the Total Weight of live Insects in the Treat Insect Activity of Dig-84 Insect Toxin Produced in ment, TNIT is the Total Number of Insects in the Treatment Pseudomonas Fluorescens TWIBC is the Total Weight of live Insects in the Background 45 DIG-84 insect toxin was tested for activity on larvae of a Check (Buffer control), and TNIBC is the Total Number of Colepteran insect, western corn rootworm (WCR, Diabrotica Insects in the Background Check (Buffer control). virgifera virgifera LeConte). DIG-84 insect toxin was further The GIs is determined to be the concentration of DIG-84 tested for activity on larvae of Lepidopteran insects, includ protein in the diet at which the GI value is 50%. The LCso ing, for example, diamondback moth (DBM; Plutella xylos 50 (50% Lethal Concentration) is recorded as the concentration tella (Linnaeus) and cry1A-resistant DBM (rDBM). of DIG-84 protein in the diet at which 50% of test insects are Sample preparation and bioassays DIG-84 samples were killed. Statistical analysis was done using JMP software prepared in 10 mM CAPS pH10 and all bioassays contained (SAS, Cary, N.C.). a control treatment consisting of this buffer, which served as Replicated bioassays demonstrated that ingestion of DIG a background check for mortality or growth inhibition. 55 84 insect toxin results in a stunting of western corn rootworm Protein concentrations in bioassay buffer were estimated larvae, as shown in Table 4. Activity against other insects by gel electrophoresis using BSA to create a standard curve tested was not observed. TABLE 4 Stunting effects of DIG-84 protein ingested by western corn rootworm larvae. Means for Oneway Analysis of Variance of average weight (mg) per insect

Treatment Number of tests Mean Std Error Lower 95% Upper 95%

Buffer CAPS 10 6 O.464032 O.O3O3O O.39979 0.52827 DIG-84 3 O.246928 O.O4285 O.15608 O.337.77 US 8,304,605 B2 31 32 TABLE 4-continued Stunting effects of DIG-84 protein ingested by western corn rootworm larvae. Water 6 O.436942 O.O3O3O 0.37270 OSO118 Comparisons for all pairs (Average weight (mg) per insect) using Tukey-Kramer HSD

Treatment Class Class Mean

Buffer CAPS 10 A. O464032 Water A. O.436942 DIG-84 B O.246928

*TREATMENTS NOT CONNECTED BY SAMELETTERARESIGNIFICANTLYDIFFERENT

EXAMPLE 6 15 antibiotics kanamycin, neomycin and G418, as well as those genes which code for resistance or tolerance to glyphosate; Agrobacterium Transformation hygromycin; methotrexate, phosphinothricin (bialaphos), imidazolinones, Sulfonylureas and triazolopyrimidine herbi Standard cloning methods are used in the construction of cides, such as chlorosulfuron, bromoxynil, dalapon and the binary plant transformation and expression plasmids. like. Restriction endonucleases and T4 DNA Ligase are obtained Electro-competent cells of Agrobacterium tumefaciens from NEB. Plasmid preparations are performed using the strain Z707S (a streptomycin-resistant derivative of Z707; NucleoSpin(R) Plasmid Preparation kit or the NucleoBondR Hepburn et al., 1985) are prepared and transformed using AXXtra Midikit (both from Macherey-Nagel), following the electroporation (Weigel and Glazebrook, 2002). After elec instructions of the manufacturers. DNA fragments are puri 25 troporation, 1 mL of YEP broth (gm/L: yeast extract, 10; fied using the QIAquick PCR Purification Kit or the QIAEX peptone, 10; NaCl, 5) are added to the cuvette and the cell II Gel Extraction Kit (both from Qiagen) after gel isolation. YEP suspension is transferred to a 15 mL culture tube for DNA fragments comprising the nucleotide sequences that incubation at 28° in a water bath with constant agitation for 4 encode the modified DIG-11 insect toxins, or fragments hours. The cells are plated on YEP plus agar (25 gm/L) with thereof, may be synthesized by a commercial vendor (e.g. 30 DNA2.0, Menlo Park, Calif.) and supplied as cloned frag spectinomycin (200 g/mL) and streptomycin (250 ug/mL) ments in standard plasmid vectors, or may be obtained by and the plates are incubated for 2-4 days at 28°. Well sepa standard molecular biology manipulation of other constructs rated single colonies are selected and streaked onto fresh containing appropriate nucleotide sequences. Unique restric YEP+agar plates with spectinomycin and streptomycin as tion sites internal to each gene may be identified and a frag 35 before, and incubated at 28° for 1-3 days. ment of each gene synthesized, each containing a specific The presence of the DIG-11 insect toxin gene insert in the deletion or insertion. The modified Cry fragments may sub binary plant transformation vector is performed by PCR cloned into other Cry fragments coding regions at a appro analysis using vector-specific primers with template plasmid priate restriction sites to obtain a coding region encoding the DNA prepared from selected Agrobacterium colonies. The desired full-length protein, fused proteins, or deleted variant 40 cell pellet from a 4 mL aliquot of a 15 mL overnight culture proteins. For example one may identify an appropriate growninYEP with spectinomycin and streptomycin as before restriction recognition site at the start of the gene and a second is extracted using Qiagen Spin Mini Preps, performed per internal restriction site specific for each gene, which may be manufacturers instructions. Plasmid DNA from the binary used to construct variant clones. vector used in the Agrobacterium electroporation transforma In a non-limiting example, a basic cloning strategy may be 45 tion is included as a control. The PCR reaction is completed to Subclone full length or modified Cry coding sequences using Taq DNA polymerase from Invitrogen per manufac (CDS) into a plant expression plasmid at NcoI and Sad ture's instructions at 0.5x concentrations. PCR reactions are restriction sites. The resulting plant expression cassettes con carried out in a MJ Research Peltier Thermal Cycler pro taining the appropriate Cry coding region under the control of grammed with the following conditions: Step 1) 94° for 3 plant expression elements, (e.g., plant expressible promoters, 50 3' terminal transcription termination and polyadenylate addi minutes; Step 2) 94° for 45 seconds; Step 3) 55° for 30 tion determinants, and the like) are subcloned into a binary seconds; Step 4) 72° for 1 minute per kb of expected product vector plasmid, utilizing, for example, Gateway(R) technology length; Step 5) 29 times to Step 2: Step 6) 72 for 10 minutes. or standard restriction enzyme fragment cloning procedures. The reaction is maintained at 4' after cycling. The amplifica LR ClonaseTM (Invitrogen) for example, may be used to 55 tion products are analyzed by agarose gel electrophoresis recombine the full length and modified gene plant expression (e.g. 0.7% to 1% agarose, w/v) and visualized by ethidium cassettes into a binary plant transformation plasmid if the bromide staining. A colony is selected whose PCR product is Gateway(R) technology is utilized. It is convenient to employ identical to the plasmid control. a binary plant transformation vector that harbors a bacterial Alternatively, the plasmid structure of the binary plant gene that confers resistance to the antibiotic spectinomycin 60 transformation vector containing the DIG-11 gene insert is when the plasmid is present in E. coli and Agrobacterium performed by restriction digest fingerprint mapping of plas cells. It is also convenient to employ a binary vector plasmid mid DNA prepared from candidate Agrobacterium isolates that contains a plant-expressible selectable marker gene that by standard molecular biology methods well known to those is functional in the desired host plants. Examples of plant skilled in the art of Agrobacterium manipulation. expressible selectable marker genes include but are not lim 65 Those skilled in the art of obtaining transformed plants via ited to the aminoglycoside phosphotransferase gene of trans Agrobacterium-mediated transformation methods will poson Tn5 (Aph II) which encodes resistance to the understand that other Agrobacterium strains besides Z7075 US 8,304,605 B2 33 34 may be used to advantage, and the choice of Strain may Those skilled in the art of dicot plant transformation will depend upon the identity of the host plant species to be trans understand that other methods of selection of transformed formed. plants are available when other plant expressible selectable marker genes (e.g. herbicide tolerance genes) are used. EXAMPLE 7 Insect Bioassays of transgenic Arabidopsis Transgenic Arabidopsis lines expressing modified Cry proteins are dem Production of DIG-11 Insect Toxins and Variants in onstrated to be active against sensitive insect species in arti Dicot Plants ficial diet overlay assays. Protein extracted from transgenic and non-transgenic Arabidopsis lines is quantified by appro Arabidopsis Transformation Arabidopsis thaliana Col-01 10 priate methods and sample Volumes are adjusted to normalize is transformed using the floral dip method (Weigel and Gla protein concentration. Bioassays are conducted on artificial Zebrook, 2002). The selected Agrobacterium colony is used diet as described above. Non-transgenic Arabidopsis and/or to inoculate 1 mL to 15 mL cultures of YEP broth containing buffer and water are included in assays as background check appropriate antibiotics for selection. The culture is incubated treatmentS. overnight at 28° with constant agitation at 220 rpm. Each 15 culture is used to inoculate two 500 mL cultures of YEP broth EXAMPLE 8 containing appropriate antibiotics for selection and the new cultures are incubated overnight at 28° with constant agita Agrobacterium Transformation for Generation of tion. The cells are pelleted at approximately 8700xg for 10 Superbinary Vectors minutes at room temperature, and the resulting Supernatant is discarded. The cell pellet is gently resuspended in 500 mL of The Agrobacterium Superbinary system is conveniently infiltration media containing: /2x Murashige and Skoog salts used for transformation of monocot plant hosts. Methodolo (Sigma-Aldrich)/Gamborg's B5 vitamins (Gold BioTechnol gies for constructing and validating Superbinary vectors are ogy, St. Louis, Mo.), 10% (w/v) sucrose, 0.044 uMbenzy well established. See, for example, European Patent No. laminopurine (10 uL/liter of 1 mg/mL stock in DMSO) and 25 EP604662B1 and U.S. Pat. No. 7,060,876. Standard molecu 300 uL/liter Silwet L-77. Plants approximately 1 month old lar biological and microbiological methods are used to gen are dipped into the media for 15 seconds, with care taken to erate superbinary plasmids. Verification/validation of the assure Submergence of the newest inflorescence. The plants structure of the Superbinary plasmid is done using method are then laid on their sides and covered (transparent or ologies as described above for binary vectors. opaque) for 24 hours, washed with water, and placed upright. 30 The plants are grown at 22, with a 16-hour light/8-hour dark EXAMPLE 9 photoperiod. Approximately 4 weeks after dipping, the seeds are harvested. Production of DIG-11 Insect Toxins and Variants in Arabidopsis Growth and Selection Freshly harvested T1 Monocot Plants seed is allowed to dry for at least 7 days at room temperature 35 in the presence of desiccant. Seed is suspended in a 0.1% Agrobacterium-Mediated Transformation of Maize Seeds agar/water (Sigma-Aldrich) solution and then stratified at 4 from a High II FI cross (Armstrong et al., 1991) are planted for 2 days. To prepare for planting, Sunshine Mix LP5 (Sun into 5-gallon-pots containing a mixture of 95% Metro-Mix Gro Horticulture Inc., Bellevue, Wash.) in 10.5 inchX21 inch 360 soilless growing medium (Sun Gro Horticulture, Belle germination trays (T.O. Plastics Inc., Clearwater, Minn.) is 40 Vue, Wash.) and 5% clay/loam soil. The plants are grown in a covered with fine vermiculite, sub-irrigated with Hoagland's greenhouse using a combination of high pressure Sodium and solution (Hoagland and Arnon, 1950) until wet, then allowed metal halide lamps with a 16:8 hour Light: Dark photoperiod. to drain for 24 hours. Stratified seed is sown onto the vermicu For obtaining immature F embryos for transformation, con lite and covered with humidity domes (KORD Products, Bra trolled sib-pollinations are performed. Immature embryos are malea, Ontario, Canada) for 7 days. Seeds are germinated and 45 isolated at 8-10 days post-pollination when embryos are plants are grown in a Conviron (Models CMP4030 or approximately 1.0 to 2.0 mm in size. CMP3244; Controlled Environments Limited, Winnipeg, Infection and co-cultivation. Maize ears are surface steril Manitoba, Canada) under long day conditions (16 hours ized by scrubbing with liquid soap, immersing in 70% ethanol light/8 hours dark) at a light intensity of 120-150 ummol/m for 2 minutes, and then immersing in 20% commercial bleach sec under constant temperature) (22) and humidity 50 (0.1% sodium hypochlorite) for 30 minutes before being (40-50%). Plants are initially watered with Hoagland's solu rinsed with sterile water. A Suspension Agrobacterium cells tion and subsequently with deionized water to keep the soil containing a Superbinary vector is prepared by transferring moist but not wet. 1-2 loops of bacteria grown on YEP solid medium containing The domes are removed 5-6 days post sowing and plants 100 mg/L spectinomycin, 10 mg/L tetracycline, and 250 are sprayed with a chemical selection agent to kill plants 55 mg/L streptomycin at 28° for 2-3 days into 5 mL of liquid germinated from nontransformed seeds. For example, if the infection medium (LS Basal Medium (Linsmaier and Skoog. plant expressible selectable marker gene provided by the 1965), N6 vitamins (Chu et al., 1975), 1.5 mg/L 2,4-Dichlo binary plant transformation vector is a pat or bar gene (Wehr rophenoxyacetic acid (2,4-D), 68.5gm/L Sucrose, 36.0gm/L mann et al., 1996), transformed plants may be selected by glucose, 6 mM L-proline, pH 5.2) containing 100 uMaceto spraying with a 1000x solution of Finale (5.78% glufosinate 60 Syringone. The Solution is Vortexed until a uniform Suspen ammonium, Farnam Companies Inc., Phoenix, Ariz.). Two sion is achieved, and the concentration is adjusted to a final subsequent sprays are performed at 5-7 day intervals. Survi density of 200 Klett units, using a Klett-Summerson colorim vors (plants actively growing) are identified 7-10 days after eter with a purple filter. Immature embryos are isolated the final spraying and transplanted into pots prepared with directly into a micro centrifuge tube containing 2 mL of the Sunshine Mix LP5. Transplanted plants are covered with a 65 infection medium. The medium is removed and replaced with humidity dome for 3-4 days and placed in a Conviron under 1 mL of the Agrobacterium solution with a density of 200 the above-mentioned growth conditions. Klett units, and the Agrobacterium and embryo Solution is US 8,304,605 B2 35 36 incubated for 5 minutes at room temperature and then trans Altschul, S.F., Gish, W., Miller, W., Myers, E. W., Lipman, D. ferred to co-cultivation medium (LS Basal Medium, N6 vita J. (1990) Basic local alignment search tool. J. Mol. Biol. mins, 1.5 mg/L 2,4-D, 30.0gm/L sucrose, 6 mM L-proline, 215:403-410. 0.85 mg/L AgNO, 100 uMacetosyringone, 3.0gm/L Gellan Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., gum (PhytoTechnology Laboratories, Lenexa, Kans.), pH Zhang, Z. Miller, W., Lipman, D. J. (1997) Gapped 5.8) for 5 days at 25°C. under dark conditions. After co-cultivation, the embryos are transferred to selec BLAST and PSI-BLAST: a new generation of protein data tive medium after which transformed isolates are obtained base search programs. Nucl. Acids Res. 25:3389-3402. over the course of approximately 8 weeks. For selection of Armstrong, C. L., Green, C. E., Phillips, R. L. (1991) Devel maize tissues transformed with a Superbinary plasmid con opment and availability of germplasm with high Type I taining a plant expressible pat or bar selectable marker gene, 10 culture formation response. Maize Genet. Coop. Newslett. an LS based medium (LS Basal medium, N6 vitamins, 1.5 65:92-93. mg/L 2,4-D, 0.5 gm/L MES (2-(N-morpholino)ethane Aronson, A. I., Han, E.-S., McGaughey, W., Johnson, D. sulfonic acid monohydrate; PhytoTechnologies Labr), 30.0 (1991) The solubility of inclusion proteins from Bacillus gm/L Sucrose, 6 mM L-proline, 1.0 mg/L. AgNO, 250 mg/L thuringiensis is dependent upon protoxin composition and cefotaxime, 2.5gm/L Gellangum, pH 5.7) is used with Biala 15 phos (Gold BioTechnology). The embryos are transferred to is a factor intoxicity to insects. Appl. Environ. Microbiol. selection media containing 3 mg/L Bialaphos until embryo 57:981-986. genic isolates are obtained. Recovered isolates are bulked up Aronson, A.I., Geng, C. Wu. L. (1999) Aggregation of Bacil by transferring to fresh selection medium at 2-week intervals lus thuringiensis Cry1A toxins upon binding to target for regeneration and further analysis. insect larval midgut vesicles. Appl. Environ. Microbiol. Those skilled in the art of maize transformation will under 65:2503-2507. stand that other methods of selection of transformed plants Arvidson, H., Dunn, P. E., Strand, S. Aronson, A. I. (1989) are available when other plant expressible selectable marker Specificity of Bacillis thuringiensis for lepidopteran lar genes (e.g. herbicide tolerance genes) are used. vae: factors involved in vivo and in the structure of a Regeneration and seed production. For regeneration, the 25 purified toxin. Molec. Microbiol. 3:1533-1543. cultures are transferred to "28 induction medium (MS salts Ausubel et al., eds. (1995) Current Protocols in Molecular and vitamins, 30 gm/L Sucrose, 5 mg/L Benzylaminopurine, Biology, Chapter 2 (Greene Publishing and Wiley-Inter 0.25 mg/L 2,4-D, 3 mg/L Bialaphos, 250 mg/L cefotaxime, 2.5 gm/L Gellan gum, pH 5.7) for 1 week under low-light science, New York). conditions (14 uEms') then 1 week under high-light con Bailey, J. M., Shenov, N. R., Ronk, M., and Shively, J. E., ditions (approximately 89 uEm’s'). Tissues are subse 30 (1992) Automated carboxy-terminal sequence analysis of quently transferred to "36' regeneration medium (same as peptides. Protein Sci. 1:68-80. induction medium except lacking plant growth regulators). Beltz, G. A., Jacobs, K. A., Eickbush, T. H., Cherbas, P. T., When plantlets grow to 3-5 cm in length, they are transferred Kafatos, F. C. (1983) Isolation of multigene families and to glass culture tubes containing SHGA medium (Schenk and determination of homologies by filter hybridization meth Hildebrandt salts and vitamins (1972); PhytoTechnologies 35 ods. In Wu, R., Grossman, L., Moldave, K. (eds.) Methods Labr.), 1.0gm/L myo-inositol, 10 gm/L Sucrose and 2.0gm/L of Enzymology, Vol. 100 Academic Press, New York pp. Gellan gum, pH 5.8) to allow for further growth and devel 266-285. opment of the shoot and roots. Plants are transplanted to the Bown, D. P. Wilkinson, H. S., Jongsma, M.A., Gatehouse, J. same soil mixture as described earlier herein and grown to A. (2004) Characterisation of cysteine proteinases respon flowering in the greenhouse. Controlled pollinations for seed 40 sible for digestive proteolysis in guts of larval western corn production are conducted. rootworm (Diabrotica virgifera) by expression in the yeast Pichiapastoris. Insect Biochem. Molec. Biol. 34:305-320. EXAMPLE 10 Bravo, A., Gill, S. S., Soberon, M. (2007) Mode of action of Bacillus thuringiensis Cry and Cyttoxins and their poten Bioassay of Transgenic Maize 45 tial for insect control. Toxicon 49:423-435. Caruthers, M. H. Kierzek, R., Tang, J.Y. (1987) Synthesis of Bioactivity of the DIG-11 insect toxin and variants pro oligonucleotides using the phosphoramidite method. Bio duced in plant cells is demonstrated by conventional bioassay active Molecules (Biophosphates Their Analogues) 3:3- methods (see, for example Huang et al., 2006). One is able to 21. demonstrate efficacy, for example, by feeding various plant 50 Christeller, J.T., Laing, W.A., Markwick, N. P. Burgess, E. P. tissues or tissue pieces derived from a plant producing a J. (1992) Midgut protease activities in 12 phytophagous DIG-11 insect toxin to target insects in a controlled feeding lepidopteran larvae: dietary and protease inhibitor interac environment. Alternatively, protein extracts may be prepared tions. Insect Biochem. Molec. Biol. 22:735-746. from various plant tissues derived from a plant producing the Chu, C. C., Wand, C. C., Sun, C. S., Hsu, C., Yin, K.C., Chu, DIG-11 insect toxin and incorporate the extracted proteins in 55 C.Y., Bi, F.Y. (1975) Establishment of an efficient medium an artificial diet bioassay as previously described herein. It is for another culture of rice through comparative experi to be understood that the results of such feeding assays are to ments on the nitrogen sources. Scientia Sinica 18:659-668. be compared to similarly conducted bioassays that employ Crameri, A. Cwirla, S., Stemmer, W. P. C. (1996a) Construc appropriate control tissues from host plants that do not pro tion and evolution of antibody-phage libraries by DNA duce the DIG-11 insect toxin or variants, or to other control 60 shuffling. Nat. Med. 2:100-103. samples. Crameri, A., Dawes, G., Rodriguez, E., Silver, S., Stemmer, W. P. C. 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SEQUENCE LISTING

<16O is NUMBER OF SEO ID NOS: 7

<210s, SEQ ID NO 1 &211s LENGTH: 3492 212. TYPE : DNA <213> ORGANISM: Bacillus thuringiensis <4 OOs, SEQUENCE: 1 atgaattgttg gagaccataa tgaatticgat attatagatg taattgaaaa caaccagact 6 O aaag catcac gacatgttaa tgagt cagac aatgtaaata gacaaaggaa titt at Ctaat 12 O acgattittitt Ctaatctato ttctaattat cct ctagoaa gcaatccaaa tacac cattt 18O caaaatatga attataaaga at atctgaat attactgaag gggggattat taaccc.gacc 24 O

Cttgcgggga gcgctattgt agttgcgcag aatgttagta agacaatcct taaaaaatta 3OO gggagtacaa ttittggggaa gatt Cttggit agtgttctag at attittatg gccaactaat 360 actgaagaaa tatggttgga attaatagat gaggtagaag aactgattaa toaaaaaata gaggaac agg taataattga tgcagaaa.ca gctittagagt cagtaaaatt aaatgttgat ttatatttala atgcacttgc agaatgggaa acaag accta ctaatgaata Cagtacagaa 54 O ctggtotata aaaggtttac tgatgcatat aattatgcqc gaact agitat gcc attittitt agtgttctgaa cittatgaagt ttct citat ta. t cagtgt atg Cacaa.gctgc taat attagt 660 ttgcttittat cga.gagatgc gcaaatatat ggagatttgt ggggatttga cgalacatgac 72 O aaagcc actt ttgatggtga acgaaaatta tittagagctg aatatataga to attgcact aaat attata aagttggact tgatagacta aaaggat citt cittacgaatc ttgggtaaat 84 O tataatcgtt atcgtagaga aatgacatta atgat attag ataccatagc agcatt.ccca 9 OO tatt atgaca ttgaagagta Cccalatagag gttag tactic agittagcaa.g agaggittitat 96.O actgat coaa taataacgt.c atttgttgaa t cagat catg gaccaagttt ttctitt catg gaaagtaacg caatticgaaa accacaccitt gttgattatt tagataatct ttatatatat a catcgagat t cagaacatt ttcaaatgaa tittcaac citg atctaaatta ttgggctgct 14 O cataaagt ca aatataaata ttctggggat cc tact t tact atgaaac acc catatatggit 2OO aatgcatcta attatgaaag tacagggaac tact cattta gaggtaatag tattt at Caa 26 O acgittatcag citccttctgc aatact taca CCCaattaca tct attatgg tatagagcaa. 32O gttgagttitt atggtaacaa aggtaatgta tattatagag gaggtaataa at accct citg agtgtggatt ctgctaatca attaccacca gatgtagaac caataacaga aaattacaat 44 O catgtttitat gtcatgctac agctgtgc ct gtaaaagatg gtggtacagt t cc tatt titt SOO t cittggacac atagaagtgc ggattatt at aataccattt atccagataa gattacgcaa. 560 ctitcctgcag tcaaaag.cac t cott citc. ca. galagtggaag ggcttaaagt gcaagaaggit c caggctitta Caggtggaga t cittgttgta gcaaaat caa gtaatcaaac tattgttagg ttaaaggitta cgg tagattic tcc.gggaa.ca caaaagtatic gtatalagact aaaatatgcg 74 O gctact agta at t t t t at ct aggtgctt at gCaggaagta atggggggala cggaatticca ggtaticagta ctgttcctaa aacaatgaat atagaagatc citct t t cata tact t cattt 86 O gcttatattg atttacctga ttcatatact tittagt caaa alagacgaggt tataagattic 92 O actataaata tatacgaatc agg.cggagcc gtatatgcag acaaagttga at titat cocci 98 O gtggatgctg attacgatga aggagttcaa ttggaaaaag Cacagaaagc cgtgaatgcc US 8,304,605 B2 43 44 - Continued ttgtttacag cgggaagaaa cgcactacaa acagatgtga Cagattacaa agtagat cag 21OO gtgtcaatitt tagtggattg tgt at Caggg gagttatacc ccalatgagaa acgcgaact a 216 O

Caaaat Ctaa tcaaatacgc aaaacgtttg agctatt coc gtaatttact cctagat coa 222 O acatt cqatt Citat caattic at Cagatgag aatggctggit acggaagtaa tgg tattgca 228O atcggcagtg ggaatattgt attcaaaggg aact acttaa. ttittct cagg taccalatgat 234 O gaacaat atc Calaccitat ct Citatcaaaaa. atagacgaat ctaagttaaa agaatataca 24 OO cgittataaac tgagaggttt tat cagagt agt caggatt tagaa.gcata cgtgatt.cgt. 246 O tatgatgcaa aaCatcaaac aatggatgta to Caataatc. tatt citcaga tat tact cott 252O gtaaatgcat gcggagaacc aaatcgttgt gcggCactac catacctgga tgaaaatcca 2580 agattagaat gtagttcgat acaagatgga attictatctg att cqcatt c gttittcticto 264 O catatagata caggttctat tgatttcaat gagaacgtag gcatttgggit gttgtttaaa 27 OO attt CCaCaC tagaaggata cgcgaaattit gggaacctag aagtgattga agatggcc.ca 276 O gtcattggag aag cattagc cc.gtgttgaaa cgc.caagaaa cgaagttggag aaacaagttg 282O acacaactgc gaacggaaac acaag.cgatt tatacaagag caaaacaagc cattgataat 288O ttattoacala atgaac agga citct cact ta. aaaat aggta cga catttgc gtcaattgttg 294 O gctgcacgaa agattgtc.ca atc catacgt. gaagcgtata tgt catggitt atctatogt c 3 OOO cCaggtgtaa attatcCtat ttttacagaa ttgaatgaga gagtacagca agcatttcaa 3 O 6 O ttatatgatg tacgaaatgt cgtgcgtaat ggc.cgatticc agagtggaac atctgattgg 312 O attgtaac ct Ctgacgtaaa ggtacaagaa gaaaatggga ataacg tatt agttctttcc 318O aattgggatg cgcaagtatt acaatgcatg acgct ct acc aag accgtgg gtatat citta 324 O cgcgta acag cacgtaagga aggactgggc galaggg tatg taacaat CaC tgatgaagaa 33 OO ggaaatacag atcaattgag atttggtgga tgttgaggaga tagatgcatc taact cqttic 3360 gtat coacag gttatgttac aaaagaacta gaatttitt co Cagatacaga gaaagtgcgt 342O atagaaattg gagaalacaga aggaatatt c Caggtgggala gtgtagaatt atttittgatg 3480 gaagat citat gt 34.92

SEQ ID NO 2 LENGTH: 1164 TYPE : PRT ORGANISM: Bacillus thuringiensis

< 4 OOs SEQUENCE: 2 Met Asn. Cys Gly Asp His Asn. Glu Phe Asp Ile Ile Asp Wall Ile Glu 1. 1O 15

Asn Asn Glin Thir Lys Ala Ser Arg His Val Asn Glu Ser Asp Asn. Wall 25

Asn Arg Glin Arg Asn Lieu. Ser Asn Thir Ile Phe Ser Asn Luell Ser Ser 35 4 O 45

Asn Tyr Pro Lieu Ala Ser ASn Pro Asn. Thir Pro Phe Glin Asn Met Asn SO 55 6 O

Tyr Glu Tyr Lieu. Asn Ile Thr Glu Gly Gly Ile Ile Asn Pro Thir 65 70 7s

Lell Ala Gly Ser Ala Ile Wal Wall Ala Glin Asn Wall Ser Thir Ile 85 90 95

Lell Leu Gly Ser Thr Ile Lieu. Gly Lys Ile Lell Gly Ser Wall 105 11 O

Lell Asp Ile Leu Trp Pro Thr Asn Thr Glu Glu Ile Trp Luell Glu Lieu. US 8,304,605 B2 45 46 - Continued

115 12 O 125

Ile Asp Glu Wall Glu Glu Lell Ile Asn Glin Lys Ile Glu Glin Glin Wall 13 O 135 14 O

Ile Ile Asp Ala Glu Thir Ala Luell Glu Ser Wall Lell Asn Wall Asp 145 150 155 160

Lell Tyr Luell Asn Ala Lell Ala Glu Trp Glu Thir Arg Pro Thir Asn Glu 1.65 17O 17s

Ser Thir Glu Lell Wall Arg Phe Thir Asp Ala Tyr Asn Tyr 18O 185 19 O

Ala Arg Thir Ser Met Pro Phe Phe Ser Wall Arg Thir Tyr Glu Wall Ser 195 2OO

Lell Luell Ser Wall Tyr Ala Glin Ala Ala Asn Ile Ser Lell Luell Luell Ser 21 O 215 22O

Arg Asp Ala Glin Ile Tyr Gly Asp Luell Trp Gly Phe Asp Glu His Asp 225 23 O 235 24 O

Ala Thir Phe Asp Gly Glu Arg Luell Phe Arg Ala Glu Tyr Ile 245 250 255

Asp His Thir Wall Gly Luell Asp Arg Luell Gly 26 O 265 27 O

Ser Ser Tyr Glu Ser Trp Wall Asn Tyr Asn Arg Arg Arg Glu Met 27s 285

Thir Luell Met Ile Lell Asp Thir Ile Ala Ala Phe Pro Asp Ile 29 O 295 3 OO

Glu Glu Tyr Pro Ile Glu Wall Ser Thir Glin Luell Ala Arg Glu Wall Tyr 3. OS 310 315

Thir Asp Pro Ile Ile Thir Ser Phe Wall Glu Ser Asp His Gly Pro Ser 3.25 330 335

Phe Ser Phe Met Glu Ser Asn Ala Ile Arg Lys Pro His Luell Wall Asp 34 O 345 35. O

Luell Asp Asn Lell Ile Tyr Thir Ser Arg Phe Arg Thir Phe Ser 355 360 365

Asn Glu Phe Glin Pro Asp Lell Asn Tyr Trp Ala Ala His Wall Lys 37 O 375

Tyr Ser Gly Asp Pro Thir Luell His Glu Thir Pro Ile Gly 385 390 395 4 OO

Asn Ala Ser Asn Tyr Glu Ser Thir Gly Asn Tyr Ser Phe Arg Gly Asn 4 OS 415

Ser Ile Glin Thir Lell Ser Ala Pro Ser Ala Ile Lell Thir Pro Asn 42O 425 43 O

Ile Tyr Gly Ile Glu Glin Wall Glu Phe Gly Asn Gly 435 44 O 445

Asn Wall Gly Gly Asn Pro Lell Ser Wall Ser 450 45.5 460

Ala Asn Glin Luell Pro Pro Asp Wall Glu Pro Ile Thir Glu Asn Asn 465 470

His Wall Luell His Ala Thir Ala Wall Pro Wall Asp Gly Gly Thir 485 490 495

Wall Pro Ile Phe Ser Trp Thir His Arg Ser Ala Asp Tyr Asn Thir SOO 505

Ile Pro Asp Ile Thir Glin Luell Pro Ala Wall Lys Ser Thir Pro 515 52O 525

Ser Pro Glu Wall Glu Gly Lell Wall Glin Glu Gly Pro Gly Phe Thir 53 O 535 54 O US 8,304,605 B2 47 48 - Continued Gly Gly Asp Lieu Val Val Ala Lys Ser Ser Asn Glin Thir Ile Val Arg 5.45 550 555 560 Lieu Lys Val Thr Val Asp Ser Pro Gly Thr Glin Llys Tyr Arg Ile Arg 565 st O sts Lieu Lys Tyr Ala Ala Thir Ser Asn. Phe Tyr Lieu. Gly Ala Tyr Ala Gly 58O 585 59 O Ser Asn Gly Gly Asn Gly Ile Pro Gly Ile Ser Thr Val Pro Llys Thr 595 6OO 605 Met Asin Ile Glu Asp Pro Leu Ser Tyr Thr Ser Phe Ala Tyr Ile Asp 610 615 62O Lieu Pro Asp Ser Tyr Thr Phe Ser Gln Lys Asp Glu Val Ile Arg Phe 625 630 635 64 O Thir Ile Asn. Ile Tyr Glu Ser Gly Gly Ala Val Tyr Ala Asp Llys Val 645 650 655 Glu Phe Ile Pro Val Asp Ala Asp Tyr Asp Glu Gly Val Glin Lieu. Glu 660 665 67 O Lys Ala Glin Lys Ala Val Asn Ala Lieu. Phe Thr Ala Gly Arg Asn Ala 675 68O 685 Lieu. Glin Thr Asp Val Thr Asp Tyr Llys Val Asp Glin Val Ser Ile Lieu. 69 O. 695 7 OO Val Asp Cys Val Ser Gly Glu Lieu. Tyr Pro Asn. Glu Lys Arg Glu Lieu 7 Os 71O 71s 72O Glin Asn Lieu. Ile Llys Tyr Ala Lys Arg Lieu. Ser Tyr Ser Arg Asn Lieu. 72 73 O 73 Lieu. Lieu. Asp Pro Thr Phe Asp Ser Ile ASn Ser Ser Asp Glu. ASn Gly 740 74. 7 O Trp Tyr Gly Ser Asn Gly Ile Ala Ile Gly Ser Gly Asn Ile Val Phe 7ss 760 765 Lys Gly Asn Tyr Lieu. Ile Phe Ser Gly Thr Asn Asp Glu Glin Tyr Pro 770 775 78O Thir Tyr Lieu. Tyr Glin Lys Ile Asp Glu Ser Lys Lieu Lys Glu Tyr Thr 78s 79 O 79. 8OO Arg Tyr Lys Lieu. Arg Gly Phe Ile Glu Ser Ser Glin Asp Lieu. Glu Ala 805 810 815 Tyr Val Ile Arg Tyr Asp Ala Lys His Glin Thr Met Asp Val Ser Asn 82O 825 83 O Asn Lieu. Phe Ser Asp Ile Thr Pro Val Asn Ala Cys Gly Glu Pro Asn 835 84 O 845 Arg Cys Ala Ala Lieu Pro Tyr Lieu. Asp Glu ASn Pro Arg Lieu. Glu. Cys 850 855 860 Ser Ser Ile Glin Asp Gly Ile Lieu. Ser Asp Ser His Ser Phe Ser Lieu. 865 87O 87s 88O His Ile Asp Thr Gly Ser Ile Asp Phe Asn. Glu Asn Val Gly Ile Trp 885 890 895 Val Lieu. Phe Lys Ile Ser Thr Lieu. Glu Gly Tyr Ala Lys Phe Gly Asn 9 OO 905 91 O Lieu. Glu Val Ile Glu Asp Gly Pro Val Ile Gly Glu Ala Lieu Ala Arg 915 92 O 925 Val Lys Arg Glin Glu Thir Lys Trp Arg Asn Llys Lieu. Thr Glin Lieu. Arg 93 O 935 94 O Thr Glu Thr Glin Ala Ile Tyr Thr Arg Ala Lys Glin Ala Ile Asp Asn 945 950 955 96.O Lieu. Phe Thr Asn Glu Gln Asp Ser His Leu Lys Ile Gly Thr Thr Phe 965 97O 97. US 8,304,605 B2 49 50 - Continued

Ala Ser Ile Val Ala Ala Arg Lys Ile Val Glin Ser Ile Arg Glu Ala 98O 985 99 O Tyr Met Ser Trp Lieu. Ser Ile Val Pro Gly Val Asn Tyr Pro Ile Phe 995 1OOO 1005 Thr Glu Lieu. Asn. Glu Arg Val Glin Glin Ala Phe Glin Lieu. Tyr Asp O1O O15 O2O Val Arg Asn Val Val Arg Asn Gly Arg Phe Glin Ser Gly. Thir Ser O25 O3 O O35 Asp Trp Ile Val Thir Ser Asp Wall Lys Val Glin Glu Glu Asn Gly O4 O O45 OSO Asn Asn Val Lieu Val Lieu. Ser Asn Trp Asp Ala Glin Val Lieu. Glin O55 O6 O O65 Cys Met Thr Lieu. Tyr Glin Asp Arg Gly Tyr Ile Lieu. Arg Val Thr Of O O7 O8O Ala Arg Lys Glu Gly Lieu. Gly Glu Gly Tyr Val Thir Ile Thr Asp O85 O9 O O95 Glu Glu Gly Asn. Thir Asp Glin Lieu. Arg Phe Gly Gly Cys Glu Glu 1 OO 105 11 O Ile Asp Ala Ser Asn Ser Phe Val Ser Thr Gly Tyr Val Thr Lys 115 12 O 125 Glu Lieu. Glu Phe Phe Pro Asp Thr Glu Lys Val Arg Ile Glu Ile 13 O 135 14 O Gly Glu Thr Glu Gly Ile Phe Glin Val Gly Ser Val Glu Lieu Phe 145 15 O 155 Lieu Met Glu Asp Lieu. Cys 16 O

<210s, SEQ ID NO 3 &211s LENGTH: 1992 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic molecule <4 OOs, SEQUENCE: 3 atgaactgtg gcgaccacaa tdagtttgat atcatcgacg tcatcgaaaa caaccagacc 6 O aaggccticca gacacgtgaa tagagcgac aatgtcaiaca gacagcgcaa Cctitt Ctaac 12 O acgatc.ttct ctaacttgtc gtccaactat cotct cqcga gcaatcc gaa caccc catt c 18O

Cagaac atga act acaagga gitat Ctcaac at Caccgagg gtggcatcat Caatc.cgaca 24 O

Ctggctggca gcgctatcgt ttggcacag aacgtgtc.ca agacaat act gaagaagttg 3OO ggaa.gcacca t cct cqgaaa gat CCtcggc agcgt.ccttg at atcttgttg gccaacgaac 360 accgaggaaa tictggcttga act cattgat gaggttgagg aactgat caa t cagaagatt gaggagcaag ticatcattga cqcagagaca gcc ct caat Ctgtgaaact gaatgtggac

Ctct atctga acgctctggc agagtgggag acgaga.ccga C caatgagta Ctccaccgag 54 O ttgg to taca aacgctt cac agacgc.ctac aactato.cga gcacct cqat gcc.gttctitt t cagtgagga cittacgaggt gttct ctdctic tictdt citatg cccaa.gctgc caa.cat atcg 660

Ctgctic ctitt C cagagatgc ccaaatct at ggcgatctitt ggggatt.cga Calacatgac 72 O aaggcgacat t catgggga gcggaagctg tittagggcag agtacatcga C cactgcacg aagtactaca aagttgggct tacagactgaaaggcagot catacgagtic atgggittaac 84 O tacaatcgct acagacggga gatgacgttg atgattctgg acacaat agc agc ctitt Coc 9 OO US 8,304,605 B2 51 - Continued tactacgaca tcgaggagta tcc cattgag gtgtcaa.ccc agctggctag ggaggtotac 96.O accgaccc.ga t cat tact to Ctttgttggaa tcc gat catg titcct tcatg gaatcgaacg c cataaggaa accocacctic gttgact atc ttgacaatct CtaCatctac accagcagat tcc.gcactitt Cagcaatgag titc.ca.gc.cag acct calacta Ctgggctgcc 14 O caca aggt ca agtacaagta Ctctggcgac c caac Cttgc acgagacacc catctacgga 2OO aatgcct caa act atgagag Cactggcaac tact cott ta. ggggaaactic gat citat cag 26 O accct citcgg citc.cgtctgc cattcttacg CCtalactaca tctactacgg gatagagcaa. 32O gtggagttct acgggaacaa gggcaacgt.c tactataggg gtggcaacaa gt atc.cgctg t cagttgact cggcaaacca gttgcctic cc gacgttgagc citat cacgga gaact acaat 44 O catgtc.ctitt gcc acgc.cac agcc.gttc cc gtcaaggatg gtggCaccgt gcctatotitt SOO t catgg actic atcggit cogc tgact actac aatacgatct atcctgataa gatalacc cag 560 Cttic cagcgg tgaagagcac gcc tt cacca gaggtggagg gtc.tcaaagt cCaagaagga cctggctt.ca Ctggtgggga tittggttgtc gcgaagt cca gcaat Cagac catcgtcaga

Ctgaaagtga Cagtggattic t cctgcacg Cagaagtaca gaat Cagact gaagtacgct 74 O gcgacCtcaa act totat ct gggagcctac gctgggit coa atggtggcaa. cgggatt CCt ggcatctoca ctgttccaaa gactatgaac attgaagatc coct ct citta cacgagctitt 86 O gcgtacattg atttgccaga cagctacact ttctoacaaa. aggacgaagt gatacgcttic 92 O actatocaa.ca tctacgaatc gggtggagcg gtgtacgctg acalaggt ca gttcatcc.ca 98 O gtggatgcag ac 992

<210s, SEQ ID NO 4 &211s LENGTH: 545 212. TYPE : PRT &213s ORGANISM: Bacillus thuringiensis

<4 OOs, SEQUENCE: 4

Lell Glu Ala Glu Ser Asp Lieu. Glu Arg Ala Glin Ala Wall Asn Ala 1. 1O 15

Lell Phe Thir Ser Ser Asn. Glin Ile Gly Lieu Lys Thir Asp Wall Thir Asp 25 3O

His Ile Asp Arg Val Ser Asn Lieu Wall Glu Lell Ser Asp Glu 35 4 O 45

Phe Cys Luell Asp Glu Lys Lys Glu Lieu. Ser Glu Lys Wall His Ala SO 55 6 O

Lys Arg Luell Ser Asp Glu Arg Asn Lieu. Lieu. Glin Asp Pro Asn Phe Arg 65 70 7s 8O

Gly Ile Asn Arg Glin Lieu. Asp Arg Gly Trp Arg Gly Ser Thir Asp Ile 85 90 95

Thir Ile Glin Gly Gly Asp Asp Val Phe Lys Glu Asn Wall Thir Lieu. 105 11 O

Lell Gly Thir Phe Asp Glu. Cys Tyr Pro Thr Tyr Lell Tyr Glin Lys Ile 115 12 O 125

Asp Glu Ser Llys Lieu Lys Ala Tyr Thr Arg Tyr Glin Lell Arg Gly Tyr 13 O 135 14 O

Ile Glu Asp Ser Glin Asp Lieu. Glu Ile Tyr Lieu. Ile Arg Asn Ala 145 150 155 160

His Glu Thir Wall Asn. Wall Pro Gly Thr Gly Ser Lell Trp Pro Leu 1.65 17O 17s

Ser Ala Pro Ser Pro Ile Gly Lys Cys Ala His His Ser His His Phe US 8,304,605 B2 53 - Continued

18O 185 19 O Ser Lieu. Asp Ile Asp Val Gly Cys Thr Asp Lieu. Asn. Glu Asp Lieu. Gly 195 2OO 2O5 Val Trp Val Ile Phe Lys Ile Llys Thr Glin Asp Gly. His Ala Arg Lieu 21 O 215 22O Gly Asn Lieu. Glu Phe Lieu. Glu Glu Lys Pro Lieu Val Gly Glu Ala Lieu. 225 23 O 235 24 O Ala Arg Val Lys Arg Ala Glu Lys Llys Trp Arg Asp Lys Arg Glu Lys 245 250 255 Lieu. Glu Trp Glu Thir Asn. Ile Val Tyr Lys Glu Ala Lys Glu Ser Val 26 O 265 27 O Asp Ala Lieu. Phe Val Asn. Ser Glin Tyr Asp Arg Lieu. Glin Ala Asp Thr 27s 28O 285 Asn. Ile Ala Met Ile His Ala Ala Asp Lys Arg Val His Ser Ile Arg 29 O 295 3 OO Glu Ala Tyr Lieu Pro Glu Lieu. Ser Val Ile Pro Gly Val Asn Ala Ala 3. OS 310 315 32O Ile Phe Glu Glu Lieu. Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu Tyr 3.25 330 335 Asp Ala Arg Asn Val Ile Lys Asn Gly Asp Phe Asn. Asn Gly Lieu. Ser 34 O 345 35. O Cys Trp Asn. Wall Lys Gly His Val Asp Val Glu Glu Glin Asn. Asn His 355 360 365 Arg Ser Val Lieu Val Val Pro Glu Trp Glu Ala Glu Val Ser Glin Glu 37 O 375 38O Val Arg Val Cys Pro Gly Arg Gly Tyr Ile Lieu. Arg Val Thir Ala Tyr 385 390 395 4 OO Lys Glu Gly Tyr Gly Glu Gly Cys Val Thir Ile His Glu Ile Glu Asn 4 OS 41O 415 Asn. Thir Asp Glu Lieu Lys Phe Ser Asn. CyS Val Glu Glu Glu Val Tyr 42O 425 43 O Pro Asn Asn Thr Val Thr Cys Asn Asp Tyr Thr Ala Thr Glin Glu Glu 435 44 O 445 Tyr Glu Gly Thr Tyr Thr Ser Arg Asn Arg Gly Tyr Asp Gly Ala Tyr 450 45.5 460 Glu Ser Asn. Ser Ser Val Pro Ala Asp Tyr Ala Ser Ala Tyr Glu Glu 465 470 47s 48O Lys Ala Tyr Thir Asp Gly Arg Arg Asp Asn Pro Cys Glu Ser Asn Arg 485 490 495 Gly Tyr Gly Asp Tyr Thr Pro Leu Pro Ala Gly Tyr Val Thr Lys Glu SOO 505 51O Lieu. Glu Tyr Phe Pro Glu Thr Asp Llys Val Trp Ile Glu Ile Gly Glu 515 52O 525 Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu Lieu Lleu Lleu Met Glu 53 O 535 54 O

Glu 5.45

<210s, SEQ ID NO 5 &211s LENGTH: 1209 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic molecule

<4 OOs, SEQUENCE: 5 US 8,304,605 B2 55 56 - Continued

Met Asn Gly Asp His Asn Glu Phe Asp Ile Ile Asp Val Ile Glu 15

Asn Asn Glin Thir Lys Ala Ser Arg His Wall ASn Glu Ser Asp Asn Wall 2O 25 3O

Asn Arg Glin Arg Asn Lell Ser Asn Thir Ile Phe Ser Asn Luell Ser Ser 35 4 O 45

Asn Tyr Pro Luell Ala Ser Asn Pro Asn Thir Pro Phe Glin Asn Met Asn SO 55 6 O

Tyr Glu Lell Asn Ile Thir Glu Gly Gly Ile Ile Asn Pro Thir 65 70

Lell Ala Gly Ser Ala Ile Wall Wall Ala Glin ASn Wall Ser Thir Ile 85 90 95

Lell Luell Gly Ser Thir Ile Luell Gly Ile Lell Gly Ser Wall 105 11 O

Lell Asp Ile Luell Trp Pro Thir Asn Thir Glu Glu Ile Trp Luell Glu Luell 115 12 O 125

Ile Asp Glu Wall Glu Glu Lell Ile Asn Glin Ile Glu Glin Glin Wall 13 O 135 14 O

Ile Ile Asp Ala Glu Thir Ala Luell Glu Ser Wall Lell Asn Wall Asp 145 150 155 160

Lell Luell Asn Ala Lell Ala Glu Trp Glu Thir Arg Pro Thir Asn Glu 1.65 17O 17s

Ser Thir Glu Lell Wall Arg Phe Thir Asp Ala Tyr Asn 18O 185 19 O

Ala Arg Thir Ser Met Pro Phe Phe Ser Wall Arg Thir Tyr Glu Wall Ser 195 2OO

Lell Luell Ser Wall Tyr Ala Glin Ala Ala Asn Ile Ser Lell Luell Luell Ser 21 O 215 22O

Arg Asp Ala Glin Ile Tyr Gly Asp Luell Trp Gly Phe Asp Glu His Asp 225 23 O 235 24 O

Ala Thir Phe Asp Gly Glu Arg Luell Phe Arg Ala Glu Tyr Ile 245 250 255

Asp His Thir Lys Wall Gly Luell Asp Arg Luell Gly 26 O 265 27 O

Ser Ser Tyr Glu Ser Trp Wall Asn Asn Arg Arg Arg Glu Met 27s 285

Thir Luell Met Ile Lell Asp Thir Ile Ala Ala Phe Pro Asp Ile 29 O 295 3 OO

Glu Glu Pro Ile Glu Wall Ser Thir Glin Luell Ala Arg Glu Wall Tyr 3. OS 310 315

Thir Asp Pro Ile Ile Thir Ser Phe Wall Glu Ser Asp His Gly Pro Ser 3.25 330 335

Phe Ser Phe Met Glu Ser Asn Ala Ile Arg Pro His Luell Wall Asp 34 O 345 35. O

Luell Asp Asn Lell Ile Tyr Thir Ser Arg Phe Arg Thir Phe Ser 355 360 365

Asn Glu Phe Glin Pro Asp Lell Asn Trp Ala Ala His Wall 37 O 375

Tyr Ser Gly Asp Pro Thir Luell His Glu Thir Pro Ile Gly 385 390 395 4 OO

Asn Ala Ser Asn Tyr Glu Ser Thir Gly Asn Ser Phe Arg Gly Asn 4 OS 41O 415

Ser Ile Glin Thir Lell Ser Ala Pro Ser Ala Ile Lell Thir Pro Asn US 8,304,605 B2 57 58 - Continued

425 43 O

Ile Tyr Gly Ile Glu Glin Wall Glu Phe Gly Asn Gly 435 44 O 445

Asn Wall Gly Gly Asn Pro Lell Ser Wall Asp Ser 450 45.5 460

Ala Asn Glin Luell Pro Pro Asp Wall Glu Pro Ile Thir Glu Asn Tyr Asn 465 470

His Wall Luell His Ala Thir Ala Wall Pro Wall Asp Gly Gly Thir 485 490 495

Wall Pro Ile Phe Ser Trp Thir His Arg Ser Ala Asp Tyr Asn Thir SOO 505

Ile Pro Asp Ile Thir Glin Luell Pro Ala Wall Lys Ser Thir Pro 515 52O 525

Ser Pro Glu Wall Glu Gly Lell Wall Glin Glu Gly Pro Gly Phe Thir 53 O 535 54 O

Gly Gly Asp Luell Wall Wall Ala Ser Ser ASn Glin Thir Ile Wall Arg 5.45 550 555 560

Lell Wall Thir Wall Asp Ser Pro Gly Thir Glin Arg Ile Arg 565 st O sts

Lell Ala Ala Thir Ser Asn Phe Luell Gly Ala Tyr Ala Gly 585 59 O

Ser Asn Gly Gly Asn Gly Ile Pro Gly Ile Ser Thir Wall Pro Thir 595 605

Met Asn Ile Glu Asp Pro Lell Ser Thir Ser Phe Ala Ile Asp 610 615

Lell Pro Asp Ser Tyr Thir Phe Ser Glin Asp Glu Wall Ile Arg Phe 625 630 635 64 O

Thir Ile Asn Ile Tyr Glu Ser Gly Gly Ala Wall Ala Asp Lys Wall 645 650 655

Glu Phe Ile Pro Wall Asp Ala Asp Luell Glu Ala Glu Ser Asp Luell Glu 660 665 67 O

Arg Ala Glin Ala Wall Asn Ala Luell Phe Thir Ser Ser Asn Glin Ile 675 685

Gly Luell Thir Asp Wall Thir Asp His Ile Asp Arg Wall Ser Asn 69 O. 695 7 OO

Lell Wall Glu Lell Ser Asp Glu Phe Luell Asp Glu Glu 7 Os

Lell Ser Glu Wall His Ala Arg Luell Ser Asp Glu Arg Asn 72 73 O 73

Lell Luell Glin Asp Pro Asn Phe Arg Gly Ile ASn Arg Glin Luell Asp Arg 740 74. 7 O

Gly Trp Arg Gly Ser Thir Asp Ile Thir Ile Glin Gly Gly Asp Asp Wall 760 765

Phe Lys Glu Asn Tyr Wall Thir Luell Luell Gly Thir Phe Asp Glu Tyr 770 775

Pro Thir Luell Tyr Glin Ile Asp Glu Ser Lell Ala Tyr 79 O 79.

Thir Arg Glin Lell Arg Gly Ile Glu Asp Ser Glin Asp Luell Glu 805 810 815

Ile Luell Ile Arg Asn Ala Lys His Glu Thir Wall Asn Wall Pro 82O 825 83 O

Gly Thir Gly Ser Lell Trp Pro Luell Ser Ala Pro Ser Pro Ile Gly 835 84 O 845 US 8,304,605 B2 59 - Continued Cys Ala His His Ser His His Phe Ser Lieu. Asp Ile Asp Val Gly Cys 850 855 860 Thir Asp Lieu. Asn. Glu Asp Lieu. Gly Val Trp Val Ile Phe Lys Ile Llys 865 87O 87s 88O Thr Glin Asp Gly His Ala Arg Lieu. Gly Asn Lieu. Glu Phe Lieu. Glu Glu 885 890 895 Llys Pro Lieu Val Gly Glu Ala Lieu Ala Arg Val Lys Arg Ala Glu Lys 9 OO 905 91 O Llys Trp Arg Asp Lys Arg Glu Lys Lieu. Glu Trp Glu Thir Asn. Ile Val 915 92 O 925 Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Lieu. Phe Val Asn. Ser Glin 93 O 935 94 O Tyr Asp Arg Lieu. Glin Ala Asp Thr Asn. Ile Ala Met Ile His Ala Ala 945 950 955 96.O Asp Lys Arg Val His Ser Ile Arg Glu Ala Tyr Lieu Pro Glu Lieu. Ser 965 97O 97. Val Ile Pro Gly Val Asn Ala Ala Ile Phe Glu Glu Lieu. Glu Gly Arg 98O 985 99 O Ile Phe Thr Ala Phe Ser Lieu. Tyr Asp Ala Arg Asn Val Ile Lys Asn 995 1OOO 1005 Gly Asp Phe Asn. Asn Gly Lieu. Ser Cys Trp Asn. Wall Lys Gly His O1O O15 O2O Val Asp Val Glu Glu Glin Asn. Asn His Arg Ser Val Lieu Val Val O25 O3 O O35 Pro Glu Trp Glu Ala Glu Val Ser Glin Glu Val Arg Val Cys Pro O4 O O45 OSO Gly Arg Gly Tyr Ile Lieu. Arg Val Thir Ala Tyr Lys Glu Gly Tyr O55 O6 O O65 Gly Glu Gly Cys Val Thir Ile His Glu Ile Glu Asn Asn Thr Asp Of O O7 O8O Glu Lieu Lys Phe Ser Asn. Cys Val Glu Glu Glu Val Tyr Pro Asn O85 O9 O O95 Asn Thr Val Thr Cys Asn Asp Tyr Thr Ala Thr Glin Glu Glu Tyr OO O5 10 Glu Gly. Thir Tyr Thir Ser Arg Asn Arg Gly Tyr Asp Gly Ala Tyr

Glu Ser Asn. Ser Ser Val Pro Ala Asp Tyr Ala Ser Ala Tyr Glu

Glu Lys Ala Tyr Thr Asp Gly Arg Arg Asp Asn Pro Cys Glu Ser

Asn Arg Gly Tyr Gly Asp yr Thr Pro Leu Pro Ala Gly Tyr Val

Thr Lys Glu Lieu. Glu Tyr Phe Pro Glu Thir Asp Llys Val Trp Ile

Glu e Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser Val Glu

Lieu. Lieu. Lieu Met Glu Glu 2O5

<210s, SEQ ID NO 6 &211s LENGTH: 1635 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic molecule

US 8,304,605 B2 63 64 - Continued cc cacgitatic tictato agaa gattgacgag tocaa.gctica aagcc tacac acgctat cag 42O citcagaggct acattgagga ct citcaagac ct cqaaatct acttgat cag atacaacgc.c 48O alagcacgaga C9gtgaacgt. C cctgggact gggtcactgt ggccactgtc. g.gcaccctic 54 O c caatcggaa agtgcgctica ccacagocac cacttct coc ttgacataga tigttgggtgt 6OO acggacttga atgaggat.ct gggtgttgttgg gtgat ctitta agat Caaga C C Caagatggit 660 Catgcgaggc titggcaacct tagttcCtt galagaga agc Ctttggtcgg agaggcactg 72 O gctcgc.gtga agagggctga gaagaaatgg aggga Calaga giggagaaact gagtgggag 78O accalacatag titacaagga ggccalaggag ticagtggacg cactgtttgt caatt CC cag 84 O tatgat aggc tccaag.cgga cacgalacatc gcc atgatcc atgcagcgga Caagagggitt 9 OO Cact coat aa gggaggccta t ct tccggag Ctgtcagtga titcCtggggit Caacgcagcc 96.O atctttgagg aattggaagg gaggat.ct tc accgctttct Ctctgtacga cqctcggaac O2O gtcatcaaga atggtgattt Caacaatgga Ctcagctgct ggaacgtgaa agggcatgtc. O8O gatgttgaag alacagaacaa t caccgcago gtgctggtgg ttc.cggagtg ggaag.ccgag 14 O gtct cacaag aagttcagagt gtgcc ctggg aggggttaca tottgcgggt cacagcctac 2OO aaggaaggtt atggcgaagg Ctgtgtcacg atc catgaga t caaaacaa Cacagacgag 26 O Ctgaagttitt C caactgtgt taggaggag gtctat ccta acaat actgt tacgtgcaac 32O gact acacag C cact Caaga ggagtacgag ggc acttaca Cct ct cqcala cagaggctac 38O gacggtgcct acgagt caaa cagct cogtg C cagcggact acgc.ctcggc titacgaagag 44 O aaggcgtaca CC9acggtC9 gagggataac CC9tgcgaga gcaatagagg Ctatggcgac SOO tacact cotc. tcc cagctgg ctacgtgacc aaggagttgg agtacttitcc ggagacagac 560 aaagttctgga ttgagattgg agagacagaa ggcacgttca togtggactic tittgaactic 62O ttgctgatgg aggag 635

We claim: 11. An isolated nucleic acid that encodes a polypeptide of 1. An isolated polypeptide comprising residues 142 to 664 40 claim 2. of SEQ ID NO:2, wherein said polypeptide has insecticidal 12. An isolated nucleic acid that encodes a polypeptide of activity. claim 3. 13. An isolated nucleic acid that encodes a polypeptide of 2. The isolated polypeptide of claim 1 comprising residues claim 4. 1 to 664 of SEQ ID NO:2, wherein said polypeptide has 45 14. The isolated nucleic acid of claim 10 having a sequence insecticidal activity. of SEQID NO: 1 or SEQID NO:3. 3. The isolated polypeptide of claim 1 comprising residues 15. The polypeptide of claim 1 comprising an amino acid 142 to 1164 of SEQID NO:2, wherein said polypeptide has sequence of SEQID NO: 2 or SEQID NO:5. insecticidal activity. 16. A DNA construct comprising the nucleotide sequence 4. The isolated polypeptide of claim 1 comprising the 50 of claim 10 operably linked to a promoter that is not derived amino acid sequence of SEQID NO:2. from Bacillus thuringiensis and is capable of driving expres 5. A plant comprising the polypeptide of claim 1. sion in a plant. 6. A plant comprising the polypeptide of claim 2. 17. A transgenic plant that comprises the DNA construct of 7. A plant comprising the polypeptide of claim 3. claim 16 stably incorporated into its genome. 8. A plant comprising the polypeptide of claim 4. 55 18. A method for protecting a plant from a pest comprising 9. A method for controlling a pest population comprising introducing into said plant the construct of claim 16. contacting said population with a pesticidally effective 19. The polypeptide of claim 1, claim 2, claim 3, or claim amount of the polypeptide of claim 1. 4, wherein said polypeptide has insecticidal activity against 10. An isolated nucleic acid that encodes a polypeptide of COrn rootWOrm. claim 1.