OA17138A - Proteins toxic to hemipteran insect species - Google Patents

Abstract

The present invention discloses hemipteran insect inhibitory proteins, methods of using such proteins, nucleotide sequences encoding such proteins, methods of detecting and isolating such proteins, and their use in agricultural systems.

Description

|0001( This application claims priority to U.S. Provisional Application Serial No. 61/621,436 filed April 6,2012, which is incorporated herein by reference in its entirety.

INCORPORATION OF SEQUENCE LISTING [0002( The Sequence Listing contained in the file named “38_21_56191_APCT_Sequence Listing_ST25.txt”, which is 529,794 bytes in size (measured in operating System MSWindows) and was created on April 4, 2013, is contemporaneously filed by electronic submission (using the United States Patent Office EFS-Web filing System) and is incorporated herein by reference în its entirety.

FIELD OF THE INVENTION

100031 The présent invention generally relates to the field of insect inhibitory proteins. In particular, the présent invention relates to proteins exhibiting insect inhibitory activity against agriculturally relevant pests of crop plants and seeds, particularly Hemipteran species of insect pests.

BACKGROUND OFTHE INVENTION [0004( Insect inhibitory proteins derived from Bacillus thuringicnsts (Bt) are non-toxic to humans, vertebrates, and plants. These proteins are also biodégradable, safe, and effective in controlling pest insects. Some of these proteins hâve been and are being used to control agriculturally relevant pests of crop plants by spraying plants with formulations containing these proteins or with microorganisms that express them, treating seeds with treatments containing these proteins, or expressing these proteins in crop plants and seeds of crop plants as plant-incorporated protectants.

[00051 Certain Hemiptera species, particularly Amrasca, Empoasca and Lygus bugs, are pests of cotton and alfalfa, and typically are only controlled using broad spectrum chemistries, e.g., endosulfan, acephate, and oxamyl, which can persist in and are harmful to the environment. A few Bt proteins hâve been developed in formulations or as transgenic traits in crop plants for commercial use by farmers to control Coleopteran and Lepidopteran pest species, but no Bt proteins hâve been developed for use in commercial control of Hemipteran pest species.

[0006( Hemipteran spécifie toxic proteins hâve been reported in the art. TIC807 is a Bacillus thuringiensis protein disclosed in U.S. Patent Application Publication No. US 2008-0295207

Al as being toxic to Hemipteran pest species. A Cry5lAal protein reported as toxic to Lepidopteran species that closely resembles the amino acid sequence of TIC807 has also been disclosed (Huang et al., (2007) J. Invertebr. Pathol. 95(3), 175-180), but no Hemipteran spécifie activity was reported. Baum et al. disclosed TIC853, a protein reported to be toxic to Lygus pest species (U.S. Patent Application Publication No. US 2010-0064394 Al). A protein referred to as AXMI-I71 was reported to exhibit some limited inhibition of Hemipteran insects (U.S. Patent Application Publication No. US2010-0298207 AI, example 18), particulariy Lygus hesperus.

100071 Ail of these proteins exhibit a narrow range of toxicity only against Lygus hesperus and exhibit toxic effects against other Lygus pest species only in high doses which are not considered to be achievable by expression in plants. Compared to the Hemipteran toxic proteins in the prior art, there is a need for toxin proteins that can be used on and in plants that exhibit a broad host range against Hemipteran pest species and at low concentration effective doses.

BRIEF SUMMARY OF THE INVENTION [0008! Recombinantly engineered Hemipteran toxic proteins described herein (referred to herein as “engineered toxin proteins’’, “engineered toxic proteins”, “engineered Hemipteran toxic proteins”, or “engineered Hemipteran toxin proteins”, are also referred to herein in truncated form as “eHTP’s” when referred to in groups of two or more such proteins, and “eHTP” when referred to singularly) are dérivatives of naturally occurring Bacillus thuringiensis insectiddai toxins, TIC807 (SEQ ID NO:2), TIC807_M2 (SEQ ID NO:8), CrySlAal (SEQ ID NO:182), TIC853 (SEQ ID NO:184), and AXMI-171 (SEQ ID NO:206) hâve been described previously to exhibit bio-control activity directed to Hemipteran pest species, particulariy Lygus hesperus insect species (references cited elsewhere herein). The recombinant Hemipteran insect toxic proteins of the present invention are particulariy toxic to insects of the Amrasca, Empoasca and Lygus species of insect pests and to other insect pest species that are phylogenetically related to each of these species of insect pests, and additionally to insect pests that feed on plants using a piercing and sucking mechanism used by the pest species Amrasca, Empoasca and Lygus species of the order Hemiptera. Unlike the precursor insectiddai toxins TIC807 (SEQ ID NO:2), TIC807_M2 (SEQ ID NO:8), CrySlAal (SEQ ID NO:182), TIC853 (SEQ ID NO:184), and AXMI-171 (SEQ ID NO:206) from which they are derived, which each require moderately high to high doses of protein to achieve toxic effects upon one Lygus species and exhibit very low or virtually undetectable toxic effects upon a second closely related species of Lygus, the eHTP proteins of the présent invention exhibit surprising and unexpected low dose toxic effects against insect pests of the order Hemiptera, including host range toxic effects that span the spectrum of pests within the order.

[0009] The eHTP’s of the présent invention each contain at least one amino acid substitution, one amino acid addition, or one amino acid délétion compared to the primary amino acid sequence of one or more of the toxin proteins set forth in any of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:l82, or SEQ ID NO:l84. In certain embodiments, an eHTP is provided that contains at least from about 2 to about 260 fold greater inhibitory activity against a Lygus pest species than any one or more of the toxins set forth in any of SEQ ID NO:2 (TIC807), SEQ ID NO:8 (TIC807_M2), SEQ ID NO:l82 (Cry5lAal), SEQ ID NO:l84 (TIC853), and/or SEQ ID NO:206 (AXMI-171). Optionally the eHTP exhibits at least about 95% amino acid sequence identity to the toxin protein selected from the group consisting of SEQ ID NO:2 (TIC807) and SEQ ID NO:l82 (Cry5lAal). In certain embodiments, an eHTP is provided that contains at least one amino acid substitution, at least one amino acid addition, or at least one amino acid délétion when compared to the amino acid sequence of any of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:I82, or SEQ ID NO:l84. The eHTP exhibits an increased or greater Lygus inhibitory activity and target pest species spectrum compared to the activity and target pest species spectrum of the Bacillus thuringiensis proteins of set forth in SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:l82, and SEQ ID NO-.I84. Each of the aforementioned eHTP’s contain at least, collectively or in the alternative: (i) the amino acid substitution, addition, or délétion in a solvent accessible amino acid residue of SEQ ID NO:2;

(ii) the amino acid substitution, addition, or délétion within 3 consecutive residues of a solvent accessible amino acid residue of SEQ ID NO:2; or, (iii) an amino acid sequence as set forth in SEQ ID NO:l80. The aforementioned eHTP’s will each contain at least, with reference to the amino acid sequence positions as numbered according to the amino acid positions of TIC807, one substitution or délétion selected from the group consisting of asparagine at position I2 replaced by aspartic acid, phenylalanine at position 46 replaced by serine, isoleucine at position 52 replaced by méthionine, tyrosine at position 54 replaced by histidine, threonine at position 68 replaced by alanine, glutamine at position 70 replaced by alanine, alanine at position 87 replaced by serine, threonine at position 93 replaced by alanine, serine at position 95 replaced by alanine, glycines at position 105 replaced by alanine, serine at position 117 replaced by alanine, serine at position 119 replaced by alanine, glutamate at position 125 replaced by cysteine, histidine, arginine, phenylalanine, serine, glutamine, lysine, threonine, asparagine, alanine, leucine, valine, méthionine, aspartic acid, or tyrosine, glycines at position 128 replaced by alanine, threonine at position 133 replaced by glutamic acid, tyrosine, or tryptophan, isoleucine at position 134 replaced by alanine, valine, leucine, phenylalanîne, lysine, cysteine, or méthionine, glutamate at position 135 replaced by serine, alanine, valine, tryptophan, or threonine, asparagine at position 137 replaced by histidine, tyrosine, threonine, glutamic acid, serine, alanine, glutamine, glycine, isoleucine, tryptophan, lysine, cysteine, méthionine, aspartic acid, phenylalanîne, or arginine, phenylalanîne at position 138 replaced by valine, Alal39 replaced by serine, Thrl45 replaced by alanine, Phel47 replaced by serine, valine, threonine, cysteine, leucine, aspartic acid, alanine, glycine, glutamic acid, isoleucine, tyrosine, méthionine, asparagine, glutamine, hystidine, alanine, arginine, tryptophan, or proline, glutamine at position 148 replaced by alanine, glutamine at position 149 replaced by aspartic acid, glutamic acid, cysteine, alanine, or phenylalanîne, alanine at position 150 replaced by serine, leucine, valine, glycine, aspartic acid, tryptophan, glutamic acid, asparagine, tyrosine, phenylalanîne, proline, lysine, threonine, glutamine, or arginine, seroine at position I5l replaced by alanine, aspartate at position 153 replaced by alanine, glutamate at position 155 replaced by cysteine, isoleucine, lysine, aspartic acid, histidine, tyrosine, glutamine, lysine, asparagine, threonine, alanine, phenylalanîne, arginine, méthionine, proline, tryptophan, serine, or valine, asparagine at position 157 replaced by cysteine, aspartic acid, tryptophan, tyrosine, méthionine, alanine, phenylalanîne, valine, leucine, proline, glutamic acid, threonine, glycine, isoleucine, or arginine, isoleucine at position 158 replaced by alanine, serine at position 159 replaced by alanine or threonine, serine at position 167 replaced by arginine or alanine, valine at position 175 replaced by alanine, méthionine at position 177 replaced by alanine, asparagine at position 180 replaced by aspartic acid, threonine at position 182 replaced by alanine, leucine at position 187 replaced by alanine, histidine at position 196 deleted, tyrosine at position 197 deleted, serine at position 198 deleted, histidine at position 199 deleted, tyrosine at position 200 replaced by alanine, tyrosine at position 200 deleted, Ser20l replaced by alanine, serine at position 201 délétion, tryptophan at position 208 replaced by alanine, serine at position 217 replaced by asparagine, proline at position 219 replaced by arginine, tryptophan at position 223 replaced by tyrosine, phenylalanîne at position 235 replaced by alanine, asparagine at position 239 replaced by alanine, aspartate at position 241 replaced by alanine, threonine at position 243 replaced by alanine, valine at position 244 replaced by isoleucine, threonine at position 245 replaced by alanine, tyrosine at position 246 replaced by phenylalanîne, threonine at position 247 replaced by alanine or lysine, serine at position 249 replaced by alanine or arginine, valine at position 250 replaced by alanine, valine at position 251 replaced by alanine, serine at position 252 replaced by alanine, arginine at position 273 replaced by tryptophan, threonine at position 274 replaced by alanine, isoleucine at position 275 replaced by alanine, arginine at position 282 replaced by alanine, histidine at position 287 replaced by alanine or phenylalanine, serine at position 293 replaced by alanine, asparagine at position 295 replaced by alanine, glutamate at position 299 replaced by alanine, méthionine at position 300 replaced by alanine, threonine at position 303 replaced by alanine, proline at position 305 replaced by alanine, isoleucine at position 306 replaced by alanine, and threonine at position 308 replaced by alanine, or wherein the protein comprises any combination of the referenced substitutions and/or délétions. eHTP’s contain at least one amino acid substitution, one amino acid addition, or one amino acid délétion at an amino acid residue of SEQ ID NO:2, or the corresponding amino acid position of SEQ ID NO:8, SEQ ID NO:l82, or SEQ ID NO:I84, selected from the group consisting of (i) an amino acid residue having a relative solvent-accessibility of from at least about 15% to at least about 36%; and (ii) an amino acid residue located within a distance of about 3 consecutive residues from an amino acid having from at least about 15% to at least about 36% relative solventaccessibility. An eHTP of the présent invention contains at least one amino acid substitution, addition, or délétion at an amino acid residue selected from the group consisting of Thr93, Ser95, Ser97, PheJ47, Glnl49, Serl5l, Asnl80, Thrl82, VaJ25l, Gln253, and Ser255 of SEQ ID NO:2. Any of the aforementioned eHTP’s can contain at least one additional amino acid substitution, addition, or délétion at an amino acid residue selected from the group consisting of VallO, Ilel4, Asn22, Asn23, Gly24, Ile25, Gln26, Gly27, Phe30, Gln38, Ile39, Asp40, Thr4l, Ile43, Serl93, Thrl94, Glul95, Hisl96, Tyrl97, Serl98, Hisl99, Tyr200, Ser20l, Gly202, Tyr203, Pro204, Ile2O5, Leu206, Thr207, Trp208, Ile209, Ser2l0, Tyr2l6, Ser2I7, Gly2l8, Pro2l9, Pro220, Met22l, Ser222, Trp223, Tyr224, Phe225, Asn239, and Val244 of SEQ ID NO: 2 or the corresponding amino acid residue position of SEQ ID NO:8, SEQ ID NO:I82, or SEQ ID NO:I84. Any of the aforementioned eHTP’s may contain one or more modifications selected from the group consisting of S95A, F147A, Q149E, V251A, P219R, and a délétion of any three consecutive amino acids from amino acid residues I96201 as set forth in SEQ ID NO:2. Any of the eHTP’s of the présent invention can be further modifîed to exhibit increased solubility compared to the underfying naturally occurring Bacillus ihurirtgiensis protein as set forth in any of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:l82, or SEQ ID NO:l84 in which the eHTP contains at least one or more amino acid sequence modifications relative to the amino acid sequence as set forth in SEQ ID NO:2.

The modification(s) contain at least a lysine substitution at one or more of the amino acid positions defined as 58, 59, 198, 199, 201, or 202 in SEQ ID NO:2; a glutamic acid residue substitution at one or more ofthe amino acid positions defined as 198, 248, or 301 in SEQ ID NO:2; or an arginine residue substitution at one or more of the amino acid positions defined as 246,250, or 253 in SEQ ID NO:2. An eHTP having an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:lO, SEQ ID NO:l2, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:4l, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NOMS, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:5l, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:6l, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:7I, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:8l, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:9I, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:lOO, SEQ ID NO:lOl, SEQ ID NO:l02, SEQ ID NO:I03, SEQ ID NO:l04,

SEQ ID NO:l05, SEQ ID NO:I06, SEQ ID NO:l07, SEQ ID NO:I08, SEQ ID NO:l09,

SEQ ID NO:llO, SEQ ID NO:lll, SEQ ID NO:lI2, SEQ ID NO:Il3, SEQ ID NO:II4,

SEQ ID NO:ll5, SEQ ID NO:ll6, SEQ ID NO:ll7, SEQ ID NO:ll8, SEQ ID NO:ll9,

SEQ ID NO:l20, SEQ ID NO:l2l, SEQ ID NO:l22, SEQ ID NO:I3, SEQ ID NO:l24, SEQ ID NO:I25, SEQ ID NO:l26, SEQ ID NO:I27, SEQ ID NO:l28, SEQ ID NO:l29, SEQ ID NO:l30, SEQ ID NO:l3I, SEQ ID NO:l32, SEQ ID NO:l33, SEQ ID NO:l34, SEQ ID

NO:l35, SEQ ID NO:l36, SEQ ID NO:l37, SEQ ID NO:I38, SEQ ID NO:l39, SEQ ID

NO:l40, SEQ ID NO:l4l, SEQ ID NO:l42, SEQ ID NO:l43, SEQ ID NO:I44, SEQ ID

NO:l45, SEQ ID NO:l46, SEQ ID NO:l47, SEQ ID NO:I48, SEQ ID NO:I49, SEQ ID

NO:l50, SEQ ID NO:l5l, SEQ ID NO:l52, SEQ ID NO:l53, SEQ ID NO:l54, SEQ ID

NO:l55, SEQ ID NO:I56, SEQ ID NO:l57, SEQ ID NO:l58, SEQ ID NO:I59, SEQ ID

NO:l60, SEQ ID NO:I6l, SEQ ID NO:l62, SEQ ID NO:l63, SEQ ID NO:l64, SEQ ID

NO:l65, SEQ ID NO:I66, SEQ ID NO:l67, SEQ ID NO:l68, SEQ ID NO:l69, SEQ ID

NO:l70, SEQ ID NO:I7l, SEQ ID NO:l72, SEQ ID NO:l73, SEQ ID NO:l74, SEQ ID

NO:l75, SEQ ID NO:176, SEQ ID NO:I77, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:202, and SEQ ID NO:204, or an insect inhibitory fragment thereof, is a preferred embodiment of the présent invention. The target Hemipteran pest species inhibited by the eHTP’s of the présent invention include at least Lygus hesperus, Lygus lineolaris, Empoasca fabae and Amrasca dévastons, as well as other pests within the order Hemiptera that are phylogenetically related to each other or which use a piercing and sucking approach for feeding on plants.

[0010] Methods of controlling a Hemipteran pest by contacting the pest with a Hemipteran inhibitory amount of a eHTP of the présent invention, as well as an insect inhibitory composition that contains at Ieast a Hemipteran controlling amount (or Hemipteran inhibitory amount) of one or more of the eHTP’s of the présent invention, are also provided. In certain embodiments, an insect inhibitory composition comprising any of the eHTP’s disclosed herein is provided. In certain embodiments of these methods, the Hemipteran pest is in a cotton field, a soybean field or an alfalfa field. Hemipteran toxic or Hemipteran controlling compositions can contain at Ieast one or more eHTP along with a supplémentai agent that is selected from the group consisting of an insect inhibitory protein, an insect inhibitory dsRNA molécule, and an insect inhibitory chemistry. Each of these agents can exhibit Hemipteran controlling properties, can exhibit properties for controlling pests unrelated to Hemipteran species such as Lepidopteran species or Coleopteran species, or may exhibit dual mode of action properties in which one or more Hemipteran species and one or more Lepidopteran or Coleopteran species are simultaneously controlled.

[0011] Recombinant polynucleotides that encode eHTP’s of the présent invention are provided. Microbes are also provided that contain the polynucleotides of the présent invention, and such polynucleotides within such microbes are functionally positioned within expression cassettes designed to express the eHTP’s of the présent invention from operabiy linked functional genetic regulatory éléments. Microbes are intended to include bacterial cells, as well as transgenic plant cells. Such transgenic plant cells can be regenerated into whole plants, or plant parts that also contain the recombinant polynucieotide. Methods of controlling a Hemipteran pest by exposing the pest to the microbe, whether bacterial cell or transgenic plant cell, plant or plant part, each of which expresses a Hemipteran inhibitory amount of an eHTP are also provided. The recombinant polynucieotide may contain a nudeotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:1I, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25,

SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:3I, SEQ ID NO:33, SEQ ID NO:35, SEQID

NO:186, SEQ ID NO:187, SEQ ID NO:I88, SEQ ID NO:189, SEQ ID NO:190, SEQID

NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQID

NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID N0:200, SEQID

NO:201, and SEQ ID NO:203, or other sequences that can be assembled to encode one or more of the proteins of the présent invention. In certain embodiments, the recombinant polynucleotide can further comprise a nucléotide sequence encoding one or more insect inhibitory agents that are different from the eHTP encoded by the recombinant polynucleotide. The transgenic plant part is a seed, a boll, a leaf, a flower, pollen, a stem, a root, or any portion thereof. The transgenic plant part may be a non-regenerable portion of the seed, boll, leaf, flower, stem, or root. Also provided are methods of controlling a Hemipteran pest, comprising exposing the transgenic microbe, bacteria, plant cell, plant or plant part to the target pest, wherein the microbe, bacteria, plant cell, plant or plant part expresses a Hemipteran inhibitory amount of a eHTP encoded by the recombinant polynucleotide.

[0012[ Processed plant products that contain a détectable amount of a recombinant polynucleotide encoding an eHTP or any distinguîshing Hemipteran controlling portion thereof are also provided. Such processed products include, but are not limited to, plant biomass, oil, meal, animal feed, flour, flakes, bran, lint, hulls, and processed seed. The processed product may be non-regenerable.

100131 Methods of making a transgenic plant by introducing the recombinant polynucleotide into a plant cell and selecting a transgenic plant that expresses an insect inhibitory amount of an eHTP encoded by a recombinant polynucleotide are also provided. The methods include introducing the recombinant polynucleotide encoding any of the eHTP’s provided herein into a plant cell and selecting a transgenic plant that expresses an insect inhibitory amount of the eHTP encoded by the recombinant polynucleotide.

(00141 Other embodiments, features, and advantages of the invention will be apparent from the following detailed description, the examples, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

100151 Figure 1 illustrâtes the mortality of Lygus species plotted against eHTP protein concentration. Figure l A illustrâtes the mortality of Lygus hesperus populations in response to various concentrations of four different eHTP’s compared to a control sample containing the naturally occurring TIC807 protein. Figure IB illustrâtes the mortality of Lygus Uneolaris populations in response to various protein concentrations of three different eHTP’s compared to a control sample containing the naturally occurring TIC807 protein.

[0016] Figure 2 illustrâtes a ribbon dîagram of the atomic structure of a Hemîpteran toxic protein of the présent invention showing the relative positions of the resuit effective changes increasing toxic effects and/or broadenïng host range specificity compared to the relative position of the same amino acid position within a TIC807 or related protein. Two surface patches are illustrated by spheres encircling particular residue positions within the atomic structure in the ribbon diagram: [1] one sphere has an atomic radius of from about 9.2 to about 12.2 Angstroms from the beta carbon atom of S95 (relative to the S95 position as set forth in SEQ ID NO:2); [2] another sphere has an atomic radius of from about 9.2 to about

12.2 Angstroms from the beta carbon atom of P219 (relative to the P219 position as set forth in SEQ ID NO:2). Changes to the amino acids within the ribbon structure that fall within these spheres are resuit effective in causing increased toxic propertles and broader host range toxic effects compared to a protein having a naturally occurring amino acid at that particular position.

[0017| Figure 3 is a chart view illustrating the population mortality of Lygus species for thirteen different eHTP’s compared to each other and to the naturally occurring TIC807 protein.

DETAILED DESCRIPTION [0018| This application describes eHTP’s (engineered Hemîpteran species toxic proteins). The eHTP’s of the présent invention are to be distinguished from proteins such as TIC807, TIC853, CrySlAal and AXMI-171, which are known in the art and are not to be considered to be within the scope or définition of the term eHTP, as the prior art proteins are not engineered to exhibit improved toxic properties directed to one or more Hemîpteran pest species and do not exhibit broad host range levels of inhibitory activity. eHTP’s surpsisingly and unexpectedly exhibit high levels of toxic activity against Hemîpteran and related pest species. An additional feature of these eHTP’s that is even more unexpected and surprising is the finding that these proteins exhibit broader host range toxic properties compared to progenitor proteins which provide the foundational basis for the eHTP’s of the présent invention. The foundational or baseline scaffold toxin proteins, such as TIC807 (SEQ ID NO:2), CrySlAal (SEQ ID NO:8), TIC853 (SEQ ID NO: 184), and AXMI-171 (SEQ ID NO:206) do not exhibit the breadth and scope of biological anti-Hemipteran activity or host range of the eHTP proteins of the présent invention.

(0019] More than 2000 different amino acid sequence variants of Hemipteran toxic proteins derived from Bacillus thuringiensis species were tested to identify the spécifie amino acid insertions, substitutions, or délétions described herein which confer expanded Hemipteran species host range inhibitory spectrum and also provide dramatically increased Hemipteran species inhibitory activity when compared to the spectrum and activity of the baseline scaffold protein, TIC807, TIC853, and CrySlAal. Amino acid residues are identifîed in the baseline scaffold proteins that (a) can be modified to yield enhanced Hemipteran inhibitory spectrum and/or improved Lygus inhibitory activity relative to one or more of the scaffold proteins, (b) accumulate in surface patches of a folded insect inhibitory protein exhîbitîng the fold structure of one or more of the scaffold proteins, and /or (c) occur in spécifie positions of one or more fo the scaffold protein amino acid sequence that are resuit effective in decreasing the resulting eHTP proteins’ mean effective dose for controlling a Hemipteran species and broadening the range of Hemipteran species that are affected by the eHTP protein.

|0020| The Hemipteran pest species are intended to mean insects that feed upon plants and plant tissues by slashing or piercing the outer surface of the target plant, and then consume macerated plant exudates poolîng in the slash or pierce location by sucking or wicking the pooled exudates. Such insects include adults and nymphs, including but not limited to the following listing of plant bugs: the Family Miridae, cicadas from the Family Cicadidae, leafhoppers (e. g., Empoasca spp., Amrasca spp.) from the Family Cicadellidae, planthoppers from the families Fulgoroidea and Delphacidae, treehoppers from the Family Membracidae, psyllids from the Family Psyllidae, whiteflies from the Family Aleyrodidae, aphids from the Family Aphidïdae, phylloxéra from the Family Phylloxeridae, mealybugs from the Family Pseudococcidae, scales from the families Coccidae, Diaspididae and Margarodidae, lace bugs from the Family Tingidae, stink bugs from the Family Pentatomidae, cinch bugs (e. g., Blissus spp.) and other seed bugs from the Family Lygaeidae, spittlebugs from the Family Cercopidae squash bugs from the Family Coreidae, and red bugs and cotton stainers from the Family Pyrrhocoridae. Other pests from the order Hemiptera include Acrostemum hilare (green stink bug), A nas a tris fis (squash bug), Blissus leucopterus leucopterus (chinch bug), Corythuca gossypii (cotton lace bug), Cyrtopeltis modesta (tomato bug), Dysdercus suturellus (cotton stainer), Euschistus servus (brown stink bug), Euschistus variolarius (onespotted stink bug), Graptostethus spp. (complex of seed bugs), Leptoglossus corculus (leaffooted pine seed bug), Lygus lineolaris (tamished plant bug), Lygus hesperus (Western tamish plant bug), Nezara viridula (southem green stink bug), Oebalus pugnax (rice stink bug), Oncopeltus fasciatus (large milkweed bug), and Pseudatomoscelis seriatus (cotton fleahopper). More specifically, the Family Cicadellidae inciudes, but is not limited to the tribe Empoascini, e.g. Amrasca biguttula, Amrasca dévastons, Austroasca viridigrisea, Asymmetrasca decedens, Empoasca decipiens, Empoasca distinguenda, Empoasca dolichi, Empoasca fabae, Empoasca kerrï, Empoasca kraemeri, Empoasca onukii, Empoasca sakaii, Empoasca smithi, Empoasca vitis, Jacobiasca lybica, Sonasasca Solana, tribe Erythroneurini, e.g. Empoascanara nagpurensis, Thaiaassamensis, Zygnidia quyumi, tribe Nirvaniae, e.g. Sophonia rufofascia, Family Delphacidae, e.g. Nilapoarvata lugens, Sogatella furcifera, Unkanodes sapporonus, and Family Lophopidae, e.g. Zophiuma lobulata.

(0021( eHTP’s of the présent invention contain one or more amino acid sequence modifications compared to one or more of the scaffold proteins, including substitutions and délétions, of amino acid residues at seventy-two (72) different amino acid positions. Such modifications pro vide eHTP’s with increased toxicity and/or an enhanced inhibitory spectrum against Hemipteran insects when compared to one or more of the scaffold proteins which include but are not limited to TIC807 (SEQ ID NO:2), or related protein such as TIC807_M2 (SEQ ID NO:8), CrySlAal (SEQ ID NO: 182), and TIC853(SEQ ID NO: 184). eHTP’s include, but are not limited to, modifications of at least one amino acid substitution or one amino acid délétion at any of these seventy-two positions, described as “X” in the amino acid sequence set forth as SEQ ID NO: 180 but do not include the amino acid sequences ofSEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:182, or SEQ ID NO:184. eHTP’s of the présent invention also exhibit enhanced Hemipteran inhibitory spectrum and/or improved Hemipteran inhibitory activity when compared to the spectrum and activity of the baseline or scaffold proteins.

(00221 eHTP’s include at least one amino acid modification of the relative positions of TIC807 (SEQ ID NO:2) as set forth above in paragraph [0009]. eHTP’s can also include at least two, three, four, or more of these aforementioned amino acid substitutions and/or délétions and can also include at least two, three, four, or more of these amino acid substitutions and/or délétions as well as a délétion of any three contiguous amino acids within residues 196-201 ofSEQ ID NO:2. Accordingly, eHTP's include proteins set forth as SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51,

SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:7I, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:9I, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO: 100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID

NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID

NO:111, SEQ ID N0:112, SEQ ID NO:113, SEQ ID NO:1I4, SEQ ID N0;115, SEQ ID

NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID

NO:12I, SEQ ID NO:122, SEQ ID NO:I23, SEQ ID NO:124, SEQ ID NO:125, SEQ ID

NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID

NO:131, SEQ ID NO:132, SEQ ID NO:I33, SEQ ID NO:I34, SEQ ID NO:I35, SEQ ID

NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID

NO:14I, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID

NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID

NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO:154, SEQ ID NO: 155, SEQ ID

NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID

NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO:164, SEQ ID NO: 165, SEQ ID

NO:166, SEQ ID NO:I67, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:170, SEQ ID

NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID

NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO:202, and SEQ ID NO:204, and insect inhibitory fragments thereof.

[00231 eHTP’s of the présent invention exhibit any amino acid sequence different from any one or more of the scaffold proteins, including SEQ ID NO:2 (TIC8O7), in at least one amino acid position where the different amino acid residue either (i) has a relative amino acid solvent-accessibility of at least from about 15% to at least about 36% compared to the same residue positions in any one or more of the scaffold proteins; and/or (ii) is located within a distance of about 3 consecutive amino acid residues from an amino acid having at least from about 15% to at least about 36% relative solvent-accessibility compared to the corresponding amino acid residue positions in the primary amino acid sequence of one or more of the scaffold proteins, and exhibits broadened Hemipteran inhibîtory spectrum and/or increased Hemipteran inhibîtory activity when compared to the acvitity correlated with one or more of the scaffold proteins. The words “increased spectrum” are intended to mean, with reference to two different proteins exhibiting toxic effects to a particular single pest, the protein exhibiting increased spectrum exhibits toxic effects to that particular single pest as well as to one or more other pests within the same phylogenetic order or to one or more other pests in one or more different phylogenetic orders other than the order to which the particular single pest belongs. The words “increased Hemipteran inhibîtory activity” are intended to mean that a particular protein exhibiting such increased activity requires, under standardized conditions, a lower amount of that protein to achieve a particular affect, such as mortality, stunting, morbidity, cessation of feeding, or another measureable phenotypic effect upon a particular single pest, than a control protein.

[0024| eHTP’s exhibit an amino acid sequence that diffère from one or more of the scaffold proteins, including particulariy TIC807, in at least one amino acid residue located within at least one of the two different surface patches of a folded insect inhibîtory protein (see Figure 2 and Table 3 data). One surface patch is defined as including the amino acid residues encompassed within a sphère having an atomic radius of from about 9.2 to about 12.2 Angstroms (Figure 2, sphère [1]) relative to the beta-carbon (Cb) atom of Ser95 as set forth in SEQ ID NO:2 when that protein is folded into a three dimensional structure under physiological conditions; which includes residues Thr93, Ser95, Ser97, Phe 147, Glnl49, Serl5l, Asnl80, Thrl82, Val251, Gln253, and Ser255. As used herein, the phrase “Cb atom” refers to the beta-carbon atom in the amino acid residue side chain. The Cb atom is thus the first carbon in the protein side chain that is présent in ail amino acid residues with the exception of Glycyl residues. With reference to Figure 1, eHTP’s can include, but are not limited to, one or more conservative or non-conservative substitutions of surface patch [1] amino acid residues T93, S95, S97, F147, Q149, S151, N180, Tl82, V25I, Q253, and S255 or the équivalent amino acids within one or more of the scaffold proteins, particulariy SEQ ID NO:2 (TIC807). eHTP’s can include, but are not limited to, one or more substitutions of surface patch [1] amino acid residues such as: T93A; S95A, S95V, S95L, or S95I; F147T, F147C, F147D, FI47G, F147E, F147Y, F147M, F147N, F147Q, F147H, FI47R, F147W, F147P, F147A, F147V, F147L, or F147I; Q149A, Q149C, Q149F, Q149E or Q149D; S151A; N180D; T182A; V251E or V251A, and/or Q253R. The other or second surface patch that has been identified as amino acid residues that are réceptive to modifications which are resuit effective in conferring improved Hemipteran inhibîtory bioactivity in the form of eHTP’s of the présent invention is defined as including the amino acid residues encompassed within a sphere having an atomic radius of from about 9.2 to about 12.2 Angstroms (Figure 2, sphere [2]) relative to the beta-carbon atom of Pro2l9 or the équivalent amino acid position ΐη one or more of the scaffold proteine, particularly as set forth in SEQ ID NO:2, when any one of the applicable scaffold proteins is folded into a three dimensional structure under physiological conditions, which includes residues VallO, Ilel4, Asn22, Asn23, Gly24, Ile25, Gln26, G!y27, Phe30, Gln38, I!e39, Asp40, Thr4l, I!e43, Serl93, Thr194, Glul95, His!96, Tyr!97, Ser!98, Hisl99, Tyr200, Ser201, G!y202, Tyr203, Pro204,1!e205, Leu206, Thr207, Trp208,Ile209, Ser2!0, Tyr2l6, Ser2!7, Gly2!8, Pro2!9, Pro220, Met22l, Ser222, Trp223, Tyr224, Phe225, Asn239, and Va!244. Such eHTP’s can include, but are not limited to, one or more conservative or non-conservative amino acid residues substitutions and/or one or more amino acid délétions within surface patch [2] including VallO, Ilel4, Asn22, Asn23, Gly24, I!e25, Gln26, Gly27, Phe30, Gln38, lle39, Asp40, Thr4l, Ile43, Serl93, Thrl94, Glul95, Hisl96, Tyrl97, Serl98, Hisl99, Tyr200, Ser20l, Gly202, Tyr203, Pro204, Ile205, Leu206, Thr207, Trp208,1!e209, Ser2l0, Tyr2l6, Ser2l7, G!y2l8, Pro2l9, Pro220, Met22l, Ser222, Trp223, Tyr224, Phe225, Asn239, and Val244 of SEQ ID NO:2 (TIC807). eHTP’s can include, but are not limited to, one or more substitutions and/or délétions witin the amino acid residues located within surface patch [2] such as: a délétion of any three contiguous amino acid residues in the sequence Hisl96, Tyrl97, Ser!98, Hisl99, Tyr200, Ser20l; Ser2l7Asn, Ser2l7Gln, Ser2l7Arg; and/or Pro2l9Arg, Pro2l9Asn, Pro2l9Gln. eHTP’s can include, but are not limited to, one or more amino acid residue substitutions and/or délétions within surface patch [2] such as: a délétion of any three contiguous HisTyrSer residues in the sequence Hisl96, Tyrl97, Serl98, Hisl99, Tyr200, Ser20l; Ser2l7Asn, Ser2l7Gln, Ser217Arg; and/or Pro2l9Arg, Pro2l9Asn, Pro2l9Gln. An eHTP can hâve at least one amino acid modification in each of the two aforementioned surface patches of the folded insect inhibitory protein. eHTP can hâve one, or a combination of more than one modification at residues T93, S95, F147, Q149, Sl5l, N180, T182, H196, Y197, S!98, H199, Y200, S20l, W208, S217, P219, W223, N239, V244, or V25l relative to SEQ ID NO:2 (TIC807). Conservative amino acid changes can be made by substituting an acidic, basic, neutral polar, or neutral non-polar -type amino acid with another amino acid of the same type. Non-conservative amino acid changes can be made by substituting an acidic, basic, neutral polar, or neutral non-polar amino acid-type with an amino acid of a different type. Furthermore, of the eHTP proteins listed in Table 4B, ail 267 are amino acîd sequence variants that exhibit increased toxicity to Lygus spp. when compared to one or more of the scaffold proteins, inciuding scaffold protein TIC807. Only ten of these amino acid sequence variants exhibit modified amino acid residues compared to one or more of the scaffold proteins that are positioned outside of the two referenced surface patches.

|0025| The prior art teaches solubility problems associated with the scaffold proteins. eHTP’s exhibit improved solubility compared to the scaffold proteins, and generally exhibit increased solubility at a pH of less than 9.0, in contrast to the observed solubility profile of one or more of the scaffold proteins. This increased solubility at more physiological pH is évident when the eHTP is expressed in £. coli, in a plant cell, in a plant cell cytoplasm, a plant cell apoplast, or in or targeted for import into a plastid of a plant cell. Amino acid modifications that improve solubility relative to one or more of the scaffold proteins, inciuding SEQ ID NO:2 (TIC807) include but are not limited to, substitution of a lysine amino acid residue at one or more of the following amino acid positions in TIC807 or the applicable residue in any of the other scaffold proteins: 58, 59, 198, 199, 201, or 202; or, substitution of a glutamic acid amino acid residue at one or more of amino acid positions I98, 248 or 301; or, substitution of a arginine amino acid residue at one or more of amino acid positions 246,250 or 253.

[00261 Insect inhibitory compositions comprising the above described eHTP’s are also provided. Such compositions may further comprise at least one additional insect inhibitory agent different from the eHTP induded in the composition. The insect inhibitory agent is selected from any number of insect inhibitory agents induding an insect inhibitory protein, an insect inhibitory dsRNA molécule, and one or more chemical agents useful in controlling insect pests. Examples of additional inhibitory agents indudes, but are not limited to, a TIC1415 protein, a dsRNA directed towards Hemipteran orthologs of Nilaparvata lugens VATPase-E, 21E01, a dsRNA directed towards Hemipteran orthologs of actin ortholog, ADP/ATP translocase, α-tubulin, ribosomal protein L9 (RPL9) or V-ATPase A subunit, AXMI-I71 (US20100298207A1), Cry3A, Cry4Aa, CrylIAa, and CytlAa, DIGII, DIG5, Cry7, eCry3.IAb, mCry3A, Cry8, Cry34/Cry35, Cry3, DIG2, Cryl, CrylA.105, Cry2, CrylF, VIP3, 5307, and Cry9. Chemical agents useful in controlling Hemipteran species indude but are not limited to pyrethrins and synthetic pyrethroids; oxadizine dérivatives; chloronicotinyls; nitroguanidine dérivatives; triazoles; organophosphates; pyrrols; pyrazoles; phenyl pyrazoles; diacylhydrazines; biological/fermentation products; and carbamates. Known pesticides within these categories are listed in The Pesticide Manual, 1 Ith Ed., C. D. S. Tomlin, Ed., British Crop Protection Council, Famham, Surry, UK (1997).

(00271 Pyrethroids that are useful in the présent composition include pyrethrins and synthetic pyrethroids. The pyrethrins that are preferred for use in the présent method include, without limitation, 2-allyl-4-hydroxy-3-methyl-2-cyclopenten-l-one ester of 2,2-dimethyl-3-(2methyl propenyl)-cyctopropane carboxylic acid, and/or (2-methyt-l-propenyt)-2-methoxy-4-oxo-3(2 propenyl)-2-cyclopenten-l-yt ester and mixtures of cis and trans isomers thereof (Chemical Abstracts Service Registry Number (CAS RN) 8003-34-7).

|0028] Synthetic pyrethroids that are preferred for use in the présent invention include (s)cyano(3-phenoxyphenyl)methyl 4-chîoro alpha (l-methylethyl)benzeneacetate (fenvalerate, CAS RN 51630-58-1), (S)-cyano (3-phenoxyphenyl) methyl (S)-4-chloro-alpha-(lmethylethyl) benzeneacetate (esfenvaterate, CAS RN 66230-04-4), (3-phenoxyphenyt)methyl(+)cis-trans-3-(2,2-dichoroethenyl)-2,2-dimethylcyclopropanecarboxylate (permethrin, CAS RN 52645-53-1), (±) alpha-cyano-(3-phenoxyphenyl) methyl(+)-cis,trans-3-(2,2dichtoroethenyt)-2,2-dimethyt-cyclopropane carboxylate (cypermethrin, CAS RN 52315-078), (beta-cypermethrin, CAS RN 65731-84-2), (thêta cypermethrin, CAS RN 71697-59-1), Scyano (3-phenoxyphenyl) methyl (±) cis/trans 3-(2,2-dichtoroethenyt) 2,2 dimethytcyclopropane carboxylate (zeta-cypermethrin, CAS RN 52315-07-8), (s)-aiphacyano-3-phenoxybenzyl (IR,3R)-3-(2,2-dibromovinyl)-2,2-dimethyl cyclopropanecarboxylate (deltamethrin, CAS RN 52918-63-5), alpha-cyano-3phenoxybenzyl 2,2,3,3,-tetramethyl cyctopropoanecarboxylate (fenpropathrin, CAS RN 64257-84-7), (RS)-alpha-cyano-3-phenoxybenzyl(R)-2-[2-chloro-4-(trifluoromethyl)anilino]-

3-methylbutanoate (tau-fluvalinate, CAS RN 102851-06-9), (2,3,5,6-tetrafluoro-4- methylphenyl)-methyl-(l alpha, 3 alpha)-(Z)-(±)-3-(2-chloro-3,3,3-trifluoro-t-propenyl)-2,2dimethylcyclopropanecarboxylate (tefluthrin, CAS RN 79538-32-2), (±)-cyano (3phenoxyphenyl) methyl (±)-4-(difluoromethoxy)-alpha-(l -methyl ethyi) benzeneacetate (flucythrinate, CAS RN 70124-77-5), cyano(4-fluoro-3-phenoxyphenyl)methyl 3-[2-chloro2-(4-chlorophenyt)ethenyl]-2,2-dimethylcyclopropanecaiboxylate (flumethrin, CAS RN 69770-45-2), cyano(4-fluoro-3-phenoxyphenyl) methyl 3-(2,2-dichloroethenyt)-2,2-dimethylcyclopropanedarboxytate (cyfluthrin, CAS RN 68359-37-5), (beta cyfluthrin, CAS RN 68359-37-5), (transfluthrin, CAS RN 118712-89-3), (S)-alpha-cyano-3-phenoxybenzyl(Z)(lR-cis)-2,2-dimethyl-3-[2-(2,2,2-trifluoro-trifluoromethylethoxycarbonyl)vinyl]cyctopropane carboxylate (acrinathrin, CAS RN 101007-06-1), (IR cis) S and (IS cis) R enantiomer isomer pair of alpha-cyano-3-phenoxybenzyl-3(2,2dichlorovinyl)-2,2-dimethylcyclopropane carboxylate (alpha-cypermethrin, CAS RN 67375-30-8), [IR,3S)3(rRS)(l’,2^,,2^,,2'-tetrabromoethyl)]-2,2-dimethyl cyclopropanecaiboxylic acid (s)-alpha-cyano-3-phenoxybenzyI ester (tralomethrin, CAS RN

66841-25-6), cyano-(3-phenoxyphenyI) methyi 2,2-dichloro-I- (4ethoxyphenyl)cyclopropane caiboxylate (cycloprothrin, CAS RN 63935-38-6), [la, 3a(Z)](±)-cyano-(3-phenoxyphenyl)methyl 3-(2-chloro-3,3,3-trifluoro-l-propenyl)-2,25 cimethylcyclopropanecarboxylate (cyhalothrin, CAS RN 68085-85-8), [l alpha (s), 3 alpha(z)]-cyano(3-phenoxyphenyl) methyl-3-(2-chloro-3,3,3-trifluoro-l-propenyI)-2,2dimethylcyclopropane caiboxylate (lambda cyhalothrin, CAS RN 91465-08-6), (2-methyl [l,l’-biphenyl]-3-yl) methyi 3-(2-chloro-3,3,3-trifluoro-l-propenyl)-2,2-dimethylcyclopropanecarboxylate (bifenthrin, CAS RN 82657-04-3), 5-l-benzyl-3-furylmethyl-d10 cis(lR,3S,E)2,2-dimethyl-3-(2-oxo,-2,2,4,5 tetrahydro thiophenylidenemethyl)cyclopropane caiboxylate (kadethrin, RU15525, CAS RN 58769-20-3), [5-(phenyl methyl)-3-furanyl]-3furanyl 2,2-dimethyl-3-(2-methyl-1-propenyl) cyclopropane carboxylate (resmethrin, CAS RN 10453-86-8), (lR-trans)-[5-(pheny!methyI)-3-furanyl]methyI 2,2-dimethyl-3-(2-methyll-propenyl)cyclopropanecaiboxylate (bioresmethrin, CAS RN 28434-01-7), 3,4,5,6-tetra 15 hydro-phthalimidomethyl-(IRS)-cis-trans-chrysanthemate (tetramethrin, CAS RN 7696-120), 3-phenoxybenzyl-d,l-cis,trans 2,2-dimethyI-3-(2-methylpropenyl) cyclopropane caiboxylate (phenothrin, CAS RN 26002-80-2); (empenthrin, CAS RN 54406-48-3); (cyphenothrin; CAS RN 39515-40-7), (prallethrin, CAS RN 23031-36-9), (imiprothrin, CAS RN 72963-72-5), (RS)-3-allyl-2-methyl-4-oxcyclopent-2-enyl-(IA,3R; lR,3S)-2,2-dimethyl20 3- (2-methylprop-l-enyl) cyclopropane caiboxylate (allethrin, CAS RN 584-79-2), (bioallethrin, CAS RN 584-79-2), and (ZXI890I, CAS RN 160791-64-0). It is believed that mixtures of one or more of the aforementioned synthetic pyrethroids can also be used in the présent invention. Particularly preferred synthetic pyrethroids are tefluthrin, lambda cyhalothrin, bifenthrin, permethrin and cyfluthrin. Even more preferred synthetic pyrethroids 25 are tefluthrin and lambda cyhalothrin, and yet more preferred is tefluthrin.

[0029] Insecticides that are oxadiazine dérivatives are useful in the subject invention. The . oxadizine dérivatives that are preferred for use in the présent invention are those that are identified in U.S. Patent No. 5,852,012. More preferred oxadiazine dérivatives are 5-(2chloropyrid-5-ylmethyl)-3-methyl-4-nitroiminoperhydro-1,3,5-oxadiazine, 5-(230 chlorothiazol-5-yImethyl)-3-methyl-4-nitroiminoperhydro-1,3,5-oxadiazine, 3-methyi-4nitroimino-5-(I-oxido-3-pyridinomethyI) perhydro-l,3,5-oxadiazine, 5-(2-chloro-l-oxido-5pyridiniomethyl)-3-methyl-4-nitroiminoperhydro- 1,3,5-oxidiazine; and 3-methyl-5-(217 methylpyrid-5-ylmethyl)-4-nitroiminoperhydro-l,3,5-oxadiazine. Even more preferred is thiamethoxam (CAS RN 153719-23-4).

[0030] Chloronicotinyl insecticides are also useful in the subject invention. Chloronicotinyls that are preferred for use in the subject composition are described in U.S. Patent No. 5,952,358, and include acetamiprid ((E)-N-[(6-chloro-3-pyridinyl)methyl]-N'-cyano-Nmethyleneimidamide, CAS RN 135410-20-7), imidacloprid (l-[(6-chloro-3pyridinyl)methol]-N-nitro-2-imidazolidinimime, CAS RN 138261-41-3), and nitenpyram (N[(6-chloro-3-pyridinyl)methyl]-N-ethyl-N'-methyl-2-nitro-l,l-ethenediamine, CAS RN 120738-89-8).

[0031] Nitroguanidine insecticides are useful in the présent invention. Such nitroguanidines can include those described in U.S. Patent Nos. 5,633,375, 5,034,404 and 5,245,040.

[0032] Pyrrols, pyrazoles and phenyl pyrazoles that are useful in the présent invention include those that are described in U.S. Patent 5,952,358. Preferred pyrazoles include chlorfenapyr (4-bromo-2-(4-chlorophenyl)-l-ethoxymethyl-5-trifIuoromethylpyrrole-3carbonitrile, CAS RN 122453-73-0), fenpyroximate ((E)-l,l-dimethylethyl-4[[[[(l,3dimethyl-5-phenoxy-1 H-pyrazole-4-yl)methylene]amino]oxy]methyl]benzoate, CAS RN 111812-58-9), and tebufenpyrad (4-chloro-N[[4-l,l-dimethylethyl)phenyl]methyl]-3-ethyl-lmethyl-lH-pyrazole-5-carboxamide, CAS RN 119168-77-3). A prefered phenyl pyrazole is fipronil (5-amino-[2,6-dichloro-4-(trifIuoromethyl)phenyl]-4-[(l R,S)(trifIuoromethyl)sulfinyl]-lH-pyrazole-3-carbonitrile, CAS RN 120068-37-3).

[0033] Diacylhydrazines that are useful in the présent invention indude halofenozide (4chlorobenzoate-2-benzoyl-2-(l,l-dimethylethyl)-hydrazide, CAS RN 112226-61-6), methoxyfenozide (RH-2485; N-tert-butyl-N'-(3-methoxy-o-toluoyl)-3,5-xylohydrazide, CAS RN 161050-58-4), and tebufenozide (3,5-dimethylbenzoic acid l-(l,l-dimethylethyl)-2,(4ethylbenzoyl)hydrazide, CAS RN 112410-23-8).

[00341 Triazoles, such as amitrole (CAS RN 61-82-5) and triazamate are useful in the nethod of the présent invention. A preferred triazole is triazamate (ethyl [[1[(dimethylamino)carbonyl]-3-( 1,1-dimethyl ethyl)-1 H-l ,2,4-triazol-5-yl]thio]acetate, CAS RN 112143-82-5).

[0035] Biological/fermentation products, such as avermectin (abamectin, CAS RN 7175141-2) and spinosad (XDE-105, CAS RN 131929-60-7) are useful in the présent invention.

[0036] Organophosphate insecticides are also useful as one of the components of the présent invention. Preferred organophophate insecticides include acephate (CAS RN 30560-19-1), chlorpyrifos (CAS RN 2921-88-2), chlorpyrifos-methyl (CAS RN 5598-13-0), diazinon (CAS RN 333-41-5), fenamiphos (CAS RN 22224-92-6), and malathion (CAS RN 121-75-5). [0037] In addition, carbamate insecticides are useful in the subject invention. Preferred carbamate insecticides are aldicarb (CAS RN 116-06-3), carbaryl (CAS RN 63-25-2), carbofuran (CAS RN 1563-66-2), oxamyl (CAS RN 23135-22-0) and thiodicarb (CAS RN 59669-26-0).

[0038] When a chemical insecticide is described herein, it is to be understood that the description is intended to include sait forms of the insecticide as well as any isomeric and/or tautomeric form of the insecticide that exhibits the same insecticidal activity as the form of the insecticide that is described.

[0039] The chemical insecticides that are useful in the present invention can be of any grade or purity that pass in the trade as such insecticide. Other materials that accompany the insecticides in commercial préparations as impurities can be tolerated in the subject invention and compositions, as long as such other materials do not destabilize the composition or significantly reduce or destroy the activity of any of the insecticide components or the transgenic event against the target pest(s). One of ordinary skill in the art of the production of insecticides can readily identify those impurities that can be tolerated and those that cannot.

[0040] eHTP’s are related by amino acid modifications such that the modified proteins exhibit enhanced Hemipteran inhibitory spectrum and/or improved Hemipteran inhibitory activity against Lygus spp., Empoasca spp. and/or Amrasca spp. compared to the parent protein, T1C807. The phrases “more active”, “improved activity, “enhanced specificity”, “increased toxic potency”, “increased toxicity”, “improved Hemipteran inhibitory activity, “enhanced Hemipteran inhibitory activity”, “improved Lygus, Empoasca and/or Amrasca inhibitory activity”, “greater Lygus, Empoasca and/or Amrasca inhibitory activity”, “greater Hemipteran inhibitory activity” and “enhanced Lygus, Empoasca and/or Amrasca inhibitory spectrum” and “enhanced Hemipteran inhibitory spectrum” refer to a comparison of the activity of an eHTP and of the activity of a TIC807 (SEQ ID NO:2), TlC807_M2 (SEQ ID NO:8), Cry51Aal (SEQ ID ΝΟ.Ί82), TIC853 (SEQ ID NO:184), and/or a AXMI-171 (SEQ ID NO:206) protein against a Hemipteran insect, wherein activity attributed by the eHTP of the present invention is greater than the activity attributed to the TIC807 protein (SEQ ID NO:2), TIC807_M2 (SEQ ID NO:8), Cry51Aal (SEQ ID NO:182), T1C853 (SEQ ID NO: 184, and/or a AXMI-171 (SEQ ID NO:206) protein. eHTP’s provided herein exhibit enhanced Hemipteran inhibitory spectrum and/or improved or greater Hemipteran inhibitory activity when compared to the Bacillus thuringiensis proteins of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:I82, and SEQ ID NO:I84, where the Hemîpteran pest species include Lygus hesperus, Lygus lineolaris, Empoasca fabae, and Amrasca dévastons. Amrasca dévastons is also called Amrasca biguttula biguttula. eHTP’s exhibiting enhanced insect inhibitory spectrum and/or improved insect inhibitory activity compared to TIC807 can be identlfied by many different methods. In general, exemplary and non-limiting methods for identifying eHTP proteins can comprise :

(!) administering identical amounts of a test eHTP and of control TIC807 (SEQ ID NO:2), TIC807_M2 (SEQ ID NO:8), CrySlAal (SEQ ID NO:l82), TIC853 (SEQ ID NO;l84), and/or an AXMM71 (SEQ ID NO:206) protein to a test insect under controlled assay conditions; and, measuring and comparing the potency of the test and control proteins; and/or, (2) determining the protein doses (e.g., protein concentration in dîet) of a test eHTP and of control TIC807 (SEQ-ID NO:2), TIC807_M2 (SEQ ID NO:8), CrySlAal (SEQ ID NO:l82), TIC853 (SEQ ID NO:l84), and/or an AXMI-I71 (SEQ ID NO:206) protein which elicit équivalent insect population responses under controlled assay conditions (i.e. obtaining a dose response curve).

In the second approach, a statistically robust dose response value used for comparison wouid be the médian léthal concentration (LC50) required to kill 50% of a test population. However, in certain embodiments, other values including but not limited to, a médian inhibitory concentration (“IC50”) required to resuit in 50% growth inhibition of a test population can be used. In this context, “growth inhibition” can comprise stunting and/or inhibition of Hemipteran development.

(0028( As used herein, the phrase “an insect inhibitory amount”, refers to an amount of a composition containing an agent that is effective in achieving any measurable inhibition of insect viability, growth, insect development, insect reproduction, insect feeding behavior, insect matîng behavior and/or any measurable decrease in the adverse effects caused by insect feeding on a composition containing the agent. Sîmilarly, a “Hemipteran inhibitory amount” refers to an amount of a protein of the présent invention alone or with other agents targeting the applicable Hemipteran species for control, that results in any measurable inhibition of target insects belonging to the order Hemiptera related to viability, growth, development, reproduction, feeding behavior, mating behavior, and or any measurable decrease in the adverse effects caused by Hemipteran insects feeding on a plant. Likewise, “Lygus, Empoasca and/or Amrasca inhibitory amount” refers to an amount of a composition containing one or more proteins of the présent invention, i.e., eHTP’s, or other agent that results in any measurable inhibition, viability, growth, development, reproduction, feeding behavior, mating behavior and/or any measurable decrease în the adverse effects caused by Lygus, Empoasca and/or Amrasca feeding on a composition containg that eHTP. As used herein in the context of an eHTP, an “enhanced Hemipteran inhibitory activity or “greater enhanced Hemipteran inhibitory activity” refers to any measurable increase in the inhibition of Hemipteran viability, growth, development, reproduction, feeding behavior, mating behavior and/or any measurable decrease in the adverse effects caused by Hemipteran feeding on a composition containing that eHTP relative to the corresponding inhibitory activity observed with any one or more of the scafïold proteins, including TIC807, Cry51Aal(SEQ ID NO:182), TIC853 (SEQ ID NO:184), and/or AXMI-171 (SEQ ID NO:206) proteins. Likewise, “enhanced Lygus, Empoasca and/or Amrasca inhibitory activity” or “greater enhanced Lygus, Empoasca and/or Amrasca inhibitory activity” refers to any measurable increase in the inhibition, viability, growth, development, reproduction, feeding behavior, mating behavior and/or any measurable decrease in the adverse effects caused by the presence of one or more eHTP of the présent invention in a composition or plant provided in the diet of Lygus, Empoasca and/'or Amrasca relative to the corresponding inhibitory activity observed with an équivalent composition or plant containing only an applicable amount of one or more of the scafïold proteins, including but not limited to TIC807 (SEQ ID NO:2), Cry51Aal(SEQ ID NO:182), TIC853 (SEQ ID NO:184), and/or AXMI-171 (SEQ IDNO:206) proteins.

10029] As used herein in the context of an eHTP, an “enhanced Lygus, Empoasca and/or Amrasca inhibitory spectrum” refers to any measurable increase in the inhibition of a spécifie Lygus spp., Empoasca spp. and/or Amrasca spp. viability, growth, development, reproduction, feeding behavior, mating behavior and/or any measurable decrease in the adverse effects caused by that Lygus spp., Empoasca spp. and/or Amrasca spp. feeding on a plant relative to the corresponding inhibition of that spécifie Lygus spp., Empoasca spp. and/or Amrasca spp. observed with the TIC807 protein. In certain embodiments, eHTP provided herein exhibit an enhanced Lygus inhibitory spectrum relative to TIC807 in that those eHTP’s can provide increased inhibition of Lygus lineolaris.

(00301 An eHTP provided herein can exhibit from about 2 to about 260 fold greater Lygus, Empoasca and/or Amrasca inhibitory activity against a Lygus, Empoasca and/or Amrasca pest species than a protein of SEQ ID NO:2 (TIC807), SEQ ID NO:8 (TIC807_M2), SEQ ID

NO:182 (CrySIAal), SEQ ID NO:I84 (TIC853), and SEQ ID NO:206 (AXMI-171). An eHTP provided herein can exhibit from about 3, 4, 5, 7, 8, 10, 12, 15, 20, 25, 27, 30, 38, 46, 50, 52, 54, 66, 91, 122, 186, 243, or 262 fold greater Lygus, Empoasca and/or Amrasca inhibitory activity against a Lygus, Empoasca and/or Amrasca pest species than a protein of SEQ ID NO:2 (TIC807), SEQ ID NO:8 (TIC807_M2), SEQ ID NO: 182 (CrySlAal), SEQ ID NO: 184 (TIC853), and SEQ ID NO:206 (AXMI-171).

[0031| eHTP’s can exhibit an enhanced target pest inhibitory spectrum and/or improved target pest inhibitory activity over SEQ ID NO:2 (T1C807), SEQ [D NO:8 (TIC807_M2), SEQ ID NO: 182 (Cry51 Aal), SEQ ID NO:184 (TIC853), and/or a SEQ ID NO:206 (AXMI171 ) by causing mortality:

(i) at a dose of about 0.3 pg/mL to about 70 pg/mL against aLygus hesperus insect species, (ii) at a dose of about 0.85 pg/mL to about 100 pg/mL against a Lygus lineolaris insect species, (iii) measuring at an LC50 value of about 0.3 to about 70 pg/mL against Lygus hesperus, (iv) measuring at an LC50 value of about 0.85 to about 100 pg/mL against Lygus lineolaris, or (v) measuring at an LC50 value of more than two-fold Iower the LC50 value of TIC807, SEQ ID NO:8, SEQ ID NO: 182 (Cry51Aal), SEQ ID NO: 184 (TIC853), and/or a SEQ ID NO:206 (AXMI-171) against Lygus spp, Emrasca spp. and/or Amrasca spp., or (vi) at a dose of about 0.69 pg/mL to about 500 μ/mL against a Amrasca dévastons or Empoasca fabae insect species, or (vii) measuring at an LC50 value of about 3.5 to about 15 pg/mL against Amrasca de vas tans and/or Empoasca fabae.

|0032| Table 4A and 4B tabulate the exemplary eHTP’s of the présent invention with

Amrasca and Lygus spp. mortality data. Mortality data avaîlable for Lygus spp. and Amrasca spp. are reported either as (a) a pg/mL LC50 value, or as (b) a % mortality at doses of about 1 to about 3 pg/mL for L. hesperus or about 100 pg/mL protein for L. lineolaris, and about 0.69 to 500 pg/mL for Amrasca dévastons. The fold increased toxicity compared to T1C807 (SEQ

ID NO:2) and TIC807_M2 (SEQ ID NO:8) is provided for exemplary eHTP’s where LC50 values were determined.

10033] The eHTP’s of the présent invention are particularly useful în controlling insects of the order Hemiptera compared to the scaffoîd proteins. Lygus lineolaris required high doses of TIC807 protein (e.g., in excess of 100 pg/mL) to elicit mortality. The dose response curve for one eHTP of the présent invention TIC807_M8 (SEQ ID NO: 16), an eHTP that exhibits remarkably improved toxic effects against both L. lineolaris and L. hesperus, but against L. lineolaris the eHTP exhibits a calculated LC50 value of 223 pg/mL. It has not been possible previously to achieve a protein concentration toxic dose that can elicit greater than 50% mortality against L. lineolaris species because providing signifîcantly large doses of TIC807 and TIC807_M2 protein in excess of 1000 pg/mL in the diet has not been possible. Therefore, LC50 values against L. lineolaris for TIC807 and TIC807_M2 (SEQ ID NO:8) proteins were not determined, but rather estimated as greater than (>) 223 pg/mL (See Tables I and 3, Example 4, and Figure IB).

[0034( Itérative design refers to a semi-random approach for developing and selecting eHTP’s including a combination of engineering, testing, and selecting (not necessarily in that order) (see Examples 1 through 4). The word “engineering” is intended to include identifying relevant residues to modify, cloning, and expressing eHTP’s described herein. The word “testing” is intended to refer to comparing the Hemipteran activity of an eHTP to the activity of a scafTold protein such as TIC807 (SEQ ID NO:2), TIC807_M2 (SEQ ID NO:8), Cry51Aal (SEQ ID NO: 182), and/or TIC853 (SEQ ID NO: 184); or, comparing an eHTP of the présent invention against another protein such as AXM1-171 (SEQ ID NO:206). The word “selecting” îs intended to refer to the act of identifying improved variant proteins of the présent invention, i.e., eHTP’s, and the applicable amino acid residues for “engineering”.

10035] Itérative design includes the élucidation of the atomic structure of proteins of the présent invention (for example, as set forth in Figure 2) and the use of the atomic structure to guide and complément semi-random approaches of “selecting” amino acid residues to modify for “engineering”, and in this case, has included the identification of amino acid residues at loops and at surface exposed régions of a folded insect inhibitory scaffoîd protein such as TIC807, TIC853, and Cry5IAaI that can be modified to confer improvements to insect inhibitory spectrum and activity. Such amino acid residues at loops and at surface exposed régions are selected for “engineering”. In this case, itérative design has included the identification of two different régions within the three dîmensional structure of the scaffoîd protein that harbor an accumulation of relevant amino acid residues that, when modified to contain amino acid residues other than those appearing at those positions in the naturaîly occurring scaffold protein, resuit in one or more of the eHTP proteins of the présent invention.

100361 Initially the scaffold protein T1C807 (SEQ ID NO:2) used in this process of itérative design, and 267 different eHTP’s were discovered that exhibited increased Lygus spp.

inhibitory activity compared to the scaffold protein T1C807. TtC807_M8 (SEQ ID NO: 16) was discovered in early rounds of the design process. Subséquent rounds of itérative engineering-testing-selecting led to the discovery of other eHTP proteins that exhibited yet greater levels of toxicity against Lygus species and also exhibited a broader host range of toxic effects when compared to the scaffold protein. Seven variants (eHTP’s) exhibited significantly higher levels of increased toxicity against both Lygus species (£. hesperus and L. lineolaris) when compared to TIC807. LC50 values for these seven, and other, eHTP’s constructed herein were determined against Lygus hesperus and Lygus lineolaris species and compared to LC50 values for scaffold proteins, particularly T1C807. The results are shown in Table 1, and Figure 3 is a bar chart showing graphically the results observed as tabulated in

Table 1.

Table 1. LC50 values of select eHTP’s compared to T1C807

		Lygus hesperus		Lygus lineolaris
SEQ 1DNO:	Toxin		LC50 value (pg/mL)	Toxicity (fold increase)	LC50 value (Mg/mL)	Toxicity (fold increase)
2	TIC807	73	1	>223*	1
6	TIC807 MI	23	3	100	>2
8	TIC807 M2	5.9	12	>223*	~ 1
10	TIC807 M3	2.9	25	ND	-
12	T1C807 M4	2.4	30	ND	-
14	T1C807 M5	1.1	66	ND	-
18	TIC807 M6	1.45	50	ND	-
20	TIC807 M7	1.4	52	ND	-
16	TIC807 M8	0.8	91	223	>1
28	T1C807 M9	9.9	7	8.3	>27
30	TIC807 M10	0.6	122	4.8	>46
32	TIC807 MH	1.35	54	5.9	>38
36	TIC807 Ml2	0.4	182	1.2	>186
34	TIC807 MI3	0.3	243	0.85	>262

ND = Not Determined. LC50 values are determined by presenting 8-10 different protein concentrations to a population of newly hatched Lygus nymphs, allowing nymphs to feed for 5 days, and then scoring for mortality over the dose range provided.

* Toxicity, displayed in terms of a multiple of increased activity compared to the level observed against Lygus hesperus using the observed LD50 for TIC807 as the baseline value of 1. Significantly large amounts of protein in excess of 1000 pg/mL hâve not been possible to provide in Lygus diet in order to complété the high range of toxicity dose response to Lygus lineolaris. Therefore, an LC50 value was not determined for TIC807 or TIC807_M2. Instead, a 4-dose LC50 estimation in the low range was performed verifying that expected LC50 values for TIC807 and TIC807_M2 are greater than 223 pg/mL.

(0037J reference to Table 1, the itérative design process has provided a means for identifying proteins exhibiting improved toxic properties, not only to Lygus hesperus, but also to Lygus lineolaris.

[0038| Recombinant polynucleotide compositions that encode eHTP’s are also provided. In certain embodiments, eHTP’s can be expressed with recombinant DNA constructs in which a polynucleotide moiecule with the open reading frame encoding the protein is operably linked to éléments such as a promoter and any other regulatory element functional for expression in the system for which the construct is intended. For example, plant-functiona! promoters can be operably linked to an applicable eHTP coding sequence to enable expression of the protein in plants. Promoters functional in bacteria are also contemplated for use in expression cassettes. Promoters functional in an applicable bacterium, for example, in an E. coli or in a Bacillus thuringiensis species can be operably linked to the eHTP coding sequences for expression of the applicable protein in the applicable bacterial strain. Other useful éléments that can be operably linked to the eHTP coding sequences include, but are not limited to, enhancers, introns, leaders, encoded protein immobilization tags (HIS-tag), encoded subcellular translocation peptides (i.e. plastid transit peptides, signal peptides), encoded polypeptide sites for post-translational modifying enzymes, ribosomal binding sites, and segments designed for use as RNAi triggers for suppression of one or more genes either in plants or in a particular target pest species.

[0039] Exemplary recombinant polynucleotide molécules provided herein include, but are not limited to, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO:I89, SEQ

ID NO:!90, SEQ ID NO:!91, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID

NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID

N0:200, SEQ ID NO:35, and SEQ ID NO:201. These sequences encode the respective proteins each having the amino acid sequence as set forth in SEQ ID NO:4 (TIC807_4), SEQ ID NO:6 (TIC807_MI), SEQ ID NO:8 (TIC807_M2), SEQ ID NO: 10 (TIC807_M3), SEQ ID NO: 12 (TIC807_M4), SEQ ID NO: 14 (TIC807_M5), SEQ ID NO: 16 (TIC807_M8), SEQ ID NO: 18 (TIC807_M6), SEQ ID NO:20 (TIC807_M7), SEQ ID NO:22 (TIC807_22), SEQ ID NO:24 (TIC807_24), SEQ ID NO:26 (TIC807_26), SEQ ID NO:28 (TIC807_M9), SEQ ID NO:30 (TIC807_M10), SEQ ID NO:32 (TIC807_Mll), SEQ ID NO:36 (TIC807_M12), and SEQ ID NO:34 (TIC807_M13). Because of the redundancy of the genetic code, the codons of a recombinant poiynucieotide molécule encoding for proteins of the présent invention may be substituted for synonymous codons (also called a silent substitution); and are within the scope of the présent invention. Recombinant polynucleotides encoding any of the eHTP’s disclosed herein are thus provided.

[0040] A recombinant DNA construct comprising eHTP coding sequences can also further comprise a région of DNA that codes for one or more insect inhibitory agents which can be configured to be co-expressed along with a DNA sequence encoding an applicable eHTP, a protein different from an eHTP, or an insect or plant gene inhibitory dsRNA molécule. A recombinant DNA construct can be assembled so that ail agents designed to be expressed from a particular construct are expressed from one promoter or so that separate agents are each under separate promoter control, or some combination thereof. The proteins of this invention can be expressed from a multi-gene expression system in which one or more proteins are expressed from a common nucléotide segment on which is also contained other open reading frames and/or promoters depending on the type of expression system selected. 10041( Recombinant poiynucieotide or recombinant DNA construct comprising an eHTP encoding sequence can be delivered to host cells by vectors, e.g., a plasmid, baculovirus, artificial chromosome, virion, cosmid, phagemid, phage, or viral vector. Such vectors can be used to achieve stable or transient expression of an eHTP encoding sequence in a host cell; and, if the case may be, subséquent expression to polypeptide. An exogenous recombinant poiynucieotide or recombinant DNA construct that comprises an eHTP encoding sequence and that is introduced into a host cell is also referred to herein as a “transgene”.

(0042] Also provided herewith are transgenic bacteria, transgenic plant cells, transgenic plants, and transgenic plant parts that contain any a recombinant poiynucieotide (i.e.

transgene) that expresses any one or more eHTP encoding sequence. It is intended that “bacterial cell” or “bacterium” can include, but are not limited to, an Agrobacterium, a

Bacillus, an Escherichia, a Salmonella, a Pseudomonas, or a Rhizobium cell. It is intended that “plant cell” or “plant” include an alfalfa, almont, banana, barley, bean, beet, broccoli, cabbage, brassica, brinjal, carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, celery, citrus, coconut, coffee, com, cio ver, cotton, a cucurbit, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grape, guar, hops, leek, legumes, lettuce, Loblolly pine, millets, melons, nectarine, nut, oat, okra, olive, onion, omamental, palm, pasture grass, papaya, pea, peach, peanut, pepper, pigeonpea, pine, potato, poplar, pumpkin, Radiata pine, radish, rapeseed, rice, rootstocks, rye, safïlower, shrub, sorghum, Southem pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweet com, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon, and wheat plant cell or plant. In certain embodiments: transgenic plants and transgenic plant parts regenerated from a transgenic plant cell are provided; transgenic plants can be obtained from a transgenic seed; transgenic plant parts can be obtained by cutting, snapping, grinding or otherwise disassociating the part from the plant; the plant part can be a seed, a boll, a leaf, a flower, a stem, a root, or any portion thereof; and a transgenic plant part provided herein is a non-regenerable portion of a transgenic plant part. As used in this context, a “nonregenerabte” portion of a transgenic plant part is a portion that can not be induced to form a whole plant or that can not be induced to form a whole plant that is capable of sexual and/or asexual reproduction. A non-regenerable portion of a plant part is a portion of a transgenic pollen, ovule, seed, boll, leaf, flower, stem, or root.

(00431 Also provided herein are methods of making transgenic plants that contain insect or Lygus and/or Amrasca inhibitory amounts of an eHTP. Such plants can be made by introducing a recombinant polynucleotide that encodes any of the eHTP proteins provided herein into a plant cell, and selecting a plant derived from said plant cell that expresses an insect or Hemipteran inhibitory amount of the eHTP’s. Plants can be derived from the plant cells by régénération, seed, pollen, or meristem transformation techniques.

[0044! Transgenic plants and host cells are provided that expresse an insect or Hemipteran inhibitory amount of the eHTP to control an insect or Hemipteran infestation. Any of the aforementîoned plant species can be used for protecting a plant from insect or Hemipteran infestation provided herein as long as the plant is transformed with a polynucleotide construct designed to express the applicable eHTP.

[0045] Additional aspects of the invention include antibodies, kits, methods for detecting polynucleotides that encode eHTP’s or distinguishing fragments thereof, or eHTP’s or distinguishing fragments thereof, methods for identifying additional insect inhibitory members of the protein genus of the présent invention, formulations and methods for controlling insect growth and/or infestation, and methods for providing such control to plants and other récipient hosts. Each composition, construct, cell, plant, formulation, method or kit provides for the industrial application of the proteins of the présent invention, for example, by increasing plant productivity through the commercial use of any of these proteins to inhibit insects.

|0046i A plant product, other than a seed or a fruit or vegetable, is intended as a commodity or other products which move through commerce and are derived from a transgenic plant or transgenîc plant part, in which the commodity or other products can be tracked through commerce by detecting nucléotide segments, RNA or proteins that corresponding to an eHTP of the présent invention and are produced in or maintained in the plant or plant tissue or part from which the commodity or other product has been obtained. Such commodity or other products of commerce include, but are not limited to, plant parts, biomass, oil, meal, sugar, animal feed, flour, flakes, bran, lint, processed seed, and seed. Plant parts include but are not limited to a plant seed, boll, leaf, flower, stem, pollen, or root. In certain embodiments, the plant part is a non-regenerable portion of said seed, boll, leaf, flower, stem, pollen, or root. Cotton and flax plant bolls and non-regenerable portions thereof that contain the eHTP’s are also provided.

100471 Also provided herewith are processed plant products that contain a détectable amount of an eHTP, an insect inhibîtory fragment thereof, or any distinguishing portion thereof. Without seekîng to be limited by theory, it is believed that such processed plant products containing a détectable amount of one or more of the eHTP’s provided herein can in certain embodiments exhibit réductions in undesirable microorganisms that can be transmitted by Hemiptera and/or réductions in the undesirable side products of such microorganisms. In certain embodiments, a distinguishing portion thereof can comprise any polypeptide of at least from about 20 to about 100 or more contiguous amino acids as set forth in SEQ ID NO:180, in particular in which the polypeptide does not contain a corresponding polypeptide of contiguous amino acids présent in SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:182, or SEQ ID NO:184, and wherein the polypeptide comprises at least one amino acid substitution, addition, or délétion in the corresponding amino acid sequence as set forth in SEQ ID NO:2. Such substitutions, délétions or additions are those as set forth above in paragraph [0009].

[0048] Processed plant products are provided that contain a détectable amount of a recombinant polynucleotide encoding an eHTP, an eHTP or an insect inhibîtory fragment thereof, or any distinguishing portion thereof. The processed product îs selected from the group consisting of plant biomass, oil, meal, animal feed, flour, flakes, bran, tint, hulls, and processed seed.

[00491 Hemiptera infestations of crop plants are controlled by providing in the crop plants a recombinant polynucleotide sequence encoding one or more of the eHTP’s of the présent invention. Such transgenic crops produce or are treated to contain an insect or Hemiptera inhibitory amounst of an applicable eHTP, and such crops are imbued with sufficient eHTP by (î) applying any composition comprising or encoding an eHTP to the plant or a seed that gives rise to the plant; and/or (H) transforming the plant or a plant cell that gives rise to the seed and ultimately, the plant, with a polynucleotide encoding an eHTP. The plant may be a transi ently or stably transformed transgenic plant comprising a transgene that expresses an insect or Hemiptera inhibitory amount of an eHTP. The plant may be a non-transgenic plant to which a composition comprising an eHTP has been applied. In such methods, the plant is a dicot plant, and more specifically may be a cotton, soybean or alfalfa plant. The Hemipteran insects include adults and nymphs, such as but not limited to the listing of bugs that is set forth above in paragraph [0020].

[0050] Preferably, the Lygus spp. is Lygus hesperus or Lygus lineolaris, the Empoasca spp. is Empoasca fabae, and the Amrasca spp. is Amrasca dévastons.

[0051] Other features and advantages of the invention will be apparent from the following detailed description, examples, and claims.

EXAMPLES [0052] [n view ofthe foregoing, those ofskill in the art should appreciate that changes can be made in the spécifie aspects which are disclosed and still obtain a like or similar resuit without departing from the spîrit and scope of the invention. Thus, spécifie details disclosed herein are not to be interpreted as limiting. The U.S. Provisional Application Serial No. 61/621,436 to which this application claims the benefit of priority, the Sequence Listing referenced in paragraph [0002], as well as ali references material to the inventions disclosed and claimed, particularly references and published patent applications cited in this application, are incorporated herein by reference in their entirety.

Example lî Itérative Engineering-Testing-Selecting Approach

10053] This example illustrâtes the random, combinatorial, and inventive aspects of the itérative (also can be referred to as “recursive”) engineering-testing-selecting approach used to identify and describe insect inhibitory proteins exhibiting Coleopteran and/or nematicidal activity or increased toxicity to Hemipteran insect species compared to TIC807 (SEQ ID

NO:2). Several design approaches were employed to engineer for eHTP’s with greater inhibitory activity against Lygus species; approaches that induded but were not limited to semi-random modifications, directed modifications of variances in an alignment of TIC807 with other native Bt proteins, and structure/function assisted design. Numerous rounds of engineering and testing were conducted (both consecutively and concurrently) to select for TIC807 protein variants exhibiting increased toxicity. Design approaches were adjusted as data was collected. This itérative engineering-testing-selection approach also induded, but was not limited to steps induding cloning, expressing, purifying, and bioassay testing of TIC807 control protein compared to the eHTP’s.

100541 About 267 exemplary eHTP’s having exhibited increased Lygus toxicity compared to TIC807 were obtained from more than 2000 groups of candidate eHTP’s (i.e. “test” proteins) that were assayed for improved insect inhibitory activity. The actual total number of candidate eHTP’s tested was much greater than 2000 because testing induded recombinant nucléotide segments encoding a number of candidate eHTP’s derived from library mutagenesis that were not sequenced in the sélection process.

[0055] Protein stocks of various amounts and purity were prepared depending on the purpose of the test and the testing throughput desired. For example, lower quantity and lower purity protein préparations were prepared for screening higher numbers of variants in bioassay. Larger quantity and higher purity protein stocks were prepared for hîgh-powered bioassays. Testing trended towards the high-powered bioassays as principally relevant residue positions of the improved variants were elucidated. Inîtially, about 2000 variants were tested on Lygus hesperus. Based on data from L. hesperus approximately 600 variants were designed and then further tested on Lygus lineolaris. Of these, about 267 variants (Table 4B) demonstrated increased toxicity against Lygus hesperus and/or Lygus lineolaris when compared to TIC807. These 267 variants induded twenty-two (22) variants that were confirmed to demonstrate increased toxicity against both Lygus species. Further confirmation and dose response testing narrowed the sélection to seven (7) variants that were subsequently characterized using an 8dose replicated bioassay to détermine LC50 values against both Lygus species.

[0056] The sélection process induded dynamic updates of testing data, constantly adjusting engineering approaches, and performing itérative rounds. Concurrently, labor intensive cloning, protein expression, protein purification, and bioassay experiments were employed test the candidate eHTP’s.

Example 2: Engineering Approaches

Alignment based approaches [0057| A multiple sequence alignment of protein members of Cry51: CrySlAal (SEQ ID NO: 182), TIC853 (SEQ ID NO: 184), and TIC807 (SEQ ID NO:2) were used to identify régions of variability, e.g., positions 195 to 201 and positions 211 to 219, relative to SEQ ID NO:2 (TIC807). These régions were targeted for saturation mutagenesis through use of degenerate oligonucleotide primers encoding random amino acid residues in these régions. Construct libraries were prepared for subséquent protein expression in host cells.

(0058| A multiple sequence alignment of CrySlAal (SEQ ID NO: 182), TIC853 (SEQ ID NO:I84), and TIC807 (SEQ ID NO:2) was used in combination with a BLOSUM 80 substitution matrix to calculate average pair-wise distances for each position variant to TIC807. Residue positions with lower average pair-wise distances were substituted with alternative amino acid residues using degenerate oligonucleotide primers encoding for alternative amino acid residues, e.g.» G28X, G31X, F46X, E125X, FI38X, F147X, S167X, Y216X, P218X, G234X, T247X, D268X, and T308X. Construct libraries were prepared for subséquent protein expression in host cells.

Scanning approaches [0059J Polynucleotide constructs were engineered to express a single Alanine substitution or a double Alanine substitution (AIanine-<parent residue>-Alanine) at every possible position over the full-length of SEQ ID NO:2 (T1C807). See Table 2 for a hypothetical example.

Table 2. A hypothetical example of single and double Alanine scans on a scaffold protein containing the amino acid sequence XXXXAXX.

	Single Alanine Scan	Double Alanine Scan
I	AXXXaXX	AXAXaXX
2	XAXXaXX	XAXAaXX
3	XXAXaXX	XXAXSXX
4	XXXAaXX	XXXAaAX
5	XXXXSXX	XXXXSXA
6	XXXXaAX	-
7	XXXXaXA	-

X = parent residue a = parent residue is an Alanine residue A = Modified to an Alanine residue S = Modified to a Serine residue [0060] Where an Alanine residue was already présent in TIC807, a Serine was substituted instead. Protein variants that exhibited increased toxicity compared to TIC807 were further tested by combination and saturation mutagenesis at those Alanine-substituted residues that conferred increased toxicity. Scanning approaches were also performed on improved combination variants having accumulated modifications from previous itérative rounds of engineering-testing-selecting, e. g., TlC807_M2 (SEQ ID NO:8) having mutations F46S, Y54H, S167R, S217N, and a contiguous triple délétion in residue range 196-201 was further engineered by an additional round of single Alanine substitutions to further improve upon the improved TIC807_M2. Principally relevant residues were identified and further tested by combination and saturation mutagenesis (e. g., A150X, E125X, EI55X, F147X, I134X, NI57X, Q149X, TI33X, E135X, and N137X). Variants engineered by these combined approaches exhibited further improvements to increased toxicity compared to TIC807 and were further combined with other design approaches that took advantage of the atomic structure of TIC807 (SEQ ID NO:2).

Surface exposed residues [0061J The atomic structure of proteins of the présent invention was determined in the mîdst of the Itérative Engineering-Testing-Selecting approach; and, the relative solventaccessibility (%SA) of each residue was determined using Molsoft’s ICM-Browser (Molsoft L.L.C., 11199 Sorrento Valley Road, S209, San Diego, CA 92121). Shown in Table 3 in columns (A) and (B), actual %SA was calculated for proteins having the respective amino acid sequences set forth as SEQ ID NO: 185 (TIC807_LllM) and SEQ ID NO:8 (TIC807_M2). The predicted %SA for residues of TIC807 and TIC853 are listed in Table 3 in columns (A) and (C), respectively. Altogether, the %SA values reported in Table 3 are calculated as a percentage of the sol vent-accessible surface area probed by a water molécule over the maximal solvent accessible area in standard extended conformation (Gly-XXX-Gly) for each residue in each position of the atomic structure. Table 3 aligns the residues of each protein by aligned residues in a Clustal W alignment. %SA greater than 100 can occur when maximal solvent accessible area in standard extended conformation (Gly-XXX-Gly) for each residue is less than the actual solvent accessible area probed by a water molécule. %SA greater than 100 are reported in the table as 100%.

I0062J Combined engineering-testing-selecting approaches described herein resulted in a number of principaîly relevant residues that accumulate in a surface patch ([2] of Figure 2) of residues having a radius of about 9.2 - 12.2 Angstroms around the Cb atom of P219 ofSEQ

ID NO:2 (TIC807): VIO, 114, N22, N23, G24,125, Q26, G27, F30, Q38,139, D40, T41,143,

S193, TI94, E195, H196, Y197, S198, HI99, Y200, S201, G202, Y2O3, P204, 1205, L206,

T207, W2O8,1209, S210, Y216, S217, G218, P219, F220, M221, S222, W223, Y224, F225,

N239, and V244 of SEQ ID NO:2 (TIC807). At least half of these residues exhibit %SA values of greater or equal to fifteen (15).

Table 3. Relative % Solvent Accessability (SA) of Amino Acids of eHTP’s & Scaffold Proteins.

(A) TIC807 LIIM (SEQ IDNO: 185)	(B) TIC807 M2 (SEQ ID NO:8)	(C) TIC853 (SEQ IDNO: 184)
Position amino residue	and acid	Calculated %SA per residue	Position amino residue	and add	Calculated %SA per residue	Position amino residue	and acid	Estimated %SA per residue
2ALA	82.1#	2ALA	64.8#	2ALA	60.9#
31LE	23.3	3 ILE	28.9	3 ILE	24.3
4 LEU	26.4	4LEU	31.9	4LEU	27.9
5ASP	26.0	5ASP	22.7	5ASP	29.9
6LEU	1.0	6LEU	3.4	6LEU	3.7
7LYS	25.1	7LYS	16.2	7LYS	17.1
8SER	46.4#	8SER	37.6#	8SER	44.9#
9LEU	8.9	9LEU	5.2	9LEU	6.3
10VALpl	0.3	10VAL	0.6	10 VAL	0.0
11MET	25.0	H LEU	17.0	liLEU	16.6
12 ASN’	49.8#	12 ASN	43.2#	12 ASP	39.7#
13ALA	0.0	13 ALA	0.0	13 ALA	0.0
I4ILEpl	0.0	141LE	0.0	MILE	0.0
15 ASN	23.7	15ASN	24.9	15ASN	19.2
I6TYR	29.5	16TYR	47.1#	I6TYR	52.5#
I7TRP	14.1	17TRP	18.2	17TRP	20.1
I8GLY	4.3	18GLY	1.4	I8GLY	LO
19PRO	63.6#	19PRO	57.3#	19PRO	59.0#
20LYS	57.3#	20LYS	77.2#	20LYS	100#
21 ASN	36.3#	21 ASN	28.4	21 ASN	61.5#
22ASNpl	16,9	22ASN	10.1	22ASN	15.2
23 ASN2'	0.3	23ASN	0.8	23ASN	0.0
24GLY”1	42.0#	24GLY	43.2#	24GLY	43.3#
25ILEpl	10.1	25ILE	13.8	25 ILE	7.6
26GLN”1	92.4#	26GLN	86.2#	26GLN	94.7#
27GLY”1	62.0#	27GLY	73.9#	27GLY	62.8#
28 GLY	49,0#	28GLY	50.6#	28TYR	47.7#
29ASP	66.0#	29ASP	68.1#	29ASN	80.7#
30PHEpl	4.5	30PHE	4.1	30PHE	1.5
31GLY	37.2#	31 GLY	41.4#	3IASN	61.1#

(A) TIC807L1IM (SEQ ÎD ND: 185)	(B)	(C) TIC853 (SEQ ID NO: 184)
TIC807. M2 (SEC	ID NO:8)
Position amino residue	and add	Calculated •/•SA per residue	Position amino residue	and acid	Calculated %SA per residue	Position amino residue	and add	Estlmated •/•SA per residue
32TYR	25.2	32TYR	25.3	32TYR	21.5
33PRO	70.7#	33 PRO	76.0#	33PRO	78.5#
34ILE	4.8	34ILE	5.5	341LE	2.6
35SER	42.2#	35SER	29.1	35SER	27.0
36GLU	54.2#	36GLU	47.2#	36GLU	50.2#
37LYS	81.0#	37LYS	79.5#	37ARG	87.5#
38GLNpl	12.8	38GLN	14.5#	38GLN	9.0
39ILEpl	7.8	391LE	7.9	391LE	5.1
40ASPpl	52.4#	40ASP	55.3#	40ASP	49.8#
41THRpl	0.3	41THR	0.0	41THR	0.2
42SER	53.1#	42SER	56.0#	42SER	53.0#
431LEpl	13.1	431LE	23.5	431LE	25.1
441LE	8.3	441LE	12.0	441LE	8.1
45THR	30.7	45THR	37.8#	45THR	45.7#
46PHE*	20.0	46SER	43.7#	46SER	40.5#
47THR	48.1#	47THR	45.2#	47THR	43.7#
48HIS	73.5#	48FIIS	65.3#	48HIS	78.3#
49PRO	9.4	49PRO	12.6	49SER	9.0
50ARG	58.7#	50ARG	53.7#	50ARG	61.5#
51 LEU	13.7	51 LEU	8.1	51 LEU	3.0
52ILE*	32.4	52ILE	31.5	52MET	43.7#
53PRO	22.2	53PRO	26.5	53PRO	22.8
54TYR’	52.7#	54H1S	42.2#	54FIIS	45.5#
55ASP	57.5#	55ASP	59.2#	55ASP	55.5#
56LEU	15.0	56LEU	18.6	56LEU	15.1
57THR	62.0#	57TF1R	73.2#	57THR	80.0#
58ILE	67.6#	581LE	60.9#	581LE	68.0#
59PRO	26.6	59PRO	21.9	59PRO	20.7
60GLN	28.8	60GLN	20.7	60GLN	21.3
61ASN	71.9#	61 ASN	73.6#	61ASN	74.6#
62LEU	13.4	62LEU	11.7	62LEU	10.0
63GLU	62.2#	63GLU	66.0#	63GLU	66.4#
64THR	51.2#	64THR	51.1#	64TFIR	49.0#
65ILE	46.1#	65 ILE	41.5#	65ILE	38.4#
66PHE	27.0	66PHE	26.3	66PHE	29.6
67THR	52.5#	67TFIR	55.5#	67THR	62.2#
68THR’	31.6	68TF1R	35.8#	68THR	35.3
69THR	54.3#	69TFIR	51.1#	69TF1R	50.3#
70GLN*	31.0	70GLN	36.5#	70GLN	34.3
71 VAL	53.4#	71 VAL	53.2#	71 VAL	53.1#
72LEU	7.9	72LEU	11.8	72 LEU	8.1
73THR	40.5#	73TF1R	44.0#	73TFIR	47.0#
74ASN	0.6	74ASN	0.0	74ASN	0.0
75ASN	69.8#	75ASN	63.2#	75ASN	65.9#
76THR	36.7#	76TFIR	40.5#	76THR	44.0#
77ASP	80.2#	77ASP	63.0#	77ASP	62.1#
78LEU	62.6#	78LEU	62.5#	78VAL	63.8#

(A)		(B)		(C)
TIC807 LIIM (SEQ ID NO:I85)	TIC807 M2 (SEÇ	ID NO:8)	TIC853 (SEQ ID	NO:184)
Position and	Calculated	Position and	Calculated	Position and	Estimated
amino acid	%SA per	amino acid	%SA per	amino acid	%SA per
residue	residue	residue	residue	residue	residue
79GLN	74.4#	79GLN	54.7#	79GLN	43.5#
80GLN	33,3	80GLN	32.1	80GLN	40.1#
81SER	81.0#	81SER	68.6#	8ISER	75.2#
82GLN	19.0	82GLN	23.9	82GLN	24.6
83T11R	62.7#	83THR	63.6#	83TI1R	63.7#
84V AL	1.8	84VAL	0.9	84VAL	0.0
85SER	50.8#	85SER	55.7#	85SER	54.0#
86PHE	7.2	86PHE	5.8	86P11E	4.0
87ALA'	58.5#	87ALA	61.9#	87SER	68.0#
88LYS	30.5	88 LYS	30.6	88LYS	32.6
89LYS	69.8#	89LYS	67.8#	89LYS	67.9#
90THR	19.9	90TI1R	23.1	90THR	16.7
91THR	54.1#	91TI1R	55.1#	91THR	48.1#
92THR	1.8	92THR	1.8	92THR	0.0
93THRp2’	40.3#	93THR	36.4#	93THR	29.4
94THR	0.0	94THR	0.6	94THR	0.2
95SERp2’	14.4	95SER	15.7	95SER	18.1
96THR	5.5	96T11R	1.5	96THR	0.0
97SERp2	16.6	97SER	18.5	97SER	29.9
98THR	8.2	98T11R	5.9	98THR	1.9
99THR	41.8#	99THR	46.4#	99THR	49.3#
100ASN	37.7#	100 ASN	34.1	100ASP	20.1
IOIGLY	1.0	IOIGLY	1.9	IOIGLY	0.0
102TRP	3.6	102TRP	10.4	102TRP	6.6
I03THR	8.1	103THR	8.1	103THR	3.9
I04GLU	9.7	104GLU	21.9	I04GLU	14.8
105GLY’	35.3	105GLY	46.8#	I05GLY	31.4
106GLY	57.0#	106GLY	68.6#	I06GLY	61.8#
107LYS	52.4#	IO7LYS	57.2#	107ARG	54.6#
108 ILE	61.8#	1081LE	63.5#	108ILE	67.1#
109SER	43.5#	109 S ER	47.9#	I09SER	47.7#
11OASP	83.1#	110ASP	83.5#	ÜOASP	65.7#
1I1THR	43.4#	111THR	41.3#	111THR	39.9#
112LEU	26.7	112LEU	29.6	112LEU	31.3
113GLU	53.8#	H3GLU	64.1#	113GLU	62.5#
114 GLU	34.8	114GLU	30.9	114GLU	32.6
I15LYS	62.2#	115LYS	55.2#	115ASN	54.6#
116VAL	6.4	I16VAL	8.6	II6VAL	10.4
117SER*	46.6#	1I7SER	48.9#	1I7SER	51.2#
118 VAL	0.9	II8VAL	2.5	U 8 VAL	1.3
1I9SER’	20.2	1I9SER	23.4	1I9SER	23.7
120ILE	0.8	1201LE	0.3	120ILE	0.0
121PRO	5.9	121 PRO	10.7	12IPRO	8.0
I22PHE	0.2	122PHE	1.4	I22P1IE	0.3
I23ILE	19.1	123 ILE	20.8	I23ILE	18.3
124GLY	4.3	124GLY	6.2	I24GLY	3.3
125GLU*	59.7#	125GLU	56.2#	I25ALA	57.8#

(A)		(B)		(C)
TIC807LIIM (SEQ ID NO: 185)	TIC807 M2 (SEÇ	MD NO:8)	TIC853 (SEQ ID	NO: 184)
Position and	Calculated	Position and	Calculated	Position and	Estlmated
amino acid	%SA per	amino add	%SA per	amino acid	%SA per
residue	residue	residue	residue	residue	residue
I26GLY	50.0#	126GLY	52.5#	126GLY	49.6#
I27GLY	47.2#	127GLY	56.7#	127GLY	38.6#
128GLY*	34.7	I28GLY	30.3	128 ALA	23.0
129LYS	68.8#	129LYS	73.9#	129LYS	78.4#
I30ASN	16.2	130 ASN	14.6	130 ASN	10.1
131SER	78.2#	131SER	77.9#	131SER	80.3#
132THR	9.8	132THR	10.3	132TIIR	12.3
133THR'	45.7#	133THR	42.0#	133THR	44.3#
1341LE'	1.1	134ILE	0.8	I34ILE	0.0
I35GLU’	51.5#	I35GLU	45.2#	135GLU	48.5#
136ALA	0.0	136ALA	1.3	136ALA	2.4
137ASN'	18.1	137ASN	15.5	137ASN	15.6
138PHE'	1.9	138PHE	0.9	138VAL	2.5
139 ALA'	2.8	139 ALA	6.3	139 ALA	4.1
I40HIS	2.3	140HIS	2.1	140HIS	0.0
141 ASN	5.3	14IASN	6.5	141 ASN	2.8
142SER	5.4	142SER	4.4	I42SER	6.6
143SER	7.7	143SER	10.6	143SER	7.0
144THR	23.5	144THR	17.3	144TIIR	16.6
I45THR'	48.3#	145TIIR	52.7#	145THR	55.2#
I46THR	50.2#	I46THR	49.7#	146THR	53.6#
147PHEp2’	49.9#	147PHE	61.6#	147SER	51.7#
148GLN'	12.9	148GLN	17.8	148GLN	18.4
I49GLNp2'	59.5#	149GLN	65.1#	I49GLN	69.1#
150 ALA'	6.9	150 AL A	8.7	150 AL A	9.1
151SERpI*	51.0#	151SER	51.7#	151SER	57.9#
152THR	9.9	152THR	8.7	152THR	12.3
153ASP'	83.5#	153 ASP	84.5#	I53GLU	63.3#
154ILE	11.2	1541LE	6.1	1541LE	6.3
155GLU'	49.5#	155GLU	63.9#	155GLU	49.7#
156TRP	1.7	156TRP	3.8	I56TRP	1.8
I57ASN*	59.1#	157ASN	59.1#	157ASN	53.4#
158ILE'	13.1	158ILE	5.9	158ILE	0.8
I59SER*	60.2#	159SER	52.9#	159SER	52.2#
160GLN	29.2	160GLN	19.3	I60GLN	9.3
161 PRO	54.0#	161 PRO	63.6#	161PRO	62.6#
162 VAL	0.6	162 VAL	4.0	162VAL	2.4
163LEU	53.8#	163 LEU	56.6#	163LEU	64.5#
164VAL	0.0	164 VAL	0.0	164 VAL	0.0
165PRO	22.8	I65PRO	22.1	I65PRO	26.9
166PRO	30.7	166PRO	36.1#	166PRO	39.7#
167SER'	31.0	167 ARG	32.8	167ARG	36.7#
168LYS	18.2	168 LYS	18.5	I68LYS	19.9
169GLN	17.4	I69GLN	15.1	169GLN	10.7
170 VAL	0.0	170 VAL	0.0	170 VAL	0.0
I71VAL	13.2	17IVAL	13.8	17IVAL	12.2
I72ALA	0.0	I72ALA	0.0	I72ALA	0.0

(A) T1C807 L11M (SEQ ID NO:I8S)	(B)	(C) TICS 53 (SEQ ID NO:I84)
TIC807 M2 (SEÇ	ID NO:8)
Position and amino acid residue	Calculated %SA per residue	Position amino residue	and add	Calculated •/.SA per residue	Position amino residue	and acid	Estimated •/.SA per residue
173THR	9.8	173THR	9.2	173THR	6.5
174LEU	1.3	174LEU	2.6	174LEU	0.2
175 VAL'	17.2	175 VAL	17.8	175 VAL	13.4
1761LE	0.0	1761LE	0.0	1761LE	0.0
177MET'	7.0	177MET	7.7	177MET	17.3
I78GLY	1.6	178GLY	0.5	178GLY	0.0
179GLY	15.9	179GLY	22.2	179GLY	16.5
18OASN”2*	60.0#	180ASN	60.1#	I80ASP	44.9#
181PHE	0.7	181PHE	2.8	181PHE	1.8
182THRp2'	50.6#	182THR	44.3#	182THR	40.8#
183 ILE	0.0	183 ILE	1.1	183 VAL	0.0
184PRO	36.6#	184PRO	34.2	184PRO	34.5
185MET	4.4	185MET	2.1	185MET	1.8
186ASP	52.4#	186ASP	23.5	186ASP	20.3
187LEU	0.8	187LEU	0.0	187LEU	0.0
188MET	25.9	188MET	12.7	1881LE	24.9
189THR	1.4	189THR	2.9	189THR	0.5
190THR	26.1	190THR	26.2	190THR	24.1
191ILE	4.0	191ILE	6.2	191ILE	1.8
192ASP	25.9	192ASP	29.2	192ASP	21.4
193SERpl	7.4	193SER	7.7	193SER	2.7
194THRpl	66.2#	I94THR	60.2#	194THR	59.9#
195GLU”1	38.5#	195GLU	35.0	195GLN	35.5#
196H1S*	37.7#	-	100	-	100#
197TYR’	32.2	-	100#	•	100#
198SER*	35.5#	-	100#	-	100#
199HISpl	64.3#	196HIS	55.5#	1961 IIS	51.7#
200TYRpP	85.3#	197TYR	56.2#	197PHE	45.8#
201SERpp	50.3#	198SER	68.3#	I98THR	64.5#
202GLY”1	32.8	199GLY	50.0#	199GLY	51.1#
203TYRpl	21.5	200TYR	22.6	200TYR	26.0
204PRO”'	1.4	201PRO	1.0	201 PRO	LO
205IL˔1	1.1	2021LE	0.3	2021LE	0.0
206LEU”'	1.8	203LEU	2.6	203LEU	0.0
207THRpl	0.0	204THR	0.0	204THR	0.0
208TRPpP	38.8#	205TRP	35.6#	205TRP	22.5
209ILEpl	0.0	2061LE	0.0	206ILE	0.0
21 OSER”1	22.5	207SER	20.1	207GLU	17.0
211SER	3.1	208SER	3.4	208ASN	4.6
212PRO	56.5#	209PRO	58.4#	209PRO	56.2#
213ASP	68.0#	210ASP	55.2#	210GLU	60.5#
2I4ASN	65.5#	211ASN	66.4#	211H1S	64.4#
215SER	67.2#	212SER	74.1#	2I2ASN	74.2#
216TYRpl	42.5#	213TYR	39.8#	213VAL	29.4
217SERpP	43.2#	214ASN	46.3#	214ARG	57.1#
218GLY”1	1.2	215GLY	4.1	215GLY	6.1
219PROpP	14.4	216PRO	14.7	216ARG	33.7

(A)		(B)		(C)
TIC8O7 L11M (SEQ ID N0:I8S)	T1C8O7 MÏ (SEC	»IDNO:8)	TIC853(SEQ IDNO:184)
Position and	Calculated	Position and	Calculated	Position and	Estima ted
amino acid	%SA per	amino acid	%SA per	amino acid	•/•SA per
residue	residue	residue	residue	residue	residue
220PHEpi	0.0	217PHE	0.0	217PHE	0.0
22IMETpl	15.2	218MET	i6.1	218LEU	8.4
222SER1	3.3	219SER	3.3	219SER	0.0
223TRPpl*	35.6#	220TRP	34.3	220TRP	42.5#
224TYRpl	13.5	221TYR	15.9	221PHE	11.3
225PHEpl	0.9	222PHE	i.4	222PHE	0.0
226ALA	15.9	223ALA	13.1	223ALA	7.8
227ASN	40.7#	224ASN	41.9#	224ASN	43.2#
228TRP	9.0	225TRP	8.9	225TRP	7.3
229PRO	56.3#	226PRO	61.5#	226PRO	65.5#
230ASN	67.6#	227ASN	67.3#	2 27ASN	67.3#
23! LEU	21.i	228LEU	16.1	228LEU	16.1
232PRO	23.7	229PRO	23.0	229PRO	23.6
233SER	97.0#	230SER	95.8#	230SER	88.1#
234GLY	23.0	231GLY	19.5	231 GLU	i3.0
235PHE'	8.3	232PHE	9.0	232PHE	6.1
236GLY	26.1	233GLY	18.3	233GLY	28.5
237PRO	72.6#	234PRO	70.8#	234SER	81.9#
238LEU	27.7	235LEU	28.i	235LEU	25.9
239ASN*	33.2	236ASN	26.5	23 6ASN	42.2#
240SER	100#	237SER	100#	237SER	100#
241 ASP’	62.2#	238ASP	61.7#	238ASP	55.2#
242ASN	15.1	239ASN	20.8	239ASN	21.7
243THR'	3.3	240THR	2.4	240THR	2.6
244VAL'	1.8	241 VAL	3.1	24iILE	0.0
245THR'	19.0	242THR	23.8	242THR	30.1
246TYR'	8.6	243TYR	4.8	243TYR	0.4
247THR’	36.8#	244THR	40.8#	244LYS	58.8#
248GLY	2.5	245GLY	1.4	245GLY	0.0
249SER'	20.7	246SER	23.9	246SER	27.0
250VAL'	6.0	247VAL	1.4	247VAL	0.0
25iVALp1'	32.6	248VAL	30.0	248VAL	29.7
252SER'	0.0	249SER	0.0	249SER	0.0
253GLNpï	40.2#	250GLN	37.6#	250ARG	51.3#
254VAL	1.2	251 VAL	1.5	251ILE	2.7
255SER2	35.3	252SER	37.3#	252SER	43.7#
256ALA	6.1	253ALA	2.4	253ALA	2.1
257GLY	4.1	254GLY	6.1	254GLY	1.2
258VAL	0.3	255VAL	0.0	255VAL	0.0
259TYR	2.2	256TYR	L!	256TYR	1.0
260ALA	0.7	257ALA	0.7	257ALA	0.0
261THR	9.2	258THR	9.3	258THR	5.0
262VAL	3.6	259V AL	L5	259VAL	0.2
263ARG	26.6	260ARG	29.8	260ARG	26.9
264PHE	0.5	261 PHE	3.8	261PHE	1.5
265ASP	6.9	262ASP	7.2	262ASP	10.8
266GLN	5.6	263GLN	5.8	263GLN	2.6
267TYR	16.1	264TYR	14.5	264TYR	12.4

(A) TIC8O7 L11M (SEQ ID NO:185)	(B) TIC807 M2 (SEQ ID NO:8)	(C) T1C853 (SEQ IDNO:184)
Position amino residue	and acid	Calculated •/•SA per residue	Position amino residue	and acid	Calculated %SA per residue	Position amino residue	and acid	Estimated %SA per residue
268ASP	29.8	265ASP	31.4	265ALA	19.3
269ILE	25.4	2661LE	18.2	2661LE	14.5
270H1S	85.5#	267H1S	72.2#	267ASN	92.0#
27IASN	43.4#	268ASN	46.9#	268ASN	64.0#
272LEU	40.3#	269LEU	43.1#	269LEU	39.8#
273ARG*	86.3#	270ARG	63.1#	270ARG	84.4#
274THR’	52.0#	27ITHR	66.1#	27ITHR	76.8#
2751LE*	41.0#	2721LE	37.9#	272 ILE	32.4
276GLU	47.9#	273GLU	50.1#	273GLU	53.0#
277LYS	49.8#	274LYS	47.2#	274LYS	70.2#
278THR	46.3#	275THR	51.2#	275THR	53.7#
279TRP	25.2	276TRP	25.0	276TRP	33.4
280TYR	35.5	277TYR	30.7	277TYR	21.3
281 ALA	6.6	278ALA	7.9	278ALA	4.4
282ARG*	77.6#	279ARG	80.6#	279ARG	86.1#
283HIS	45.2#	280HIS	36.6#	280HIS	35.8#
284ALA	0.8	281 ALA	0.8	281 GLY	0.6
285THR	14.7	282TIIR	8.6	282THR	2.0
286LEU	3.6	283LEU	5.9	283LEU	2.3
287HIS'	8.4	2841IIS	16.5	284HIS	11.9
288ASN	40.4#	285ASN	43.9#	285ASN	38.7#
289GLY	61.0#	286GLY	53.5#	286GLY	61.1#
290LYS	61.7#	287LYS	61.6#	287LYS	73.8#
291 LYS	68.4#	288LYS	66.2#	288LYS	51.9#
292ILE	19.2	2891LE	19.5	2891LE	21.0
293SER'	40.3#	290SER	47.9#	290SER	45.7#
294ILE	3.4	2911LE	4.8	291 ILE	5.1
295ASN'	29.3	292ASN	21.9	292ASN	i8.0
296ASN	38.2#	293ASN	40.4#	293ASN	37.4#
297VAL	1.3	294VAL	1.4	294VAL	0.7
298THR	10.1	295THR	9.5#	295THR	4.3
299GLU*	77.1#	296GLU	72.7#	296GLU	68.8#
300MET*	48.6#	297MET	46.4#	297MET	42.8#
301 ALA	65.3#	298ALA	54.1#	298ALA	60.4#
302PRO	66.0#	299PRO	73.0#	299PRO	77.8#
303THR'	83.7#	300THR	85.8#	300THR	94.1#
304SER	77.4#	301SER	76.1#	301SER	84.9#
305PRO'	81.1#	302PRO	65.7#	302PRO	83.4#
3061LE*	78.1#	3O31LE	81.6#	303 ILE	91.3#
307LYS	81.9#	304LYS	99.3#	304GLU	100#
308THR*	89.4#	305THR	100#	305ARG	100#
309ASN	100#	306ASN	100#	306ASN	100#

P1 désignâtes an amino acid in surface patch [ 1 ] of Figure 2.

P2 désignâtes an amino acid ïn surface patch [2] of Figure 2.

* désignâtes one of the 72 principaiiy relevant amino acids described herein (see Figure 2).

Shown are residues of TIC8O7_L1 IM, TIC807_M2, andT1C853 aligned by Clustal W.

Numbers marked with H represent %SA of at least about 36%.

Receptor Binding [0063] A surface patch ([1] of Figure 2) of residues having %SA values greater than 36% or within about 3 residues of a restdue having %SA greater than 36% in a radius of about 9.2 - 12.2 Angstroms from the Cb atom of S95 of SEQ ID NO:2 (TIC807) was identified as a région comprising residues of a T1C807 protein that can be substituted to provide for eHTP’s that exhibit enhanced Lygus inhibitory spectrum and/or improved Lygus inhibitory activity, This surface patch région may be associated with target insect receptor btnding activity; and, includes residues T93, S95, S97, F147, Q149, S151, NI 80, Tl 82, V251, Q253, and S255 of SEQ ID NO:2 (TIC807). eHTP’s can include, but are not limited to, one or more substitutions of surface patch 1 amino acid residues such as S95A, F147A, Q149E, and/or, V251A.

[0064) The combined engineering-testing-selecting approaches described herein identified residues located in surface patch 1 that can provide for eHTP’s when substituted or otherwise modified. These residues may be important for productive binding of eHTP’s to receptors in Lygus insect gut to provide for enhanced Lygus inhibitory spectrum and/or improved Lygus inhibitory activity when compared to T1C807. Modifications of the surface patch I amino acid residues that can provide for eHTP’s include substitutions that provide aromatic groups and/or hydrogen-bonding groups which favoring binding to sugar groups found on glycosylated receptors of insects.

Membrane Binding |0065| Certain amino acid residues located in beta-sheet régions of the protein were identified from the atomic structure of T1C807 and were substituted with aromatic residues. More specifically, amino acids L78, 1123, H270, R273,1275of the folded T1C807 beta sheet régions were substituted with Phenyalanine, Tyrosine, or Tryptophan. Aromatic amino acid substitutions of R273 and 1275 were amongst those residues that provided for an enhanced Lygus inhibitory spectrum and/or improved Lygus inhibitory activity (See Table 4, data for SEQ ID NOs:32, 34, 68, 92, and 122). Amino acid side chains of residues in these positions may be likely to interact with the membrane of target insects.

Proteolytic activation sites [0066] Glycine residues generally thought to be involved in proteolysis were substituted with Serines to alter proteolytic cleavage dynamics. The presence of a glycine residue in a loop région can impart more flexibility and therefore susceptibility to proteolysis, which can 5 either increase insect inhibîtory activity or decrease insect inhibîtory activity. Residues in structurally identified loop régions were substituted with a glycine residue, and no improvements were observed. Positions in loops that were already glycines, (e.g. GI8, G24, G27) were substituted with a serine, a small residue in an attempt to reduce proteolytic susceptibility, and no improvements were observed.

Combined Structure design approaches [00671 The atomic structure of TIC807 (SEQ ID NO:2) was used to identify loop régions for library mutagenesis followed by testing of the engineered variants. A loop at amino acid positions 211-216 of SEQ ID NO:2 (TIC807) was library-mutagenized and tested.

Consecutive loops in close proximity at amino acid positions 75-83, 161-167, and 267-276 of SEQ ID NO:2 (TIC807) was library-mutagenized and tested.

[0068] Analysis of the atomic structure of TIC807 suggests that a structural loop résides at residues 113-138 of SEQ ID NO:2, and variants were engineered to stabilize and destabilize the loop.

10069] In another région spanning two beta-strands connected by a short loop, the two betastrands exhibited an altemating pattem of hydrophobie and hydrophilic amino acid residues at positions 116 to 121 and at positions 133 to 138 relative to SEQ ID NO:2, characteristic of pore-forming loops. An expression library was engineered to modify both beta-strand segments replacing residues VI16, VI18, and 1120 with respective combinations

II6V/Y/I7H/F/D, 118V/Y/L/H/F/D, and 120I/D/F/H/L/N/V/Y for a total of 288 possible variants in the library. This procedure was repeated for: residues SI 17, SI 19, and P121 with respective combinations 117S/A/D/E/G/K/N/R/T, 119S/A/D/E/G/K/N/R/T, and 121P/S/T for 243 potential variants; residues 1133, A135, and FI37 with respective combinations 133I/D/F/N/V/Y, I35A/D/F/H/UV/Y, and 137F/D/H/L/V/Y for 252 possible variants; and residues T134, E136, and N138 with respective combinations 134T/A/D/E/G/K/N/R/S,

136E/A/D/G/K/N/R/S/T, and 138N/A/D/G/S/T for 486 possible variants. An enhanced

Lygus inhibîtory spectrum and/or improved Lygus inhibîtory activity was associated with certain of these substitutions as shown in Table 4.

Structure-Function Relatïonship

10070) Altogether, more than 2000 clones (including mixed library clones) expressing variants of T1C807 were tested for enhanced Lygus inhibitory spectrum and/or improved Lygus inhibitory activity against Lygus spp. compared to TIC807. Semi-random modifications, directed modifications, and prédictive structure-function modifications, including structure modeling, receptor binding potential, métal binding potential, oligomérization potential, uniformity of surface charge distribution, pore formation potential, ion channel function, and identification of surface exposed patches to with an objective of identifying eHTP’s with an enhanced Lygus inhibitory spectrum and/or improved Lygus inhibitory activity compared to TIC807. These clones were expressed for bioassay testing.

Example 3: Protein expression and purification of TIC807, Including variants and fragments [0071] Control protein TIC807 is a protein of 309 amino acids in length that can be expressed in crystalline form in Bacillus thuringiensis (Bt) or aggregate form in E. coli. Test variants thereof were recombinantly expressed in Bt. An expression characteristic of TIC807 and variants of TIC807 is the prédominant crystalline and aggregate forms extracted from Bt and E. coli cells, respectively. To test for Lygus bioactivity, test and control samples were made suitable for Lygus bioassay by solubilizing samples in 25 mM Sodium Carbonate buffer and removing unsolubilized materiels by centrifugation. The amount of protein in test and control samples were measured using total protein methods, e.g.s, a Bradford assay, an ELIS A method, or similar. Gel electrophoresis was used to détermine the purity and stock concentration of the sotubitized recombinant protein. C-terminal HIS-tagged TIC807 protein was engineered to facilitate détection, purification, and quantification of large amounts of TIC807 control protein. C-terminal HIS-tagged TIC807 and un-tagged TIC807 test samples were separately assayed and confirmed to hâve équivalent activity against Lygus (see Examples 4,5, and 6).

(0072] Site-directed amino acid substitutions were made to TIC8O7__M13 (SEQ ID NO:34) to elevate expressionof a soluble form. Inventors postulate that more readily soluble variants of the proteins of the présent invention can facilitate expression and purification, e.g., expressed in E. coli host cells; and can increase insect inhibitory efficacy when expressed in plant host cells. Recombinant DNA constructs encoding TIC807_M13 (SEQ ID NO:34) were engineered three different ways to reflect three different variants: Relative to

TIC807_M13, the modifications were for Variant #1:158K and P59K, for Variant #2: S198K and G199K, and for Variant #3: S246R, V248E, and Q250R. Relative to TIC807 (SEQ ID NO:2), the modifications can be altematively described as follows for Variant #1:158K and P59K, for Variant #2: S201K and G202K, and for Variant #3: S249R, V251E, and Q253R;

this positional différence is congruent due to a contiguous triple délétion of SEQ ID NO:2 (TIC807) in residue range 196-201 that is reflected in TIC8O7_M13 (SEQ ID NO:34). The four engineered recombinant DNA constructs were each cloned and expressed in E. coli. The soluble fraction from the four E. coli préparations were evaluated by coomassie-stained SDSPAGE, which showed that TIC807_M13 (SEQ ID NO:34) was not détectable in the soluble 10 fraction; but, in contrast, Variant #s 1, 2, and 3 were soluble. Similar amino acid substitutions either singly or in combination are made to proteins of the présent invention to elevate their solubility in non-Bt or plant host cells. Recombinant DNA constructs were engineered to encode for and express TIC807_M13 variant #3 (renamed TIC807_MI4; nucléotide SEQ ID NO:203 and amino acid SEQ ID NO:204). Prepared E. coli lysate was 15 clarified, and the recombinant protein purified and enriched-for on a sériés of columns, inciuding ion-exchange and gel filtration methods. Pooled protein fractions were quantifîed and determined to be active against Lygus insects (See Example 4, Table 4B).

(0073] Proteins of the présent invention, inciuding but not limited to proteins having the amino acid sequence as set forth as SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, or SEQ 20 ID NO:36, are engineered to elevate expression of a soluble form when expressed în a host cell, e.g., expressed în Bt, E. coli, or in a plant cell or in a compartment of a plant cell. Engineering includes substituting a lysine amino acid residue at one or more of the following positions 58, 59, 198, 199, 201, or 202; or, a Glutamic acid at one or more of the following positions 198, 248, or 301; or, an Arginine at one or more of the following positions 246, 25 250, or 253.

[00741 The C-terminal région protrudes away from the monomeric core of the protein (See Figure 2). A recombinant DNA construct was engineered to encode for and express a protein having the amino acid sequence of SEQ ID NO:202, which is a protein fragment (amino acids 1 to 301) of TIC807_M8 (SEQ ID NO:16); and, the expressed protein was purified, 30 quantifîed, and determined active against Lygus insects (See Example 4, Table 4B).

Recombinant DNA constructs were designed to encode for and express TIC8O7 fragments exhibiting varying troncations off of the C-terminus end of the proteins of the présent invention at the respective TIC807 positions A281, G289, S293, A301, and S304. Protein fragments are engineered to encode for and express proteins having the amino acid sequences set forth as SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, and SEQ ID

NO:36; and, the expressed protein fragments are used as test samples against Lygus insects.

Example 4: Hemipteran activity of Engineered proteins |0075] This example illustrâtes eHTP’s to hâve improved insecticidal activity or enhanced insecticidal specificity against Hemipteran insects when provided in the diet of Hemipteran insects, including but not limited to members of the Heteroptera miridae, induding the genus Lygus, e.g., Lygus hesperus and Lygus lineolaris, and the famîly Cîcadellidae, induding the genus Amrasca, e.g. Amrasca dévastons, and Empoasca, e.g. Empoasca fabae. This example with Table 4B illustrâtes the feeding assay used to détermine the enhanced Lygus inhibitory spectrum and/or improved Lygus inhibitory activity of a Bt expressed recombinant proteins of the présent invention against both Lygus hesperus and Lygus lineolaris. Proteins expressed in recombinant bacterium host cells were solubilized in carbonate buffer and analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE); and, protein concentrations determined by densitometry using bovine sérum albumin (BSA) as a standard. Protein stock (2X) prepared this way were mixed with diet for feeding assays.

(0076] Feeding assays with the Hemipteran species Lygus hesperus and Lygus lineolaris were based on a 96 well micro-titer plate format with Lygus diet encapsulated between stretched Parafilm® and Mylar sheets. Artificial diet was obtained from Bio-Serv ® (Bîo-Serv ® Diet F9644B, Frenchtown, NJ). Autoclaved, boiling water (518 mL) was combined with 156.3 grams of Bio-Serv ® diet F9644B in a surface-sterilized blender. The contents of four surface-sterilized chicken eggs were added and the mixture blended until smooth, then adjusted to one liter total volume and allowed to cool to room température, this being the 2X diet. Test samples were prepared by mixing in a i:i ratio of 2X diet and 2X sample. A sheet of Parafilm® (Pechiney Plastic Packing, Chicago, IL) was placed over a vacuum manifold designed for 96-well format (Analytical Research Systems, Gainesville, FL) and a vacuum of approximately -20 millimeters mercury was applied, suffîcient to cause extrusion of the Parafilm® into the wells. Twenty to forty microliters of test sample were added to the Parafilm® extrusions. A sheet of Mylar film (Clear Lam Packaging, Inc., Elk Grave Village, IL) was placed over the sample filled Parafilm® extrusions and sealed with a tacking iron (Bienfang Sealector II, Hunt Corporation, Philadelphia, PA), thus forming diet filled Parafilm® sachets. These Parafilm® sachets were positioned over a flat-bottom 96-well plate containing Lygus eggs suspended in a dilute agarose solution. Upon hatching, Lygus nymphs feed ori the diet by piercing the diet filled Parafilm® sachets. Altematively, newly hatched

Lygus nymphs instead of eggs were manually infested into each well. Stunting and mortality scores were determined on day 5 and compared to contrats. Data were analyzed using JMP4 statistical software. For each protein at a test concentration, three populations of eight nymphs were subjected to this bioassay, and mortality scores reported in Table 4B.

[0077] For LC50 déterminations listed in Table 1 and Table 4B, proteins were presented to newly hatched Lygus nymphs at 8-10 concentrations and the nymphs allowed to feed for 5 days before scoring for mortality over the dose range. For each concentration, three populations of eight nymphs were subjected to this bioassay, and ail LC50 déterminations in Table 1 and Table 4B were repeated at least once.

[0078| For LC50 estimations, proteins were presented to newly hatched Lygus lineolaris nymphs at 4 concentrations and the nymphs allowed to feed for 5 days before scoring for mortality over the dose range. Lygus lineolaris LC50 estimations were performed on TIC807 and TlC807_M2 because significantly large amounts of these proteins in excess of 1000 pg/mL hâve not been possible to provide in Lygus diet in order to complété the high range of toxicity dose response to Lygus lineolaris; and therefore, an LC50 value was not determined for TIC807 or TIC807_M2. instead, a 4-dose LC50 estimation in the low range was performed, and reported in Table 1 and Table 4B. The estimated Lygus lineolaris LC50 for TlC807_M14 is 4.4 pg/mL. For each concentration, three populations of eight nymphs were subjected to this bioassay.

(0079) This example with Tables 4A and 4B illustrate the feeding assay used to détermine the enhanced inhibitory spectrum and/or improved inhibitory activity of a Bt expressed recombinant protein disclosed herein against Amrasca devastans. TIC807 variants with improved insecticidal activity or enhanced insecticidal specificîty against Lygus hesperus and Lygus lineolaris exhibit improved insecticidal activity against Amrasca devastans.

[0080] T1C807, and TlC807_M 13 were dissolved in 25mM sodium carbonate buffer, pH 10. Amrasca devastans eggs were collected on Okra leaf and incubated in a petriplate containing 2% agar. Upon hatching the neonates were used for biossays using the diluted (1:5) Lygus diet. The proteins and diet were mixed at equal proportion (bringing final concentration of protein to 500 pg/mL) and dispensed into test arena. Untreated control was prepared by mixing the buffer with the diet. Individual neonates were infested into the test arena, the assays were incubated at 25°C, 60% RH. Twenty neonate nymphs were tested for each concentration, protein and in 2 replicates. A control was maintained with 25mM Sodium Carbonate buffer, pH 10, in 1:5 diluted Lygus diet. Mortality ofthe insects was determined on the fifth day. Mortality values were calculated by the following formula: (% mortality in treatment - % mortality in control)/(100 - % mortality in control) x 100. Table 4A tabulâtes

Amrasca activity for TIC807 and TIC807_M13 at 5 different concentrations.

Table 4A. TIC807 and T1C8O7_M13 Percent Mortality Directed to Amrasca species

SEQ IDNO:	Protein Name	Mortality (%)
500 pg/mL	166.66 pg/mL	55.55 pg/mL	18.51 pg/mL	6.17 pg/mL
2	TIC807	100%	55.88 %	17.64%	0	0
34	TIC807_M13	100%	88.23 %	73.52 %	44.11%	26.47 %

[00811 LC50 values were determined for TIC807 and TlC807_M13 in a separate test. SEQ ID NO:2 (TIC807) exhibited a LC50 value of 116.79 pg/mL and LC90 of 437.27 pg/mL. SEQ ID NO:34 (TIC8O7_M13) exhibited a LC50 value of 7.59 pg/mL and LC90 value of

239.8 pg/mL.

[0082] A feeding assay as described for Amrasca dévastons is used to test eHTP’s for improved insecticidal activity and/or enhanced insecticidal specificity against Empoasca fabae. TIC807 variants with improved insecticidal activity or enhanced insecticidal specificity against Lygus hesperus and Lygus lineolaris exhibit improved insecticidal activity against Empoasca fabae.

[00831 The LC50 values of CrySlAal (SEQ ID NO: 182), for T1C807 (SEQ ID NO:2), TIC807_M2 (SEQ ID NO:8), TIC807_M10 (SEQ ID NO:30) and TIC807_M13 (SEQ ID NO:34) against Lygus hesperus and Lygus lineolaris were determined in one testset. TIC807_M2, TIC8O7_M1O and TIC807_M12 exhibit improved LC50 values compared to CrySlAal.

[00841 It should be apparent to those skilled in the art that variations to this procedure can exist that should not affect results.

Table 4B. Itérative engineering-testing-selecting of eHTP’s against Lygus spp. resulted in 267 proteins with enhanced Lygus inhibitory spectrum and/or improved Lygus inhibitory activity against Lygus spp. compared to T1C807.

	Amino add différences compared to T1C807 parent protein
SEQ ID NO	Protein Name	Amino add difTertncefs) relative to SEQ ID NO:2 (TÏC807)	Amiao add difrennct(s) la tbe first patch	Amino add dlfTerencefs) la the second patch
2	TIC807	Parent	Parent	Parent
6	TIC8O7 Ml	FI47A	FI47A	None
8	T1C807 M2	F46S, Y54H, SI67R, S217N, then a contiguous triple ddction in residue lange 196-201	none	S2I7N, then contiguous triple ddction in residue range 196-201
iO	T1C807 M3	F46S. Y54H. T93A, SI67R, S217N, then a contiguous triple ddction in residue range 196-201	T93A	S217N, then a contiguous triple ddction in residue range 196-201
12	TIC807 M4	F46S. Y 54 H, S95A, SI67R. S2I7N, then a contiguous triple ddction in residue range 196-201	S95A	S217N, then a contiguous triple ddction in residue range 196-201
14	TIC807 M5	F46S. Y54H. Π47Α, S167R. S217N, then a contiguous triple dclction in residue range 196-201	F147A	S217N. then a contiguous triple ddction in residue range 196-201
16	TIC807 M8	F46S, Y54 H. S95A, FI47A, SI67R. S217N. then a contiguous triple dclction in residue range 196-201	S95A, Fl 47 A	S2I7N, then a contiguous triple ddction in residue range 196-201
18	110807 M6	F46S, Y 54 H, T93A, F147A, SI67R. S217N, then a contiguous triple dclction în residue range 196-201	T93A, F147A	S217N, then a contiguous triple ddction in residue range 196-201
20	T1C807 M7	F46S, Y54H, QI49E, Si 67 R. S2I7N, then a contiguous triple ddction in residue range 196-201	QI49E	S2I7N. then a contiguous triple ddction in residue range 196-201
28	TIC807 M9	F46S, Y54H, S95A. F147A. S167R. P2I9R. then a contiguous triple	S95A, F147A	P2I9R, then a contiguous triple dclction in residue

LC50 value (pg/mL)	fold Increased toildty (1X50)
73	1
23	3
60	12
29	25
24	30
1 1	66
08	91
1.5	50
1.4	52
99	7

*/* Mortality at about 1-3 pg/mL protria*

'ta lirteolaris

LC50 value (pg/mL)	fold increased toildty (LC50)
>223	1
100	at least 2
>223
ND
ND
ND
223	at least 1
ND
ND
83	at least 27

% Mortality at about 100 pg/mL protriu*

Xk

U3

Amine add difTerencet compared to TIC807 parent protein

ZnfHsArs

perMf

LygHS lineotarit

SEQ ID NO:

Protêt· Ni me

Amino acid difTerence(s) relative to SEQ ID NO:2 (T1C807)

Amino add différence^) ln the first patch

Amino aeid difierence(s) lu the second patch

LC50 value (jtg/mL)

foid Increased toxidty (LC50)

% Mortality at about 1-3 pgtaiL protein*

LC50 value (pg/mL)

foid Increased toxicity (LC50)

% Mortality at about 100 pgfaiL protein*

ddetion m residue range 196-201

range 196-201

30

T1C807 MIO

F46S, Y54H. S95A, Fl47A, SI67R, P219R, V251A, then a contiguous tnple ddetion in residue range 196-201

S95A, F147A, V251A

P2I9R, then a contiguous triple ddetion in residue range 196-201

06

122

ND

48

at least 46

ND

32

T1C807 Mil

F46S, Y54H. S95A, FI47A, S167R, P2I9R, R273W, then a contiguous triple ddetion in residue range 196-201

S95A, F147A

P2I9R, then a contiguous triple ddetion in residue range 196-201

1 4

54

ND

59

at least 38

ND

34

TIC807 Ml 3

F46S, Y54H, S95A, FI47S, QI49E. SI67R, P219R, R273W. then a contiguous triple ddetion in residue range 196-201

S95A, F147A, Q149E

P2I9R, then a contiguous tnple ddetion in residue range 196-201

03

243

ND

085

at least 262

ND

36

TIC807 M12

F46S, Y54H, S95A, F147A, S167 R, P2I9R, N239A, V25IA, then a contiguous triple ddetion in residue range 196-201

S95A, F147A, V25IA

P2I9R, then a contiguous triple ddetion in residue range 196-201

04

182

ND

12

at least 186

ND

37

TIC807 37

T1C807 HYS ddetion

nonc

a contiguous tnple ddetion in HYSHYS residues (positions 196-201)

224

3

ND

ND

ND

38

TIC807 38

F46S, Y54H, FI38V, SI67R, S2I7N, then a contiguous triple ddetion in residue range 196-20)

nonc

S217N, then a contiguous tnple ddetion in residue range 196-201

ND

41

ND

44

39

T1C8O7 39

F46S, Y54H, SI67R, S217N, H287F, then a contiguous triple ddetion in residue range 196-201

nonc

S217N, then a contiguous tnple ddetion in residue range 196-201

ND

57

ND

31

40

T1C807 40

F46S, 152M, Y54H. S167 R, S2I7N, then contiguous triple

none

S217N, then a contiguous triple ddetion in residue

ND

52

ND

36

m

Ο

Amino add différences compared to T1C807 parent protein

Znrushev

vents

Lfgiu lineoiarii

SEQ ID NO·

Protri· Nome

Amino add dlfferencefs) relative to SEQ ID NO:2 (TICS07)

Amino add dlfTerencef1) la the first patch

Amino add différence/·) ln the second patch

LC50 value (pg/mL)

fold Increased toxidty (LC50)

% Mortality at about 1-3 pg/mL protda*

ucsa value (pg/mL)

fold lucres sed toxlclty (LC50)

% Mortality at about 100 pg/mL protda*

ddction in residue range 196-201

range 196-201

41

T1C807 41

N12D, F46S. Y54H, S167R, S217N, then a contiguous triple ddction in residue range 196-20!

none

S217N. then a contiguous triple ddetion in residue range 196-201

ND

52

ND

31

42

T1C807 42

F46S, Y54H, SI67R, N180D, S217N, then a contiguous triple ddction in residue range 196-201

NI80D

S217N, then a contiguous triple ddction in residue range 196-20!

ND

63

ND

50

43

T1C807 43

F46S, Y54H, SI67R, then a contiguous triple ddction in residue range 196-20!

none

a cortiguous tnple ddction m HYSHYS residues (positions 196-201)

ND

46

ND

87

44

T1C807 44

F46S, Y54H, SI67R, S217N. P219R, then a contiguous triple ddetion m residue range 196-20!

none

S217N, P2I9R, then a contiguous triple ddetion in residue range 196-201

ND

30

ND

94

45

T1C807 45

F46S, Y54H, SI59T, SI67R, S217N, then a contiguous triple ddetion m residue range 196-201

none

S217N, then a contiguous triple ddction in residue range 196-201

ND

67

ND

21

46

TIC807 46

F46S, Y54H, SI67R, S217N, T247K. then a contiguous triple délétion in residue range 196-201

none

S2I7N, then a contiguous triple ddetion m residue range 196-201

ND

52

ND

31

47

TIC807 47

F46S, Y54H, S167R, S2I7N, V244I, then a contiguous triple ddetion in residue range 196-201

none

S217N, then a contiguous triple ddction in residue range 196-201

ND

59

ND

31

48

TIC807 48

F46S, Y54H. S167R, S2I7N. V244I, T247K. then a contiguous triple ddction in residue range 196-201

none

S2I7N, then a contiguous triple ddetion in residue range 196-201

ND

58

ND

34

49

T1C807 49

F46S. Y54H, S167R, S2I7N, W223Y, then a

none

S2I7N. W223Y. then a cortiguous

ND

*

17

ND

13

Amine add différences compared to T1C807 parent protein

Lma het,

Lnut lineotaris

SEQ ID NO-

Protdn Name

Amiao add différence^) relative to SEQ ID NO:2 (TIC807)

Amine add differcncc(s) in the first patch

Amin· add difference(i) ln the second patch

LCSD value (jig/mL)

fold Increased toxidty (LC50)

% Mortality at about 1-3 Pg/mL protdu*

LC50 value (pg/mL)

fold Increased toxic) ty (LC50)

% Mortality at about 100 pgfaiL protdu*

contiguous triple dcletion in residue range 196-201

tnpte ddetion in residue range i 96201

50

TIC807 50

F46S, Y54H. SI67R, S2I7N, Y246F, then a contiguous triple dcletion in residue range 196-201

none

S2I7N, then a contiguous tnplc ddetion in residue range 196-201

ND

48

ND

19

51

TTC807 SI

F46S. Y54H. FI47A, GI28A, SI67R, S217N, then a contiguous triple dcletion in residue range 196-201

FI 47 A

S2I7N, then a contiguous triple ddetion in residue range 196-201

ND

TJ

ND

ND

52

TIC807 52

F46S, Y54H, FI47A, SI67R, S217N, M300A, then a contiguous triple dcletion tn residue range 196-201

FI 47 A

S217N, then a contiguous triple dcletion in residue range 196-201

ND

73

ND

ND

53

T1C807 53

F46S, Y54H. F147 A, S167R, S2I7N. S293A, then contiguous triple dcletion in residue range 196-201

F147A

S2I7N, then a contiguous triple ddetion in residue range 196-201

ND

73

ND

ND

54

TIC807 54

F46S. Y54H, F147 A, SI67R, S2I7N, H287A, then a contiguous triple dcletion in residue range 196-201

FI47A

S2I7N, then a contiguous triple dcletion in residue range 196-201

ND

67

ND

ND

55

TÏC807 55

F46S, Y54H, FI47A, SI67R, S2I7N, T274A, then a contiguous triple dcletion in residue range 196-201

Fl 47 A

S2I7N, then a contiguous triple dcletion in residue range 196-201

ND

65

ND

ND

56

T1C807 56

F46S, Y54H. F147 A, SI67R, S2I7N, R282A, then a contiguous triple ddetion in residue range 196-201

Fl 47 A

S2I7N, then a contiguous triple ddetion in residue range 196-201

ND

64

ND

ND

57

TIC807 57

F46S, Y54H, T93A, SI67R, S2I7N, T3O8A, then contiguous triple dcletion in residue range 196-201

T93A

S217N, then a contiguous triple dcletion in residue range 196-201

ND

63

ND

ND

V)

Amino add différence* compared lo TIC807 parent protein

Lygushex

penrj

Lygus iineolarit

SEQ ID NO:

Protêt· Name

Amino add différence^) relative to SEQ ID NO;2 (T1C807)

Amino add difTerencefs) 1· the fi rat patch

Amino add difTcrenceO) la the second patch

LC50 value (pg/mL)

fold lacreaicd toiidty (LC50)

% Mortality •t about 1-3 pgfaiL protein*

LC50 velue (pg/mL)

fold Increased toiidty (LC50)

% Mortality at about 100 pg/mL protein*

58

TTC807 58

F46S, Y54H, Q70A, T93A, SI67R, S217N, then a contiguous triple dctetion in residue range 196-201

T93A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

61

ND

ND

59

TIC807 59

F46S, Y54H, EI25A, F147A, S167R, S217N, then * contiguous triple ddetion in residue range 196-201

F147A

S2I7N, then a contiguous triple ddetion in residue range 196-201

ND

61

ND

ND

60

T1C807 60

F46S, Y54H, FI47A, S167R, S217N, T247A, then a contîgwus triple ddetion in residue range 196-201

Fl 47 A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

61

ND

ND

61

TIC807 61

F46S, Y54H, Τ’) 3 A, S167R. S217N, P3O5A, then a contiguous triple ddetion ïn residue range 196-201

T93A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

59

ND

ND

62

TIC807 62

F46S, Y54H. Fl 47A, S167R. S217N, 1306A, then a contiguous triple ddetion in residue range 196-201

FI47A

S217N. then a contiguous triple dctetion in residue range 196-201

ND

57

ND

ND

63

T1C807 63

F46S. Y54H. T93A, SI67R. S217N, R282A, then a contiguous tnple ddetion m residue range 196-201

T93A

S2I7N, then a contiguous tnple dctetion in residue range 196-201

ND

56

ND

ND

64

TIC807 64

F46S, Y54H, T93A, SI67R. S217N, T308A, then a contiguous tnple ddetion in residue range 196-201

T93A

S2I7N, then a contiguous triple ddetion in residue range 196-201

ND

55

ND

ND

65

T1C807 65

F46S, Y54H, T93A, S167R, S217N, M300A, then a contiguous triple délétion in residue range 196-201

T93A

S2I7N, then a contiguous tnple délétion in residue range 196-201

ND

55

ND

ND

66

T1C807 66

F46S, Y54H, T93A, S167R, S2I7N, H287A, then a contiguous triple

T93A

S2I7N, then a contiguous triple ddetion in residue

ND

53

ND

ND

un u>

Amino add différence* compared toTIC807 parent protein

Lygushet.

Lfgui tlneolaris

SEQ ID NO-

Protri· Name

Amino add différence!*) relative to SEQ ID NO:Î (T1C807)

Ami io add différence!*) In the first patch

Amino acid différence!*) in the second patch

LC50 vaine (pg/mL)

fold Increased toiidty (tC50)

% Mortallty at about 13 pg/mL protein*

LC50 vaine (pg/mL)

foid increased toiidty (LC50)

% Mortallty at about 100 pg/mL protein*

ddeuon m residue range 196-201

range 196-201

67

T1C807 67

F46S, Y54 H. S95A. S167R, S2I7N, M300A, then a contiguous triple ddetïon in residue range 196-201

S95A

S217N, then a contîguous triple ddetïon in residue range 196-201

ND

52

ND

ND

68

TIC807 68

F46S, Y54H, F147A, S167R, S217N, 1275a. then a contîguous triple ddetïon in residue range 196-201

F147A

S217N, then a contiguous triple ddetïon in residue range 196-201

ND

52

ND

ND

69

T1C807 69

F46S, Y54H. S95A, S167R, S217N, T247A, then a contiguous triple deietion in residue range 196-201

S95A

S217N, then a contiguous triple deietion in residue range 196-201

ND

51

ND

ND

70

TIC807 70

F46S, Y54H. F147A. S167R, V175A. S2I7N, then a contîguous triple ddetïon in residue range 196-201

F147A

S217N, then a contiguous triple ddeuon in residue range 196-201

ND

50

ND

ND

71

T1C807 71

F46S. Y54H. F147A, S159A. SI67R, S217N, then a contîguous triple deietion in residue range 196-201

F147A

S217N, then a contiguous triple ddeuon in residue range 196-201

ND

50

ND

ND

72

TIC807 72

F46S, Y54H. S95A, F147A, S167R, S217N, then a contîguous triple deietion in residue range 196-201

S95A.FI47A

S217N, then a contiguous triple ddeuon in residue tange 196-201

ND

48

ND

ND

73

T1C807 73

F46S, Y54H. F147A, S167R, LI87A, S217N. then a contîguous triple deietion ïn residue range 196-201

F147A

S217N, then a contiguous triple ddeuon ïn residue range 196-201

ND

47

ND

ND

74

T1C807 74

F46S. Y54H. T93A, SI67R, Tl 82A, S217N. then a contîguous triple ddetïon in residue range 196-201

T93A

S2I7N, then a contiguous triple deirtion in residue range 196-201

ND

47

ND

ND

75

TIC807 75

F46S, Y54H, FI47A,

FI47A

S2I7N, then a

ND

-

46

ND

-

ND

LH

4*

Amin· add différences compared t· TIC807 parent protein

Lygus hey

Lygus tiueolaris

SEQ ID NO-

Protda Name

Amino add differenceft) relative to SEQ ID NO:2 (T1C807)

Amino add différence^) la the first patch

Amin· add differencefs) la the second patch

LC50 value (pg/mL)

fold increased toxicity (LC50)

% Mortality at about 1-3 pg/mL protein*

LC50 value (pg/mL)

fold increased toxicity (LC50)

% Mortality at about 100 pghnL protda*

SI67R, S217N, T245A, then a contiguous triple ddetion in residue range 196-201

contiguous triple ddetion in residue range 196-201

76

T1C807 76

F46S, Y54H, T93A, SI67R, S217N, S249A, then a contiguous triple ddetion in residue range 196-201

T93A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

46

ND

ND

77

T1C807 77

F46S, Y54H, T93A, QI49A, SI67R, S217N, then a contiguous triple ddetion in residue range 196-201

T93A.QI49A

S2I7N, then a contiguous triple ddetion in residue range 196-201

ND

45

ND

ND

78

TTC807 78

F46S. Y54H. T93A, SI5ÎA, SI67R, S217N, then a contiguous triple ddetion in residue range 196-201

T93A.SI5IA

S2I7N, then a contiguous triple ddetion in residue range 196-201

ND

45

ND

ND

79

TIC807 79

F46S, Y54 H, Q70A, FI47A, S167R, S2I7N, then a contiguous triple ddetion m residue range 196-201

F147A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

45

ND

ND

80

T1C807 80

F46S, Y54H, S95A, QI48A, SI67R, S2I7N, then a contiguous triple ddetion in residue range 196-201

S95A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

44

ND

ND

81

TTC807 81

F46S. Y54H, T93A, SÎ67R, S217N, T274A, then contiguous triple ddetion in residue range 196-201

T93A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

44

ND

ND

82

T1C807 82

F46S, Y54H, T93A, S167R, S217N. then a contiguous triple ddetion in residue range 196-201

T93A

S2I7N, then a contiguous triple ddetion in residue range 196-201

ND

43

ND

ND

83

17C807 83

F46S, Y54H, T93A, S167R, MI77A, S217N. then a contiguous triple ddetion in residue range

T93A

S2I7N, then a contiguous triple ddetion in residue range 196-201

ND

42

ND

ND

U! U!

Amino acid différence* compared to TIC807 parent protein

Lygus 6«

ΡΛΤίΙ

Lygus lineolaris

SEQ ID NO:

Protein N*me

Amino ocid différence^) relative to SEQ 1D NO:2 (T1C807)

Amin· acid différence^) In the first patch

Amin· acid differencefs) in the second patch

LCS0 vaine (pg/mL)

fold increased toxicity (LC50)

% Mortality at aboat 1-3 pg/mL proteia*

LC50 vaine (pg'mL)

fold increased toxicity (LC50)

% Mortality at about 100 pg/mL protein*

196-201

84

T1C807 84

F46S, Y54H, S9SA. SI67R, S217N, V250A. then a contiguous triple dcietion in residue range 196-201

S95A

S2I7N. then a contiguous triple dcietion in residue range 196-201

ND

42

ND

ND

85

T1C807 85

F46S, Y54H, T93A. EI55A, S167R, S217N. then a contiguous triple dcietion in residue fange 196-201

T93A

S2I7N, then a contiguous triple dcietion în residue range 196-201

ND

42

ND

ND

86

T1C807 86

F46S, Y54H, II34A, F147A, SI67R, S217N, then a contiguous triple dcietion în residue range 196-201

F147A

S217N, then contiguous tnple dcietion in residue range 196-201

ND

41

ND

ND

87

T1C807 87

F46S. Y54 H, T93A, SI67R, S2I7N, T245A, then a contiguous triple dcietion in residue range 196-201

T93A

S217N, then a contiguous tnple dcietion în residue range 196-201

ND

41

ND

ND

88

TIC807 88

F46S. Y54 H, T93A, S167 R, S217N, then a contiguous triple dcietion in residue range 196-201 Followed by a S198 A

T93A

S2I7N, then contiguous triple dcietion in residue range 196-201 Followed by SI98A

ND

40

ND

ND

89

T1C807 89

F46S, Y54H, F147A. SI67R, S2I7N. N295A, then a contiguous triple dcietion in residue range 196-201

FI47A

S2I7N, then a contiguous tnple dcietion in residue range 196-201

ND

40

ND

ND

90

T1C807 90

F46S. Y54H, A87S. F147A. S167R, S217N, then a contiguous triple dcietion în residue range 196-201

Fl 47 A

S217N, then a contiguous tnple dcietion in residue range 196-201

ND

38

ND

ND

91

T1C807 91

F46S, Y54H, FI47A, SJ67R, S2I7N. S249A. then a contiguous triple deleuon in residue range 196-201

FI47A

S217N, then a contiguous tnple dcietion în residue range 196-201

ND

38

ND

ND

92

TIC807 92

F46S. Y54H, S95A,

S95A

S2I7N, then a

ND

-

37

ND

-

ND

Ui σι

Amino add differente* compared to TIC807 parent protein

Lnuthm

PCTVt

Lrgus lineolaris

SEQ ID NO-

Protêt· Name

Amino add différence/·) relative to SEQ ID NO:2 (11C807)

Amino add différence/*) in the first patch

Amino add difference/t) in the secoud patch

LC 50 value (pg/mL)

fold iacreaied loxidty (LC50)

% Mortality at about 1-3 pg/mL protein*

LC50 vaine (pg/mL)

fold increitcd toxicity (LC50)

·/· Mortality et about 100 pg/mL protein*

SI67R, S2I7N, G75A, then a contiguous triple ddetion in residue range 196-201

contiguous triple ddetion in residue range 196-201

93

T1C807 93

F46S. Y54 H, T93A, A139S, SI67R, S2I7N, then a contiguous tnple délétion in residue range 196-201

T93A

S2I7N, then a contiguous triple ddetion in residue range 196-201

ND

37

ND

ND

94

T1C807 94

F46S, Y54H, F147A, SI67R, S2I7N, P3O5A, then a contiguous triple ddetion in residue range 196-201

F147A

S2I7N, then a contiguous triple ddetion in residue range 196-201

ND

36

ND

ND

95

T1C807 95

F46S, Y54 H, FI47A, S167R, S217N, then a contiguous triple ddetion in residue range 196-201 followed by a S198A

FI47A

S2I7N, then a contiguous triple ddetion in residue range 196-201 followed by S198 A

ND

36

ND

ND

96

T1C807 96

F46S. Y54H, FI47A, S167R, S217N, S252A, then a contiguous triple ddetion in residue range 196-201

F147A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

36

ND

ND

97

TÏC807 97

F46S. Y54H, T9JA, F147A, S167R, S217N, then a contiguous triple ddetion in residue range 196-201

T93A.F147A

S217N. then a contiguous triple ddetion in residue range 196-201

ND

36

ND

ND

98

T1C8O7 9g

F46S. Y54H, FI47A, SI67R, S2I7N. V250A, then a contiguous triple ddetion in residue range 196-201

FI47A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

36

ND

ND

99

1X807 99

F46S, Y54H, FI47A, S167R, S217N, T243A, then a contiguous triple ddetion in residue range 196-201

FI47A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

35

ND

ND

100

1X807 100

F46S. Y54H, F147A, S167R, S2I7N. then a contiguous triple

F147A

S2I7N. then a contiguous triple ddetion in residue

ND

35

ND

ND

ιη Μ

Amino acid différences compared to T1C807 parent protein

LjTuAes

pénis

Lyxus lùwlarb

SEQ ID NO

Protêt· Ni me

Amino add différence^) relative to SEQ ID NO:Î (TIC807)

Amin· add différence^) » the first patch

Amin· add dlffertncefs) la the second patch

LC50 value (PR/mL)

fold Incrensed toxlclty (LCS0)

% Mortality at about 1-3 pg/mL protdn*

LC50 value (pgfoiL)

fold Increased toxldty (LCS0)

% Mortality at about 100 pg/mL protdn*

ddetion in residue range 196-201 followed by a YI97A

range 196-201 followed by Yl 97 A

loi

TIC807 101

F46S, Y54H. S95A, S167R, S217N, N295A, then a contiguous triple ddetion în residue range 196-201

S95A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

35

ND

f

ND

102

TIC807 102

F46S, Y54H, F147A, S167R, S2I7N, then * contiguous tnple ddetion in residue range 196-201 '

Fl 47 A

S217N, then a conttguous triple ddetion in residue range 196-201

ND

34

ND

•

ND

103

TIC807 103

F46S, Y54H, FI47A, S167R, S217N, E299A, then · contiguous triple ddetion in residue range 196-201

« « Fl 47 A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

32

ND

•

ND

104

T1C807 104

F46S, Y54H, S95A, SI67R, S217N, R282A, then a contiguous triple ddetion in residue range 196-20!

S9SA

S217N, then * contiguous triple ddetion in residue range 196-20!

ND

32

ND

ND

105

T1C807 I0S

F46S, Y 54 H, T93A, SI67R, S2I7N. 1306A, then a contiguous triple ddetion in residue range 196-201

T93A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

32

ND

ND

106

T1C8O7 106

F46S. Y54H, S95A, SI67R, S2I7N, S249A, then a contiguous triple ddetion in residue range 196-201

S95A

S217N. then a contiguous triple ddetion in residue range 196-201

ND

30

ND

ND

107

TIC807 107

F46S. Y54H, A87S, T93A, S167R, S2I7N, then a contiguous triple ddetion tn residue range 196-201

T93A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

30

ND

ND

108

T1C807 108

F46S, Y54H, T93A, SI59A, SI67R, S217N. then a contiguous triple ddetion tn residue range 196-201

T93A

S217N, then a contiguous tnple ddetion in residue range 196-201

ND

29

ND

ND

LH

Amin· add differente* compared te TIC807 parent protda

Lyjpn ht\

perw

Lnpa lineolarit

SEQ ID NO

Protdn Name

Amin· add dlfferencefr) relative t· SEQ ID NO:2 (TIC807)

Amine add diHerenccf*) la the first patch

Amin· add differtnetfs) la the second patch

LC50 value (pg/mL)

fold Increawd toxidty (LCS0)

% Mortality at about 1-3 pg/mL protda·

LCS0 value (pg/mL)

fold Increattd toxidty (LC50)

% Mortality at about 100 pgfmL protda*

109

77C807 109

F46S, Y54H, FI47A, S167R, S217N, T303A. then a contiguous triple ddetion in residue range 196-201

F147A

S217N. then a contiguous triple ddetion in residue range 196-201

ND

28

ND

ND

110

TIC807 110

F46S. Y54H, T93A, Q148A, S167R. S217N, then a contiguous triple ddetion in residue range 196-201

793 A

S217N, then a contiguous tnpie ddetion in residue range 196-201

ND

27

ND

ND

111

T1C807 111

F46S. Y54H, S95A. SI67R, V175A, S217N. then a contiguous triple ddetion in residue range 196-201

S95A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

27

ND

ND

112

T1C807 112

F46S, Y54H, F147A, SI67R, S217N. D241A, then a contiguous triple ddetion in residue range 196-201

FI47A

S217N. then a contiguous triple ddetion in residue range 196-201

ND

27

ND

ND

113

T1C807 113

F46S, Y54H, S95A, FI47A. SI67R, S2I7N. then a contiguous triple ddetion in residue range 196-201

S95A.FI47A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

26

ND

ND

11*

T1C8O7 114

F46S, Y54H, F147A, S167R, M177A, S217N. then a contiguous triple ddetion in residue range 196-201

FI47A

S2I7N, then a contiguous triple ddetion in residue range 196-201

ND

26

ND

ND

115

TIC807 115

F46S. Y54 H, F147A, SI67R, S217N, M300A, then a contiguous tnpie ddetion in residue range 196-201

FI47A

S2I7N, then a contiguous triple ddetion in residue range 196-201

ND

26

ND

ND

116

TIC807 116

F46S. Y54H, S95A, S167R, S217N, P305A, then a contiguous tnpie ddetion in residue range 196-201

S95A

S2I7N, then a contiguous triple ddetion in residue range 196-201

ND

26

ND

ND

117

T1C807 117

F46S, Y54H, FI47A, S167R, S2I7N, then a contiguous triple

Fl 47 A

S2I7N, then a contiguous triple ddetion in residue

ND

25

ND

ND

VI <O

Amino add différence* compared to TIC807 parent protein

Lygus hn

penrf

Lygus lineolaris

SEQ ID NO:

Proie!· Name

Amino add différence!·) relative to SEQ ID NO:2 (TIC807)

Amino add diffcrenccO) la the first patch

Amino add differencc(s) la the second patch

LC 50 value ipg/mL)

fold Increased toxicity (LC50)

% Mortality at about 1-3 pg/mL protdn*

LC50 value (pg/mL)

fold Increased toxidty (LC 50)

% Mortality at about 100 pg/mL protdn*

ddction m residue range 196-201 followed by H196 A

range 196-201 followed by H196 A

118

T1C807 ilS

F46S, Y54H, A139S, FI47A, S167R, S2I7N. then a contiguous triple délétion in residue range 196-201

FI47A

S217N, then a contiguous triple dclction in residue range 196-201

ND

25

ND

ND

119

T1C807 119

F46S. Y54H. T93A, SI67R, S217N, N295A, then a contiguous triple délétion in residue range 196-201

T93A

S2I7N, then a contiguous triple ddction in residue range 196-201

ND

25

ND

ND

120

1TC807 120

F46S. Y54H. T93A, T145A, SI67R, S2I7N, then a contiguous triple ddction in residue range 196-201

T93A

S217N. then a contiguous triple ddction in residue range 196-201

ND

24

ND

ND

121

T1C807 121

F46S. Y54H, SI17A, FI47A, S167R, S2i7N, then a contiguous triple dclction in residue range 196-201

FI47A

S217N, then a contiguous triple ddction in residue range 196-201

ND

24

ND

ND

122

TIC807 122

F46S. Y54H. T93A, SI67R, S217N, Π75Α, then a contiguous triple dclction in residue range 196-201

T93A

S217N, then a contiguous triple ddction in residue range 196-201

ND

23

ND

ND

123

TX7807 123

F46S, Y54H. S95A, S167R, S217N, H287A. then a contiguous triple dclction in residue range 196-201

S95A

S217N. then a contiguous triple ddction in residue range 196-201

ND

23

ND

ND

124

T1C807 124

F46S, Y54H, S95A, GI05A, SI67R. S2I7N, then a contiguous triple dclction in residue range 196-201

S95A

S217N, then a contiguous triple ddction in residue range 196-201

ND

21

ND

ND

125

T1C807 125

F46S, Y54H, S95A, S167R, S217N, then a contiguous triple dclction in residue range 196-201

S95A

S217N. then a contiguous triple ddction m residue range 196-201

ND

20

ND

ND

σι

Amino acid dilTcrencci compared to T1C807 parent protein

Lygus Aet

Lygus tineolaris

SEQ ID NO

Protdn N» me

Amino acid difTertnctfi) relative lo SEQ ID NO:Î (T1C807)

Amino add difierencef*) ta the tint patch

Amino add différences) ’he secoad patch

LC50 valve (pg/mL)

fold Increased toxldty (LC50) .

% Mortaiity at about 1-3 pg/mL proteia*

LCS9 value (pg/mL)

fold increased toxldty (LC50)

% Mortaiity at about 100 pg/mL protdn*

126

T1C807 126

F46S, Y54H, S95A, Il 34A, S167R, S217N. then a contiguous tripie délétion în residue range 196-201

S9SA

S217N, then a contiguous triple ddetion in residue range 196-201

ND

20

ND

ND

127

T1C807 127

F46S, Y54H, T93A, S167R, S217N, M300A, then a contiguous triple ddetion in residue range 196-201

T93A

S217N, then a contiguous tripie ddetion in residue range 196-201

ND

20

ND

ND

128

T1C807 128

F46S, Y54H. T93A, S167R, S217N, T3O3A. then a contiguous triple ddetion in residue range 196-201

T93A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

18

ND

ND

129

T1C807 129

F46S. Y54H. T93A, A1S0S, S167R, S217N, then a contiguous triple ddetion in residue range 196-201

T93A

S217N, then a contiguous triple deietion in residue range 196-201

ND

18

ND

ND

130

T1C807 130

F46S. Y54H, S9SA. E15SA. S167 R, S217N, then a contiguous triple ddetion in residue range 196-201

S9SA

S2I7N. then a contiguous triple ddetion in residue range 196-201

ND

18

ND

ND

131

T1C807 131

F46S, Y54H, T93A, T145A, S167 R, S217N, then a contiguous triple ddetion in residue range 196-201

T93A

S2I7N, then a contiguous triple ddetion in residue range 196-201

ND

17

ND

ND

132

TIC807 132

F46S, Y54H, S95A, S167R, W208A. S217N, then a contiguous triple ddetion in residue range 196-201

S9SA

W2O8A, S217N, then a contiguous triple ddetion in residue range 196201

ND

17

ND

ND

133

TIC807 133

F46S, Y54H, S95A, S167R, TI82A. S217N, then a contiguous triple ddetion in residue range 196-201

S95A.T182A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

17

ND

ND

134

TIC807 134

F46S, Y54H, T93A, S167R, S217N, T243A, then a contiguous triple

T93A

S217N, then a contiguous triple ddetion in residue

ND

17

ND

ND

σι

Amino acid différences compared to Tl0807 parent protein

Lniuhn

pen»

Lvxus llnrolarii

SEQ ID NO:

Protri· Nam*

Amino acid dlfferencefs) relative to SEQ1DNO:2 (TIC807)

Amino add différences) 1» the Ont patch

Amino acid différence^) the second patch

LC50 value (pg/mL)

fold Increased toxicity (LC50)

% Mortality at about 1-3 pg/mL protein*

LC50 value (pg/mL)

fold Increased toxicity (LC50)

% Mortality at about 100 pgfrnL protrio*

dclction in residue range 196-201

range 196-201

135

T1C807 I3S

F46S, Y54H, S95A. S167R, S217N, I306A. then a contiguous triple ddetion in residue range 196-201

S95A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

16

ND

ND

136

TIC807 136

F46S, Y54H. S95A. SI17A. S167R, S217N, then a contiguous triple dclction in residue range 196-201

S9SA

S2I7N, then a contiguous tnple ddetion in residue range 196-201

ND

16

ND

ND

137

T1C807 137

F46S. Y54H, T93A. SI19A, SI67R, S2I7N, then a contiguous triple dclction in residue range 196-201

T93A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

IS

ND

ND

138

T1C807 138

F46S, Y54H, T68A. FI47A. SI67R, S217N, then a contiguous triple ddetion in residue range 196-201

Fl 47 A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

15

ND

ND

139

1TC807 139

F46S, Y54H, GI05A. FI47A. SI67R, S217N. then a contiguous triple ddetion in residue range 196-201

FI47A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

15

ND

ND

140

7TC807 140

F46S. Y54H, S95A. El 25 A, SI67R, S217N, then a contiguous triple ddetion in residue range 196-201

S95A

S2I7N, then a contiguous triple ddetion in residue range 196-201

ND

15

ND

ND

141

T1C807 141

F46S, Y54H. T93A, EI55A, S167R, S217N. then a contiguous triple ddetion in residue range 196-201

T93A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

14

ND

ND

142

T1C807 142

F46S, Y54H. T93A, S167 R, S217N, P3O5A, then a contiguous triple ddetion in residue range 196-201

T93A

S2I7N, then a contiguous tnple dclction in residue range 196-201

ND

14

ND

ND

143

TIC807 143

F46S, Y54H, S95A,

S95A

S2I7N, then a

ND

-

13

ND

-

ND

σι

Amino add différence· compared to T1C807 parent protdn

Lyxul hn.

pentr

Lnpu lineolaris

SEQ ID NO:

Protda Name

Amino add dlfferenccfi) rdatlve to SEQ ID NO:2 (TIC807)

Amino add dlffercnccft) la the first patch

Amine add dlfferenccfa) la the second patch

LC50 value (pg/mL)

fold increaied toxldty (LC50)

·/. Mortality at about 1-3 Pg/mL protda*

LC50 vaine (pg/mL)

fold lacreaied toxldty (LC50)

*/· Mortality at about 100 pg/mL protdn*

SI67R, MI77A, S217N, then a contiguous triple ddetion in residue range 196-201

contiguous triple ddetion in residue range 196-201

144

T1C807 144

F46S, Y54 H, T93A, S167R, S2I7N, then a contiguous triple ddetion in residue range 196-201 followed by H196A

T93A

S217N, then a contiguous triple ddetion in residue range 196-201 followed by H196A

ND

13

ND

ND

145

TIC807 145

F46S, Y54H, T93A. D153A, S167R, S2I7N, then a contiguous triple ddetion in residue range 196-20I-T93A-DI53A

T93A

S2I7N, then a contiguous triple ddetion in residue range 196-201

ND

11

ND

ND

146

T1C807 146

F46S, Y54H, F147 A, SI67R, S217N, W208A, then a contiguous triple ddetion in residue range 196-201

F147A

W2O8A, S217N, then a contiguous triple ddetion in residue range 196201

ND

10

ND

ND

147

TIC807 147

F46S. Y54H. T93A, SI67R, S217N, 1306A, then a contiguous triple ddetion in residue range 196-201

T93A

S2I7N, then a contiguous triple ddetion in residue range 196-201

ND

9

ND

ND

148

T1C807 148

F46S, Y54H, S95A, SI67R, S217N, then a contiguous triple ddetion in residue range 196-201 followed by HI96A

S95A

S2I7N, then a contiguous triple ddetion in residue range 196-201 followed by H196 A

ND

9

ND

ND

149

T1C807 149

F46S, Y54H. S95A. QI49A, S167R, S2I7N, then a contiguous triple ddetion in residue range 196-201

S95A.QI49A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

9

ND

ND

150

TIC807 150

F46S, Y54H. S95A, S167R, S217N, S293A. then a contiguous triple ddetion in residue range 196-201

S95A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

8

ND

ND

151

TIC807 151

F46S, Y54H, S95A. AI50S,SI67R. S217N,

S95A

S217N, then a contiguous triple

ND

8

ND

ND

<n u»

Amino ndd difFerences compared toTIC807 parent protria

Zygitshesi

pmts

SEQ ID NO·

Protri· Name

Amiao add difTerenecH) relative to SEQ ID NO:2 (TIC807)

Amino add differencefs) 1· <he first patch

Amino add differencd1) 1» the second patch

LC 50 vaine (pg/mL)

fold Increased toxldty (LC50)

% Mortality at about 1-3 pg/mL protein*

LC50 value (jig/mL)

Ibid increased toxldty (LC50)

*/· Mortality at about 100 pg/mL protri n*

then a contiguous triple ddetion in residue range 196-201

ddetion m residue range 196-201

IS2

T1C807 152

F46S, Y54H, SII9A. F147A. SI67R, S217N. then a contiguous triple ddetion in residue range 196-201

Fl 47 A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

7

ND

ND

153

T1C807 153

F46S, ¥54 H, S95A. S167A. S217N, then a contiguous triple ddetion in residue range 196-201

S95A

S217N, then a contiguous tnple ddetion in residue range 196-201

ND

7

ND

ND

154

nCR07 154

F46S, Y54H, Fl 47 A. I158A, SI67R, S2I7N, then a contiguous triple délétion ïn residue range 196-201

Fl 47 A

S2I7N. then a contiguous triple ddetion in residue range 196-201

ND

7

ND

ND

155

T1C807 155

F46S. Y54H, S95A, S167R, S217N. M300A. then a contiguous triple ddetion in residue range 196-201

S95A

S2I7N, then a contiguous triple délétion in residue range 196-201

ND

7

ND

ND

156

T1C807 156

F46S, Y54H. F147 A. T182A. S167R, S217N, then a contiguous triple ddetion in residue range 196-201

F147A.T182A

S217N, then a contiguous triple ddetion m residue range 196-201

ND

6

ND

ND

157

T1CS07 157

F46S. Y54H. F147 A. SI67R. S217N, F235A, then a contiguous triple ddetion in residue range 196-201

FI47A

S217N, then a contiguous triple ddetion ïn residue range 196-201

ND

5

ND

ND

ISS

TIC807 158

F46S. Y54H. F147A. S167A, S2I7N. then a contiguous triple ddetion in residue range 196-201

FI47A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

5

ND

ND

159

T1C807 159

F46S, Y54H, T93A. S167R, S217N, D24IA. then a contiguous triple ddetion in residue range 196-201

T93A

S217N, then a contiguous triple ddetion ra residue range 196-201

ND

5

ND

ND

σι

And no add dUTertnces compared to TIC807 parent protein

Lygtahest

Lfjia lineo/aris

SEQ ID NO1

Protêt· Ni me

Amino add difTerencefs) relative te SEQ ID NO:2 (T1C807)

Amine acid dilTerencefs) in the Ont patch

Amine acid diflerencefs) ln the second patch

LC50 vaine (pg/mL)

fold Incrtased toxldty (LC50)

% Mortality at abont 1-3 pg/mL protein*

LC50 vaine (pg/mL)

fold iacreased toxldty (LC50)

% Mortality at about 100 pg/mL protein*

160

TIC807 160

F46S, Y54H, S95A, S167R, S217N. T274A, tbcn a contiguous triple ddetion in residue range 196-201

S95A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

4

ND

ND

161

T1C807 161

F46S, Y54H, T9ÎA, Q148A, S167R, S2I7N, tbcn a contiguous triple ddetion in residue range 196-201

T93A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

4

ND

ND

162

T1C807 162

F46S, Y54H, S95A, S167R. S217N, D241A, then a contiguous triple ddetion m residue range 196-201

S95A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

4

ND

ND

163

T1C807 163

F46S, Y54H, S95A, E155A, S167R, S2I7N, then a contiguous triple ddetion in residue range 196-201

S95A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

4

ND

ND

164

T1C807 164

F46S. Y54H, T93A, S167R, U 87A, S217N, tbcn a contiguous triple ddetion in residue range 196-201

T93A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

4

ND

ND

163

TIC807 165

F46S, Y54H, T93A, S167R, S217N, T303A. then a contiguous triple ddetion in residue range 196-201

T93A

S217N, then a contiguous triple ddetion in residue range 196-201

ND

4

ND

ND

166

TIC807 166

F46S, Y54H, T93A, El 55 A, S167R, S217N, then a contiguous triple ddetion in residue range 196-201

T93A

S2I7N, then a contiguous triple ddetion in residue range 196-201

ND

3

ND

ND

167

TIC807 167

F46S, Y54H, T93A, D153A, SI67R, S217N, then a contiguous triple ddetion in residue range 196-201

T93A

S2I7N, tbcn a contiguous triple ddetion in residue range 196-201

ND

3

ND

ND

I6S

T1C807 168

F46S, Y54H, T93A, 1134A, S167R, S217N. then a contiguous triple

T93A

S2I7N, tbcn a contiguous triple ddetion in residue

ND

3

ND

ND

Amin· acid différences compared to TIC807 parent protein

Lyftahts,

Lypts liitnlirii

SEQ ID NO-

Protdn Name

Amino acid differcnccf») relative to SEQ ID NO-.2 (T1C807)

Amino add différence^) In the first patch

Amino add differencefi) ln the second patch

LC50 value (pg/mL)

fold lacreaied toxldty (LC50)

% Mortality at about 1-9 pg/mL protein*

LC50 value (pg/mL)

fold Increased toxldty (LC50)

% Mortality at about 100 pg/mL protdn*

delctton in residue range 196-201

range 196-201

169

T1C807 169

N137T

none

None

ND

44*

ND

ND

169

TIC807 169

E135S

none

None

ND

20*

ND

ND

169

TIC807 169

NI37T.E135S

none

None

ND

34*

ND

ND

169

TIC807 169

E135T, NI37D

none

None

ND

16*

ND

ND

169

T1C807 169

T133E

none

None

ND

60*

ND

ND

169

TIC807 169

E135A.NI37G

none

None

ND

50*

ND

ND

170

TIC807 170

EI25C

none

None

ND

12

ND

ND

170

TIC807 170

EI25H

none

None

ND

38

ND

ND

170

TIC807 170

E125R

none

None

ND

14

ND

ND

170

TIC807 170

EI25F

none

None

ND

33

ND

ND

170

TIC807 170

E125S

none

None

ND

24

ND

ND

170

TIC807 170

E125Q

none

None

ND

21

ND

ND

170

TIC807 170

EI25K

none

None

ND

20

ND

ND

170

T1C807 170

EI25T

none

None

ND

33

ND

ND

170

TIC807 170

EI25N

none

None

ND

19

ND

ND

170

TIC807 170

EI25A

none

None

ND

41

ND

ND

170

TIC807 170

EI25L

none

None

ND

13

ND

ND

170

T1C807 170

E125V

none

None

ND

14

ND

ND

170

T1C807 170

EI25M

none

None

ND

13

ND

ND

170

TIC8O7 170

EI25D

none

None

ND

15

ND

ND

170

T1C8O7 170

EI25Y

none

None

ND

38

ND

ND

171

TICS 07 171

TI33E

none

None

ND

23

ND

ND

171

TIC8O7 171

TI33Y

none

None

ND

17

ND

ND

171

TIC807 171

TI33W

none

None

ND

13

ND

ND

172

T1C807 172

II34 V

none

None

ND

18

ND

ND

172

TIC807 172

II34L

none

None

ND

10

ND

ND

172

T1C807 172

1134F

none

None

ND

18

ND

ND

172

TIC8O7 172

Il 34 K

none

None

ND

13

ND

ND

172

TIC807 172

II34C

none

None

ND

30

ND

ND

172

T1C807 172

II34M

none

None

ND

33

ND

ND

173

TIC807 173

EI35V

none

None

ND

13

ND

ND

173

TIC807 173

EI35W

none

None

ND

13

ND

ND

173

TIC807 173

EI35T

none

None

ND

39

ND

ND

174

T1C807 174

NI37H

none

None

ND

42

ND

ND

174

T1C807 174

NI37Y

none

None

ND

17

ND

ND

174

TIC807 174

N137T

none

None

ND

31

ND

ND

174

T1C807 174

N137E

none

None

ND

32

ND

ND

174

TIC807 174

N137S

none

None

ND

24

ND

ND

174

TIC807 174

NI37A

none

None

ND

24

ND

ND

σ> σ>

Amino add différence* compared toTIC807 parent protein

Lygtuhm

i)T«ï Himkrit

SEQ ID Να

Protêt· Name

Amino acid differeocef*) relative to SEQ ID NO:Ï (T1C807)

Amino add différence!*) in lhe first patch

Amino add différence!*) in the tecood patch

LC50 vaine (pg/mL)

fold Increased toxidty (LC50)

% Mortality at abont 1-3 Pg/mL proteia*

LC50 vaine (pg/mL)

fold Increased toxidty (LCSA)

% Mortality at about 100 pg/mL protda*

174

T1C807 174

NI37Q

none

None

ND

21

ND

•

ND

174

TIC8O7 174

NI37G

none

None

ND

18

ND

ND

174

TIC807 174

N1371

no ne

None

ND

10

ND

ND

174

TIC807 174

NI37W

none

None

ND

17

ND

ND

174

TIC807 174

NI37K.

none

None

ND

66

ND

ND

174

TIC8O7 174

N137C

none

None

ND

19

ND

ND

174

TIC8O7 174

NI37M

none

None

ND

41

ND

ND

174

TIC8O7 174

NI37D

none

None

ND

69

ND

ND

174

TIC807 174

N137F

none

None

ND

13

ND

ND

174

TIC8O7 174

NI37R

none

None

ND

37

ND

ND

175

TIC8O7 175

F147V

FI47V

None

ND

87

ND

ND

175

TIC8O7 175

FI47T

FI47T

None

ND

68

ND

ND

175

TIC8O7 175

FI47C

FI47C

None

ND

74

ND

ND

175

TIC8O7 175

F147L

FI47L

None

ND

62

ND

ND

175

TIC8O7 175

FI47D

FI47D

None

ND

51

ND

ND

175

TIC8O7 175

FI47A

FI47A

None

ND

57

ND

ND

175

TIC807 175

F147G

FI47G

None

ND

56

ND

ND

175

TIC807 175

FI47E

F147E

None

ND

50

ND

ND

175

T1C8O7 175

FI47I

Fl 471

None

ND

69

ND

ND

175

TIC8O7 175

FI47Y

F147Y

None

ND

67

ND

ND

175

TIC8O7 175

F147M

FJ47M

None

ND

64

ND

ND

175

TIC8O7 175

FI47N

FJ47N

None

ND

64

ND

ND

175

TIC8O7 175

F147Q

FI47Q

None

ND

50

ND

ND

175

TIC8O7 175

FI47H

FJ47H

None

ND

60

ND

ND

175

TIC807 175

FI47R

F147R

None

ND

20

ND

ND

175

TIC8O7 175

F147W

F147W

None

ND

82

ND

ND

175

TIC8O7 175

FI47P

F147P

None

ND

7

ND

ND

176

TIC8O7 176

Q149D

Q149D

None

ND

92

ND

ND

176

TIC807 176

Q149E

QJ49E

None

ND

89

ND

ND

176

T1C8O7 176

QI49C

Q149C

None

ND

87

ND

ND

176

TIC807 176

QI49A

Q149A

None

ND

76

ND

ND

176

TIC807 176

QI49F

Q149F

None

ND

54

ND

ND

177

TIC8O7 177

Al SOS

none

None

ND

34

ND

ND

177

TIC8O7 177

AI50L

none

None

ND

24

ND

ND

177

TIC8O7 177

A150 V

none

None

ND

25

ND

ND

177

TIC8O7 177

A150G

none

None

ND

28

ND

ND

177

T1C807 177

AJ50D

none

None

ND

19

ND

ND

177

TIC8O7 177

AI50W

none

None

ND

13

ND

ND

177

TIC8O7 177

AJ50E

none

None

ND

24

ND

ND

177

TIC807 177

A) SON

none

None

ND

18

ND

ND

177

TIC807 177

Al SOY

none

None

ND

H

ND

ND

σ> Μ

Amino add différences compared to TIC807 parent protdn

Lygus Ars

perut

Lygus Bneoiaris

SEQ ID NO:

Protein Name

Amino add difference(s} relative to SEQ ID NO:2 (TIC807)

Amino add differenccfi) la the first patch

Ami» acid differencefs) la the second patch

LC50 vaine Gig/mL)

fold increased toxldty (LC50)

% Mortality at abont 1-3 pg/mL protein*

LC50 value (pg/mL)

fold Increased toxldty (LC50)

% Mortality at about 100 pg/mL protda*

177

TIC807 177

A150F

none

None

ND

11

ND

ND

177

TIC8O7 177

A150P

none

None

ND

II

ND

ND

177

TIC807 177

A150K

none

None

ND

17

ND

ND

»77

T1C807 177

Al SOT

none

None

ND

17

ND

ND

177

TIC8O7 177

A150Q

none

None

ND

II

ND

ND

177

TIC8O7 177

A150 R

none

None

ND

11

ND

ND

178

TIC8O7 178

EI5SC

none

None

ND

82

ND

ND

178

TIC807 178

EI551

none

None

ND

36

ND

ND

178

TIC8O7 178

EI55K.

none

None

ND

28

ND

ND

178

TIC807 178

EI55D

none

None

ND

22

ND

ND

178

TIC807 178

EI55H

none

None

ND

22

ND

ND

178

TIC8O7 178

EI55Y

none

None

ND

16

ND

ND

178

TIC8O7 178

EI55Q

none

None

ND

16

ND

ND

178

TIC807 178

EI55L

none

None

ND

15

ND

ND

178

TIC807 178

EI55N

none

None

ND

14

ND

ND

178

TIC807 178

E155T

none

None

ND

13

ND

ND

178

TIC807 178

E155A

none

None

ND

II

ND

ND

178

TIC807 178

E155F

none

None

ND

7

ND

ND

178

TIC807 178

E155R

none

None

ND

6

ND

ND

178

T1C807 178

EI55M

none

None

ND

6

ND

ND

178

TIC807 178

E155P

none

None

ND

5

ND

ND

178

TIC807 178

EI55W

none

None

ND

5

ND

ND

178

TIC807 178

EI55S

none

None

ND

4

ND

ND

178

T1C807 178

El 55 V

none

None

ND

4

ND

ND

179

T1C807 179

NI57C

none

None

ND

86

ND

ND

179

TIC807 179

N157D

none

None

ND

64

ND

ND

179

TIC807 179

N157W

none

None

ND

52

ND

ND

179

TIC807 179

N157 Y

none

None

ND

39

ND

ND

179

TIC807 179

NI57M

none

None

ND

22

ND

ND

179

T1C807 179

N157 A

none

None

ND

22

ND

ND

179

TIC807 179

NI57F

none

None

ND

20

ND

ND

179

TIC8O7 179

N157V

none

None

ND

18

ND

ND

179

T1C807 179

N157L

none

None

ND

18

ND

ND

179

TIC807 (79

NI57P

none

None

ND

18

ND

ND

179

TIC807 179

NI57E

none

None

ND

17

ND

ND

179

TIC807 179

N157T

none

None

ND

8

ND

ND

179

TIC807 179

NI57G

none

None

ND

7

ND

ND

179

TIC807 179

N1571

none

None

ND

7

ND

ND

179

TIC8O7 179

NI57R

none

None

ND

6

ND

ND

202

T1C807 M8 dSC

F46S. Y54H. S95A, F147A, SI67R. S217N,

S95A, F147A

S217N, then a contiguous triple

ND

92*

ND

ND

Amino add différences compared to TIC807 parent protein

lygnsAn

pma

lygns lineoiaris

SEQ ID NO-

Protein Name

Amino add differente!») relative ta SEQ ID NO:2 (TICJ07)

Amino add différence!·) In tbe first patch

Amino acid différence!·) in the second patch

LC50 vaine ÎPR/mL)

foid Increased toxidty ÎLC50)

% Mortality at abont 1-3 pg/mL protein*

LC50 vaine ÎPg/mL)

foid Increased toxicity (LC50)

% Mortality at about 100 pg/mL protein*

then a five amino acid ddetion at 305-309, then a contiguous tnple ddetion în residue range 196-201

ddetion in residue range 196-201

204

1 TIC807 MI4

F46S, Y54H. S95A. FI47S, QI49E, SI67R. P2I9R, S249R. V25IE, Q253R. R273W, then a contiguous triple ddetion in residue range 196-201

S95A. FI47A, QI49E, V25IE, Q253R

P219R, then a contiguous triple deietion în residue range 196-201

ND

ND

60*

ND = Not Determined; *tested at about 5 pg/mL.

Ch

Example 5: Insect inhibitory activities of protein members of the présent invention |0085] Proteins of the présent invention, such as but not limited to TIC807_Ml (SEQ ID NO:6), TlC807_M2 (SEQ ID NO:8), TIC807_M3 (SEQ ID NO: 10), TIC807.M4 (SEQ (D NO: 12), TIC807_M5 (SEQ ID NO: 14), TIC807_M6 (SEQ ID NO: 18), TIC807.M7 (SEQ ID NO:20), TIC807_M8 (SEQ ID NO: 16), TIC807_M9 (SEQ ID NO:28), TIC807_Mll (SEQ ID NO:32), TIC8O7_M13 (SEQ ID NO:34), and TIC8O7_M12 (SEQ ID NO:36), are prepared and tested for bioactivity against pests of plants other than from Lygus.

)0086( Proteins TIC807_Mt0 (SEQ ID NO:30), TiC807_MU (SEQ ID NO:32), TIC807_M12 (SEQ ID NO:36), and TIC807_Ml3 (SEQ ID NO:34) were prepared and tested for bioactivity against pests from the order Lepidoptera, Coleoptera, Heteroptera, and Homoptera. Protein TIC807_M5 (SEQ ID NO: 14) was prepared and tested for bioactivity against Coleopteran pests. Bioassays were conducted to evaluate the effects of these proteins on insects as shown in Table 5. Feeding assays were conducted on an artificial diet containing the insecticidal protein. The insecticidal protein was prepared as described in example 3 and topically applied using an insect-specific artificial diet, depending on the insect being tested. The toxin was suspended in a buffer and applied at a rate of 500 pg/mL of sample per well, and in the case of TIC807_M5 of 1000 pg/mL, and then aliowed to dry. Mean stunting scores and population mortalities were determined on three populations of 8 insects per insect species tested. Results were expressed as positive (+) for insect reactions such as stunting and mortality that were statistically significant compared to the untreated control. Results were expressed as négative (-) if the insects were similar to the UTC, that is, feeding diet to which the above buffer only has been applied.

Table 5. eHTP’s demonstrate additional insect inhibitory activities against pests other than Lygus spp.

Protein	pg/mL	CPB	WCR	ECB	SWCB	CEW	FAW	SGSB	NBSB	GPA
UTC	0	-		-	-	-	-	-	-	-
TIC807 M5	1000	+		ND	ND	ND	ND	ND	ND	ND
TlC807 Ml0	500	-		-	-	-	-	-	-	-
T1C8O7 M1I	500	+		-	-	-	-	-	-	-
T1C8O7 M12	500	-		-	-	-	-	-	-	-
T1C8O7 M13	500	+		-	-	-	-	-	-	-

UTC = UnTreated Control; ND = Not Determined

CPB = Colorado potato beetle (Leptinotarsa decemlineata); WCR = western corn rootworm (Diabratica virgifera); ECB = European corn borer (Ostrinia nubilalis); southwestern corn borer (Dlatraea grandiosella); CEW = corn earworm (Helicoverpa zeo); FAW = Fall armyworm (Spodaptera frugiperda); SGS0 = southern green stink bug (Nezara virudula); NBSB = neotropical brown stïnk bug (Euschistus heras); GPA = Green peach aphid (Myzus persicae).

[0087] The proteins of the présent invention are also tested for bioactivity against a pest from the phylum Nematoda.

Example 6: Plants expressing proteins of the présent invention exhlblt insect inhibitory activity [0088] This example illustrâtes expression of proteins of the présent invention in plants, and demonstrates that cotton plants expressing proteins of the présent invention exhibit insect inhibitory activity.

[0089] Polynucleotide segments for use in expression of the proteins of the présent invention in plants are made according to the methods set forth in US Patent No. 7,741,118. For example, toxin proteins having the amino acid sequence as set forth in SEQ ID NO:4 (TlC807_4), SEQ ID NO:6 (TIC807_Mi), SEQ ID NO:8 (TIC807_M2), SEQ ID NO: 10 (TlC807_M3), SEQ ID NO: 12 (TIC807_M4), SEQ ID NO: 14 (TIC807_M5), SEQ ID NO:i6 (TIC807_M8), SEQ ID NO: 18 (TIC807_M6), SEQ ID NO:20 (TIC807.M7), SEQ ID NO:22 (T1C807 22), SEQ ID NO:24 (TlC807_24), SEQ ID NO:26 (TIC807_26), SEQ ID NO:28 (TIC807_M9), SEQ ID NO:30 (TIC807_M10), SEQ ID NO:32 (TlC807_MiI), and SEQ ID NO:34 (TIC8O7_M13), are expressed from polynucleotide segments designed for use in plants and encoding the proteins of the présent invention, including the polynucleotide sequences as set forth in SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:I9I, SEQ ID NO:I92, SEQ ID NO:I93, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO:200, and SEQ ID NO:201, respectively.

[0090] It is intended that polynucleotide segments (or polynucleotide molécules) encoding each of the variant proteins or insect inhibitory fragments thereof, be used alone or in combination with each other, or in combination with other insect inhibitory proteins or insect inhibitory agents such as dsRNA mediated gene suppression molécules. Such combinations designed to work in synergistic or compatible mechanism with the proteins of the présent invention. The intention of these combinations is to achieve plants and plant cells protected from pest, particulariy insect pest, infestation. The spécifie variant proteins within the scope of the invention include the proteins corresponding to SEQ ID NOs listed in Table 4B and described throughout the application as filed.

[0091] Polynucleotide segments from SEQ ID NO: 188 (encodes for TIC807_M2, SEQ ID NO: 8) and from SEQ ID NO: 192 (encodes for TIC807_M8, SEQ ID NO: 16) were each recombinantly engineered into expression constructs for cotton transformation.

]0092] Transgenic cotton plants (recombinant cotton plants) were produced and tested for efficacy. Regenerated (RO) transgenic plants were selected that were low in copy number and high in expression of the respective variant protein, as determined by various quantitative and semi-quantitative methods, e.g. PCR, ELISAs and Westerns. Expression levels in RO cotton leaf tissue typically ranged from 0.5 to 500 ppm fresh weight. R0 plants expressing high levels of protein were transferred to soil and selfed. Thirty seed from each of the selfed R0 plants were planted and progeny homozygous for the transgene were grown to flowering. Eleven to 18 plants per 4 to 5 events per each construct of this example were tested for efficacy against Lygus (Tables 6A, 6B, and 6C). The untransformed cotton cultivar, plants from the pooled négative segregate population (progeny not containing the transgene), and plants expressing TIC807 parent protein served as négative controls. A branch of a flowering stage cotton plant was enclosed in a mesh bag made from breathable plastic ‘pollination* sleeves (Vilutis and Co. Inc., Frankfort, IL), and multiple branches set up in similar fashion. Each mesh bag was secured at the stem using a twist tie. About 4-6 Lygus hesperus nymphs (<24 hours post-hatch) were placed into a 1.4 ml conica! tube (Matrix Technologies Corp., NH). The branch inside a mesh bag was infested with nymphs by sliding the uncapped conical tube into the mesh bag. Insects were allowed to feed for a period of 10-11 days before ail surviving insects in the mesh bag were collected on dry ice. Survivors were weighed to obtain a gross mass. Percent mortality and mean survivor mass were calculated. Missing insects were inciuded in the mortality percent mortality calculation. As shown in Tables 6A, 6B, and 6C, cotton plants expressing the variant proteins TIC807_M2 and TIC807_M8 significantly impacted the growth and development of Lygus hesperus nymphs. Based on these results, these plants, seed, expression constructs were advanced for further development.

Table 6A. Mean % mortality determined from flowering stage Lygus feeding assays with

cotton plants	expressing t	ie variant TIC807 proteins TIC807 M2 and TIC807 M8.
Plant Event ID	Protein	N	Mean % Mortality	Std Dev	SEM	Lo95%	L!p95·/.	Mortality t group
64	T1C807 M8	18	78X89	19 967	4 706	68 959	88 818	A
49	T1C807 M8	18	75X56	22 288	5253	64 472	86 639	A
91	T1C807 M2	18	74.444	20 356	4 798	64 321	84 567	A
20	T1C807 M8	18	73X33	19 403	4 573	63 685	82 982	A
15	TIC807 M8	18	66.667	25 668	6 050	53 902	79 431	AB
58	T1C807 M2	18	65X56	19 166	4517	56 025	75 086	AB
48	TIC807 M2	18	«4.444	21.206	4 998	53 899	74 990	AB
19	TIC807 M2	18	53X33	25 668	6 050	40,569	66 098	BC

Plant Event ID 68	Protein TIC807 M2	N 18	Mean % Mortallty 47.771	Std Dev 25 795	SEM 6 080	Lo95% 34 950	Up95% 60 605	Mortallty t group C
Négative		24	41.667	22 001	4 491	32.376	50 957	C

Table 6B. Mean Instar determined from flowering stage Lygus feeding assays with cotton

plants expressing the variant T1C807 proteins Ί	riC807 M2andTIC807 M8.
Plant Event ID	Contint et	N	Mean Inttar	Std Dev	SEM	Lo95%	Up 95%	Initar t group
64	TIC807 M8	II	3.636	0 552	0 166	3 266	4 007	C
68	TIC8O7 M2	16	3.949	0 803	0301	3 521	4 377	BC
48	TIC8O7 M2	16	4.042	0604	0151	3 720	4 364	BC
58	TIC8O7 M2	17	4.069	0 802	0 194	3 657	4 481	BC
15	TIC8O7 M8	15	4.094	0 747	0 193	3 681	4 508	BC
19	TIC8O7 M2	17	4.100	0 698	0 169	3 741	4 459	BC
91	TIC807 M2	12	4.12S	0 829	0339	3 598	4 652	ABC
49	TIC8O7 M8	12	4.139	0 762	0 220	3 655	4 623	ABC
20	TIC807 M8	14	4398	0918	0345	3 768	4 828	AB
Négative		24	4599	0 774	0158	4 273	4 926	A

Table 6C. Mean Survival Mass determined from flowering stage Lygus feeding assays with

cotton plants expressing the variant '	[1C807 proteins TIC807 M2 and T1C807 M8.
Plant Event ID	Contint et	N	Mean Survival Ma»	Std Dev	SEM	Lo 95%	Up 95%	Survivor Man t group
64	TIC807 M8	il	23IS	1 489	0 449	1 314	3315	C
68	TIC807 M2	16	3548	1.325	0331	2 843	4 254	B
58	TIC807 M2	17	3561	1.348	0 327	2 868	4 255	B
48	TIC807 M2	16	3596	1 436	0 359	2831	4 362	B
91	TIC807 M2	12	3.77S	1 775	0512	2 647	4 902	AB
49	TIC807 M8	12	3537	2 135	0616	2481	5.193	AB
19	TIC8O7 M2	17	3.908	1 467	0 356	3 154	4 662	AB
20	TIC807 M8	14	3.918	1 950	0 521	2 792	5044	AB
15	TIC807 M8	15	3.937	1.906	0 492	2 881	4 993	AB
Négative		24	4.73S	1 179	024)	4 237	5 233	A

Std Dev standard déviation

SEM = Standard error on the mean

Lo 95% - Lower limit at 95% confidence interval

Up 95% - Upper limil al 95% confidence interval

T grouping Using a leasl significant différence test, F value - 101.1756, df= 15,44, Pr< 0.0001

100931 In another example, cotton plants from five transgenic events expressing TIC807_Mll were tcsted in a field trial having naturel Lygus infestation pressures. These plants demonstrated field efficacy compared to the non-transgenic récipient line (DP393 germplasm used for transformation). The average number of Lygus lineolaris insects on five plants per event was significantly lower than the average number of Lygus lineolaris insects on plants from the non-transgenic control. Seed cotton yield from plants from the five events was statistically comparable to seed cotton yield of the non-transgenic control, e.g. seasonlong square rétention.

[0094| In another similar field trial, cotton plants from seven transgenic events expressing

TIC807_M10 demonstrated field efficacy compared to the non-transgenic control. The average number of Lygus lineolaris insects on five plants per event was significantly lower than the average number of Lygus lineolaris insects on plants from the non-transgenic control. Seed cotton yieid from plants from three of the seven events was statistically higher than to seed cotton yieid of the non-transgenic control.

[00951 In another example, cotton plants from thirty-four transgenic events expressing TIC807_M13 demonstrated growth chamber efficacy compared to the non-transgenic control. Mesh bags were placed around the whole cotton plants at flowering stage (instead of just around single branches described earlier in this example). Five plants per event were evaluated and the average number of Lygus lineolaris insects recovered (nymphs to adults to 2™* génération Lygus) per plant was significantly lower than the average number of Lygus lineolaris insects per non-transgenic plant.

[0096| Similar experiments are performed with plants expressing proteins listed in Table 1 and in Tables 4A and 4B.

Example 7: Tissue from alfalfa plants expressing proteins of the présent invention exhibit insect inhibitory activity [0097] This example illustrâtes expression of proteins of the present invention in alfalfa plants, and demonstrates that tissue from alfalfa plants expressing proteins of the present invention exhibit insect inhibitory activity.

100981 Polynucleotide segment from SEQ ID NO: 192 (encodes for TIC807_M8, SEQ ID NO: 16) was recombinantly engineered into three differently configured expression constructs for alfalfa transformation. For purposes of data reporting, the three recombinant constructs are coded [ER], [ES], and [ET].

[0099J Transgenic alfalfa plants (recombinant alfalfa plants) were recovered from transformants that were outerossed and then selfed. Recombinant alfalfa plants were selected that were low in copy number and high in TIC807 expression as determined by RT-PCR and semi-quantitative Western methods, respectively. Alfalfa plant tissue from ten separate events were pooled, lyophilized, ground, and resuspended in stock buffer, 25 mM NaCarb, pH10.5. Plant tissue from Alfalfa having no TIC807_M8 expressing transgene was prepared for use as control. Stock préparations were serially diluted 100, 300, and 900 fold for incorporation into Lygus diet. Using the feeding assay method of Example 4, mortality and stunting scores were determined on day 5 and compared to contrôla (See Tables 7A and 7B; data were analyzed using JMP4 statistical software). For each test saniple and each dilution, three populations of eight nymphs were subjected to this bioassay. Stunting scores correspond to visual mass ratings where 0 = no différence to négative control, 1 = about 25% less mass, 2 = about 50% less mass, and 3 = about 75% less mass. The average of the stunting scores for each population of eight nymphs is reported.

Table 7A. Mean % mortality determined from Lygus feeding assays with diet incorporated with tissue from alfalfa plants expressing the variant T1C807 protein T1C807 M8.

Construct	Sample Source	Dilution fold	Mean Stunting Score	SidDev	SEM	Lower 95%	Upper 95%	t Grouplng
[ER] TIC807_M8		100	200	000	000	200	2.00	CD
300	000	000	000	000	000	E
900	000	000	000	000	000	E
[ES] TtC807_M8	Pooled Alfalfa tissue from 10 events per construct	100	300	000	000	300	300	A
300	2.33	0 58	033	090	3 77	BC
900	000	000	000	000	000	E
[ET] TIC807_M8		100	2 67	0 58	033	1 23	4 10	AB
300	167	0 58	0 33	0 23	310	D
900	000	000	000	000	000	E
None	Control Alfalfa	100	200	000	000	200	200	CD
300	000	000	000	000	000	E
900	000	000	000	000	000	E
No Alfalfa incorporated in the diet	0	000	000	000	000	000	E

Table 7B. Mean stunting determined from Lygus feeding assays with diet incorporated with tissue from alfalfa plants expressing the variant TIC807 protein TIC807 M8.

Construct	Sample Source	Dilution fold	Mean Percent mortality	Sld Dev	SEM	Lower 95%	Upper 95%	t Grouplng
[ER] TIC8O7_M8	Pooled Alfalfa tissue from 10 events pcr construct	100	4 17	122	4 17	-13.76	2209	CD
300	000	000	000	000	000	CD
900	13 10	12 54	724	• 1806	44 25	CD
[ES] TIC807_M8	100	56 55	627	3 62	40 97	7212	AB
300	41 67	1909	1102	•5 77	8910	B
900	000	000	000	000	000	CD
[et] TIC8O7_M8	100	64 8 8	1991	11.50	1542	114 34	A
300	16 67	1909	Il 02	-30.77	6410	C
900	12 50	12 50	722	-18 55	43 55	CD
None	Control Alfalfa	100	12 50	12 50	722	• 18 55	43.55	CD
300	000	000	000	000	000	CD
900	8 33	14 43	8 33	-27 52	4419	CD
No Alfalfa incorporated in the diet	0	2 50	7 01	181	-1 38	6.38	D

Example 8; Plants co-expressing an eHTP and a second insect Inhibitory protein exhibiting Lygus species inhibitory activity |00100| Protein samples were prepared containing various mixtures of TIC 1415 and

TIC807_M13 and tested in bioassay. The TIC 1415 protein and other Lygus inhibitory 15 proteins are described in PCT Patent Application Publication No. WO 2012/139004. Sample mixtures were fed to Lygus lineolaris using bioactivity assay. TIC 1415 protein alone and

TIC807_M13 alone were also prepared as positive controls. Buffer was used as négative control. Samples from ail three types of préparations exhibited mortality against Lygus lineolaris and survivors were stunted. Mortality and stunting scores were significant compared to bioactivity scores of insects fed with buffer (see Table 8A). The data suggests that there are no antagonistic effects. Additional bioassay tests are performed on mixtures to demonstrate synergistic and/or additive effects.

Table8A. Bioassay data for proteinmix: T1C1415 combined withTIC807_M13

SAMPLE	TIC1415 (pg/mL)	TIC807_M13 (pg/mL)	Meant Population mortality	T Grouplng on mort	Meant stunting* score	T Grouplng on stunting
TIC1415 + TIC807 M13	4.35	1	21.79	AB*	0.60	AB*
TIC1415 + TIC807 M13	2.175	1	20.36	B*	0.60	AB*
TIC1415 + TIC8O7 M13	1.0875	1	12.50	BC	0.60	AB*
TIC1415 + TIC8O7 M13	4.35	0.5	32.50	A*	0.80	A*
TIC1415 + TIC8O7 M13	1.75	0.265	7.86	CD	0.40	ABC
TIC1415 + TIC8O7 M13	0.875	0.265	0.00	D	0.00	C
TIC1415 + TIC807 M13	0.4375	0.265	5.36	CD	0.00	C
TIC1415 + TIC8O7 M13	4.35	0.25	13.21	BC	0.40	ABC
TIC1415 + TIC8O7 M13	1.75	0.1325	0.00	D	0.00	C
TIC1415 + TIC807 M13	1.75	0.06625	0.00	D	0.00	C
TIC1415	4.35	0	12.50	BC	0.40	ABC
TIC1415	1.75	0	7.86	CD	0.00	C
TIC807 M13	0	1	0.00	D	0.20	BC
TIC807 M13	0	0.265	2.50	CD	0.00	C
Buffer (négative) control	0	0	0.00	D	0.00	C
tAverage (mean) of 5 populal	ions of 8 nymphs per population.

♦ Stunting scores correspond to visual mass ratlngs where 0 = no différence to négative control, 1 = about 25% less mass, 2 = about 50% less mass, and 3 = about 75% less mass. The average of the stunting scores for each population of eight nymphs Is reported.

* At 95% confidence interval.

[00101] Cotton plants comprising events with transgenic DNA were designed to co-express respective proteins TIC 1415 and TIC807_M13. Such plants were evaluated in a caged whole plant assay infested with Lygus lineolaris. Five plants each from ten events were caged and infested with 2 pairs of male and female L. lineolaris per plant. The assay was incubated in a growth chamber under normal environmental conditions for cotton plant development for 21 days. DP393 négative control plants were grown in similar manner. At the end of the 3 week period, Lygus of various stages of development were counted. The mean number per plant of

Lygus hesperus insects at each stage in development were calculated (see Table 8B).

Table 8B. In-planta data for for protein mix: TIC1415 combined with TIC807_Ml3

Mean Mean

	Mean 3rd Instar	Mean 4th Instar	Mean 5th Instar	Llve 2nd Gen.	Total 2nd Gen.	Tukey
Construct	Event	N	or<	Nymphs	Nymphs	Adults	Lygus	5EM	G ro u ping
12	021	5	0.00	0.00	0.00	0.00	0.00	0.00	B
	625	5	0.20	0.20	0.20	0.00	0.60	0.24	B
	830	5	2.20	0.20	0.00	0.00	2.40	1.12	AB
	890	5	4.40	0.00	0.20	0.00	4.60	2.62	AB
	521	5	4.60	0.60	0.00	0.00	5.20	4.27	AB
	980	5	3.40	1.20	1.20	0.00	5.80	4.86	AB
13	426	5	0.00	0.00	0.00	0.00	0.00	0.00	B
	611	5	0.60	0.00	0.00	0.00	0.60	0.60	B
	999	5	0.40	0.00	0.40	0.00	0.80	0.37	B
	356 DP393	5	6.20	0.00	0.40	0.00	6.60	4.73	AB
Inbred	(Négative)	10	7.00	2.50	0.80	0.00	10.30	3.75	A

Claims (20)

1. WHAT IS CLAIMED IS:

   1. An eHTP engineered from a scafïold toxin protein, wherein said eHTP exhibits

   Hemipteran insect species inhibitory activity against a Hemipteran pest species that is from about 2 to about 260 times greater than the Hemipteran inhibitory activity of said scaffold toxin; and optionally wherein said eHTP exhibits at least about 95% amino acid sequence identity to said toxin protein.
2. 2. The eHTP of claim 1, said eHTP having the amino acid sequence as set forth in any of the proteins selected from the group consisting of SEQ ID NO:34, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:I2, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:7I, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:8I, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID N0:100, SEQ ID NO:I0l, SEQ ID NO:I02, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:1I I, SEQ ID NO:I12, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO:II6, SEQ ID NO: 117, SEQ ID NO:1I8, SEQ ID NO:1I9, SEQ ID NO:120, SEQ ID NO:I2I, SEQ ID NO:I22, SEQ ID NO:I23, SEQ ID NO:I24, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:I27, SEQ ID NO:128, SEQ ID NO:I29, SEQ ID NO:I30, SEQ ID NO:I31,

   SEQ ID NO:t32, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID N0:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID N0:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID N0:161, SEQ ID NO:162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID ΝΟ.Ί71, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:202, and SEQ ID NO:204, or an insect inhibitory fragment thereof.
3. 3. The eHTP of claim 1, wherein said scaffold toxin protein is selected from the group consisting of SEQ ID NO:2 (TIC807), SEQ ID NO.-8 (TIC807_M2), SEQ ID NO: 182 (Cry51Aal), SEQ ID NO: 184 (TIC853), and SEQ ID NO:206 (AXMI171).
4. 4. The eHTP of claim 1, further comprising at least one amino acid substitution, at least one amino acid addition, or at least one amino acid délétion in said scaffold toxin protein selected from the group consisting of SEQ ID NO:2 (TIC807), SEQ ID NO:8 (TIC807.M2), SEQ ID NO: 182 (Cry51Aal), SEQ ID NO: 184 (TIC853), and SEQ ID NO:206 (AXMI-171).
5. 5. The eHTP ofclaim 4, wherein in the alternative:

   (i) said amino acid substitution, addition, or délétion is in a solvent accessible amino acid residue ofSEQ ID NO:2; .

   (ii) said amino acid substitution, addition, or délétion is within 3 consecutive amino acid residues of a solvent accessible amino acid residue of SEQ ID NO:2; or .

   (iii) said eHTP has the amino acid sequence as set forth in SEQ ID NO: 180.
6. 6. The eHTP of claim 5, wherein said eHTP comprises at least one substitution or délétion selected from the group consisting of asparagine at position 12 replaced by aspartic acid, phenylalanine at position 46 replaced by serine, isoleucine at position 52 replaced by méthionine, tyrosine at position 54 replaced by histidine, threonine at position 68 replaced by alanine, glutamine at position 70 replaced by alanine, alanine at position 87 replaced by serine, threonine at position 93 replaced by alanine, serine at position 95 replaced by alanine, glycines at position 105 replaced by alanine, serine at position 117 replaced by alanine, serine at position 119 replaced by alanine, glutamate at position 125 replaced by cysteine, histidine, arginine, phenylalanine, serine, glutamine, lysine, threonine, asparagine, alanine, leucine, valine, méthionine, aspartic acid, or tyrosine, glycines at position 128 replaced by alanine, threonine at position 133 replaced by glutamic acid, tyrosine, or tryptophan, isoleucine at position 134 replaced by alanine, valine, leucine, phenylalanine, lysine, cysteine, or méthionine, glutamate at position 135 replaced by serine, alanine, valine, tryptophan, or threonine, asparagine at position 137 replaced by histidine, tyrosine, threonine, glutamic acid, serine, alanine, glutamine, glycine, isoleucine, tryptophan, lysine, cysteine, méthionine, aspartic acid, phenylalanine, or arginine, phenylalanine at position 138 replaced by valine, Alal39 replaced by serine, Thrl45 replaced by alanine, Phel47 replaced by serine, valine, threonine, cysteine, leucine, aspartic acid, alanine, glycine, glutamic acid, isoleucine, tyrosine, méthionine, asparagine, glutamine, hystidine, alanine, arginine, tryptophan, or proline, glutamine at position 148 replaced by alanine, glutamine at position 149 replaced by aspartic acid, glutamic acid, cysteine, alanine, or phenylalanine, alanine at position 150 replaced by serine, leucine, valine, glycine, aspartic acid, tryptophan, glutamic acid, asparagine, tyrosine, phenylalanine, proline, lysine, threonine, glutamine, or arginine, seroine at position 151 replaced by alanine, aspartate at position 153 replaced by alanine, glutamate at position 155 replaced by cysteine, isoleucine, lysine, aspartic acid, histidine, tyrosine, glutamine, lysine, asparagine, threonine, alanine, phenylalanine, arginine, méthionine, proline, tryptophan, serine, or valine, asparagine at position 157 replaced by cysteine, aspartic acid, tryptophan, tyrosine, méthionine, alanine, phenylalanine, valine, leucine, proline, glutamic acid, threonine, glycine, isoleucine, or arginine, isoleucine at position 158 replaced by alanine, serine at position 159 replaced by alanine or threonine, serine at position 167 replaced by arginine or alanine, valine at position 175 replaced by alanine, méthionine at position 177 replaced by alanine, asparagine at position 180 replaced by aspartic acid, threonine at position 182 replaced by alanine, leucine at position 187 replaced by alanine, histidine at position 196 deleted, tyrosine at position 197 deleted, serine at position 198 deleted, histidine at position 199 deleted, tyrosine at position 200 replaced by alanine, tyrosine at position 200 deleted, Ser20l replaced by alanine, serine at position 201 délétion, tryptophan at position 208 replaced by alanine, serine at position 217 replaced by asparagine, proline at position 219 replaced by arginine, tryptophan at position 223 replaced by tyrosine, phenylalanine at position 235 replaced by alanine, asparagine at position 239 replaced by alanine, aspartate at position 241 replaced by alanine, threonine at position 243 replaced by alanine, valine at position 244 replaced by isoleucine, threonine at position 245 replaced by alanine, tyrosine at position 246 replaced by phenylalanine, threonine at position 247 replaced by alanine or lysine, serine at position 249 replaced by alanine or arginine, valine at position 250 replaced by alanine, valine at position 251 replaced by alanine, serine at position 252 replaced by alanine, arginine at position 273 replaced by tryptophan, threonine at position 274 replaced by alanine, isoleucine at position 275 replaced by alanine, arginine at position 282 replaced by alanine, histidine at position 287 replaced by alanine or phenylalanine, serine at position 293 replaced by alanine, asparagine at position 295 replaced by alanine, glutamate at position 299 replaced by alanine, méthionine at position 300 replaced by alanine, threonine at position 303 replaced by alanine, proline at position 305 replaced by alanine, isoleucine at position 306 replaced by alanine, and threonine at position 308 replaced by alanine;

   wherein said substitution or délétion is made with reference to the scaffold toxin protein T1C807 as set forth in SEQ ID NO:2, or to the TIC807 corresponding amino acid residue position of TIC807_M2 as set forth in SEQ ID NO:8, CrySlAal as set forth in SEQ ID NO: 182, or TIC853 as set forth in SEQ ID NO: 184; and wherein said eHTP comprises any combination of said substitutions or délétions.
7. 7. The eHTP of claim 6, wherein said eHTP comprises at least one amino acid substitution, one amino acid addition, or one amino acid délétion at an amino acid residue of SEQ ID NO:2 selected from (i) an amino acid residue having a relative solvent-accessibility of at least from about 15% to about 36%; and (ii) an amino acid residue located within a distance of about 3 consecutive residues from an amino acid having at least from about 15% to about 36% relative solventaccessibility.
8. 8. The eHTP ofclaim 7, wherein said eHTP comprises optionally:

   a. at least one amino acid substitution, one amino acid addition, or one amino acid délétion at an amino acid residue of SEQ ID NO:2 selected from the group consisting of Thr93, Ser95, Ser97, Phel47, Glnl49, Serl51, Asnl80, Thrl82, Val251, Gln253, and Ser255;

   b. at least one additional amino acid substitution, one amino acid addition, or one amino acid délétion at an amino acid residue of SEQ ID NO: 2 selected from the group consisting of Val 10, llel4, Asn22, Asn23, Gly24, Ile25, Gln26, Gly27, Phe30, Gln38, Ile39, Asp40, Thr41, Ile43, Serl93, ThrI94, Glul95, Hisl96, Tyrl97, Serl98, HisI99, Tyr200, Ser201, Gly202, Tyr203, Pro204, Ile205, Leu206, Thr207, Trp208, Ile209, Ser2I0, Tyr216, Ser217, Gly218, Pro219, Pro220, Met221, Ser222, Trp223, Tyr224, Phe225, Asn239, and Val244;

   c. at least one amino acid substitution, one amino acid addition, or one amino acid délétion at an amino acid residue of SEQ ID NO: 2 selected from the group consisting of VallO, Ilel4, Asn22, Asn23, Gly24, Ile25, Gln26, Gly27, Phe30, Gln38, Ile39, Asp40, Thr41, Ile43, Serl93, Thrl94, Glul95, Hisl96, Tyrl97, Ser 198, His 199, Tyr200, Ser201, Gly202, Tyr203, Pro204, Ile2O5, Leu206, Thr207, Trp208, Ile209, Ser210, Tyr216, Ser217, Gly218, Pro219, Pro220, Met221, Ser222, Trp223, Tyr224, Phe225, Asn239, and Val244;

   d. one or more mutations in SEQ ID NO:2 selected from the group consisting of S95A, F147A, Q149E, V251 A, P219R; or

   e. a contiguous triple délétion in SEQ ID NO:2 in residue range 196-201.
9. 9. The eHTP of claim 8, further modified to exhibit increased solubility compared to the native Bacillus thuringiensis protein as set forth in SEQ ID NO:2, wherein said eHTP comprises at least one modification relative to SEQ ID NO:2, said modification comprising a lysine substitution at one or more of the following amino acid positions: 58, 59, 198, 199, 201, or 202; or a glutamic acid substitution at one or more of the amino acid positions 198, 248, or 301; or an arginine substitution at one or more ofthe amino acid positions 246, 250, or 253.
10. 10. The eHTP of claim 9, wherein said Hemipteran pest species is selected from the group consisting of Lygus hesperus, Lygus lineo loris, and Amrasca dévastons.
11. 11. An insect inhibitory composition comprising the eHTP of claim 10.
12. 12. The insect inhibitory composition of claim 11, further comprising at least one insect inhibitory agent different from said eHTP, wherein said insect inhibitory agent is selected from the group consisting of an insect inhibitory protein, an insect inhibitory dsRNA molécule, and an insect inhibitory chemistry, wherein said insect inhibitory protein different from said eHTP is selected from the group consisting of a Cryl toxin, a Cry2 toxîn, a Cry 5 toxin, a Cry7 toxin, a Cry9 toxin, and a VIP3 toxin; and wherein said insect inhibitory chemistry is selected from the group consisting of pyrethrins and synthetic pyrethroids; oxadizine dérivatives; chloronicotinyls; nitroguanidine dérivatives; triazoles; organophosphates; pyrrols; pyrazoles; phenyl pyrazoles; diacylhydrazines; biological/fermentation products; and carbamates.
13. 13. A method of controlling a Hemipteran pest, comprising contacting said pest with a

    Hemipteran inhibitory amount of the eHTP of claim 10, wherein said Hemipteran pest is in a cotton field.
14. 14. A recombinant polynucleotide encoding the eHTP of claim 10, and optionally comprising:

    a. the nucléotide sequence selected from the group consisting of SEQ ID NO:33, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO: 186, SEQ ID

    NO: 187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID

    NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID

    NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID

    NO: 199, SEQ ID N0:200, SEQ ID NO:201, and SEQ ID NO:203; or

    b. a nucléotide sequence encoding one or more insect inhibitory agents that are different from the eHTP encoded by said recombinant polynucleotide.
15. 15. A transgenic plant cell, plant or plant part comprising the recombinant polynucleotide of claim 14, and wherein optionally:

    a. said plant part is a seed, a boll, a leaf, a flower, a stem, a root, or any portion thereof; or

    b. said plant part is a non-regenerable portion of said seed, boll, leaf, flower, stem, or root.
16. 16. A method of controliing a Hemipteran pest, comprising exposing the pest to the transgenic plant cell, plant or plant part of claim 15, wherein said plant cell, plant or plant part expresses a Hemipteran inhibitory amount of said eHTP.
17. 17. A commodity product derived from the plant cell, plant or plnat part of claim 15, wherein said product comprises a détectable amount of said recombinant polynucleotide; and wherein said product is selected from the group consisting of plant biomass, oil, meal, animal feed, flour, flakes, bran, tint, hulls, and processed seed; and wherein optionally said product is non-regenerable.
18. 18. A method of making a plant résistant to Hemipteran pest infestation, comprising the steps of introducing a recombinant polynucleotide encoding an eHTP into a plant cell; regenerating from said plant cell a transgenic plant expressing an insect inhibitory amount of the eHTP; and demonstrating Hemipteran pest infestation résistance as a property of said transgenic plant;

    wherein said plant optionally:

    a. is selected from the group consisting ofa dicot plant and a monocot plant;

    b. is selected from the group consisting of alfalfa, almont, banana, barley, bean, beet, broccoli, cabbage, brassica, brinjal, carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, celery, citrus, coconut, coffee, com, clover, cotton, a cucurbît, cucumber, Douglas fir, eggplant, eucalyptus, flax, garlic, grapc, guar, hops, leek, legumes, lettuee, Loblolly pine, millets, melons, nectarine, nut, oat, okra, olive, onion, ornemental, palm, pasture grass, papaya, pea, peach, peanut, pepper, pigeonpea, pi ne, potato, poplar, pumpkin, Radiata pîne, radish, rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum, Southem pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweet com, sweet gum, sweet potato, switchgrass, tea, tobacco, tomato, triticale, turf grass, watermelon, and wheat; or

    c. comprises a supplémentai agent selected from the group consisting of an insect inhibitory protein different from said eHTP, an insect inhibitory dsRNA molécule, and an insect inhibitory chemistry, wherein said insect inhibitory protein different from said eHTP is selected from the group consisting of a Cryl toxin, a Cry2 toxin, a Cry 5 toxin, a Cry7 toxin, a Cry9 toxin, and a V1P3 toxin; and wherein said insect inhibitory chemistry is selected from the group consisting of pyrethrins and synthetic pyrethroids; oxadizine dérivatives; chloronicotinyls; nitroguanidine dérivatives; triazoles; organophosphates; pyrrols; pyrazoles; phenyl pyrazoles; diacylhydrazines; biological/fermentation products; and carbamates.
19. 19. The method of claim 18, wherein said Hemipteran insect pest is selected from the group consisting of plant bugs (including the Family Miridae), cicadas from the Family Cicadidae, leafhoppers (e. g., Empoasca spp., Amrasca spp. from the Family Cicadellidae including the tribe Empoascini, e.g. Amrasca biguttula, Amrasca dévastons, Austroasca viridigrisea, Asymmetrasca decedens, Empoasca decipiens, Empoasca distïnguenda, Empoasca dolichi, Empoasca fabae, Empoasca kerri, Empoasca kraemeri, Empoasca onukii, Empoasca sakaii, Empoasca smithi, Empoasca vitis, Jacobiasca lybica, Sonasasca Solana, tribe Erythroneurini, e.g. Empoascanara nagpurensis, Thaiaassamensis, Zygnidia quyumi, tribe Nirvaniae, Sophonia rufofascia, Family Delphacidae, e.g. Nilapoarvata lugens, Sogatella furcifera, Unkanodes sapporonus, and Family Lophopidae, e.g. Zophiuma lobtilata), planthoppers (from the familles Fulgoroidea and Delphacidae), treehoppers (from the Family Membracidae), psyllids (from the Family Psyllidae), whiteflies (from the Family Aleyrodidae), aphids (from the Family Aphididae), phylloxéra (from the Family Phylloxeridae), mealybugs (from the Family Pseudococcidae), scales (from the famïlies Coccidae, Diaspididae and Margarodidae), lace bugs (from the Family Tingidae), stink bugs (from the Family Pentatomidae), cinch bugs (e. g., Blissus spp. and other seed bugs from the Family Lygaeidae), spittlebugs (from the Family Cercopidae), squash bugs (from the Family Coreidae), red bugs and cotton stainers (from the Family Pyrrhocoridae), Acrostemum hilare (green stink bug), Anasa tristis (squash bug), Blissus leucopterus leucopterus (chinch bug), Corythuca gossypii (cotton lace bug), Cyrtopeltis modes ta (tomato bug), Dysdercus suture II us (cotton stainer), Eus ch is tus servus (brown stink bug), Euschistus variolarius (one-spotted stink bug), Graptostethus spp. (complex of seed bugs), Leptoglossus corculus (leaf-footed pine seed bug), Lygus lineolaris (tamished plant bug), Lygus hesperus (Western tamish plant bug), Nexara viridula (southem green stink bug), Oebalus pugnax (rice stink bug), Oncopeltus fasciatus (large milkweed bug), and Pseudatomoscelis seriatus (cotton fleahopper).
20. 20. An eHTP (engineered Hemipteran toxic protein) selected from the group consisting of SEQ ID NO:34, of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:lO, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO;40, SEQ ID NO:4l, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:5l, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:6I, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:7l, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ IDNO:80, SEQ ID NO:8I, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:9l, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:lOO, SEQ ID NO:IOl, SEQ ID NO:l02, SEQ ID NO:l03, SEQ ID NO:l04, SEQ ID NO:l05, SEQ ID NO:l06, SEQ ID NO:I07, SEQ ID NO:I08, SEQ ID NO:l09, SEQ ID NO:llO, SEQ ID NO:lll, SEQ ID NO:H2, SEQ ID NO:II3, SEQ ID NO:II4, SEQ ID NO:ll5, SEQ ID NO:Il6, SEQ ID NO:Il7, SEQ ID NO:ll8, SEQ ID NO:lI9, SEQ ID NO:l20,

    SEQ ID N0:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID

    NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129,

    SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ (D

    NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138,

    5 SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ (D NO: 142, SEQ (D

    NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:15I, SEQ ID NO:I52, SEQ ID NO: 153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156,

    SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID 10 NO:161, SEQ ID NO:162, SEQ ID NO:I63, SEQ ID NO:164, SEQ ID NO:165,

    SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174,

    SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID

    NO: 179, SEQ ID NO:202, and SEQ ID NO:204, or an insect inhibitory fragment 15 thereof.