# Patent

## Title
Proteins toxic to hemipteran insect species

## 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.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 13/857,196, filed Apr. 5, 2013, now U.S. Pat. No. 9,322,033, issued Apr. 26, 2016, which claims priority to U.S. Provisional Application Ser. No. 61/621,436, filed Apr. 6, 2012, all of which are incorporated herein by reference in their entireties.


INCORPORATION OF SEQUENCE LISTING
The Sequence Listing contained in the file named “P34307US02.txt”, which is 529,835 bytes in size (measured in operating system MS-Windows) and was created on Feb. 4, 2016, is contemporaneously filed by electronic submission (using the United States Patent Office EFS-Web filing system) and is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
The present invention generally relates to the field of insect inhibitory proteins. In particular, the present invention relates to proteins exhibiting insect inhibitory activity against agriculturally relevant pests of crop plants and seeds, particularly Hemipteran species of insect pests.
BACKGROUND OF THE INVENTION
Insect inhibitory proteins derived from Bacillus thuringiensis (Bt) are non-toxic to humans, vertebrates, and plants. These proteins are also biodegradable, safe, and effective in controlling pest insects. Some of these proteins have 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.
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 have 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 have been developed for use in commercial control of Hemipteran pest species.
Hemipteran specific toxic proteins have been reported in the art. TIC807 is a Bacillus thuringiensis protein disclosed in U.S. Patent Application Publication No. US 2008-0295207 A1 as being toxic to Hemipteran pest species. A Cry51Aa1 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 specific 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 A1). A protein referred to as AXMI-171 was reported to exhibit some limited inhibition of Hemipteran insects (U.S. Patent Application Publication No. US2010-0298207 A1, example 18), particularly Lygus hesperus. 
All 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
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 derivatives of naturally occurring Bacillus thuringiensis insecticidal toxins, TIC807 (SEQ ID NO:2), TIC807_M2 (SEQ ID NO:8), Cry51Aa1 (SEQ ID NO:182), TIC853 (SEQ ID NO:184), and AXMI-171 (SEQ ID NO:206) have been described previously to exhibit bio-control activity directed to Hemipteran pest species, particularly Lygus hesperus insect species (references cited elsewhere herein). The recombinant Hemipteran insect toxic proteins of the present invention are particularly 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 insecticidal toxins TIC807 (SEQ ID NO:2), TIC807_M2 (SEQ ID NO:8), Cry51Aa1 (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 present 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.
The eHTP's of the present invention each contain at least one amino acid substitution, one amino acid addition, or one amino acid deletion 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:182, or SEQ ID NO:184. 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:182 (Cry51Aa1), SEQ ID NO:184 (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:182 (Cry51Aa1). 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 deletion when compared to the amino acid sequence of any of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:182, or SEQ ID NO:184. 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:182, and SEQ ID NO:184. Each of the aforementioned eHTP's contain at least, collectively or in the alternative: (i) the amino acid substitution, addition, or deletion in a solvent accessible amino acid residue of SEQ ID NO:2; (ii) the amino acid substitution, addition, or deletion 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:180. 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 deletion 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 methionine, 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, methionine, 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 methionine, 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, methionine, aspartic acid, phenylalanine, or arginine, phenylalanine at position 138 replaced by valine, Ala139 replaced by serine, Thr145 replaced by alanine, Phe147 replaced by serine, valine, threonine, cysteine, leucine, aspartic acid, alanine, glycine, glutamic acid, isoleucine, tyrosine, methionine, 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, methionine, proline, tryptophan, serine, or valine, asparagine at position 157 replaced by cysteine, aspartic acid, tryptophan, tyrosine, methionine, 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, methionine 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, Ser201 replaced by alanine, serine at position 201 deletion, 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, methionine 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 deletions. eHTP's contain at least one amino acid substitution, one amino acid addition, or one amino acid deletion at an amino acid residue of SEQ ID NO:2, or the corresponding amino acid position of SEQ ID NO:8, SEQ ID NO:182, or SEQ ID NO:184, 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 solvent-accessibility. An eHTP of the present invention contains at least one amino acid substitution, addition, or deletion at an amino acid residue selected from the group consisting of Thr93, Ser95, Ser97, Phe147, Gln149, Ser151, Asn180, Thr182, Val251, 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 deletion at an amino acid residue selected from the group consisting of Val10, Ile14, Asn22, Asn23, Gly24, Ile25, Gln26, Gly27, Phe30, Gln38, Ile39, Asp40, Thr41, Ile43, Ser193, Thr194, Glu195, His196, Tyr197, Ser198, His199, Tyr200, Ser201, Gly202, Tyr203, Pro204, Ile205, Leu206, Thr207, Trp208, Ile209, Ser210, Tyr216, Ser217, Gly218, Pro219, Pro220, Met221, 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:182, or SEQ ID NO:184. 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 deletion of any three consecutive amino acids from amino acid residues 196-201 as set forth in SEQ ID NO:2. Any of the eHTP's of the present invention can be further modified to exhibit increased solubility compared to the underlying naturally occurring Bacillus thuringiensis protein as set forth in any of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:182, or SEQ ID NO:184 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 of the 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: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:71, 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: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 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 NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:13, 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 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 NO: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 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: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 thereof, is a preferred embodiment of the present invention. The target Hemipteran pest species inhibited by the eHTP's of the present invention include at least Lygus hesperus, Lygus lineolaris, Empoasca fabae and Amrasca devastans, 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.
Methods of controlling a Hemipteran pest by contacting the pest with a Hemipteran inhibitory amount of a eHTP of the present invention, as well as an insect inhibitory composition that contains at least a Hemipteran controlling amount (or Hemipteran inhibitory amount) of one or more of the eHTP's of the present 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 least one or more eHTP along with a supplemental agent that is selected from the group consisting of an insect inhibitory protein, an insect inhibitory dsRNA molecule, 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.
Recombinant polynucleotides that encode eHTP's of the present invention are provided. Microbes are also provided that contain the polynucleotides of the present invention, and such polynucleotides within such microbes are functionally positioned within expression cassettes designed to express the eHTP's of the present invention from operably linked functional genetic regulatory elements. 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 polynucleotide. 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 polynucleotide may contain a nucleotide 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: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:33, 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 NO:200, SEQ ID NO:201, and SEQ ID NO:203, or other sequences that can be assembled to encode one or more of the proteins of the present invention. In certain embodiments, the recombinant polynucleotide can further comprise a nucleotide 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.
Processed plant products that contain a detectable amount of a recombinant polynucleotide encoding an eHTP or any distinguishing 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.
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.
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
FIG. 1 illustrates the mortality of Lygus species plotted against eHTP protein concentration. FIG. 1A illustrates 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. FIG. 1B illustrates the mortality of Lygus lineolaris populations in response to various protein concentrations of three different eHTP's compared to a control sample containing the naturally occurring TIC807 protein.
FIG. 2 illustrates a ribbon diagram of the atomic structure of a Hemipteran toxic protein of the present invention showing the relative positions of the result effective changes increasing toxic effects and/or broadening 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 result effective in causing increased toxic properties and broader host range toxic effects compared to a protein having a naturally occurring amino acid at that particular position.
FIG. 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
This application describes eHTP's (engineered Hemipteran species toxic proteins). The eHTP's of the present invention are to be distinguished from proteins such as TIC807, TIC853, Cry51Aa1 and AXMI-171, which are known in the art and are not to be considered to be within the scope or definition of the term eHTP, as the prior art proteins are not engineered to exhibit improved toxic properties directed to one or more Hemipteran pest species and do not exhibit broad host range levels of inhibitory activity. eHTP's surprisingly and unexpectedly exhibit high levels of toxic activity against Hemipteran 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 present invention. The foundational or baseline scaffold toxin proteins, such as TIC807 (SEQ ID NO:2), Cry51Aa1 (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 present invention.
More than 2000 different amino acid sequence variants of Hemipteran toxic proteins derived from Bacillus thuringiensis species were tested to identify the specific amino acid insertions, substitutions, or deletions 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 Cry51Aa1. Amino acid residues are identified 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 exhibiting the fold structure of one or more of the scaffold proteins, and/or (c) occur in specific positions of one or more of the scaffold protein amino acid sequence that are result 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.
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 pooling 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 Aphididae, phylloxera 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 Acrosternum hilare (green stink bug), Anasa tristis (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 (one-spotted stink bug), Graptostethus spp. (complex of seed bugs), Leptoglossus corculus (leaf-footed pine seed bug), Lygus lineolaris (tarnished plant bug), Lygus hesperus (Western tarnish plant bug), Nezara viridula (southern green stink bug), Oebalus pugnax (rice stink bug), Oncopeltus fasciatus (large milkweed bug), and Pseudatomoscelis seriatus (cotton fleahopper). More specifically, the Family Cicadellidae includes, but is not limited to the tribe Empoascini, e.g. Amrasca biguttula, Amrasca devastans, Austroasca viridigrisea, Asymmetrasca decedens, Empoasca decipiens, Empoasca distinguenda, 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, e.g. Sophonia rufofascia, Family Delphacidae, e.g. Nilapoarvata lugens, Sogatella furcifera, Unkanodes sapporonus, and Family Lophopidae, e.g. Zophiuma lobulata. 
eHTP's of the present invention contain one or more amino acid sequence modifications compared to one or more of the scaffold proteins, including substitutions and deletions, of amino acid residues at seventy-two (72) different amino acid positions. Such modifications provide 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), Cry51Aa1 (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 deletion 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 of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:182, or SEQ ID NO:184. eHTP's of the present 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.
eHTP's include at least one amino acid modification of the relative positions of TIC807 (SEQ ID NO:2) as set forth above. eHTP's can also include at least two, three, four, or more of these aforementioned amino acid substitutions and/or deletions and can also include at least two, three, four, or more of these amino acid substitutions and/or deletions as well as a deletion of any three contiguous amino acids within residues 196-201 of SEQ 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:71, 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: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 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 NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO: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 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 NO: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 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: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, and insect inhibitory fragments thereof.
eHTP's of the present invention exhibit any amino acid sequence different from any one or more of the scaffold proteins, including SEQ ID NO:2 (TIC807), 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 inhibitory spectrum and/or increased Hemipteran inhibitory activity when compared to the activity 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 inhibitory 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.
eHTP's exhibit an amino acid sequence that differs from one or more of the scaffold proteins, including particularly TIC807, in at least one amino acid residue located within at least one of the two different surface patches of a folded insect inhibitory protein (see FIG. 2 and Table 3 data). One surface patch 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 (FIG. 2, sphere [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, Phe147, Gln149, Ser151, Asn180, Thr182, 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 present in all amino acid residues with the exception of Glycyl residues. With reference to FIG. 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, T182, V251, Q253, and S255 or the equivalent amino acids within one or more of the scaffold proteins, particularly 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, F147G, F147E, F147Y, F147M, F147N, F147Q, F147H, F147R, F147W, F147P, F147A, F147V, F147L, or F1471; 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 receptive to modifications which are result effective in conferring improved Hemipteran inhibitory bioactivity in the form of eHTP's of the present 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 (FIG. 2, sphere [2]) relative to the beta-carbon atom of Pro219 or the equivalent amino acid position in one or more of the scaffold proteins, 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 Val10, Ile14, Asn22, Asn23, Gly24, Ile25, Gln26, Gly27, Phe30, Gln38, Ile39, Asp40, Thr41, Ile43, Ser193, Thr194, Glu195, His196, Tyr197, Ser198, His199, Tyr200, Ser201, Gly202, Tyr203, Pro204, Ile205, Leu206, Thr207, Trp208, Ile209, Ser210, Tyr216, Ser217, Gly218, Pro219, Pro220, Met221, Ser222, Trp223, Tyr224, Phe225, Asn239, and Val244. 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 deletions within surface patch [2] including Val10, Ile14, Asn22, Asn23, Gly24, Ile25, Gln26, Gly27, Phe30, Gln38, Ile39, Asp40, Thr41, Ile43, Ser193, Thr194, Glu195, His196, Tyr197, Ser198, His199, Tyr200, Ser201, Gly202, Tyr203, Pro204, Ile205, Leu206, Thr207, Trp208, Ile209, Ser210, Tyr216, Ser217, Gly218, Pro219, Pro220, Met221, 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 deletions within the amino acid residues located within surface patch [2] such as: a deletion of any three contiguous amino acid residues in the sequence His196, Tyr197, Ser198, His199, Tyr200, Ser201; Ser217Asn, Ser217Gln, Ser217Arg; and/or Pro219Arg, Pro219Asn, Pro219Gln. eHTP's can include, but are not limited to, one or more amino acid residue substitutions and/or deletions within surface patch [2] such as: a deletion of any three contiguous HisTyrSer residues in the sequence His196, Tyr197, Ser198, His199, Tyr200, Ser201; Ser217Asn, Ser217Gln, Ser217Arg; and/or Pro219Arg, Pro219Asn, Pro219Gln. An eHTP can have at least one amino acid modification in each of the two aforementioned surface patches of the folded insect inhibitory protein. eHTP can have one, or a combination of more than one modification at residues T93, S95, F147, Q149, S151, N180, T182, H196, Y197, S198, H199, Y200, S201, W208, S217, P219, W223, N239, V244, or V251 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, all 267 are amino acid sequence variants that exhibit increased toxicity to Lygus spp. when compared to one or more of the scaffold proteins, including 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.
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 evident when the eHTP is expressed in E. 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, including 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 198, 248 or 301; or, substitution of a arginine amino acid residue at one or more of amino acid positions 246, 250 or 253.
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 included in the composition. The insect inhibitory agent is selected from any number of insect inhibitory agents including an insect inhibitory protein, an insect inhibitory dsRNA molecule, and one or more chemical agents useful in controlling insect pests. Examples of additional inhibitory agents includes, but are not limited to, a TIC1415 protein, a dsRNA directed towards Hemipteran orthologs of Nilapoarvata lugens V-ATPase-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-171 (US20100298207A1), Cry3A, Cry4Aa, Cry11Aa, and Cyt1Aa, DIG11, DIG5, Cry7, eCry3.1Ab, mCry3A, Cry8, Cry34/Cry35, Cry3, DIG2, Cry1, Cry1A.105, Cry2, Cry1F, VIP3, S307, and Cry9. Chemical agents useful in controlling Hemipteran species include but are not limited to pyrethrins and synthetic pyrethroids; oxadizine derivatives; chloronicotinyls; nitroguanidine derivatives; triazoles; organophosphates; pyrrols; pyrazoles; phenyl pyrazoles; diacylhydrazines; biological/fermentation products; and carbamates. Known pesticides within these categories are listed in The Pesticide Manual, 11th Ed., C. D. S. Tomlin, Ed., British Crop Protection Council, Farnham, Surry, UK (1997).
Pyrethroids that are useful in the present composition include pyrethrins and synthetic pyrethroids. The pyrethrins that are preferred for use in the present method include, without limitation, 2-allyl-4-hydroxy-3-methyl-2-cyclopenten-1-one ester of 2,2-dimethyl-3-(2methyl propenyl)-cyclopropane carboxylic acid, and/or (2-methyl-1-propenyl)-2-methoxy-4-oxo-3-(2 propenyl)-2-cyclopenten-1-yl ester and mixtures of cis and trans isomers thereof (Chemical Abstracts Service Registry Number (“CAS RN”) 8003-34-7).
Synthetic pyrethroids that are preferred for use in the present invention include (s)-cyano(3-phenoxyphenyl)methyl 4-chloro alpha (1-methylethyl)benzeneacetate (fenvalerate, CAS RN 51630-58-1), (S)-cyano (3-phenoxyphenyl) methyl (S)-4-chloro-alpha-(1-methylethyl)benzeneacetate (esfenvalerate, CAS RN 66230-04-4), (3-phenoxyphenyl)-methyl(+)cis-trans-3-(2,2-dichoroethenyl)-2,2-dimethylcyclopropanecarboxylate (permethrin, CAS RN 52645-53-1), (±) alpha-cyano-(3-phenoxyphenyl) methyl(+)-cis,trans-3-(2,2-dichloroethenyl)-2,2-dimethyl-cyclopropane carboxylate (cypermethrin, CAS RN 52315-07-8), (beta-cypermethrin, CAS RN 65731-84-2), (theta cypermethrin, CAS RN 71697-59-1), S-cyano (3-phenoxyphenyl) methyl (±) cis/trans 3-(2,2-dichloroethenyl) 2,2 dimethylcyclopropane carboxylate (zeta-cypermethrin, CAS RN 52315-07-8), (s)-alpha-cyano-3-phenoxybenzyl (IR,3R)-3-(2,2-dibromovinyl)-2,2-dimethyl cyclopropanecarboxylate (deltamethrin, CAS RN 52918-63-5), alpha-cyano-3-phenoxybenzyl 2,2,3,3,-tetramethyl cyclopropoanecarboxylate (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-(1 alpha, 3 alpha)-(Z)-(±)-3-(2-chloro-3,3,3-trifluoro-1-propenyl)-2,2-dimethylcyclopropanecarboxylate (tefluthrin, CAS RN 79538-32-2), (±)-cyano (3-phenoxyphenyl) methyl (±)-4-(difluoromethoxy)-alpha-(1-methyl ethyl) benzeneacetate (flucythrinate, CAS RN 70124-77-5), cyano(4-fluoro-3-phenoxyphenyl)methyl 3-[2-chloro-2-(4-chlorophenyl)ethenyl]-2,2-dimethylcyclopropanecarboxylate (flumethrin, CAS RN 69770-45-2), cyano(4-fluoro-3-phenoxyphenyl)methyl 3-(2,2-dichloroethenyl)-2,2-dimethyl-cyclopropanedarboxylate (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)-(IR-cis)-2,2-dimethyl-3-[2-(2,2,2-trifluoro-trifluoromethyl-ethoxycarbonyl)vinyl]cyclopropane 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(1′RS)(1′,2′,2′,2′-tetrabromoethyl)]-2,2-dimethyl cyclopropanecarboxylic acid (s)-alpha-cyano-3-phenoxybenzyl ester (tralomethrin, CAS RN 66841-25-6), cyano-(3-phenoxyphenyl)methyl 2,2-dichloro-1-(4-ethoxyphenyl)cyclopropane carboxylate (cycloprothrin, CAS RN 63935-38-6), [1α,3α(Z)]-(±)-cyano-(3-phenoxyphenyl)methyl 3-(2-chloro-3,3,3-trifluoro-1-propenyl)-2,2-cimethylcyclopropanecarboxylate (cyhalothrin, CAS RN 68085-85-8), [1 alpha (s), 3 alpha(z)]-cyano(3-phenoxyphenyl) methyl-3-(2-chloro-3,3,3-trifluoro-1-propenyl)-2,2-dimethylcyclopropane carboxylate (lambda cyhalothrin, CAS RN 91465-08-6), (2-methyl [1,1′-biphenyl]-3-yl) methyl 3-(2-chloro-3,3,3-trifluoro-1-propenyl)-2,2-dimethyl-cyclopropanecarboxylate (bifenthrin, CAS RN 82657-04-3), 5-1-benzyl-3-furylmethyl-d-cis(1R,3S,E)2,2-dimethyl-3-(2-oxo,-2,2,4,5 tetrahydro thiophenylidenemethyl)cyclopropane carboxylate (kadethrin, RU15525, CAS RN 58769-20-3), [5-(phenyl methyl)-3-furanyl]-3-furanyl 2,2-dimethyl-3-(2-methyl-1-propenyl)cyclopropane carboxylate (resmethrin, CAS RN 10453-86-8), (1R-trans)-[5-(phenylmethyl)-3-furanyl]methyl 2,2-dimethyl-3-(2-methyl-1-propenyl)cyclopropanecarboxylate (bioresmethrin, CAS RN 28434-01-7), 3,4,5,6-tetra hydro-phthalimidomethyl-(IRS)-cis-trans-chrysanthemate (tetramethrin, CAS RN 7696-12-0), 3-phenoxybenzyl-d,l-cis,trans 2,2-dimethyl-3-(2-methylpropenyl) cyclopropane carboxylate (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-(1A,3R; 1R,3 S)-2,2-dimethyl-3-(2-methylprop-1-enyl)cyclopropane carboxylate (allethrin, CAS RN 584-79-2), (bioallethrin, CAS RN 584-79-2), and (ZXI8901, 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 present invention. Particularly preferred synthetic pyrethroids are tefluthrin, lambda cyhalothrin, bifenthrin, permethrin and cyfluthrin. Even more preferred synthetic pyrethroids are tefluthrin and lambda cyhalothrin, and yet more preferred is tefluthrin.
Insecticides that are oxadiazine derivatives are useful in the subject invention. The oxadizine derivatives that are preferred for use in the present invention are those that are identified in U.S. Pat. No. 5,852,012. More preferred oxadiazine derivatives are 5-(2-chloropyrid-5-ylmethyl)-3-methyl-4-nitroiminoperhydro-1,3,5-oxadiazine, 5-(2-chlorothiazol-5-ylmethyl)-3-methyl-4-nitroiminoperhydro-1,3,5-oxadiazine, 3-methyl-4-nitroimino-5-(1-oxido-3-pyridinomethyl)perhydro-1,3,5-oxadiazine, 5-(2-chloro-1-oxido-5-pyridiniomethyl)-3-methyl-4-nitroiminoperhydro-1,3,5-oxidiazine; and 3-methyl-5-(2-methylpyrid-5-ylmethyl)-4-nitroiminoperhydro-1,3,5-oxadiazine. Even more preferred is thiamethoxam (CAS RN 153719-23-4).
Chloronicotinyl insecticides are also useful in the subject invention. Chloronicotinyls that are preferred for use in the subject composition are described in U.S. Pat. No. 5,952,358, and include acetamiprid ((E)-N-[(6-chloro-3-pyridinyl)methyl]-N′-cyano-N-methyleneimidamide, CAS RN 135410-20-7), imidacloprid (1-[(6-chloro-3-pyridinyl)methol]-N-nitro-2-imidazolidinimime, CAS RN 138261-41-3), and nitenpyram (N-[(6-chloro-3-pyridinyl)methyl]-N-ethyl-N′-methyl-2-nitro-1,1-ethenediamine, CAS RN 120738-89-8).
Nitroguanidine insecticides are useful in the present invention. Such nitroguanidines can include those described in U.S. Pat. Nos. 5,633,375, 5,034,404 and 5,245,040.
Pyrrols, pyrazoles and phenyl pyrazoles that are useful in the present invention include those that are described in U.S. Pat. No. 5,952,358. Preferred pyrazoles include chlorfenapyr (4-bromo-2-(4-chlorophenyl)-1-ethoxymethyl-5-trifluoromethylpyrrole-3-carbonitrile, CAS RN 122453-73-0), fenpyroximate ((E)-1,1-dimethylethyl-4[[[[(1,3-dimethyl-5-phenoxy-1H-pyrazole-4-yl)methylene]amino]oxy]methy]benzoate, CAS RN 111812-58-9), and tebufenpyrad (4-chloro-N[[4-1,1-dimethylethyl)phenyl]methyl]-3-ethyl-1-methyl-1H-pyrazole-5-carboxamide, CAS RN 119168-77-3). A preferred phenyl pyrazole is fipronil (5-amino-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(1R, S)-(trifluoromethyl)sulfinyl]-1H-pyrazole-3-carbonitrile, CAS RN 120068-37-3).
Diacylhydrazines that are useful in the present invention include halofenozide (4-chlorobenzoate-2-benzoyl-2-(1,1-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 1-(1,1-dimethylethyl)-2,(4-ethylbenzoyl)hydrazide, CAS RN 112410-23-8).
Triazoles, such as amitrole (CAS RN 61-82-5) and triazamate are useful in the method of the present invention. A preferred triazole is triazamate (ethyl [[1-[(dimethylamino)carbonyl]-3-(1,1-dimethylethyl)-1H-1,2,4-triazol-5-yl]thio]acetate, CAS RN 112143-82-5).
Biological/fermentation products, such as avermectin (abamectin, CAS RN 71751-41-2) and spinosad (XDE-105, CAS RN 131929-60-7) are useful in the present invention.
Organophosphate insecticides are also useful as one of the components of the present 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).
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).
When a chemical insecticide is described herein, it is to be understood that the description is intended to include salt 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.
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 preparations 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.
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, TIC807. The phrases “more active”, “improved activity”, “enhanced specificity”, “increased toxic potenc ” “increased ” “ ” y, 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), TIC807_M2 (SEQ ID NO:8), Cry51Aa1 (SEQ ID NO:182), 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), Cry51Aa1 (SEQ ID NO:182), TIC853 (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:182, and SEQ ID NO:184, where the Hemipteran pest species include Lygus hesperus, Lygus lineolaris, Empoasca fabae, and Amrasca devastans. Amrasca devastans is also called Amrasca biguttula biguttula. eHTP's exhibiting enhanced insect inhibitory spectrum and/or improved insect inhibitory activity compared to TIC807 can be identified by many different methods. In general, exemplary and non-limiting methods for identifying eHTP proteins can comprise:

    
    
        (1) administering identical amounts of a test eHTP and of control TIC807 (SEQ ID NO:2), TIC807_M2 (SEQ ID NO:8), Cry51Aa1 (SEQ ID NO:182), TIC853 (SEQ ID NO:184), and/or an AXMI-171 (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 diet) of a test eHTP and of control TIC807 (SEQ ID NO:2), TIC807_M2 (SEQ ID NO:8), Cry51Aa1 (SEQ ID NO:182), TIC853 (SEQ ID NO:184), and/or an AXMI-171 (SEQ ID NO:206) protein which elicit equivalent 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 would be the median lethal concentration (LC50) required to kill 50% of a test population. However, in certain embodiments, other values including but not limited to, a median inhibitory concentration (“IC50”) required to result 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.

    
    


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 mating behavior and/or any measurable decrease in the adverse effects caused by insect feeding on a composition containing the agent. Similarly, a “Hemipteran inhibitory amount” refers to an amount of a protein of the present 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 present 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 in the adverse effects caused by Lygus, Empoasca and/or Amrasca feeding on a composition containing 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 scaffold proteins, including TIC807, Cry51Aa1(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 present 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 equivalent composition or plant containing only an applicable amount of one or more of the scaffold proteins, including but not limited to TIC807 (SEQ ID NO:2), Cry51Aa1(SEQ ID NO:182), TIC853 (SEQ ID NO:184), and/or AXMI-171 (SEQ ID NO:206) proteins.
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 specific 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 specific 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. 
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 (Cry51Aa1), SEQ ID NO:184 (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 (Cry51Aa1), SEQ ID NO:184 (TIC853), and SEQ ID NO:206 (AXMI-171).
eHTP's can exhibit an enhanced target pest inhibitory spectrum and/or improved target pest inhibitory activity over SEQ ID NO:2 (TIC807), SEQ ID NO:8 (TIC807_M2), SEQ ID NO:182 (Cry51Aa1), SEQ ID NO:184 (TIC853), and/or a SEQ ID NO:206 (AXMI-171) by causing mortality:

    
    
        (i) at a dose of about 0.3 μg/mL to about 70 μg/mL against a Lygus hesperus insect species,
        (ii) at a dose of about 0.85 μg/mL to about 100 μg/mL against a Lygus lineolaris insect species,
        (iii) measuring at an LC50 value of about 0.3 to about 70 μg/mL against Lygus hesperus, 
        (iv) measuring at an LC50 value of about 0.85 to about 100 μg/mL against Lygus lineolaris, or
        (v) measuring at an LC50 value of more than two-fold lower the LC50 value of TIC807, SEQ ID NO:8, SEQ ID NO:182 (Cry51Aa1), SEQ ID NO:184 (TIC853), and/or a SEQ ID NO:206 (AXMI-171) against Lygus spp., Empoasca spp. and/or Amrasca spp., or
        (vi) at a dose of about 0.69 μg/mL to about 500 μ/mL against a Amrasca devastans or Empoasca fabae insect species, or
        (vii) measuring at an LC50 value of about 3.5 to about 15 μg/mL against Amrasca devastans and/or Empoasca fabae. 
    
    


Table 4A and 4B tabulate the exemplary eHTP's of the present invention with Amrasca and Lygus spp. mortality data. Mortality data available for Lygus spp. and Amrasca spp. are reported either as (a) a μg/mL LC50 value, or as (b) a % mortality at doses of about 1 to about 3 μg/mL for L. hesperus or about 100 μg/mL protein for L. lineolaris, and about 0.69 to 500 μg/mL for Amrasca devastans. The fold increased toxicity compared to TIC807 (SEQ ID NO:2) and TIC807_M2 (SEQ ID NO:8) is provided for exemplary eHTP's where LC50 values were determined.
The eHTP's of the present invention are particularly useful in controlling insects of the order Hemiptera compared to the scaffold proteins. Lygus lineolaris required high doses of TIC807 protein (e.g., in excess of 100 μg/mL) to elicit mortality. The dose response curve for one eHTP of the present 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 μg/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 significantly large doses of TIC807 and TIC807_M2 protein in excess of 1000 μg/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 μg/mL (See Tables 1 and 3, Example 4, and FIG. 1B).
Iterative 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 scaffold protein such as TIC807 (SEQ ID NO:2), TIC807_M2 (SEQ ID NO:8), Cry51Aa1 (SEQ ID NO:182), and/or TIC853 (SEQ ID NO:184); or, comparing an eHTP of the present invention against another protein such as AXMI-171 (SEQ ID NO:206). The word “selecting” is intended to refer to the act of identifying improved variant proteins of the present invention, i.e., eHTP's, and the applicable amino acid residues for “engineering”.
Iterative design includes the elucidation of the atomic structure of proteins of the present invention (for example, as set forth in FIG. 2) and the use of the atomic structure to guide and complement 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 regions of a folded insect inhibitory scaffold protein such as TIC807, TIC853, and Cry51Aa1 that can be modified to confer improvements to insect inhibitory spectrum and activity. Such amino acid residues at loops and at surface exposed regions are selected for “engineering”. In this case, iterative design has included the identification of two different regions within the three dimensional structure of the scaffold 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 naturally occurring scaffold protein, result in one or more of the eHTP proteins of the present invention.
Initially the scaffold protein TIC807 (SEQ ID NO:2) used in this process of iterative design, and 267 different eHTP's were discovered that exhibited increased Lygus spp. inhibitory activity compared to the scaffold protein TIC807. TIC807_M8 (SEQ ID NO:16) was discovered in early rounds of the design process. Subsequent rounds of iterative 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 (L. 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 TIC807. The results are shown in Table 1, and FIG. 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 TIC807












Lygus hesperus
Lygus lineolaris












SEQ 

LC50 

LC50 



ID 

value
Toxicity
value
Toxicity


NO:
Toxin
(μg/mL)
(fold increase)
(μg/mL)
(fold increase)





 2
TIC807
73
 1
>223*
   1


 6
TIC807_M1 
23
 3
  100
 ≧2


 8
TIC807_M2 
 5.9
 12
>223*
 ~1


10
TIC807_M3 
 2.9
 25
ND
—


12
TIC807_M4 
 2.4
 30
ND
—


14
TIC807_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
TIC807_M9 
 9.9
 7
   8.3
 ≧27


30
TIC807_M10
 0.6
122
   4.8
 ≧46


32
TIC807_M11
 1.35
 54
   5.9
 ≧38


36
TIC807_M12
 0.4
182
   1.2
≧186


34
TIC807_M13
 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 μg/mL have not been possible to provide in Lygus diet in order to complete 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 μg/mL.






With reference to Table 1, the iterative design process has provided a means for identifying proteins exhibiting improved toxic properties, not only to Lygus hesperus, but also to Lygus lineolaris. 
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 molecule with the open reading frame encoding the protein is operably linked to elements such as a promoter and any other regulatory element functional for expression in the system for which the construct is intended. For example, plant-functional 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 elements 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 sub-cellular 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.
Exemplary recombinant polynucleotide molecules provided herein include, but are not limited to, 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 NO: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_M1), 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_M11), 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 polynucleotide molecule encoding for proteins of the present invention may be substituted for synonymous codons (also called a silent substitution); and are within the scope of the present invention. Recombinant polynucleotides encoding any of the eHTP's disclosed herein are thus provided.
A recombinant DNA construct comprising eHTP coding sequences can also further comprise a region 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 molecule. A recombinant DNA construct can be assembled so that all 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 nucleotide segment on which is also contained other open reading frames and/or promoters depending on the type of expression system selected.
Recombinant polynucleotide 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, subsequent expression to polypeptide. An exogenous recombinant polynucleotide 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”.
Also provided herewith are transgenic bacteria, transgenic plant cells, transgenic plants, and transgenic plant parts that contain any a recombinant polynucleotide (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, corn, clover, 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, ornamental, palm, pasture grass, papaya, pea, peach, peanut, pepper, pigeonpea, pine, potato, poplar, pumpkin, Radiata pine, radish, rapeseed, rice, rootstocks, rye, safflower, shrub, sorghum, Southern pine, soybean, spinach, squash, strawberry, sugar beet, sugarcane, sunflower, sweet corn, 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 “non-regenerable” 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.
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 regeneration, seed, pollen, or meristem transformation techniques.
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 aforementioned 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.
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 present invention, formulations and methods for controlling insect growth and/or infestation, and methods for providing such control to plants and other recipient hosts. Each composition, construct, cell, plant, formulation, method or kit provides for the industrial application of the proteins of the present invention, for example, by increasing plant productivity through the commercial use of any of these proteins to inhibit insects.
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 transgenic plant part, in which the commodity or other products can be tracked through commerce by detecting nucleotide segments, RNA or proteins that corresponding to an eHTP of the present 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.
Also provided herewith are processed plant products that contain a detectable amount of an eHTP, an insect inhibitory fragment thereof, or any distinguishing portion thereof. Without seeking to be limited by theory, it is believed that such processed plant products containing a detectable amount of one or more of the eHTP's provided herein can in certain embodiments exhibit reductions in undesirable microorganisms that can be transmitted by Hemiptera and/or reductions 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 present 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 deletion in the corresponding amino acid sequence as set forth in SEQ ID NO:2. Such substitutions, deletions or additions are those as set forth above.
Processed plant products are provided that contain a detectable amount of a recombinant polynucleotide encoding an eHTP, an eHTP or an insect inhibitory fragment thereof, or any distinguishing portion thereof. The processed product is selected from the group consisting of plant biomass, oil, meal, animal feed, flour, flakes, bran, lint, hulls, and processed seed.
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 present invention. Such transgenic crops produce or are treated to contain an insect or Hemiptera inhibitory amount of an applicable eHTP, and such crops are imbued with sufficient eHTP by (i) applying any composition comprising or encoding an eHTP to the plant or a seed that gives rise to the plant; and/or (ii) 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 transiently 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.
Preferably, the Lygus spp. is Lygus hesperus or Lygus lineolaris, the Empoasca spp. is Empoasca fabae, and the Amrasca spp. is Amrasca devastans. 
Other features and advantages of the invention will be apparent from the following detailed description, examples, and claims.
EXAMPLES
In view of the foregoing, those of skill in the art should appreciate that changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Thus, specific details disclosed herein are not to be interpreted as limiting. The U.S. Provisional Application Ser. No. 61/621,436 to which this application claims the benefit of priority, the Sequence Listing, as well as all 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 1
Iterative Engineering-Testing-Selecting Approach
This example illustrates the random, combinatorial, and inventive aspects of the iterative (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 included 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 iterative engineering-testing-selection approach also included, but was not limited to steps including cloning, expressing, purifying, and bioassay testing of TIC807 control protein compared to the eHTP's.
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 included recombinant nucleotide segments encoding a number of candidate eHTP's derived from library mutagenesis that were not sequenced in the selection process.
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 preparations were prepared for screening higher numbers of variants in bioassay. Larger quantity and higher purity protein stocks were prepared for high-powered bioassays. Testing trended towards the high-powered bioassays as principally relevant residue positions of the improved variants were elucidated. Initially, 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 included twenty-two (22) variants that were confirmed to demonstrate increased toxicity against both Lygus species. Further confirmation and dose response testing narrowed the selection to seven (7) variants that were subsequently characterized using an 8-dose replicated bioassay to determine LC50 values against both Lygus species.
The selection process included dynamic updates of testing data, constantly adjusting engineering approaches, and performing iterative 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
A multiple sequence alignment of protein members of Cry51: Cry51Aa1 (SEQ ID NO:182), TIC853 (SEQ ID NO:184), and TIC807 (SEQ ID NO:2) were used to identify regions of variability, e.g., positions 195 to 201 and positions 211 to 219, relative to SEQ ID NO:2 (TIC807). These regions were targeted for saturation mutagenesis through use of degenerate oligonucleotide primers encoding random amino acid residues in these regions. Construct libraries were prepared for subsequent protein expression in host cells.
A multiple sequence alignment of Cry51Aa1 (SEQ ID NO:182), TIC853 (SEQ ID NO:184), 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, F138X, F147X, S167X, Y216X, P218X, G234X, T247X, D268X, and T308X. Construct libraries were prepared for subsequent protein expression in host cells.
Scanning Approaches
Polynucleotide constructs were engineered to express a single Alanine substitution or a double Alanine substitution (Alanine-<parent residue>-Alanine) at every possible position over the full-length of SEQ ID NO:2 (TIC807). 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





1
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






Where an Alanine residue was already present 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 iterative rounds of engineering-testing-selecting, e. g., TIC807_M2 (SEQ ID NO:8) having mutations F46S, Y54H, S167R, S217N, and a contiguous triple deletion 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, E155X, F147X, I134X, N157X, Q149X, T133X, 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
The atomic structure of proteins of the present invention was determined in the midst of the Iterative Engineering-Testing-Selecting approach; and, the relative solvent-accessibility (% SA) of each residue was determined using Molsoft's ICM-Browser (Molsoft L.L.C., 11199 Sorrento Valley Road, 5209, San Diego, Calif. 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_L11M) 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 solvent-accessible surface area probed by a water molecule 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 molecule. % SA greater than 100 are reported in the table as 100%.
Combined engineering-testing-selecting approaches described herein resulted in a number of principally relevant residues that accumulate in a surface patch ([2] of FIG. 2) of residues having a radius of about 9.2-12.2 Angstroms around the Cb atom of P219 of SEQ ID NO:2 (TIC807): V10, I14, N22, N23, G24, I25, Q26, G27, F30, Q38, I39, D40, T41, I43, S193, T194, E195, H196, Y197, S198, H199, Y200, S201, G202, Y203, P204, I205, L206, T207, W208, I209, 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)
(B)
(C)


TIC807_L11M 
TIC807_M2 
TIC853 


(SEQ ID NO: 185)
(SEQ ID NO: 8)
(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





2ALA
 82.1#
2ALA
 64.8#
2ALA
 60.9#


3ILE
 23.3
3ILE
 28.9
3ILE
 24.3


4LEU
 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


10VALp1
 0.3
10VAL
 0.6
10VAL
 0.0


11MET
 25.0
1ILEU
 17.0
1ILEU
 16.6


12ASN*
 49.8#
12ASN
 43.2#
12ASP
 39.7#


13ALA
 0.0
13ALA
 0.0
13ALA
 0.0


14ILEp1
 0.0
14ILE
 0.0
14ILE
 0.0


15ASN
 23.7
15ASN
 24.9
15ASN
 19.2


16TYR
 29.5
16TYR
 47.1#
16TYR
 52.5#


17TRP
 14.1
17TRP
 18.2
17TRP
 20.1


18GLY
 4.3
18GLY
 1.4
18GLY
 1.0


19PRO
 63.6#
19PRO
 57.3#
19PRO
 59.0#


20LYS
 57.3#
20LYS
 77.2#
20LYS
100#


21ASN
 36.3#
21ASN
 28.4
21ASN
 61.5#


22ASNp1
 16.9
22ASN
 10.1
22ASN
 15.2


23ASNp1
 0.3
23ASN
 0.8
23ASN
 0.0


24GLYp1
 42.0#
24GLY
 43.2#
24GLY
 43.3#


25ILEp1
 10.1
25ILE
 13.8
25ILE
 7.6


26GLNp1
 92.4#
26GLN
 86.2#
26GLN
 94.7#


27GLYp1
 62.0#
27GLY
 73.9#
27GLY
 62.8#


28GLY
 49.0#
28GLY
 50.6#
28TYR
 47.7#


29ASP
 66.0#
29ASP
 68.1#
29ASN
 80.7#


30PHEp1
 4.5
30PHE
 4.1
30PHE
 1.5


31GLY
 37.2#
31GLY
 41.4#
31ASN
 61.1#


32TYR
 25.2
32TYR
 25.3
32TYR
 21.5


33PRO
 70.7#
33PRO
 76.0#
33PRO
 78.5#


34ILE
 4.8
34ILE
 5.5
34ILE
 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#


38GLNp1
 12.8
38GLN
 14.5#
38GLN
 9.0


39ILEp1
 7.8
39ILE
 7.9
39ILE
 5.1


40ASPp1
 52.4#
40ASP
 55.3#
40ASP
 49.8#


41THRp1
 0.3
41THR
 0.0
41THR
 0.2


42SER
 53.1#
42SER
 56.0#
42SER
 53.0#


43ILEp1
 13.1
43ILE
 23.5
43ILE
 25.1


44ILE
 8.3
44ILE
 12.0
44ILE
 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#
48HIS
 65.3#
48HIS
 78.3#


49PRO
 9.4
49PRO
 12.6
49SER
 9.0


50ARG
 58.7#
50ARG
 53.7#
50ARG
 61.5#


5ILEU
 13.7
5ILEU
 8.1
5ILEU
 3.0


52ILE*
 32.4
52ILE
 31.5
52MET
 43.7#


53PRO
 22.2
53PRO
 26.5
53PRO
 22.8


54TYR*
 52.7#
54HIS
 42.2#
54HIS
 45.5#


55ASP
 57.5#
55ASP
 59.2#
55ASP
 55.5#


56LEU
 15.0
56LEU
 18.6
56LEU
 15.1


57THR
 62.0#
57THR
 73.2#
57THR
 80.0#


58ILE
 67.6#
58ILE
 60.9#
58ILE
 68.0#


59PRO
 26.6
59PRO
 21.9
59PRO
 20.7


60GLN
 28.8
60GLN
 20.7
60GLN
 21.3


61ASN
 71.9#
61ASN
 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#
64THR
 49.0#


65ILE
 46.1#
65ILE
 41.5#
65ILE
 38.4#


66PHE
 27.0
66PHE
 26.3
66PHE
 29.6


67THR
 52.5#
67THR
 55.5#
67THR
 62.2#


68THR*
 31.6
68THR
 35.8#
68THR
 35.3


69THR
 54.3#
69THR
 51.1#
69THR
 50.3#


70GLN*
 31.0
70GLN
 36.5#
70GLN
 34.3


71VAL
 53.4#
71VAL
 53.2#
71VAL
 53.1#


72LEU
 7.9
72LEU
 11.8
72LEU
 8.1


73THR
 40.5#
73THR
 44.0#
73THR
 47.0#


74ASN
 0.6
74ASN
 0.0
74ASN
 0.0


75ASN
 69.8#
75ASN
 63.2#
75ASN
 65.9#


76THR
 36.7#
76THR
 40.5#
76THR
 44.0#


77ASP
 80.2#
77ASP
 63.0#
77ASP
 62.1#


78LEU
 62.6#
78LEU
 62.5#
78VAL
 63.8#


79GLN
 74.4#
79GLN
 54.7#
79GLN
 43.5#


80GLN
 33.3
80GLN
 32.1
80GLN
 40.1#


81SER
 81.0#
81SER
 68.6#
81SER
 75.2#


82GLN
 19.0
82GLN
 23.9
82GLN
 24.6


83THR
 62.7#
83THR
 63.6#
83THR
 63.7#


84VAL
 1.8
84VAL
 0.9
84VAL
 0.0


85SER
 50.8#
85SER
 55.7#
85SER
 54.0#


86PHE
 7.2
86PHE
 5.8
86PHE
 4.0


87ALA*
 58.5#
87ALA
 61.9#
87SER
 68.0#


88LYS
 30.5
88LYS
 30.6
88LYS
 32.6


89LYS
 69.8#
89LYS
 67.8#
89LYS
 67.9#


90THR
 19.9
90THR
 23.1
90THR
 16.7


91THR
 54.1#
91THR
 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
96THR
 1.5
96THR
 0.0


97SERp2
 16.6
97SER
 18.5
97SER
 29.9


98THR
 8.2
98THR
 5.9
98THR
 1.9


99THR
 41.8#
99THR
 46.4#
99THR
 49.3#


100ASN
 37.7#
100ASN
 34.1
100ASP
 20.1


101GLY
 1.0
101GLY
 1.9
101GLY
 0.0


102TRP
 3.6
102TRP
 10.4
102TRP
 6.6


103THR
 8.1
103THR
 8.1
103THR
 3.9


104GLU
 9.7
104GLU
 21.9
104GLU
 14.8


105GLY*
 35.3
105GLY
 46.8#
105GLY
 31.4


106GLY
 57.0#
106GLY
 68.6#
106GLY
 61.8#


107LYS
 52.4#
107LYS
 57.2#
107ARG
 54.6#


108ILE
 61.8#
108ILE
 63.5#
108ILE
 67.1#


109SER
 43.5#
109SER
 47.9#
109SER
 47.7#


110ASP
 83.1#
110ASP
 83.5#
110ASP
 65.7#


111THR
 43.4#
111THR
 41.3#
111THR
 39.9#


112LEU
 26.7
112LEU
 29.6
112LEU
 31.3


113GLU
 53.8#
113GLU
 64.1#
113GLU
 62.5#


114GLU
 34.8
114GLU
 30.9
114GLU
 32.6


115LYS
 62.2#
115LYS
 55.2#
115ASN
 54.6#


116VAL
 6.4
116VAL
 8.6
116VAL
 10.4


117SER*
 46.6#
117SER
 48.9#
117SER
 51.2#


118VAL
 0.9
118VAL
 2.5
118VAL
 1.3


119SER*
 20.2
119SER
 23.4
119SER
 23.7


120ILE
 0.8
120ILE
 0.3
120ILE
 0.0


121PRO
 5.9
121PRO
 10.7
121PRO
 8.0


122PHE
 0.2
122PHE
 1.4
122PHE
 0.3


123ILE
 19.1
123ILE
 20.8
123ILE
 18.3


124GLY
 4.3
124GLY
 6.2
124GLY
 3.3


125GLU*
 59.7#
125GLU
 56.2#
125ALA
 57.8#


126GLY
 50.0#
126GLY
 52.5#
126GLY
 49.6#


127GLY
 47.2#
127GLY
 56.7#
127GLY
 38.6#


128GLY*
 34.7
128GLY
 30.3
128ALA
 23.0


129LYS
 68.8#
129LYS
 73.9#
129LYS
 78.4#


130ASN
 16.2
130ASN
 14.6
130ASN
 10.1


131SER
 78.2#
131SER
 77.9#
131SER
 80.3#


132THR
 9.8
132THR
 10.3
132THR
 12.3


133THR*
 45.7#
133THR
 42.0#
133THR
 44.3#


134ILE*
 1.1
134ILE
 0.8
134ILE
 0.0


135GLU*
 51.5#
135GLU
 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


139ALA*
 2.8
139ALA
 6.3
139ALA
 4.1


140HIS
 2.3
140HIS
 2.1
140HIS
 0.0


141ASN
 5.3
141ASN
 6.5
141ASN
 2.8


142SER
 5.4
142SER
 4.4
142SER
 6.6


143SER
 7.7
143SER
 10.6
143SER
 7.0


144THR
 23.5
144THR
 17.3
144THR
 16.6


145THR*
 48.3#
145THR
 52.7#
145THR
 55.2#


146THR
 50.2#
146THR
 49.7#
146THR
 53.6#


147PHEp2*
 49.9#
147PHE
 61.6#
147SER
 51.7#


148GLN*
 12.9
148GLN
 17.8
148GLN
 18.4


149GLNp2*
 59.5#
149GLN
 65.1#
149GLN
 69.1#


150ALA*
 6.9
150ALA
 8.7
150ALA
 9.1


151SERp2*
 51.0#
151SER
 51.7#
151SER
 57.9#


152THR
 9.9
152THR
 8.7
152THR
 12.3


153ASP*
 83.5#
153ASP
 84.5#
153GLU
 63.3#


154ILE
 11.2
154ILE
 6.1
154ILE
 6.3


155GLU*
 49.5#
155GLU
 63.9#
155GLU
 49.7#


156TRP
 1.7
156TRP
 3.8
156TRP
 1.8


157ASN*
 59.1#
157ASN
 59.1#
157ASN
 53.4#


158ILE*
 13.1
158ILE
 5.9
158ILE
 0.8


159SER*
 60.2#
159SER
 52.9#
159SER
 52.2#


160GLN
 29.2
160GLN
 19.3
160GLN
 9.3


161PRO
 54.0#
161PRO
 63.6#
161PRO
 62.6#


162VAL
 0.6
162VAL
 4.0
162VAL
 2.4


163LEU
 53.8#
163LEU
 56.6#
163LEU
 64.5#


164VAL
 0.0
164VAL
 0.0
164VAL
 0.0


165PRO
 22.8
165PRO
 22.1
165PRO
 26.9


166PRO
 30.7
166PRO
 36.1#
166PRO
 39.7#


167SER*
 31.0
167ARG
 32.8
167ARG
 36.7#


168LYS
 18.2
168LYS
 18.5
168LYS
 19.9


169GLN
 17.4
169GLN
 15.1
169GLN
 10.7


170VAL
 0.0
170VAL
 0.0
170VAL
 0.0


171VAL
 13.2
171VAL
 13.8
171VAL
 12.2


172ALA
 0.0
172ALA
 0.0
172ALA
 0.0


173THR
 9.8
173THR
 9.2
173THR
 6.5


174LEU
 1.3
174LEU
 2.6
174LEU
 0.2


175VAL*
 17.2
175VAL
 17.8
175VAL
 13.4


176ILE
 0.0
176ILE
 0.0
176ILE
 0.0


177MET*
 7.0
177MET
 7.7
177MET
 17.3


178GLY
 1.6
178GLY
 0.5
178GLY
 0.0


179GLY
 15.9
179GLY
 22.2
179GLY
 16.5


180ASNp2*
 60.0#
180ASN
 60.1#
180ASP
 44.9#


181PHE
 0.7
181PHE
 2.8
181PHE
 1.8


182THRp2*
 50.6#
182THR
 44.3#
182THR
 40.8#


183ILE
 0.0
183ILE
 1.1
183VAL
 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
188ILE
 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


193SERp1
 7.4
193SER
 7.7
193SER
 2.7


194THRp1
 66.2#
194THR
 60.2#
194THR
 59.9#


195GLUp1
 38.5#
195GLU
 35.0
195GLN
 35.5#


196HIS*
 37.7#
—
100
—
100#


197TYR*
 32.2
—
100#
—
100#


198SER*
 35.5#
—
100#
—
100#


199HISp1*
 64.3#
196HIS
 55.5#
196HIS
 51.7#


200TYRp1*
 85.3#
197TYR
 56.2#
197PHE
 45.8#


201SERp1*
 50.3#
198SER
 68.3#
198THR
 64.5#


202GLYp1
 32.8
199GLY
 50.0#
199GLY
 51.1#


203TYRp1
 21.5
200TYR
 22.6
200TYR
 26.0


204PROp1
 1.4
201PRO
 1.0
201PRO
 1.0


205ILEp1
 1.1
202ILE
 0.3
202ILE
 0.0


206LEUp1
 1.8
203LEU
 2.6
203LEU
 0.0


207THRp1
 0.0
204THR
 0.0
204THR
 0.0


208TRPp1*
 38.8#
205TRP
 35.6#
205TRP
 22.5


209ILEp1
 0.0
206ILE
 0.0
206ILE
 0.0


210SERp1
 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#


214ASN
 65.5#
211ASN
 66.4#
211HIS
 64.4#


215SER
 67.2#
212SER
 74.1#
212ASN
 74.2#


216TYRp1
 42.5#
213TYR
 39.8#
213VAL
 29.4


217SERp1*
 43.2#
214ASN
 46.3#
214ARG
 57.1#


218GLYp1
 1.2
215GLY
 4.1
215GLY
 6.1


219PROp1*
 14.4
216PRO
 14.7
216ARG
 33.7


220PHEp1
 0.0
217PHE
 0.0
217PHE
 0.0


221METp1
 15.2
218MET
 16.1
218LEU
 8.4


222SERp1
 3.3
219SER
 3.3
219SER
 0.0


223TRPp1*
 35.6#
220TRP
 34.3
220TRP
 42.5#


224TYRp1
 13.5
221TYR
 15.9
221PHE
 11.3


225PHEp1
 0.9
222PHE
 1.4
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#
227ASN
 67.3#


23ILEU
 21.1
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
231GLU
 13.0


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
 28.1
235LEU
 25.9


239ASN*
 33.2
236ASN
 26.5
236ASN
 42.2#


240SER
100#
237SER
100#
237SER
100#


241ASP*
 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
241VAL
 3.1
241ILE
 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


251VALp2*
 32.6
248VAL
 30.0
248VAL
 29.7


252SER*
 0.0
249SER
 0.0
249SER
 0.0


253GLNp2
 40.2#
250GLN
 37.6#
250ARG
 51.3#


254VAL
 1.2
251VAL
 1.5
251ILE
 2.7


255SERp2
 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
 1.1
256TYR
 1.0


260ALA
 0.7
257ALA
 0.7
257ALA
 0.0


261THR
 9.2
258THR
 9.3
258THR
 5.0


262VAL
 3.6
259VAL
 1.5
259VAL
 0.2


263ARG
 26.6
260ARG
 29.8
260ARG
 26.9


264PHE
 0.5
261PHE
 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


268ASP
 29.8
265ASP
 31.4
265ALA
 19.3


269ILE
 25.4
266ILE
 18.2
266ILE
 14.5


270HIS
 85.5#
267HIS
 72.2#
267ASN
 92.0#


271ASN
 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#
271THR
 66.1#
271THR
 76.8#


275ILE*
 41.0#
272ILE
 37.9#
272ILE
 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


281ALA
 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
281ALA
 0.8
281GLY
 0.6


285THR
 14.7
282THR
 8.6
282THR
 2.0


286LEU
 3.6
283LEU
 5.9
283LEU
 2.3


287HIS*
 8.4
284HIS
 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#


291LYS
 68.4#
288LYS
 66.2#
288LYS
 51.9#


292ILE
 19.2
289ILE
 19.5
289ILE
 21.0


293SER*
 40.3#
290SER
 47.9#
290SER
 45.7#


294ILE
 3.4
291ILE
 4.8
291ILE
 5.1


295ASN*
 29.3
292ASN
 21.9
292ASN
 18.0


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#


301ALA
 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#


306ILE*
 78.1#
303ILE
 81.6#
303ILE
 91.3#


307LYS
 81.9#
304LYS
 99.3#
304GLU
100#


308THR*
 89.4#
305THR
100#
305ARG
100#


309ASN
100#
306ASN
100#
306ASN
100#





p1designates an amino acid in surface patch [1] of FIG. 2.


p2designates an amino acid in surface patch [2] of FIG. 2.


*designates one of the 72 principally relevant amino acids described herein (see FIG. 2).


Shown are residues of TIC807_L11M, TIC807_M2, and TIC853 aligned by Clustal W.


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






Receptor Binding

A surface patch ([1] of FIG. 2) of residues having % SA values greater than 36% or within about 3 residues of a residue 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 region comprising residues of a TIC807 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 region may be associated with target insect receptor binding activity; and, includes residues T93, S95, S97, F147, Q149, S151, N180, T182, 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.
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 TIC807. Modifications of the surface patch 1 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
Certain amino acid residues located in beta-sheet regions of the protein were identified from the atomic structure of TIC807 and were substituted with aromatic residues. More specifically, amino acids L78, I123, H270, R273, I275 of the folded TIC807 beta sheet regions were substituted with Phenyalanine, Tyrosine, or Tryptophan. Aromatic amino acid substitutions of R273 and I275 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
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 region can impart more flexibility and therefore susceptibility to proteolysis, which can either increase insect inhibitory activity or decrease insect inhibitory activity. Residues in structurally identified loop regions were substituted with a glycine residue, and no improvements were observed. Positions in loops that were already glycines, (e.g. G18, 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
The atomic structure of TIC807 (SEQ ID NO:2) was used to identify loop regions 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.
Analysis of the atomic structure of TIC807 suggests that a structural loop resides at residues 113-138 of SEQ ID NO:2, and variants were engineered to stabilize and destabilize the loop.
In another region spanning two beta-strands connected by a short loop, the two beta-strands exhibited an alternating pattern of hydrophobic 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 V116, V118, and I120 with respective combinations 116V/Y/L/H/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 S117, S119, 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 I133, A135, and F137 with respective combinations 133I/D/F/N/V/Y, 135A/D/F/H/L/V/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 inhibitory spectrum and/or improved Lygus inhibitory activity was associated with certain of these substitutions as shown in Table 4.
Structure-Function Relationship
Altogether, more than 2000 clones (including mixed library clones) expressing variants of TIC807 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 predictive structure-function modifications, including structure modeling, receptor binding potential, metal binding potential, oligomerization 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
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 predominant 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 materials by centrifugation. The amount of protein in test and control samples were measured using total protein methods, e.g.s, a Bradford assay, an ELISA method, or similar. Gel electrophoresis was used to determine the purity and stock concentration of the solubilized recombinant protein. C-terminal HIS-tagged TIC807 protein was engineered to facilitate detection, 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 have equivalent activity against Lygus (see Examples 4, 5, and 6).
Site-directed amino acid substitutions were made to TIC807_M13 (SEQ ID NO:34) to elevate expression of a soluble form. Inventors postulate that more readily soluble variants of the proteins of the present 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: I58K 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 alternatively described as follows for Variant #1: I58K and P59K, for Variant #2: S201K and G202K, and for Variant #3: S249R, V251E, and Q253R; this positional difference is congruent due to a contiguous triple deletion of SEQ ID NO:2 (TIC807) in residue range 196-201 that is reflected in TIC807_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 preparations were evaluated by coomassie-stained SDS-PAGE, which showed that TIC807_M13 (SEQ ID NO:34) was not detectable in the soluble 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 present 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_M14; nucleotide SEQ ID NO:203 and amino acid SEQ ID NO:204). Prepared E. coli lysate was clarified, and the recombinant protein purified and enriched-for on a series of columns, including ion-exchange and gel filtration methods. Pooled protein fractions were quantified and determined to be active against Lygus insects (See Example 4, Table 4B).
Proteins of the present invention, including 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 ID NO:36, are engineered to elevate expression of a soluble form when expressed in a host cell, e.g., expressed in 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, 250, or 253.
The C-terminal region protrudes away from the monomeric core of the protein (See FIG. 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, quantified, and determined active against Lygus insects (See Example 4, Table 4B). Recombinant DNA constructs were designed to encode for and express TIC807 fragments exhibiting varying truncations off of the C-terminus end of the proteins of the present 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
This example illustrates eHTP's to have 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, including the genus Lygus, e.g., Lygus hesperus and Lygus lineolaris, and the family Cicadellidae, including the genus Amrasca, e.g. Amrasca devastans, and Empoasca, e.g. Empoasca fabae. This example with Table 4B illustrates the feeding assay used to determine the enhanced Lygus inhibitory spectrum and/or improved Lygus inhibitory activity of a Bt expressed recombinant proteins of the present 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 serum albumin (BSA) as a standard. Protein stock (2×) prepared this way were mixed with diet for feeding assays.
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® (Bio-Serv® Diet F9644B, Frenchtown, N.J.). 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 temperature, this being the 2× diet. Test samples were prepared by mixing in a 1:1 ratio of 2× diet and 2× sample. A sheet of Parafilm® (Pechiney Plastic Packing, Chicago, Ill.) was placed over a vacuum manifold designed for 96-well format (Analytical Research Systems, Gainesville, Fla.) and a vacuum of approximately −20 millimeters mercury was applied, sufficient 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 Grove Village, Ill.) 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 on the diet by piercing the diet filled Parafilm® sachets. Alternatively, 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 controls. 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.
For LC50 determinations 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 all LC50 determinations in Table 1 and Table 4B were repeated at least once.
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 TIC807_M2 because significantly large amounts of these proteins in excess of 1000 μg/mL have not been possible to provide in Lygus diet in order to complete 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 TIC807_M14 is 4.4 μg/mL. For each concentration, three populations of eight nymphs were subjected to this bioassay.
This example with Tables 4A and 4B illustrate the feeding assay used to determine 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 specificity against Lygus hesperus and Lygus lineolaris exhibit improved insecticidal activity against Amrasca devastans.
TIC807, and TIC807-M13 were dissolved in 25 mM 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 μg/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 25 mM Sodium Carbonate buffer, pH 10, in 1:5 diluted Lygus diet. Mortality of the 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)×100. Table 4A tabulates Amrasca activity for TIC807 and TIC807_M13 at 5 different concentrations.







TABLE 4A







TIC807 and TIC807_M13 Percent Mortality Directed to Amrasca species









Mortality (%)













SEQ ID NO:
Protein Name
500 μg/mL
166.66 μg/mL
55.55 μg/mL
18.51 μg/mL
6.17 μg/mL
















2
TIC807
100%
55.88%
17.64%
0
0


34
TIC807_M13
100%
88.23%
73.52%
44.11%
26.47%









LC50 values were determined for TIC807 and TIC807_M13 in a separate test. SEQ ID NO:2 (TIC807) exhibited a LC50 value of 116.79 μg/mL and LC90 of 437.27 μg/mL. SEQ ID NO:34 (TIC807_M13) exhibited a LC50 value of 7.59 μg/mL and LC90 value of 239.8 μg/mL.
A feeding assay as described for Amrasca devastans 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. 
The LC50 values of Cry51Aa1 (SEQ ID NO:182), for TIC807 (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 test set. TIC807_M2, TIC807_M10 and TIC807_M12 exhibit improved LC50 values compared to Cry51Aa1.
It should be apparent to those skilled in the art that variations to this procedure can exist that should not affect results.







TABLE 4B







Iterative 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 TIC807.











Amino acid differences compared to TIC807 parent protein
Lygus hesperus
Lygus lineolaris



















Amino acid
Amino acid
Amino acid
LC50
fold increased
% Mortality
LC50
fold increased
% Mortality


SEQ ID

difference(s) relative to
difference(s) in the
difference(s) in the
value
toxicity
at about 1-3 μg/mL
value
toxicity
at about 100 μg/mL


NO:
Protein Name
SEQ ID NO: 2 (TIC807)
first patch
second patch
(μg/mL)
(LC50)
protein*
(μg/mL)
(LC50)
protein*




















2
TIC807
Parent
Parent
Parent
73
1
 0
>223
1
 0


6
TIC807_M1
F147A
F147A
None
23
3
ND
100
at least 2
ND


8
TIC807_M2
F46S, Y54H, S167R,
none
S217N, then a
6.0
12
39
>223
—
14




S217N, then a

contiguous triple




contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


10
TIC807_M3
F46S, Y54H, T93A,
T93A
S217N, then a
2.9
25
43
ND
—
ND




S167R, S217N, then a

contiguous triple




contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


12
TIC807_M4
F46S, Y54H, S95A,
S95A
S217N, then a
2.4
30
20
ND
—
ND




S167R, S217N, then a

contiguous triple




contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


14
TIC807_M5
F46S, Y54H, F147A,
F147A
S217N, then a
1.1
66
34
ND
—
ND




S167R, S217N, then a

contiguous triple




contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


16
TIC807_M8
F46S, Y54H, S95A,
S95A, F147A
S217N, then a
0.8
91
ND
223
at least 1
ND




F147A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


18
TIC807_M6
F46S, Y54H, T93A,
T93A, F147A
S217N, then a
1.5
50
ND
ND
—
ND




F147A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


20
TIC807_M7
F46S, Y54H, Q149E,
Q149E
S217N, then a
1.4
52
62
ND
—
49




S167R, S217N, then a

contiguous triple




contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


28
TIC807_M9
F46S, Y54H, S95A,
S95A, F147A
P219R, then a
9.9
7
ND
8.3
at least 27
ND




F147A, S167R, P219R,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


30
TIC807_M10
F46S, Y54H, S95A,
S95A, F147A,
P219R, then a
0.6
122
ND
4.8
at least 46
ND




F147A, S167R, P219R,
V251A
contiguous triple




V251A, then a

deletion in residue




contiguous triple

range 196-201




deletion in residue range




196-201


32
TIC807_M11
F46S, Y54H, S95A,
S95A, F147A
P219R, then a
1.4
54
ND
5.9
at least 38
ND




F147A, S167R, P219R,

contiguous triple




R273W, then a

deletion in residue




contiguous triple

range 196-201




deletion in residue range




196-201


34
TIC807_M13
F46S, Y54H, S95A,
S95A, F147A,
P219R, then a
0.3
243
ND
0.85
at least
ND




F147S, Q149E, S167R,
Q149E
contiguous triple




262




P219R, R273W, then a

deletion in residue




contiguous triple

range 196-201




deletion in residue range




196-201


36
TIC807_M12
F46S, Y54H, S95A,
S95A, F147A,
P219R, then a
0.4
182
ND
1.2
at least
ND




F147A, S167R, P219R,
V251A
contiguous triple




186




N239A, V251A, then a

deletion in residue




contiguous triple

range 196-201




deletion in residue range




196-201


37
TIC807_37
TIC807_HYS_deletion
none
a contiguous triple
22.4
3
ND
ND
—
ND






deletion in HYSHYS






residues (positions 196-201)


38
TIC807_38
F46S, Y54H, F138V,
none
S217N, then a
ND
—
41
ND
—
44




S167R, S217N, then a

contiguous triple




contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


39
TIC807_39
F46S, Y54H, S167R,
none
S217N, then a
ND
—
57
ND
—
31




S217N, H287F, then a

contiguous triple




contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


40
TIC807_40
F46S, I52M, Y54H,
none
S217N, then a
ND
—
52
ND
—
36




S167R, S217N, then a

contiguous triple




contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


41
TIC807_41
N12D, F46S, Y54H,
none
S217N, then a
ND
—
52
ND
—
31




S167R, S217N, then a

contiguous triple




contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


42
TIC807_42
F46S, Y54H, S167R,
N180D
S217N, then a
ND
—
63
ND
—
50




N180D, S217N, then a

contiguous triple




contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


43
TIC807_43
F46S, Y54H, S167R,
none
a contiguous triple
ND
—
46
ND
—
87




then a contiguous triple

deletion in HYSHYS




deletion in residue range

residues (positions




196-201

196-201)


44
TIC807_44
F46S, Y54H, S167R,
none
S217N, P219R, then
ND
—
30
ND
—
94




S217N, P219R, then a

a contiguous triple




contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


45
TIC807_45
F46S, Y54H, S159T,
none
S217N, then a
ND
—
67
ND
—
21




S167R, S217N, then a

contiguous triple




contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


46
TIC807_46
F46S, Y54H, S167R,
none
S217N, then a
ND
—
52
ND
—
31




S217N, T247K, then a

contiguous triple




contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


47
TIC807_47
F46S, Y54H, S167R,
none
S217N, then a
ND
—
59
ND
—
31




S217N, V244I, then a

contiguous triple




contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


48
TIC807_48
F46S, Y54H, S167R,
none
S217N, then a
ND
—
58
ND
—
34




S217N, V244I, T247K,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


49
TIC807_49
F46S, Y54H, S167R,
none
S217N, W223Y,
ND
—
17
ND
—
13




S217N, W223Y, then a

then a contiguous




contiguous triple

triple deletion in




deletion in residue range

residue range 196-201




196-201


50
TIC807_50
F46S, Y54H, S167R,
none
S217N, then a
ND
—
48
ND
—
19




S217N, Y246F, then a

contiguous triple




contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


51
TIC807_51
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
77
ND
—
ND




G128A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


52
TIC807_52
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
73
ND
—
ND




S167R, S217N, M300A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


53
TIC807_53
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
73
ND
—
ND




S167R, S217N, S293A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


54
TIC807_54
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
67
ND
—
ND




S167R, S217N, H287A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


55
TIC807_55
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
65
ND
—
ND




S167R, S217N, T274A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


56
TIC807_56
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
64
ND
—
ND




S167R, S217N, R282A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


57
TIC807_57
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
63
ND
—
ND




S167R, S217N, T308A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


58
TIC807_58
F46S, Y54H, Q70A,
T93A
S217N, then a
ND
—
61
ND
—
ND




T93A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


59
TIC807_59
F46S, Y54H, E125A,
F147A
S217N, then a
ND
—
61
ND
—
ND




F147A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


60
TIC807_60
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
61
ND
—
ND




S167R, S217N, T247A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


61
TIC807_61
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
59
ND
—
ND




S167R, S217N, P305A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


62
TIC807_62
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
57
ND
—
ND




S167R, S217N, I306A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


63
TIC807_63
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
56
ND
—
ND




S167R, S217N, R282A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


64
TIC807_64
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
55
ND
—
ND




S167R, S217N, T308A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


65
TIC807_65
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
55
ND
—
ND




S167R, S217N, M300A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


66
TIC807_66
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
53
ND
—
ND




S167R, S217N, H287A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


67
TIC807_67
F46S, Y54H, S95A,
S95A
S217N, then a
ND
—
52
ND
—
ND




S167R, S217N, M300A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


68
TIC807_68
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
52
ND
—
ND




S167R, S217N, I275A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


69
TIC807_69
F46S, Y54H, S95A,
S95A
S217N, then a
ND
—
51
ND
—
ND




S167R, S217N, T247A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


70
TIC807_70
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
50
ND
—
ND




S167R, V175A, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


71
TIC807_71
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
50
ND
—
ND




S159A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


72
TIC807_72
F46S, Y54H, S95A,
S95A, F147A
S217N, then a
ND
—
48
ND
—
ND




F147A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


73
TIC807_73
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
47
ND
—
ND




S167R, L187A, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


74
TIC807_74
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
47
ND
—
ND




S167R, T182A, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


75
TIC807_75
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
46
ND
—
ND




S167R, S217N, T245A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


76
TIC807_76
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
46
ND
—
ND




S167R, S217N, S249A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


77
TIC807_77
F46S, Y54H, T93A,
T93A, Q149A
S217N, then a
ND
—
45
ND
—
ND




Q149A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


78
TIC807_78
F46S, Y54H, T93A,
T93A, S151A
S217N, then a
ND
—
45
ND
—
ND




S151A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


79
TIC807_79
F46S, Y54H, Q70A,
F147A
S217N, then a
ND
—
45
ND
—
ND




F147A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


80
TIC807_80
F46S, Y54H, S95A,
S95A
S217N, then a
ND
—
44
ND
—
ND




Q148A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


81
TIC807_81
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
44
ND
—
ND




S167R, S217N, T274A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


82
TIC807_82
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
43
ND
—
ND




S167R, S217N, then a

contiguous triple




contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


83
TIC807_83
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
42
ND
—
ND




S167R, M177A, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


84
TIC807_84
F46S, Y54H, S95A,
S95A
S217N, then a
ND
—
42
ND
—
ND




S167R, S217N, V250A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


85
TIC807_85
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
42
ND
—
ND




E155A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


86
TIC807_86
F46S, Y54H, I134A,
F147A
S217N, then a
ND
—
41
ND
—
ND




F147A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


87
TIC807_87
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
41
ND
—
ND




S167R, S217N, T245A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


88
TIC807_88
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
40
ND
—
ND




S167R, S217N, then a

contiguous triple




contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201 followed by a

followed by S198A




S198A


89
TIC807_89
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
40
ND
—
ND




S167R, S217N, N295A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


90
TIC807_90
F46S, Y54H, A87S,
F147A
S217N, then a
ND
—
38
ND
—
ND




F147A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


91
TIC807_91
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
38
ND
—
ND




S167R, S217N, S249A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


92
TIC807_92
F46S, Y54H, S95A,
S95A
S217N, then a
ND
—
37
ND
—
ND




S167R, S217N, I275A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


93
TIC807_93
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
37
ND
—
ND




A139S, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


94
TIC807_94
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
36
ND
—
ND




S167R, S217N, P305A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


95
TIC807_95
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
36
ND
—
ND




S167R, S217N, then a

contiguous triple




contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201 followed by a

followed by S198A




S198A


96
TIC807_96
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
36
ND
—
ND




S167R, S217N, S252A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


97
TIC807_97
F46S, Y54H, T93A,
T93A, F147A
S217N, then a
ND
—
36
ND
—
ND




F147A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


98
TIC807_98
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
36
ND
—
ND




S167R, S217N, V250A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


99
TIC807_99
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
35
ND
—
ND




S167R, S217N, T243A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


100
TIC807_100
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
35
ND
—
ND




S167R, S217N, then a

contiguous triple




contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201 followed by a

followed by Y197A




Y197A


101
TIC807_101
F46S, Y54H, S95A,
S95A
S217N, then a
ND
—
35
ND
—
ND




S167R, S217N, N295A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


102
TIC807_102
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
34
ND
—
ND




S167R, S217N, then a

contiguous triple




contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


103
TIC807_103
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
32
ND
—
ND




S167R, S217N, E299A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


104
TIC807_104
F46S, Y54H, S95A,
S95A
S217N, then a
ND
—
32
ND
—
ND




S167R, S217N, R282A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


105
TIC807_105
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
32
ND
—
ND




S167R, S217N, I306A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


106
TIC807_106
F46S, Y54H, S95A,
S95A
S217N, then a
ND
—
30
ND
—
ND




S167R, S217N, S249A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


107
TIC807_107
F46S, Y54H, A87S,
T93A
S217N, then a
ND
—
30
ND
—
ND




T93A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


108
TIC807_108
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
29
ND
—
ND




S159A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


109
TIC807_109
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
28
ND
—
ND




S167R, S217N, T303A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


110
TIC807_110
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
27
ND
—
ND




Q148A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


111
TIC807_111
F46S, Y54H, S95A,
S95A
S217N, then a
ND
—
27
ND
—
ND




S167R, V175N, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


112
TIC807_112
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
27
ND
—
ND




S167R, S217N, D241A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


113
TIC807_113
F46S, Y54H, S95A,
S95A, F147A
S217N, then a
ND
—
26
ND
—
ND




F147A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


114
TIC807_114
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
26
ND
—
ND




S167R, M177A, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


115
TIC807_115
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
26
ND
—
ND




S167R, S217N, M300A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


116
TIC807_116
F46S, Y54H, S95A,
S95A
S217N, then a
ND
—
26
ND
—
ND




S167R, S217N, P305A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


117
TIC807_117
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
25
ND
—
ND




S167R, S217N, then a

contiguous triple




contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201 followed by

followed by H196A




H196A


118
TIC807_118
F46S, Y54H, A139S,
F147A
S217N, then a
ND
—
25
ND
—
ND




F147A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


119
TIC807_119
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
25
ND
—
ND




S167R, S217N, N295A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


120
TIC807_120
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
24
ND
—
ND




T145A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


121
TIC807_121
F46S, Y54H, S117A,
F147A
S217N, then a
ND
—
24
ND
—
ND




F147A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


122
TIC807_122
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
23
ND
—
ND




S167R, S217N, I275A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


123
TIC807_123
F46S, Y54H, S95A,
S95A
S217N, then a
ND
—
23
ND
—
ND




S167R, S217N, H287A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


124
TIC807_124
F46S, Y54H, S95A,
S95A
S217N, then a
ND
—
21
ND
—
ND




G105A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


125
TIC807_125
F46S, Y54H, S95A,
S95A
S217N, then a
ND
—
20
ND
—
ND




S167R, S217N, then a

contiguous triple




contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


126
TIC807_126
F46S, Y54H, S95A,
S95A
S217N, then a
ND
—
20
ND
—
ND




I134A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


127
TIC807_127
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
20
ND
—
ND




S167R, S217N, M300A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


128
TIC807_128
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
18
ND
—
ND




S167R, S217N, T303A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


129
TIC807_129
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
18
ND
—
ND




A150S, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


130
TIC807_130
F46S, Y54H, S95A,
S95A
S217N, then a
ND
—
18
ND
—
ND




E155A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


131
TIC807_131
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
17
ND
—
ND




T145A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


132
TIC807_132
F46S, Y54H, S95A,
S95A
W208A, S217N,
ND
—
17
ND
—
ND




S167R, W208A, S217N,

then a contiguous




then a contiguous triple

triple deletion in




deletion in residue range

residue range 196-201




196-201


133
TIC807_133
F46S, Y54H, S95A,
S95A, T182A
S217N, then a
ND
—
17
ND
—
ND




S167R, T182A, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


134
TIC807_134
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
17
ND
—
ND




S167R, S217N, T243A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


135
TIC807_135
F46S, Y54H, S95A,
S95A
S217N, then a
ND
—
16
ND
—
ND




S167R, S217N, I306A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


136
TIC807_136
F46S, Y54H, S95A,
S95A
S217N, then a
ND
—
16
ND
—
ND




S117A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


137
TIC807_137
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
15
ND
—
ND




S119A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


138
TIC807_138
F46S, Y54H, T68A,
F147A
S217N, then a
ND
—
15
ND
—
ND




F147A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


139
TIC807_139
F46S, Y54H, G105A,
F147A
S217N, then a
ND
—
15
ND
—
ND




F147A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


140
TIC807_140
F46S, Y54H, S95A,
S95A
S217N, then a
ND
—
15
ND
—
ND




E125A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


141
TIC807_141
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
14
ND
—
ND




E155A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


142
TIC807_142
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
14
ND
—
ND




S167R, S217N, P305A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


143
TIC807_143
F46S, Y54H, S95A,
S95A
S217N, then a
ND
—
13
ND
—
ND




S167R, M177A, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


144
TIC807_144
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
13
ND
—
ND




S167R, S217N, then a

contiguous triple




contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201 followed by

followed by H196A




H196A


145
TIC807_145
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
11
ND
—
ND




D153A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201-T93A-D153A


146
TIC807_146
F46S, Y54H, F147A,
F147A
W208A, S217N,
ND
—
10
ND
—
ND




S167R, S217N, W208A,

then a contiguous




then a contiguous triple

triple deletion in




deletion in residue range

residue range 196-201




196-201


147
TIC807_147
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
 9
ND
—
ND




S167R, S217N, I306A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


148
TIC807_148
F46S, Y54H, S95A,
S95A
S217N, then a
ND
—
 9
ND
—
ND




S167R, S217N, then a

contiguous triple




contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201 followed by

followed by H196A




H196A


149
TIC807_149
F46S, Y54H, S95A,
S95A, Q149A
S217N, then a
ND
—
 9
ND
—
ND




Q149A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


150
TIC807_150
F46S, Y54H, S95A,
S95A
S217N, then a
ND
—
 8
ND
—
ND




S167R, S217N, S293A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


151
TIC807_151
F46S, Y54H, S95A,
S95A
S217N, then a
ND
—
 8
ND
—
ND




A150S, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


152
TIC807_152
F46S, Y54H, S119A,
F147A
S217N, then a
ND
—
 7
ND
—
ND




F147A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


153
TIC807_153
F46S, Y54H, S95A,
S95A
S217N, then a
ND
—
 7
ND
—
ND




S167A, S217N, then a

contiguous triple




contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


154
TIC807_154
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
 7
ND
—
ND




I158A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


155
TIC807_155
F46S, Y54H, S95A,
S95A
S217N, then a
ND
—
 7
ND
—
ND




S167R, S217N, M300A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


156
TIC807_156
F46S, Y54H, F147A,
F147A, T182A
S217N, then a
ND
—
 6
ND
—
ND




T182A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


157
TIC807_157
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
 5
ND
—
ND




S167R, S217N, F235A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


158
TIC807_158
F46S, Y54H, F147A,
F147A
S217N, then a
ND
—
 5
ND
—
ND




S167A, S217N, then a

contiguous triple




contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


159
TIC807_159
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
 5
ND
—
ND




S167R, S217N, D241A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


160
TIC807_160
F46S, Y54H, S95A,
S95A
S217N, then a
ND
—
 4
ND
—
ND




S167R, S217N, T274A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


161
TIC807_161
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
 4
ND
—
ND




Q148A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


162
TIC807_162
F46S, Y54H, S95A,
S95A
S217N, then a
ND
—
 4
ND
—
ND




S167R, S217N, D241A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


163
TIC807_163
F46S, Y54H, S95A,
S95A
S217N, then a
ND
—
 4
ND
—
ND




E155A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


164
TIC807_164
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
 4
ND
—
ND




S167R, L187A, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


165
TIC807_165
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
 4
ND
—
ND




S167R, S217N, T303A,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


166
TIC807_166
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
 3
ND
—
ND




E155A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


167
TIC807_167
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
 3
ND
—
ND




D153A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


168
TIC807_168
F46S, Y54H, T93A,
T93A
S217N, then a
ND
—
 3
ND
—
ND




I134A, S167R, S217N,

contiguous triple




then a contiguous triple

deletion in residue




deletion in residue range

range 196-201




196-201


169
TIC807_169
N137T
none
None
ND
—
 44*
ND
—
ND


169
TIC807_169
E135S
none
None
ND
—
 20*
ND
—
ND


169
TIC807_169
N137T, E135S
none
None
ND
—
 34*
ND
—
ND


169
TIC807_169
E135T, N137D
none
None
ND
—
 16*
ND
—
ND


169
TIC807_169
T133E
none
None
ND
—
 60*
ND
—
ND


169
TIC807_169
E135A, N137G
none
None
ND
—
 50*
ND
—
ND


170
TIC807_170
E125C
none
None
ND
—
12
ND
—
ND


170
TIC807_170
E125H
none
None
ND
—
38
ND
—
ND


170
TIC807_170
E125R
none
None
ND
—
14
ND
—
ND


170
TIC807_170
E125F
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
E125K
none
None
ND
—
20
ND
—
ND


170
TIC807_170
E125T
none
None
ND
—
33
ND
—
ND


170
TIC807_170
E125N
none
None
ND
—
19
ND
—
ND


170
TIC807_170
E125A
none
None
ND
—
41
ND
—
ND


170
TIC807_170
E125L
none
None
ND
—
13
ND
—
ND


170
TIC807_170
E125V
none
None
ND
—
14
ND
—
ND


170
TIC807_170
E125M
none
None
ND
—
13
ND
—
ND


170
TIC807_170
E125D
none
None
ND
—
15
ND
—
ND


170
TIC807_170
E125Y
none
None
ND
—
38
ND
—
ND


171
TIC807_171
T133E
none
None
ND
—
23
ND
—
ND


171
TIC807_171
T133Y
none
None
ND
—
17
ND
—
ND


171
TIC807_171
T133W
none
None
ND
—
13
ND
—
ND


172
TIC807_172
I134V
none
None
ND
—
18
ND
—
ND


172
TIC807_172
I134L
none
None
ND
—
10
ND
—
ND


172
TIC807_172
I134F
none
None
ND
—
18
ND
—
ND


172
TIC807_172
I134K
none
None
ND
—
13
ND
—
ND


172
TIC807_172
I134C
none
None
ND
—
30
ND
—
ND


172
TIC807_172
I134M
none
None
ND
—
33
ND
—
ND


173
TIC807_173
E135V
none
None
ND
—
13
ND
—
ND


173
TIC807_173
E135W
none
None
ND
—
13
ND
—
ND


173
TIC807_173
E135T
none
None
ND
—
39
ND
—
ND


174
TIC807_174
N137H
none
None
ND
—
42
ND
—
ND


174
TIC807_174
N137Y
none
None
ND
—
17
ND
—
ND


174
TIC807_174
N137T
none
None
ND
—
31
ND
—
ND


174
TIC807_174
N137E
none
None
ND
—
32
ND
—
ND


174
TIC807_174
N137S
none
None
ND
—
24
ND
—
ND


174
TIC807_174
N137A
none
None
ND
—
24
ND
—
ND


174
TIC807_174
N137Q
none
None
ND
—
21
ND
—
ND


174
TIC807_174
N137G
none
None
ND
—
18
ND
—
ND


174
TIC807_174
N137I
none
None
ND
—
10
ND
—
ND


174
TIC807_174
N137W
none
None
ND
—
17
ND
—
ND


174
TIC807_174
N137K
none
None
ND
—
66
ND
—
ND


174
TIC807_174
N137C
none
None
ND
—
19
ND
—
ND


174
TIC807_174
N137M
none
None
ND
—
41
ND
—
ND


174
TIC807_174
N137D
none
None
ND
—
69
ND
—
ND


174
TIC807_174
N137F
none
None
ND
—
13
ND
—
ND


174
TIC807_174
N137R
none
None
ND
—
37
ND
—
ND


175
TIC807_175
F147V
F147V
None
ND
—
87
ND
—
ND


175
TIC807_175
F147T
F147T
None
ND
—
68
ND
—
ND


175
TIC807_175
F147C
F147C
None
ND
—
74
ND
—
ND


175
TIC807_175
F147L
F147L
None
ND
—
62
ND
—
ND


175
TIC807_175
F147D
F147D
None
ND
—
51
ND
—
ND


175
TIC807_175
F147A
F147A
None
ND
—
57
ND
—
ND


175
TIC807_175
F147G
F147G
None
ND
—
56
ND
—
ND


175
TIC807_175
F147E
F147E
None
ND
—
50
ND
—
ND


175
TIC807_175
F147I
F147I
None
ND
—
69
ND
—
ND


175
TIC807_175
F147Y
F147Y
None
ND
—
67
ND
—
ND


175
TIC807_175
F147M
F147M
None
ND
—
64
ND
—
ND


175
TIC807_175
F147N
F147N
None
ND
—
64
ND
—
ND


175
TIC807_175
F147Q
F147Q
None
ND
—
50
ND
—
ND


175
TIC807_175
F147H
F147H
None
ND
—
60
ND
—
ND


175
TIC807_175
F147R
F147R
None
ND
—
20
ND
—
ND


175
TIC807_175
F147W
F147W
None
ND
—
82
ND
—
ND


175
TIC807_175
F147P
F147P
None
ND
—
 7
ND
—
ND


176
TIC807_176
Q149D
Q149D
None
ND
—
92
ND
—
ND


176
TIC807_176
Q149E
Q149E
None
ND
—
89
ND
—
ND


176
TIC807_176
Q149C
Q149C
None
ND
—
87
ND
—
ND


176
TIC807_176
Q149A
Q149A
None
ND
—
76
ND
—
ND


176
TIC807_176
Q149F
Q149F
None
ND
—
54
ND
—
ND


177
TIC807_177
A150S
none
None
ND
—
34
ND
—
ND


177
TIC807_177
A150L
none
None
ND
—
24
ND
—
ND


177
TIC807_177
A150V
none
None
ND
—
25
ND
—
ND


177
TIC807_177
A150G
none
None
ND
—
28
ND
—
ND


177
TIC807_177
A150D
none
None
ND
—
19
ND
—
ND


177
TIC807_177
A150W
none
None
ND
—
13
ND
—
ND


177
TIC807_177
A150E
none
None
ND
—
24
ND
—
ND


177
TIC807_177
A150N
none
None
ND
—
18
ND
—
ND


177
TIC807_177
A150Y
none
None
ND
—
11
ND
—
ND


177
TIC807_177
A150F
none
None
ND
—
11
ND
—
ND


177
TIC807_177
A150P
none
None
ND
—
11
ND
—
ND


177
TIC807_177
A150K
none
None
ND
—
17
ND
—
ND


177
TIC807_177
A150T
none
None
ND
—
17
ND
—
ND


177
TIC807_177
A150Q
none
None
ND
—
11
ND
—
ND


177
TIC807_177
A150R
none
None
ND
—
11
ND
—
ND


178
TIC807_178
E155C
none
None
ND
—
82
ND
—
ND


178
TIC807_178
E155I
none
None
ND
—
36
ND
—
ND


178
TIC807_178
E155K
none
None
ND
—
28
ND
—
ND


178
TIC807_178
E155D
none
None
ND
—
22
ND
—
ND


178
TIC807_178
E155H
none
None
ND
—
22
ND
—
ND


178
TIC807_178
E155Y
none
None
ND
—
16
ND
—
ND


178
TIC807_178
E155Q
none
None
ND
—
16
ND
—
ND


178
TIC807_178
E155L
none
None
ND
—
15
ND
—
ND


178
TIC807_178
E155N
none
None
ND
—
14
ND
—
ND


178
TIC807_178
E155T
none
None
ND
—
13
ND
—
ND


178
TIC807_178
E155A
none
None
ND
—
11
ND
—
ND


178
TIC807_178
E155F
none
None
ND
—
 7
ND
—
ND


178
TIC807_178
E155R
none
None
ND
—
 6
ND
—
ND


178
TIC807_178
E155M
none
None
ND
—
 6
ND
—
ND


178
TIC807_178
E155P
none
None
ND
—
 5
ND
—
ND


178
TIC807_178
E155W
none
None
ND
—
 5
ND
—
ND


178
TIC807_178
E155S
none
None
ND
—
 4
ND
—
ND


178
TIC807_178
E155V
none
None
ND
—
 4
ND
—
ND


179
TIC807_179
N157C
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
N157Y
none
None
ND
—
39
ND
—
ND


179
TIC807_179
N157M
none
None
ND
—
22
ND
—
ND


179
TIC807_179
N157A
none
None
ND
—
22
ND
—
ND


179
TIC807_179
N157F
none
None
ND
—
20
ND
—
ND


179
TIC807_179
N157V
none
None
ND
—
18
ND
—
ND


179
TIC807_179
N157L
none
None
ND
—
18
ND
—
ND


179
TIC807_179
N157P
none
None
ND
—
18
ND
—
ND


179
TIC807_179
N157E
none
None
ND
—
17
ND
—
ND


179
TIC807_179
N157T
none
None
ND
—
 8
ND
—
ND


179
TIC807_179
N157G
none
None
ND
—
 7
ND
—
ND


179
TIC807_179
N157I
none
None
ND
—
 7
ND
—
ND


179
TIC807_179
N157R
none
None
ND
—
 6
ND
—
ND


202
TIC807_M8_d5C
F46S, Y54H, S95A,
S95A, F147A
S217N, then a
ND
—
 92*
ND
—
ND




F147A, S167R, S217N,

contiguous triple




then a five amino acid

deletion in residue




deletion at 305-309, then

range 196-201




a contiguous triple




deletion in residue range




196-201


204
TIC807_M14
F46S, Y54H, S95A,
S95A, F147A,
P219R, then a
ND
—
—
ND
—
 60*




F147S, Q149E, S167R,
Q149E, V251E,
contiguous triple




P219R, S249R, V251E,
Q253R
deletion in residue




Q253R, R273W, then a

range 196-201




contiguous triple




deletion in residue range




196-201





ND = Not Determined;


*tested at about 5 μg/mL.






Example 5
Insect Inhibitory Activities of Protein Members of the Present Invention
Proteins of the present invention, such as but not limited to TIC807_M1 (SEQ ID NO:6), TIC807_M2 (SEQ ID NO:8), TIC807_M3 (SEQ ID NO:10), TIC807_M4 (SEQ ID NO:12), TIC807_M5 (SEQ ID NO:14), TIC807_M6 (SEQ ID NO:16), TIC807_M7 (SEQ ID NO:18), TIC807_M8 (SEQ ID NO:20), TIC807_M9 (SEQ ID NO:22), TIC807_M14 (SEQ ID NO:32), TIC807_M15 (SEQ ID NO:34), and TIC807_M16 (SEQ ID NO:36), are prepared and tested for bioactivity against pests of plants other than from Lygus. 
Proteins TIC807_M10 (SEQ ID NO:24), TIC807_M11 (SEQ ID NO:26), TIC807_M12 (SEQ ID NO:28), and TIC807_M13 (SEQ ID NO:30) 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 μg/mL of sample per well, and in the case of TIC807_M5 of 1000 μg/mL, and then allowed 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 negative (−) 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
μg/mL
CPB
WCR
ECB
SWCB
CEW
FAW
SGSB
NBSB
GPA




















UTC
0
−
−
−
−
−
−
−
−
−


TIC807_M5
1000
+
−
ND
ND
ND
ND
ND
ND
ND


TIC807_M10
500
−
−
−
−
−
−
−
−
−


TIC807_M11
500
+
−
−
−
−
−
−
−
−


TIC807_M12
500
−
−
−
−
−
−
−
−
−


TIC807_M13
500
+
−
−
−
−
−
−
−
−





UTC = UnTreated Control;


ND = Not Determined


CPB = Colorado potato beetle (Leptinotarsa decemlineata);


WCR = western corn rootworm (Diabrotica virgifera);


ECB = European corn borer (Ostrinia nubilalis);


SWCB = southwestern corn borer (Diatraea grandiosella);


CEW = corn earworm (Helicoyerpo zea);


FAW = Fall armyworm (Spodoptera frugiperda);


SGSB = southern green stink bug (Nezara virudula);


NBSB = neotropical brown stink bug (Euschistus heros);


GPA = Green peach aphid (Myzus persicae).






The proteins of the present invention are also tested for bioactivity against a pest from the phylum Nematoda.
Example 6
Plants Expressing Proteins of the Present Invention Exhibit Insect Inhibitory Activity
This example illustrates expression of proteins of the present invention in plants, and demonstrates that cotton plants expressing proteins of the present invention exhibit insect inhibitory activity.
Polynucleotide segments for use in expression of the proteins of the present invention in plants are made according to the methods set forth in U.S. Pat. No. 7,741,118. For example, toxin proteins having the amino acid sequence as set forth in SEQ ID NO:4 (TIC807_4), SEQ ID NO:6 (TIC807_M1), 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_M11), and SEQ ID NO:34 (TIC807_M13), are expressed from polynucleotide segments designed for use in plants and encoding the proteins of the present 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: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 NO:200, and SEQ ID NO:201, respectively.
It is intended that polynucleotide segments (or polynucleotide molecules) 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 molecules. Such combinations designed to work in synergistic or compatible mechanism with the proteins of the present invention. The intention of these combinations is to achieve plants and plant cells protected from pest, particularly insect pest, infestation. The specific 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.
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.
Transgenic cotton plants (recombinant cotton plants) were produced and tested for efficacy. Regenerated (R0) 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 R0 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 negative segregate population (progeny not containing the transgene), and plants expressing TIC807 parent protein served as negative 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, Ill.), 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 conical tube (Matrix Technologies Corp., N.H.). 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 all 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 included 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 the variant TIC807 proteins TIC807_M2 and TIC807_M8.


















Mean %




Mortality


Plant Event ID
Protein
N
Mortality
Std Dev
SEM
Lo 95%
Up 95%
t group





64
TIC807_M8
18
78.889
19.967
4.706
68.959
88.818
A


49
TIC807_M8
18
75.556
22.288
5.253
64.472
86.639
A


91
TIC807_M2
18
74.444
20.356
4.798
64.321
84.567
A


20
TIC807_M8
18
73.333
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
TIC807_M2
18
65.556
19.166
4.517
56.025
75.086
AB


48
TIC807_M2
18
64.444
21.206
4.998
53.899
74.990
AB


19
TIC807_M2
18
53.333
25.668
6.050
40.569
66.098
BC


68
TIC807_M2
18
47.778
25.795
6.080
34.950
60.605
C


Negative

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 TIC807 proteins TIC807_M2 and TIC807_M8.


















Mean




Instar


Plant Event ID
Construct
N
Instar
Std Dev
SEM
Lo 95%
Up 95%
t group





64
TIC807_M8
11
3.636
0.552
0.166
3.266
4.007
C


68
TIC807_M2
16
3.949
0.803
0.201
3.521
4.377
BC


48
TIC807_M2
16
4.042
0.604
0.151
3.720
4.364
BC


58
TIC807_M2
17
4.069
0.802
0.194
3.657
4.481
BC


15
TIC807_M8
15
4.094
0.747
0.193
3.681
4.508
BC


19
TIC807_M2
17
4.100
0.698
0.169
3.741
4.459
BC


91
TIC807_M2
12
4.125
0.829
0.239
3.598
4.652
ABC


49
TIC807_M8
12
4.139
0.762
0.220
3.655
4.623
ABC


20
TIC807_M8
14
4.298
0.918
0.245
3.768
4.828
AB


Negative

24
4.599
0.774
0.158
4.273
4.926
A
















TABLE 6C







Mean Survival Mass determined from flowering stage Lygus feeding assays with


cotton plants expressing the variant TIC807 proteins TIC807_M2 and TIC807_M8.


















Mean




Survivor





Survival




Mass


Plant Event ID
Construct
N
Mass
Std Dev
SEM
Lo 95%
Up 95%
t group





64
TIC807_M8
11
2.315
1.489
0.449
1.314
3.315
C


68
TIC807_M2
16
3.548
1.325
0.331
2.843
4.254
B


58
TIC807_M2
17
3.561
1.348
0.327
2.868
4.255
B


48
TIC807_M2
16
3.596
1.436
0.359
2.831
4.362
B


91
TIC807_M2
12
3.775
1.775
0.512
2.647
4.902
AB


49
TIC807_M8
12
3.837
2.135
0.616
2.481
5.193
AB


19
TIC807_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
5.044
AB


15
TIC807_M8
15
3.937
1.906
0.492
2.881
4.993
AB


Negative

24
4.735
1.179
0.241
4.237
5.233
A





Std Dev = standard deviation


SEM = Standard error on the mean


Lo 95% = Lower limit at 95% confidence interval


Up 95% = Upper limit at 95% confidence interval


T grouping = Using a least significant difference test, F value = 101.1756, df = 15, 44, Pr <0.0001






In another example, cotton plants from five transgenic events expressing TIC807_M11 were tested in a field trial having natural Lygus infestation pressures. These plants demonstrated field efficacy compared to the non-transgenic recipient 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. season-long square retention.
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 yield from plants from three of the seven events was statistically higher than to seed cotton yield of the non-transgenic control.
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 2nd generation Lygus) per plant was significantly lower than the average number of Lygus lineolaris insects per non-transgenic plant.
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 Present Invention Exhibit Insect Inhibitory Activity
This example illustrates 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.
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].
Transgenic alfalfa plants (recombinant alfalfa plants) were recovered from transformants that were outcrossed 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 preparations 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 controls (See Tables 7A and 7B; data were analyzed using JMP4 statistical software). For each test sample and each dilution, three populations of eight nymphs were subjected to this bioassay. Stunting scores correspond to visual mass ratings where 0=no difference to negative 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 TIC807 protein TIC807_M8.


















Mean









Dilution
Stunting


Lower
Upper
t


Construct
Sample Source
fold
Score
Std Dev
SEM
95%
95%
Grouping


















[ER]
Pooled Alfalfa
100
2.00
0.00
0.00
2.00
2.00
CD


TIC807_M8
tissue from 10
300
0.00
0.00
0.00
0.00
0.00
E



events per construct
900
0.00
0.00
0.00
0.00
0.00
E


[ES]

100
3.00
0.00
0.00
3.00
3.00
A


TIC807_M8

300
2.33
0.58
0.33
0.90
3.77
BC




900
0.00
0.00
0.00
0.00
0.00
E


[ET]

100
2.67
0.58
0.33
1.23
4.10
AB


TIC807_M8

300
1.67
0.58
0.33
0.23
3.10
D




900
0.00
0.00
0.00
0.00
0.00
E


None
Control Alfalfa
100
2.00
0.00
0.00
2.00
2.00
CD




300
0.00
0.00
0.00
0.00
0.00
E




900
0.00
0.00
0.00
0.00
0.00
E














No Alfalfa incorporated in the diet
0
0.00
0.00
0.00
0.00
0.00
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.


















Mean









Dilution
Percent


Lower
Upper
t


Construct
Sample Source
fold
mortality
Std Dev
SEM
95%
95%
Grouping


















[ER]
Pooled Alfalfa
100
4.17
7.22
4.17
−13.76
22.09
CD


TIC807_M8
tissue from 10
300
0.00
0.00
0.00
0.00
0.00
CD



events per construct
900
13.10
12.54
7.24
−18.06
44.25
CD


[ES]

100
56.55
6.27
3.62
40.97
72.12
AB


TIC807_M8

300
41.67
19.09
11.02
−5.77
89.10
B




900
0.00
0.00
0.00
0.00
0.00
CD


[ET]

100
64.88
19.91
11.50
15.42
114.34
A


TIC807_M8

300
16.67
19.09
11.02
−30.77
64.10
C




900
12.50
12.50
7.22
−18.55
43.55
CD


None
Control Alfalfa
100
12.50
12.50
7.22
−18.55
43.55
CD




300
0.00
0.00
0.00
0.00
0.00
CD




900
8.33
14.43
8.33
−27.52
44.19
CD














No Alfalfa incorporated in the diet
0
2.50
7.01
1.81
−1.38
6.38
D









Example 8
Plants Co-expressing an eHTP and a Second Insect Inhibitory Protein Exhibiting Lygus Species Inhibitory Activity
Protein samples were prepared containing various mixtures of TIC1415 and TIC807_M13 and tested in bioassay. The TIC1415 protein and other Lygus inhibitory proteins are described in PCT Patent Application Publication No. WO 2012/139004. Sample mixtures were fed to Lygus lineolaris using bioactivity assay. TIC1415 protein alone and TIC807_M13 alone were also prepared as positive controls. Buffer was used as negative control. Samples from all three types of preparations 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.







TABLE 8A







Bioassay data for protein mix: TIC1415 combined with TIC807_M13
















Mean†

Mean†




TIC1415
TIC807_M13
Population
T Grouping
stunting‡
T Grouping


SAMPLE
(μg/mL)
(μg/mL)
mortality
on mort
score
on stunting
















TIC1415 +
4.35
1
21.79
AB*
0.60
AB*


TIC807_M13








TIC1415 +
2.175
1
20.36
B*
0.60
AB*


TIC807_M13








TIC1415 +
1.0875
1
12.50
BC
0.60
AB*


TIC807_M13








TIC1415 +
4.35
0.5
32.50
A*
0.80
A*


TIC807_M13








TIC1415 +
1.75
0.265
7.86
CD
0.40
ABC


TIC807_M13








TIC1415 +
0.875
0.265
0.00
D
0.00
C


TIC807_M13








TIC1415 +
0.4375
0.265
5.36
CD
0.00
C


TIC807_M13








TIC1415 +
4.35
0.25
13.21
BC
0.40
ABC


TIC807_M13








TIC1415 +
1.75
0.1325
0.00
D
0.00
C


TIC807_M13








TIC1415 +
1.75
0.06625
0.00
D
0.00
C


TIC807_M13








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 (negative)
0
0
0.00
D
0.00
C


control





†Average (mean) of 5 populations of 8 nymphs per population.


‡Stunting scores correspond to visual mass ratings where 0 = no difference to negative 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.






Cotton plants comprising events with transgenic DNA were designed to co-express respective proteins TIC1415 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 negative 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_M13






















Mean
Mean







Mean
Mean
Mean
Live
Total







3rd
4th
5th
2nd
2nd







Instar
Instar
Instar
Gen.
Gen.

Tukey


Construct
Event
N
or <
Nymphs
Nymphs
Adults
Lygus
SEM
Grouping



















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
5
6.20
0.00
0.40
0.00
6.60
4.73
AB


Inbred
DP393
10
7.00
2.50
0.80
0.00
10.30
3.75
A



(Negative)

## Claims

1. An insect inhibitory recombinant polypeptide comprising a sequence having at least 95% amino acid identity to SEQ ID NO: 2 (TIC807), wherein said insect inhibitory recombinant polypeptide exhibits about 15 fold or greater Hemipteran inhibitory activity against a Hemipteran pest species relative to the Hemipteran inhibitory activity of SEQ ID NO: 2, and wherein said insect inhibitory recombinant polypeptide comprises:
a) at least one modification at an amino acid residue selected from the group consisting of Val10, Asn12, Ile14, Asn22, Asn23, Gly24, Ile25, Gln26, Gly27, Phe30, Gln38, Ile39, Asp40, Thr41, Ile43, Ile52, Thr93, Ser95, Ser97, Phe138, Phe147, Gln149, Ser151, Ser159, Asn180, Thr182, Ser193, Thr194, Glu195, His196, Tyr197, Ser198, His199, Tyr200, Ser201, Gly202, Tyr203, Pro204, Ile205, Leu206, Thr207, Trp208, Ile209, Ser210, Tyr216, Gly218, Pro219, Met221, Ser222, Trp223, Tyr224, Phe225, Asn239, Val244, Tyr246, Thr247, Val251, Gln253, Ser255, and His287 relative to SEQ ID NO:2; and
(b) a contiguous triple deletion in residue range 196-201 relative to SEQ ID NO:2.

2. The insect inhibitory recombinant polypeptide of claim 1, wherein said insect inhibitory recombinant polypeptide exhibits from about 20 fold to about 260 fold increased Hemipteran inhibitory activity against a Hemipteran pest species relative to the Hemipteran inhibitory activity of SEQ ID NO: 2.

3. The insect inhibitory recombinant polypeptide of claim 2, wherein said insect inhibitory recombinant polypeptide exhibits about 25 fold or greater Hemipteran inhibitory activity against a Hemipteran pest species relative to the Hemipteran inhibitory activity of SEQ ID NO: 2 (TIC807).

4. The insect inhibitory recombinant polypeptide of claim 2, wherein said insect inhibitory recombinant polypeptide exhibits about 50 fold or greater Hemipteran inhibitory activity against a Hemipteran pest species relative to the Hemipteran inhibitory activity of SEQ ID NO: 2 (TIC807).

5. The insect inhibitory recombinant polypeptide of claim 1, wherein said insect inhibitory recombinant polypeptide comprises at least one mutation in SEQ ID NO:2 selected from the group consisting of N12D, I52M, T93A, S95A, F138V, F147S, F147A, Q149E, Q149A, S151A, S159T, N180D, T182A, H196A, Y197A, S198A, W208A, P219R, W223Y, Y246F, T247K, V251A, and H287F.

6. The insect inhibitory recombinant polypeptide of claim 1, wherein said insect inhibitory recombinant polypeptide comprises at least two mutations in SEQ ID NO:2 selected from the group consisting of T93A, S95A, F147S, F147A, Q149E, Q149A, S151A, N180D, T182A, H196A, Y197A, S198A, W208A, S217N, P219R, W223Y, V251A.

7. The insect inhibitory recombinant polypeptide of claim 1, wherein said insect inhibitory recombinant polypeptide comprises at least three mutations in SEQ ID NO:2 selected from the group consisting of T93A, S95A, F147S, F147A, Q149E, Q149A, S151A, N180D, T182A, H196A, S198A, Y197A, W208A, S217N, P219R, W223Y, V251A.

8. The insect inhibitory recombinant polypeptide of claim 1, wherein said insect inhibitory recombinant polypeptide comprises at least four mutations in SEQ ID NO:2 selected from the group consisting of T93A, S95A, F147S, F147A, Q149E, Q149A, S151A, N180D, T182A, H196A, Y197A, S198A, W208A, S217N, P219R, W223Y, V251A.

9. The insect inhibitory recombinant polypeptide of claim 1, wherein said Hemipteran pest species is selected from the group consisting of a Lygus sp., an Empoasca sp., an Amrasca sp, and a combination thereof; and wherein said Hemipteran pest species is selected from the group consisting of Lygus hesperus, Lygus Lineolaris, Amrasca devastans, and a combination thereof.

10. The insect inhibitory recombinant polypeptide of claim 1, comprising an enhanced inhibitory spectrum against Lygus, Empoasca, Amrasca, or a combination thereof.

11. The insect inhibitory recombinant polypeptide of claim 1, wherein said insect inhibitory recombinant polypeptide exhibits an enhanced Lygus inhibitory spectrum relative to TIC807 in that said insect inhibitory recombinant polypeptide provides increased inhibition of Lygus lineolaris.