PROTEINS TOXIC TO HEMIPTERAN INSECT SPECIES
20220356215 · 2022-11-10
Inventors
- James A. Baum (Webster Groves, MO)
- Artem G. EVDOKIMOV (St. Louis, MO, US)
- Farhad MOSHIRI (St. Louis, MO, US)
- Timothy J. RYDEL (St. Louis, MO, US)
- Eric J. STURMAN (St. Louis, MO, US)
- Moritz von RECHENBERG (Waltham, WA, US)
- Halong VU (St. Louis, MO, US)
- Andrew M. WOLLACOTT (St. Louis, MO, US)
- Meiying ZHENG (St. Louis, MO, US)
Cpc classification
C07K14/325
CHEMISTRY; METALLURGY
Y02A40/146
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
International classification
C07K14/325
CHEMISTRY; METALLURGY
C07K14/00
CHEMISTRY; METALLURGY
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.
Claims
1-20. (canceled)
21. An insect inhibitory recombinant polypeptide comprising the amino acid sequence as set forth in SEQ ID NO:16.
22. The insect inhibitory recombinant polypeptide of claim 21, wherein the polypeptide exhibits inhibitory activity against an insect species of the order Hemiptera.
23. The insect inhibitory recombinant polypeptide of claim 22, wherein the Hemipteran species is selected from the group consisting of a Lygus sp., an Emrasca sp., and an Amrasca sp.
24. The insect inhibitory recombinant polypeptide of claim 22, wherein the Hemipteran species is selected from the group consisting of Lygus Hesperus, Lygus lineolaris, and Amrasca devastans.
25. A polynucleotide encoding the insect inhibitory recombinant polypeptide of claim 21.
26. The polynucleotide of claim 25, wherein the polynucleotide comprises the nucleotide sequence as set forth in SEQ ID NO:15.
27. A host cell comprising the polynucleotide of claim 25, wherein the host cell is selected from the group consisting of a bacterial host cell and a plant host cell.
28. The host cell of claim 27, wherein the bacterial host cell is selected from the group consisting of Agrobacterium, Rhizobium, Bacillus, Escherichia, Pseudomonas, and Salmonella; and wherein the Bacillus species is a Bacillus thuringiensis, and the Escherichia is an Escherichia coli.
29. The host cell of claim 27, wherein the plant host cell is selected from the group consisting of a monocot cell and a dicot cell.
30. An insect inhibitory composition comprising the insect inhibitory recombinant polypeptide of claim 21.
31. The composition of claim 30, wherein the composition is prepared by lyophilization, extraction, filtration, or centrifugation.
32. The insect inhibitory composition of claim 30, further comprising at least one insect inhibitory agent different from the insect inhibitory recombinant polypeptide.
33. The insect inhibitory composition of claim 32, wherein the at least one insect inhibitory agent is selected from the group consisting of an insect inhibitory protein, an insect inhibitory dsRNA molecule, and an insect inhibitory chemistry.
34. The insect inhibitory composition of claim 32, wherein the at least one insect inhibitory agent exhibits activity against one or more pest species of the order Lepidoptera, Coleoptera, Hemiptera, or Homoptera.
35. A seed comprising the polynucleotide of claim 25.
36. The seed of claim 35, wherein the polynucleotide comprises the sequence as set forth in SEQ ID NO:15.
37. A method of controlling a Hemipteran pest, the method comprising presenting the Hemipteran pest with an inhibitory amount of the insect inhibitory recombinant polypeptide of claim 21.
38. The method of claim 37, wherein said presenting is via expressing the insect inhibitory recombinant polypeptide in a cotton plant.
39. A transgenic plant cell, plant, or plant part comprising the insect inhibitory recombinant polypeptide of claim 21.
40. A method of controlling a Hemipteran pest, comprising exposing the Hemipteran pest to the transgenic plant cell, plant or plant part of claim 39, wherein the plant cell, plant or plant part expresses a Hemipteran inhibitory amount of the insect inhibitory recombinant polypeptide.
41. A commodity product obtained from the plant cell, plant, or plant part of claim 39, wherein the commodity product comprises a detectable amount of the insect inhibitory recombinant polypeptide, wherein the commodity product is selected from the group consisting of plant biomass, oil, meal, animal feed, flour, flakes, bran, lint, hulls, and processed seed.
42. A method of producing a seed, the method comprising: a. planting at least one seed comprising a polynucleotide encoding the insect inhibitory recombinant polypeptide of claim 21; b. growing at least one plant from the at least one seed; and c. harvesting a seed from the at least one plant, wherein the harvested seed comprises the polynucleotide encoding the insect inhibitory recombinant polypeptide.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0015]
[0016]
[0017]
DETAILED DESCRIPTION
[0018] 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.
[0019] 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 tor controlling a Hemipteran species and broadening the range of Hemipteran species that are affected by the eHTP protein.
[0020] The Hemipteran pest species are intended to mean insects that feed upon plants and plant tissues by slashing or piercing the outer surface of the target plant, and then consume macerated plant exudates 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 Lygacidae, spittlebugs from the Family Cereopidae squash bugs from the Family Corcidae, 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), Crytopeltis modesta (tomato bugs), Dysdercus suturellus (cotton stainer), Euschistus servus (brown stink bugs), Euschistus variolarius (one-spotted stink bugs), Graptostethus spp. (complex of seed bugs), Leptoglossus corculus (leaf-footed pine seed bugs), 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 guyumi, tribe Nirvaniae, e.g. Sophonia rufofascia, Family Delphacidae, e.g. Nilapoarvata lugens, Sogatella furcifera, Unkanodes sapporanus, and Family Lophopidae, e.g. Zophiuma lobulata.
[0021] 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 era 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 or 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.
[0022] eHTP's include at least one amino acid modification of the relative positions of TIC807 (SEQ ID NO:2) as set forth above in paragraph [0009]. eHTP's 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 NO:70, SEQ ID NO:71, SEQ NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ 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, SEO 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.
[0023] 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.
[0024] 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
[0025] The prior art teaches solubility problems associated with the scaffold proteins. eHTP's exhibit improved solubility compared to the scaffold proteins, and generally exhibit increased solubility at a pH of less than 9.0, in contrast to the observed solubility profile of one or more of the scaffold proteins. This increased solubility at more physiological pH is 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.
[0026] 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 gents useful in controlling insect pests. Examples of additional inhibitory agents includes, but are not limited to, a TIC14125 protein, a dsRNA directed towards Hemipteran orthologs of Nilaparvata 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, 5307, 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).
[0027] 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).
[0028] Synthetic pyrethroids that are preferred for use in the present invention include (S)-cyan(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 (1R,3R)-3-(2,2-dibromovinyl)-2,2-dimethyl cyclopropanecarboxylate (deltamethrin, CAS RN 52918-63-5), alpha-cyano-3-phenoxybenzyl 2,2,3,3-tetramethyl cyclopropoanrcarboxylate (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-dilfuoromethoxy)-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 101007-06-01), (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), [1R,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 91465-8-6), (2-methyl[1,1′-biphenyl]-3-yl)methyl 3-(2-chloro-3,3,3-trifluoro-1-propeny)-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)cycloproane 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 hydrophthalimidomethyl-(1RS)-cis-trans-chrysanthemate (tetramethrin, CAS RN 7696-12-0), 3-phenoxybenzyl-d,1-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,3S)-2,2-dimethyl-3-(2-methylprop-1-enyl) cyclopropane carboxylate (allethrin, CAS RN 584-79-2), (bioallethrin, CAS RN 584-79-2), and (ZX18901, 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.
[0029] 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 thiamethoxan (CAS RN 153719-23-4).
[0030] Chloronicotinyl insecticides are also useful in the subject invention. Chloronicotinyls that are preferred for use in the subject composition are described in U.S. Pat. No. 5,952,358, and include acetamiprid ((E)-N-[(6-chloro-3-pyridinyl)methyl]-N′-cyano-N-methyleneimidamide, CAS RN 135140-20-7), 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).
[0031] Nitroguanidine insecticides are useful in the present invention. Such nitorguanidines can include those described in U.S. Pat. Nos. 5,633,375, 5,034,404 and 5,245,040.
[0032] 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]methyl]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).
[0033] 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).
[0034] 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).
[0035] 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.
[0036] 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).
[0037] In addition, carbamate insecticides are useful in the subject invention. Preferred carbamate insecticides are aldicarb (CAS RN 116-06-3), carbaryl (CAS RN 63-25-2), carbofuran (CAS RN 1563-66-2), oxamyl (CAS RN 23135-22-0) and thiodicarb (CAS RN 59669-26-0).
[0038] When a chemical it 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.
[0039] The chemical insecticides that are useful in the present invention can be of any grade or purity that pass in the trade as such insecticide. Other materials that accompany the insecticides in commercial 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.
[0040] eHTP's are related by amino acid modifications such that the modified proteins exhibit enhanced Hemipteran inhibitory spectrum and/or improved Hemipteran inhibitory activity against Lygus spp., Empoasca spp. and/or Amrasca spp. compared to the parent protein, TIC807. The phrases “more active”, “improved activity”, “enhanced specificity”, “increased toxic potency”, “increased toxicity”, “improved Hemipteran inhibitory activity, “enhanced Hemipteran inhibitory activity”, “improved Lygus, Empoasca and/or Amrasca inhibitory activity”, “greater Lygus, Empoasca and/or Amrasca inhibitory activity”, “greater Hemipteran inhibitory activity” and “enhanced Lygus, Empoasca and/or Amrasca inhibitory spectrum” and “enhanced Hemipteran inhibitory spectrum” refer to a comparison of the activity of an eHTP and of the activity of a TIC807 (SEQ ID NO:2), 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 lincolaris, 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: [0041] (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 a 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, [0042] (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 a 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.
[0043] 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 a 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 observe 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 a AXMI-171 (SEQ ID NO:206) proteins.
[0044] 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.
[0045] 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).
[0046] 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 SEQ ID NO:206 (AXMI-171) by causing mortality: [0047] (i) an a dose of about 0.3 μg/mL to about 70 μg/mL against a Lygus hesperus insect species, [0048] (ii) at a dose of about 0.85 μg/mL to about 100 μg/mL against a Lygus lineolaris insect species, [0049] (iii) measuring at an LC50 value of about 0.3 to about 70 μg/mL against Lygus hesperus, [0050] (iv) measuring at an LC50 value of about 0.85 to about 100 μg/mL against Lygus lineolaris, or [0051] (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 [0052] (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 [0053] (vii) measuring at an LC 50 value of about 3.5 to about 1.5 μg/mL against Amrasca devastans and/or Empoasca fabae.
[0054] 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), TIC807_M2 (SEQ ID NO:8), is provided for exemplary eHTP's where LC50 values were determined.
[0055] 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 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
[0056] 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), 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”.
[0057] Iterative design includes the elucidation of the atomic structure of proteins of the present invention (for example, as set forth in
[0058] 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
TABLE-US-00001 TABLE 1 LC50 values of select eHTP's compared to TIC807 Lygus hesperus Lygus lineolaris SEQ LC50 value Toxicity LC50 value Toxicity ID NO: Toxin ( ) (
) (
) (
) 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.
indicates data missing or illegible when filed
[0059] 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.
[0060] 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.
[0061] Exemplary recombinant polynucleotide molecules provided herein include, but are not limited to, SEQ 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:10 (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_10), 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.
[0062] 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.
[0063] Recombinant polynulceotide 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 sued 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.”
[0064] 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, almond, banana, barley, bean, beet, broccoli, cabbage, brassica, brinjal, carrot, cassava, castor, cauliflower, celery, chickpea, Chinese cabbage, celery, citrus, coconut, coffee, corn, clover, cotton, a curcurbit, 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 here 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.
[0065] 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.
[0066] Transgenic plants and host cells are provided that expressed an insect or Hemipteran inhibitory amount of the eHTP to control an insect or Hemipteran infestation. Any of the aforementioned plant species can be sued 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.
[0067] 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.
[0068] A plant product, other than a see 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.
[0069] 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:150, 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, 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.
[0070] 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.
[0071] Hemiptera infestations or 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 amounts 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.
[0072] Preferably, the Lygus spp. is Lygus hesperus or Lygus lineolaris, the Empoasca spp. is Empoasca fabae, and the Amrasca spp. is Amrasca devastans.
[0073] Other features and advantages of the invention will he apparent from the following detailed description, examples, and claims.
EXAMPLES
[0074] 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
[0075] This example illustrates the random, combinatorial, and invention 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.
[0076] 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.
[0077] 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.
[0078] 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
[0079] 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 105 to 201 and positions 211 to 291, relative 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.
[0080] A multiple sequence alignment of Cry51 Aa1 (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, F125X, F138X, F147X, S167X, Y216X, P218X, G234X, T247X, D268X, and T308X. Construct libraries were prepared for subsequent protein expression in host cells.
Scanning Approaches
[0081] 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-US-00002 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
[0082] 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
[0083] 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, S209, 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%.
[0084] Combined engineering-testing-selecting approaches described herein resulted in a number of principally relevant residues that accumulate in a surface patch ([2] of
TABLE-US-00003 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 Position Position and amino Calculated and amino Calculated and amino Estimated acid % SA per acid % SA per 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 10VAL
0.3 10VAL 0.6 10VAL 0.0 11MET 25.0 11LEU 17.0 11LEU 16.6 12ASN
49.8
12ASN 43.2
12ASP 39.7# 13ALA 0.0 13ALA 0.0 13ALA 0.0 14ILE.sup.p1 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
22ASN.sup.p1 16.9 22ASN 10.1 22ASN 15.2 23ASN.sup.p1 0.3 23ASN 0.8 23ASN 0.0 24GLY.sup.p1 42.0# 24GLY 43.2# 24GLY 43.3# 25ILE.sup.p1 10.1 25ILE 13.8 25ILE 7.6 26GLN.sup.p1 92.4# 26GLN 86.2
26GLN 94.7
27GLY.sup.p1 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
30PHE.sup.p1 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
38GLN
12.8 38GLN 14.5
38GLN 9.0 39ILE.sup.p1 7.8 39ILE 7.9 39ILE 5.1 40ASP.sup.p1 52.4
40ASP 55.3
40ASP 49.8# 41THR.sup.p1 0.3 41THR 0.0 41THR 0.2 42SER 53.1
42SER 56.0
42SER 53.0
43ILE.sup.p1 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
51LEU 13.7 51LEU 8.1 51LEU 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 71VA
53.4# 71VA
53.2
71VA
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 93THR.sup.p2
40.3
93THR 36.4# 93THR 2
.4 94THR 0.0 94THR 0.6 94THR 0.2 95SER.sup.p2
14.4 95SER 15.7 95SER 18.1 96THR 5.5 96THR 1.5 96THR 0.0 97SER.sup.p2 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 3
.
# 112LEU 26.7 112LEU 29.6 112L
U 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 120IL
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 5
.8
126GLY 50.0
126GLY 52.5
126GLY 49.6
127GLY 47.2
127GLY 56.
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
147PHE
49.9
147PHE 61.6
147SER 51.7
148GLN
12.9 148GLN 17.8 148GLN 18.4 149GLN
.sup.2
59.5
149GLN 65.1
149GLN 69.1
150ALA
6.9 150ALA 8.7 150ALA 9.1 151SER.sup.p2
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
167S
R
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 180ASN.sup.p2
60.0
180ASN 60.1
180ASP 44.9
181PHE 0.7 181PHE 2.8 181PHE 1.8 182THR.sup.p
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 193SER.sup.p1 7.4 193SER 7.7 193SER 2.7 194THR.sup.p1 66.2
194THR 60.2
194THR 59.9
195GLU.sup.p1 38.5# 195GLU 35.0 195GLN 35.5
196HIS
37.7# — 100 — 100
197TYR
32.2 — 100
— 100
198SER
35.5
— 100
— 100
199HIS
64.3
196HIS 55.5
196HIS 51.7
200TYR
85.3
197TYR 56.2
197PHE 45.8
201SER
50.3
198SER 68.3
198THR 64.5
202GLY.sup.p1 32.8 199GLY 50.0
199GLY 51.1
203TYR.sup.p1 21.5 200TYR 22.6 200TYR 26.0 204PRO.sup.p1 1.4 201PRO 1.0 201PRO 1.0 205ILE.sup.p1 1.1 202ILE 0.3 202ILE 0.0 206LEU.sup.p1 1.8 203LEU 2.6 203LEU 0.0 207THR.sup.p1 0.0 204THR 0.0 204THR 0.0 208TRP.sup.p1
38.8
205TRP 35.6
205TRP 22.5 209ILE
0.0 206ILE 0.0 206ILE 0.0 210SER
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
216TYR.sup.p1 42.5# 213TYR 39.8
213VAL 29.4 217SER.sup.p1
43.2# 214ASN 46.3
214ARG 57.1
218GLY.sup.p1 1.2 215GLY 4.1 215GLY 6.1 219PRO.sup.p1
14.4 216PRO 14.7 216ARG 33.7 220PHE.sup.p1 0.0 217PHE 0.0 217PHE 0.0 221MET.sup.p1 15.2 218MET 16.1 218LEU 8.4 222SER.sup.p1 3.3 219SER 3.3 210SER 0.0 223TRP.sup.p1
35.6
220TRP 34.3 220TRP 42.5
224TYR.sup.p1 13.5 221TYR 15.9 221PHE 11.3 225PHE.sup.p1 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
231LEU 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 251VAL.sup.p2
32.6 248VAL 30.0 248VAL 29.7 252SER
0.0 249SER 0.0 249SER 0.0 253GLN
40.2
250GLN 37.6
250ARG 51.3
254VAL 1.2 251VAL 1.5 25ILE 2.7 255SER
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
.sup.P1designates an amino acid in surface patch [1] of FIG. 2. .sup.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_L
M, TIC807_M2, and TIC853 aligned by Clustal W. Numbers marked with # represent % SA of at least about 36%.
indicates data missing or illegible when filed
Receptor Binding
[0085] A surface patch ([1] of
[0086] 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
[0087] 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
[0088] 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
[0089] 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.
[0090] 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.
[0091] 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 I133I/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 1345/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
[0092] 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
[0093] 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).
[0094] 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 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).
[0095] 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, 58, 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.
[0096] The C-terminal region protrudes away from the monomeric core of the protein (See
Example 4
Hemipteran Activity of Engineered Proteins
[0097] 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.
[0098] 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.
[0099] 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.
[0100] 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 an 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.
[0101] 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.
[0102] TIC807, and TIC807-M13 were dissolved in 25 mM sodium carbonate buffer, pH10. Amrasca devastans eggs were collected on Okra leaf and incubated in a petriplate containing 2% agar. Upon hatching the neonates were used for bioassays 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-US-00004 TABLE 4A TIC807 and TIC807_M13 Percent Mortality Directed to Amrasca species Mortality (%) SEQ Protein 500 166.66 55.55 18.51 6.17 ID NO: Name μg/mL μg/mL μg/mL μg/mL μg/mL 2 TIC807 100% 55.88% 17.64% 0 0 34 TIC807 M13 100% 88.23% 73.52% 44.11% 26.47%
[0103] 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_13) exhibited at LC50 value of 7.59 μg/mL and LC90 value of 239.8 μg/mL.
[0104] 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.
[0105] The LC50 values of Cry51Aa1 (SEQ ID NO:182), for TIC808 (SEQ ID NO:2), TIC807_M2 (SEQ ID NO:8), TIC807_M10 (SEQ ID NO:30) and TIC807-M13 (SEQ ID NO:34) against Lygus hesperus and Lygus lineolaris were determined in one testset. TIC807_M2, TIC807_M10 and TIC807_M12 exhibit improved LC50 values compared to Cry51Aa1.
[0106] It should be apparent to those skilled in the art that variations to this procedure can exist that should not affect results.
TABLE-US-00005 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. Lygus hesperus Lygus lineolaris Amino acid differences compared % % to TIC807 parent protein Mortal- Mortal- Amino acid fold ity at fold ity at difference(s) Amino acid Amino acid LC50 in- about LC50 in- about SEQ relative to difference(s) difference(s) value creased 1-3 value creased 100 ID Protein SEQ ID NO: 2 in the in the (μg/ toxicity μg/mL (μg/ toxicity μg/mL NO: Name ( C807) first patch second patch mL) (LC50) protein* mL) (LC50) protein* 2 TIC8
Parent Parent Parent 7
1 0 >2
1 0 6 TIC8
_M1 F147A F147A None 23
ND 100 at ND least 2 8 TIC807_M2 F4
S, Y54H, S16
R, none S217N, then a 6.0 12
— 14 S217N, then a five contiguous triple contiguous triple deletion in residue deletion in residue range 196-201 range 196-201 10 TIC80
_M
F46
, Y54H, T
3A, T93A S217N, then a 2.9 25 43 ND — ND S16
R, S217N, then a contiguous triple contiguous triple deletion in residue deletion in residue range 19
-201 range 196-201 12 TIC80
_M4 F46S, Y54H, S95A,
95A S217N, then a 2.4
20 ND — ND S16
R, S217N, then a contiguous triple contiguous triple deletion in residue deletion in residue range 196-201 range 196-201 1
TIC
_M5 F
S, Y5
H,
14
A, F147A S217N, then a 1.1 66
ND — ND
167R,
217N, then a contiguous triple contiguous triple deletion in residue deletion in residue range 196-201 range 196-201 16 TIC
_M
F46S, Y54H, S95A, S95A,
47A S217N, then a 0.8
1 ND 223 at ND F147A,
16
R, contiguous triple least S217N, then a deletion in residue 1 contiguous triple range 196-201 deletion in residue range 19
-201 1
TIC
_M6 F46S, Y54
, T9
A, T93A, F147A S217N, then a 1.5 5
ND ND — ND F147A, S1
7
, contiguous triple S217N, then a deletion in residue contiguous triple range 1
-201 deletion in residue range 196-201 20 TIC80
_M
6S, Y54H, Q
, Q149
S217N, then a 1.4 52 62 ND —
S1
R,
21
N,
a contiguous triple contiguous triple deletion in residue deletion in residue range 1
-201 range 196-201
_M
46S, Y
H,
95A,
95A, F1
7A P219R, then a 9.9 7 ND 8.3
ND
147A, S167R, contiguous triple P219R, then a deletion in residue contiguous triple range 19
-201 deletion in residue range 1
-201 30 TIC80
_M10 F46S, Y54H, S95A, S95A, F147A, P219R, then a 0.6 122 ND 4.8
ND F147A, S167R, P219R, V251A contiguous triple least V251A, then a deletion in residue 46 contiguous triple range 196-201 deletion in residue range 196-201 32 TIC80
_M11
46S, Y54H, S95A, S95A, F147A P21
R, then a 1.4 5
ND
.9
ND F147A, S167R, P219R, contiguous triple least R27
W, then a deletion in residue 38 contiguous triple range 196-201 deletion in residue range 196-201 34 TIC80
_M13
46S, Y54
, S95A, S95A, F147A, P219R, then a 0.3 243 ND 0.85 at ND F147S, Q14
E, S167R, Q149E contiguous triple least P219R, R27
W, then a deletion in residue 262 contiguous triple range 196-201 deletion in residue range 196-201 3
TI
807_M12
46S,
54H, S95A, S95A, F147A, P21
R, then a 0.4 182 ND 1.
at ND F147A, S167R, P219R, V251A contiguous triple least N239A, V251A, then a deletion in residue 18
contiguous triple range 1
-201 deletion in residue range 196-201
_
TI
_
_deletion none a contiguous triple 22.4 3 ND ND — ND deletion in HYSHYS residues (positions 196-201)
T
_38
46
, Y54H, F138V,
S217N, then a ND — 4
ND — 4
S1
7R, S217N, then a contiguous triple contiguous triple deletion in residue deletion in residue range 196-201 range 196-201
TI
_
F
, Y54H, S167R,
S217N, then a ND — 5
ND — 31 S21
N, H287F, then a contiguous triple contiguous triple deletion in residue deletion in residue range 196-201 range 196-201 4
TI
80
_40 F
S, I52M, Y54H,
S217N, then a ND — 52 ND — 3
S167R, S217N, then a contiguous triple contiguous triple deletion in residue deletion in residue range 19
-201 range 196-201 41 TI
N12D, F46
, Y54H,
S217N, then a ND — 52 ND — 31 S1
7R, S217N, then a contiguous triple contiguous triple deletion in residue deletion in residue range 196-201 range 196-201 42 TIC80
_42 F46
, Y54H, S167R, N180D S2
7N, then a ND — 63 ND — 50 N180D,
21
N, then a contiguous triple contiguous triple deletion in residue deletion in residue range 196-201 range 196-201
TIC
_43 F
6S, Y54H, none a contiguous triple ND — 4
ND — 87 S167R, then a deletion in contiguous triple HYSHYS deletion in residue residues (positions range 196-201 196-201) 44 TIC
_44 F46S, Y54H, S167R, none S217N, P219R, then ND — 30 ND — 9
S217N, P219R, then a a contiguous triple contiguous triple deletion in residue deletion in residue range 196-201 range 196-201 4
TI
_
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 196-201 range 196-201 46 TI
80
_4
46S, Y54H, S167R,
S217N, then a ND — 5
ND — 31 S217N, T247
, then a contiguous triple contiguous triple deletion in residue deletion in residue range 19
-201 range 196-201
T
_
S, Y5
, S1
7R,
S217N, then a ND —
ND —
1 S217N, V244I, then a contiguous triple contiguous triple deletion in residue deletion in residue range 196-201 range 1
-201
T
F46S, Y54H, S1
7R,
S217N, then a ND — 58 ND — 34 S217N, V244I, contiguous triple T24
K, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201
T
F46S, Y54H, S1
7R, none S217N, W2
Y, ND — 17 ND — 13 S217N, W22
Y, then a then a contiguous contiguous triple triple deletion in deletion in residue residue range 19
-201 range 196-201 50 TI
_
F
S, Y5
H, S167R,
S217N, then a ND — 48 ND — 19 S217N, Y24
F, then a contiguous triple contiguous triple deletion in residue deletion in residue range 196-201 range 196-201 51 TIC80
_51
46S, Y54H, F147A, F14
7A S217N, then a ND — 7
ND — ND G128A, S167R, S217N, contiguous triple then a contiguous triple deletion in residue deletion in residue range 196-201 range 196-201 5
TIC
0
_52 F46S, Y54H, F147A, F1
7A S217N, then a ND — 73 ND — ND S167R, S217N, contiguous triple M300A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201
TIC
_
F46S, Y54H, F147A, F14
A S217N, then a ND —
ND — ND S167R, S217N, contiguous triple S293A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201
TI
F46S, Y54H, F147A, F1
7A S217N, then a ND — 67 ND — ND S167R, S217N, contiguous triple H287A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201
TI
80
_
46S, Y54H, F147A, F1
A S217N, then a ND — 65 ND — ND S167R, S217N, contiguous triple T274A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 5
TIC
_56
6S, Y
H, F147A,
A S217N, then a ND —
ND — ND S167R, S217N, contiguous triple R282A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 1
-201
TIC
_
F
S, Y
, T9
A, T9
A S217N, then a ND —
ND — ND S167R, S217N, contiguous triple T
A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 5
TI
_58 F4
S, Y54H, Q
0A, T9
A S217N, then a ND — 61 ND — ND T9
A, S16
R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 19
-201
TI
_
F
6S, Y54H,
125A,
A S217N, then a ND —
ND — ND F147A, S1
7R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 60 TIC80
_60 F46S, Y54H, F147A, F1
7A S217N, then a ND — 61 ND — ND S167R, S217N, contiguous triple T247A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 61 TIC80
_61
6S, Y54H, T93A, T93A S217N, then a ND — 59 ND — ND S167R, S217N, contiguous triple P305A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 62 TIC
_62 F46S, Y54H, F14
A, F147A S217N, then a ND —
ND — ND S167R, S217N, contiguous triple I306A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201
TI
80
_
F46S, Y54H, T93A, T
3A S217N, then a ND —
ND — ND S167R, S217N, contiguous triple R282A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 64 TIC8
_
46S, Y54H, T93A, T
3A S217N, then a ND — 55 ND — ND S167R, S21
N, contiguous triple T308A, then a deletion in residue contiguous triple range 19
-201 deletion in residue range 196-201 65 TI
_
6S, Y
4H, T93A, T
3A S217N, then a ND —
ND — ND S167R, S217N, contiguous triple M300A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 19
-201
TI
_
F4
S, Y54H, T9
A, T9
A S217N, then a ND — 53 ND — ND S167R, S217N, contiguous triple H287A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201
TI
F46S, Y54H, S95A, S95A S217N, then a ND — 52 ND — ND S167R, S217N, contiguous triple M300A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 6
TI
_
F46S, Y54H, F147A,
A S217N, then a ND —
ND — ND S167R, S217N, contiguous triple I275A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 6
TIC80
_69
46S, Y
4H, S95A, S95A S217N, then a ND — 51 ND — ND S167R, S217N, contiguous triple T247A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201
TIC
_
0
46S, Y54H, F147A, F147A S217N, then a ND — 50 ND — ND S167R, V175A, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201
1 TIC8
_71 F46S, Y54H, F147A, F147A S217N, then a ND —
0 ND — ND S159A, S167R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 19
-201
2 TI
80
_72 F46S, Y54H, S95A, S95A, F14
A S217N, then a ND — 48 ND — ND F147A, S167R, contiguous triple S21
N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 19
-201
TI
_7
46S, Y54H, F147A, F14
A S217N, then a ND —
ND — ND S167R, L187A, contiguous triple S21
N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201
4 TI
_
6S, Y
4H, T93A, T
3A S217N, then a ND — 4
ND — ND S167R, T182A, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 1
-201
T
_
F4
S, Y54H,
147A, F147A S217N, then a ND — 4
ND — ND S167R, S217N, contiguous triple T245A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201
TI
_
F4
S, Y54H, T93A, T93A S217N, then a ND — 4
ND — ND S167R, S217N, contiguous triple S249A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 19
-201
TI
_
F
6S, Y54H, T9
A, T
A, Q1
9A S217N, then a ND — 45 ND — ND Q1
A, S167R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201
8 TIC80
_78 F46S, Y54H, T93A, T9
A, S151A S217N, then a ND —
5 ND — ND S151A, S167R, contiguous triple S21
N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201
9 TIC80
_
9
46S, Y
4H, Q70A, F147A S217N, then a ND — 4
ND — ND F1
A, S167R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 80 TIC
_
F46S, Y54H, S95A, S9
A S217N, then a ND — 44 ND — ND Q148A, S167R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 19
-201 81 TI
80
_81 F46S, Y54H, T
3A, T9
A S217N, then a ND — 44 ND — ND S167R, S217N, contiguous triple T274A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 19
-201
TI
80
_
2
46S, Y54H, T93A S217N, then a ND — 43 ND — ND T93A, S167R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 8
TI
_
6S, Y54H, T93A, T9
A S217N, then a ND — 42 ND — ND S167R, M177A, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 19
-201 8
TI
_
F4
S, Y54H, S95A, S9
A S217N, then a ND — 42 ND — ND S167R, S217N, contiguous triple V250A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 85 TIC
_
5 F4
S, Y54H, T93A, T93A S217N, then a ND — 42 ND — ND
155A, S167R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 19
-201 86 TI
_
6 F
6S,
5
, I134A,
A S217N, then a ND — 41 ND — ND F147A, S1
7R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201
TIC80
_8
F46S, Y
, T93A, T93A S217N, then a ND — 41 ND — ND S167R, S217N, contiguous triple T24
A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201
TIC80
_88
46S, 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 196-201 range 196-201 followed by S198A followed by a S198A
TIC
_
F46S, Y54H, F147A, F147A S217N, then a ND — 40 ND — ND S167R, S217N, contiguous triple N295A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 9
TI
_
F46S, Y54H, A87S, F1
7A S217N, then a ND — 38 ND — ND F1
A, S167R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 19
-201
1 TI
_
46S, Y54H, F147A, F147A S217N, then a ND — 38 ND — ND S1
7R, S21
N, contiguous triple S24
A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 19
-201
TI
_
46S, Y54H, S95A, S95A S217N, then a ND —
ND — ND S167R, S217N, contiguous triple I275A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 9
TI
46S, Y
H, T93A, T93A S217N, then a ND —
ND — ND A139S, S167R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 19
-201 9
TI
_
F46S, Y54H,
147A, F1
A S217N, then a ND —
ND — ND S167R, S217N, contiguous triple P
05A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201
5 TI
_
F46S, Y54H,
147A,
A S217N, then a ND —
ND — ND S167R, S217N, then a contiguous triple contiguous triple deletion in residue deletion in residue range 196-201 range 196-201 followed by S198A followed by a S19
A 9
TIC
07_9
F46S, Y54H,
147A, F147A S217N, then a ND —
ND — ND S167R, S217N, contiguous triple S252A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201
TIC807_9
F46S, Y54H, T
3A, T93A, F147A S217N, then a ND — 36 ND — ND F147A, S167R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 9
TIC80
_98 F46S, Y54H, F147A, F1
A S217N, then a ND — 36 ND — ND S167R, S21
N, contiguous triple V250A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 99 TI
_99 F46S, Y54H, F147A, F1
A S217N, then a ND —
ND — ND S167R, S217N, contiguous triple T2
3A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 100 TI
_100 F46S, Y5
H, F1
7A, F1
A S217N, then a ND —
ND — ND
167R, S217N, then a contiguous triple contiguous triple deletion in residue deletion in residue range 196-201 range 19
-201 followed by Y197A followed by a Y1
A 101 TI
_101 F46S, Y54H, S95A, S95A S217N, then a ND —
ND — ND S167R, S217N, contiguous triple N295A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 19
-201 102 TI
_102 F46S, Y54H, F147A, F147A S217N, then a ND —
ND — ND S167R, S217N, then a contiguous triple contiguous triple deletion in residue deletion in residue range 196-201 range 19
-201 10
TI
_103
S, Y5
H, F147A, F147A S217N, then a ND —
2 ND — ND S167R, S217N, contiguous triple E299A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 1
TIC80
_10
F46S, Y54H, S95A, S95A S217N, then a ND — 32 ND — ND S167R, S217N, contiguous triple R282A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 105 TIC
_105 F46S, Y54H, T93A, T9
A S217N, then a ND —
ND — ND S167R, S217N, contiguous triple I306A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 19
-201 10
TIC80
_1
F4
S, Y54H, S95A, S9
A S217N, then a ND — 30 ND — ND S167R, S217N, contiguous triple S249A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 107 TIC
_1
F46S, Y54H, A87S, T9
A S217N, then a ND —
ND — ND T9
A, S167R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 108 TI
_108
6S, Y
4H, T93A, T93A S217N, then a ND —
ND — ND S159A, S1
7R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 10
TI
_10
46S, Y54H,
147A, F1
7A S217N, then a ND —
ND — ND S167R, S217N, contiguous triple T
03A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 110 TI
_110
46S, Y54H, T93A, T9
A S217N, then a ND —
ND — ND Q148A, S167R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 19
-201 111 TI
_111 F46S, Y54H, S95A, S9
A S217N, then a ND — 27 ND — ND S167R, V175N, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 19
-201 11
TI
_11
S, Y5
H, F147A, F14
7A S217N, then a ND — 27 ND — ND S167R, S217N, contiguous triple
241A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 11
TI
_113 F46S, Y54H, S95A, S95A, F147A S217N, then a ND — 2
ND — ND F147A, S167R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 114 TIC
_114 F46S, Y54H, F147A, F147A S217N, then a ND — 26
ND — ND S167R, M177A, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 19
-201 11
TIC80
_11
F4
S, Y54H, F147A, F147A S217N, then a ND — 26 ND — ND S167R, S217N, contiguous triple M300A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 116 TIC
_11
F46S, Y54H, S95A, S95A S217N, then a ND — 26 ND — ND S1
7R, S217N, contiguous triple
30
A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 117 TI
_11
6S, Y
4H, F147A, F147A S217N, then a ND —
ND — ND S167R, S217N, then a contiguous triple contiguous triple deletion in residue deletion in residue range 196-201 range 196-201 followed by H196A followed by
19
A 11
TI
_11
46S, Y54H, A139S,
1
7A S217N, then a ND — 25 ND — ND F147A, S167R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 119 TI
_11
6S, Y54H, T93A, T9
A S217N, then a ND — 2
ND — ND S167R, S217N, contiguous triple N295A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 12
TIC80
_120 F4
S, Y54H, T93A, T93A S217N, then a ND — 24 ND — ND T1
A, S1
7R, contiguous triple S2
7N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 19
-201 1
1 TI
121 F46S, Y54H, S117A, F1
7A S217N, then a ND —
ND — ND F14
A, S16
R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 1
TIC
_122 F46S, Y54H, T93A, T93A S217N, then a ND — 23 ND — ND S167R, S217N, contiguous triple I275A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 123 TIC
_12
F46S, Y54H, S95A, S9
A S217N, then a ND — 23 ND — ND S167R, S217N, contiguous triple H287A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 124 TIC807_1
F46S, Y54H, S95A, S95A S217N, then a ND — 2
ND — ND G105A, S167R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 1
5 TIC80
_12
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 196-201 range 196-201 12
TI
_126
6S, Y
H, S95A, S
A S217N, then a ND — 20 ND — ND I1
A, S167R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 1
TI
_127
S, Y
H, T9
A, T93A S217N, then a ND — 20 ND — ND S167R, S217N, contiguous triple M300A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 12
TIC807_1
F4
S, Y54H, T93A, T93A S217N, then a ND — 18 ND — ND S167R, S217N, contiguous triple T30
A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 19
-201 129 TIC80
_12
F4
S, Y54H, T
3A, T93A S217N, then a ND — 18 ND — ND A150S, S1
7R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 19
-201 1
0 TI
_1
0
46S, Y54H, S95A, S95A S217N, then a ND — 18 ND — ND
155A, S167R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 1
1 TIC80
_131 F46S, Y54H, T93A, T93A S217N, then a ND — 17 ND — ND T145A, S167R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 1
2 TIC
_1
2 F46S, Y54H, S95A, S9
A W208A, S217N, ND — 17 ND — ND S167R, W20
A, then a contiguous S217N, then a triple deletion in contiguous triple residue deletion in residue range 196-201 range 196-201 133 TIC807_133 F46S, Y54H, S95A, S95A, T1
2A S217N, then a ND — 17 ND — ND S167R, T182A, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 1
TIC80
_134 F46S, Y54H, T93A, T
3A S217N, then a ND — 17 ND — ND S167R, S217N, contiguous triple T243A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 1
TI
_1
5
6S, Y
H, S95A, S
A S217N, then a ND — 1
ND — ND S167R, S217N, contiguous triple I30
A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 1
6 TI
_13
F4
S, Y54H, S95A, S
5A S217N, then a ND — 1
ND — ND S117A, S167R, contiguous triple S
17N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 1
TIC8
_1
F4
S, Y54H, T
A, T93A S217N, then a ND — 15 ND — ND S119A, S1
7R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 19
-201 1
8 TIC807_13
F4
S, Y
4H, T
8A, F147A S217N, then a ND — 15 ND — ND F147A, S167R, contiguous triple S21
N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 19
-201 1
9 TI
_1
9
46S, Y54H, G105A, F14
A S217N, then a ND — 15 ND — ND F147A, S167R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 140 TIC
_1
0 F46S, Y54H, S95A, S95A S217N, then a ND — 15 ND — ND
125A, S167R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 141 TIC
_1
1 F46S, Y5
H, T93A, T9
A S217N, then a ND — 14 ND — ND E155A, S167R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201
2 TIC807_1
2 F46S, Y54H, T9
A, T93A S217N, then a ND — 14 ND — ND S167R, S217N, contiguous triple P305A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 14
TIC80
_143 F46S, Y54H, S95A, S95A S217N, then a ND — 13 ND — ND S167R, M177A, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 144 TIC80
_144
6S, Y
4H, T93A, T93A S217N, then a ND — 13 ND — ND S167R, S217N, then a contiguous triple contiguous triple deletion in residue deletion in residue range 196-201 range 196-201 followed by H196A followed by
196A 14
TI
_14
6S, Y54H, T9
A, T9
A S217N, then a ND — 11 ND — ND D153A, S167R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range
-201-T
A- D15
A 1
6 TI
_1
6S, Y54H,
147A, F147A W208A, S217N, ND — 10 ND — ND S167R, S21
N, then a contiguous W
08A, then a triple deletion in contiguous triple residue deletion in residue range 196-201 range 196-201 1
7 TIC
_1
F4
S, Y54H, T93A, T93A S217N, then a ND —
ND — ND S167R, S21
N, contiguous triple I
06A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 19
-201 14
TI
_14
F
6S, Y5
H, S
5A, S
5A S217N, then a ND —
ND — ND S1
7R, S217N, then a contiguous triple contiguous triple deletion in residue deletion in residue range 196-201 range 196-201 followed by H196A followed by
196A 149 TIC807_14
F46S, Y54H, S95A, S95A, Q149A S217N, then a ND — 9 ND — ND Q149A, S167R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 1
0 TIC80
_150 F
6S, Y
H, S95A, S95A S217N, then a ND — 8 ND — ND S167R, S217N, contiguous triple S2
3A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 151 TIC
_151 F46S, Y5
H, S95A, S9
A S217N, then a ND — 8 ND — ND A150S, S167R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 152 TIC8
_15
F46S, Y54H, S119A, F1
7A S217N, then a ND — 7 ND — ND F1
A, S167R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 19
-201 1
TIC
_15
F46S, Y54H, S95A, S95A S217N, then a ND — 7 ND — ND S1
7A, S217N, then a contiguous triple contiguous triple deletion in residue deletion in residue range 196-201 range 19
-201 154 TI
_1
4 F46S, Y54H, F147A, F1
7A S217N, then a ND — 7 ND — ND I158A, S167R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 155 TI
_155 F
6S, Y5
H, S95A, S
5A S217N, then a ND — 7 ND — ND S167R, S217N, contiguous triple M
00A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 1
-201 156 TI
_156
6S, Y54H,
147A, F1
A, T18
A S217N, then a ND —
ND — ND T182A, S167R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 157 TI
_15
S, Y54H,
147A,
A S217N, then a ND —
ND — ND S167R, S21
N, contiguous triple F235A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 158 TIC
_1
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 196-201 range 196-201 1
9 TIC80
_1
9 F
6S, Y54H, T93A, T
3A S217N, then a ND — 5 ND — ND S167R, S217N, contiguous triple D241A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 160 TIC
_160 F46S, Y5
H, S95A, S
5A S217N, then a ND — 4 ND — ND S167R, S217N, contiguous triple T27
A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 1
1 TIC
_161 F46S, Y54H, T93A, T
A S217N, then a ND — 4 ND — ND Q14
A, S16
R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 19
-201 1
2 TIC
_162 F46S, Y54H, S95A, S95A S217N, then a ND — 4 ND — ND S1
7R, S217N, contiguous triple D241A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 19
-201 16
TI
80
_1
3
46S, Y54H, S95A,
95A S217N, then a ND — 4 ND — ND
155A, S167R, contiguous triple S21
N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 1
4 TI
_164
6S, Y
4H, T93A, T9
A S217N, then a ND — 4 ND — ND S167R, L187A, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 19
-201 1
TI
_16
6S, Y54H, T9
A, T93
A S217N, then a ND — 4 ND — ND S167R, S217N, contiguous triple T
03A, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 1
TI
_166
S, Y54H, T
3A, T
3A S217N, then a ND —
ND — ND
155A, S167R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 167 TIC8
_1
F46S, Y
H, T93A, T9
A S217N, then a ND — 3 ND — ND D153A, S167R, contiguous triple S217N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 168 TIC80
_168 F46S, Y
4H, T93A, T9
A S217N, then a ND — 3 ND — ND I1
A, S16
R, contiguous triple S21
N, then a deletion in residue contiguous triple range 196-201 deletion in residue range 196-201 169 TIC80
_169 N137T none None ND —
* ND — ND 169 TIC80
_169 E135S none None ND — 20* ND — ND 169 TIC80
_169 N137T, E135S none None ND — 34* ND — ND 169 TIC80
_169 E135T, N137D none None ND — 16* ND — ND 169 TIC80
_169 T133E none None ND — 60* ND — ND 16
TIC80
_169 E1
5A, N137G none None ND — 50* ND — ND 170 TIC80
_1
0
125C none None ND — 12 ND — ND 170 TIC80
_1
0
125H none None ND — 38 ND — ND 170 TIC80
_170
125R none None ND — 14 ND — ND
TIC80
_170
125F none None ND — 33 ND — ND 1
TIC80
_170
125S none None ND — 24 ND — ND 1
TIC
_170
125Q none None ND — 2
ND — ND 1
0 TIC
_170
125K none None ND — 20 ND — ND 1
0 TI
_170
125T none None ND — 33 ND — ND 1
0 TIC
_170
125N none None ND — 19 ND — ND 170 TI
_170
125A none None ND —
ND — ND 170 TIC
_17
125
none None ND — 1
ND — ND 170 TI
_170
125V none None ND — 1
ND — ND 170 TI
_1
0
125M none None ND — 1
ND — ND 170 TI
_1
0
125D none None ND — 15 ND — ND 1
TI
_1
0
12
Y none None ND — 38 ND — ND 1
1 TI
_1
1 T1
none None ND — 23 ND — ND 171 TI
_1
1 T1
Y none None ND — 17 ND — ND 171 TI
_171 T1
W none None ND — 13 ND — ND 172 TI
_17
I1
V none None ND — 1
ND — ND 1
2 TI
_1
I1
L none None ND — 10 ND — ND 1
2 TI
_1
I1
F none None ND — 18 ND — ND 1
2 TI
_1
I1
K none None ND — 13 ND — ND 1
2 TI
_1
I1
none None ND — 30 ND — ND 1
2 TI
_1
I1
M none None ND — 33 ND — ND 1
TIC80
_1
E135V none None ND — 13 ND — ND 1
3 TIC80
_1
E135W none None ND — 13 ND — ND 1
3 TIC80
_1
E135T none None ND — 39 ND — ND 174 TIC80
_1
N137H none None ND — 42 ND — ND 1
TIC
_1
N13
Y none None ND — 17 ND — ND 1
4 TIC
_1
N13
none None ND —
ND — ND 1
4 TIC
_1
N13
E none None ND — 32 ND — ND 1
4 TIC
_1
N137S none None ND — 24 ND — ND 1
4 TIC
_1
N137A none None ND — 24 ND — ND 1
4 TIC
_1
N137Q none None ND —
1 ND — ND 1
4 TIC
_1
N137G none None ND — 1
ND — ND 1
4 TIC
_1
N137I none None ND — 10 ND — ND 1
4 TIC
_1
N137W none None ND — 17 ND — ND 1
4 TIC
_1
N137K none None ND — 66 ND — ND 1
4 TIC
_1
N137C none None ND — 19 ND — ND 1
4 TIC
_1
N137M none None ND —
1 ND — ND 1
4 TIC
_1
N137D none None ND — 69 ND — ND 1
4 TIC
_1
N137F none None ND — 13 ND — ND 1
4 TIC
_1
N137R none None ND —
7 ND — ND 175 TI
_1
147V F1
7V None ND — 87 ND — ND 1
5 TI
_1
147T F1
7T None ND — 68 ND — ND 1
5 TI
_1
F147
F147
None ND — 74 ND — ND 1
5 TI
_1
F147L F147L None ND — 62 ND — ND 1
5 TI
_1
147D F147D None ND — 5
ND — ND 1
5 TI
_1
147A F147A None ND — 57 ND — ND 1
5 TI
_1
F147G F147
None ND — 56 ND — ND 1
5 TI
_1
F147E F147E None ND — 5
ND — ND 1
5 TI
_1
F147I F147I None ND —
ND — ND 1
5 TI
_1
F147Y F147Y None ND —
ND — ND 1
5 TI
_1
F147M F147M None ND —
ND — ND 1
5 TI
_1
F147N F147N None ND —
ND — ND 1
TI
_1
F147Q F147Q None ND — 50 ND — ND 1
TI
_1
F147H F147H None ND — 60 ND — ND 1
TI
_1
147R F147R None ND — 20 ND — ND 1
TI
_1
147W F147W None ND — 82 ND — ND 1
TI
_1
147P F147P None ND — 7 ND — ND 176 TI
_1
Q149D Q1
D None ND — 92 ND — ND 176 TI
_1
Q1
9
Q1
None ND — 89 ND — ND 176 TI
_1
Q1
9C Q1
None ND — 87 ND — ND 17
TI
_1
Q1
9A Q1
A None ND — 76 ND — ND 17
TI
_1
Q1
9F Q1
F None ND — 54 ND — ND 1
TI
_1
A150S none None ND — 34 ND — ND 1
TI
_1
A150L none None ND — 24 ND — ND 1
TIC
_1
A150V none None ND — 25 ND — ND 1
TIC
_1
A150G none None ND — 28 ND — ND 1
TIC
_1
A150D none None ND — 19 ND — ND 1
TIC
_1
A150W none None ND — 13 ND — ND 1
TIC
_1
A150E none None ND — 24 ND — ND 1
TIC
_1
A150N none None ND — 18 ND — ND 1
TIC
_1
A150Y none None ND — 11 ND — ND 1
TIC
_1
A150F none None ND — 11 ND — ND 1
TIC
_1
A150P none None ND — 11 ND — ND 1
TIC
_1
A150K none None ND — 17 ND — ND 1
TIC
_1
A150T none None ND — 17 ND — ND 1
TIC
_1
A150Q none None ND — 11 ND — ND 1
TIC
_1
A150R none None ND — 11 ND — ND 1
TIC
_178 E155C none None ND — 82 ND — ND 1
TIC
_1
E155I none None ND — 36 ND — ND 1
TIC
_1
155K none None ND — 28 ND — ND 1
TIC
_1
155D none None ND — 22 ND — ND 1
TIC
_1
155H none None ND — 22 ND — ND 1
TIC
_1
E155Y none None ND — 16 ND — ND 1
TIC
_1
155Q none None ND — 16 ND — ND 1
TI
_1
155L none None ND — 15 ND — ND 1
TI
_1
155N none None ND — 14 ND — ND 1
TI
_1
155T none None ND — 13 ND — ND 1
TI
_1
155A none None ND — 11 ND — ND 1
TI
_1
155F none None ND — 7 ND — ND 1
TI
_1
155R none None ND —
ND — ND 1
TI
_1
155M none None ND —
ND — ND 1
TI
_1
155P none None ND —
ND — ND 1
TIC
_1
155W none None ND —
ND — ND 1
TI
_1
155S none None ND — 4 ND — ND 1
TI
_1
15
V none None ND — 4 ND — ND 1
TI
_179 N157
none None ND — 86 ND — ND 1
TI
_1
N157D none None ND — 64 ND — ND 1
TI
_1
N157W none None ND —
2 ND — ND 1
TI
_1
N157Y none None ND — 39 ND — ND 1
TI
_1
N1
7M none None ND —
ND — ND 179 TI
_1
N1
A none None ND —
ND — ND 179 TI
_1
N1
F none None ND — 20 ND — ND 179 TI
_1
N157V none None ND — 18 ND — ND 1
TI
_1
N157L none None ND — 18 ND — ND 1
TI
_1
N157P none None ND — 18 ND — ND 1
TI
_1
N157E none None ND — 17 ND — ND 1
TI
_1
N157T none None ND — 8 ND — ND 1
9 TIC80
_1
N157G none None ND — 7 ND — ND 1
9 TIC80
17
N157I none None ND — 7 ND — ND 1
TIC80
1
N157R none None ND — 6 ND — ND 2
TIC80
_M8_d5C F
6S, Y54H, S95A, S95A, F147A S217N, then a ND — 92* ND — ND
147A, S167R, contiguous triple S217N, then a five deletion in residue amino acid deletion range 196-201 at 305-309, then a contiguous triple deletion in residue range 196-201 204 TIC80
_M1
46S, Y54H, S95A, S95A, F1
7A, P219R, then a ND — — ND — 60* F147S, Q14
E, S16
R, Q14
E, V251E, contiguous triple P219R, S24
R, V251
, Q253R deletion in residue Q253R, R
73W, then a range 196-201 contiguous triple deletion in residue range 196-201 ND = Not Determined; *tested at about 5 μg/mL.
indicates data missing or illegible when filed
Example 5
Insect Inhibitory Activities of Protein Members of the Present Invention
[0107] 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.
[0108] Proteins TIC807_M10 (SEQ ID NO:24), TIC807_M11 (SEQ ID NO:26), TIC807_M12 (SEQ ID NO:28), and TIC807_M16 (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 μ/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-US-00006 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); southwestern corn borer (Diatraea grandiosella); CEW = corn earworm (Helicoverpa zea); FAW = Fall armyworm (Spodaptera frugiperda); SGSB = southern green stink bug (Nezara virudula); NBSB = neotropical brown stink bug (Euschistus heras); GPA = Green peach aphid (Myzus persicae).
[0109] 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
[0110] 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.
[0111] 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_M22), SEQ ID NO:24 (TIC807_M24), SEQ ID NO:26 (TIC807_M26), SEQ ID NO:28 (TIC807_M9), SEQ ID NO:30 (TIC807_M10), SEQ ID NO:32 (TIC807_M11), and SEQ ID NO:45 (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.
[0112] 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.
[0113] 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.
[0114] 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., NH). The branch inside a mesh bag was infested with nymphs by sliding the uncapped conical tube into the mesh bag. Insects were allowed to feed for a period of 10-11 days before 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-US-00007 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 Mor- Plant % tality Event Mor- Std Lo Up t ID Protein N tality Dev SEM 95% 95% group 64 TIC807_M8 18 78.88
49 TIC807_M8 18 75.556
A
1 TIC807_M2 18 74.444
A 2
TIC807_M8 18 73.333
A 1
TIC807_M8 18 66.667
AB
8 TIC807_M2 18 65.
6
AB 48 TIC807_M2 18 64.444
AB 19 TIC807_M2 18 53.333
BC
8 TIC807_M2 18 47.778
Nega- 24 41.667
tive
indicates data missing or illegible when filed
TABLE-US-00008 TABLE 6B Mean Instar determined from flowering stage Lygus feeding assays with cotton plants expressing the variant TIC807 proteins TIC807_M2 and TIC807_M8. Plant Instar Event Mean Std Lo Up t ID Construct N Instar Dev SEM 95% 95% group 4 TIC807_M8 11 3.636 0.
0.
4.
C
8 TIC807_M2 1
3.949 0.
0.
4.
48 TIC807_M
1
4.042 0.
0.
4.
8 TIC807_M
17 4.069 0.
0.
4.
B
1
TIC807_M
1
4.094 0.
0.
4.
B
19 TIC807_M2 17 4.100 0.
0.
4.
BC 91 TIC807_M2 12 4.125 0.
0.
4.
ABC 49 TIC807_M8 12 4.139 0.
0.
4.
ABC
TIC807_M8 14 4.298 0.
0.
4.
B Negative 24 4.599 0.
0.
4.
A
indicates data missing or illegible when filed
TABLE-US-00009 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. Sur- Mean vivor Plant Sur- Mass Event vival Std Lo Up t ID Construct N Mass Dev SEM 95% 95% group 64 TIC807_M8 11 2.315 1.4 0.44
1.
3.31
C 68 TIC807_M2 1
3.548 1.3
0.
2.84
4.
4 B 58 TIC807_M2 17 3.561 1.34
0.
2.
8 4.
B 48 TIC807_M2 1
3.596 1.4
0.3
2.831 4.
B 91 TIC807_M2 12 3.775 1.77
0.
12 2.647 4.9
2 AB 49 TIC807_M
1
3.837 2.1
0.61
2.481 5.1
AB 1
TIC807_M2 17 3.908 1.4
7 0.3
6
.1
4 4.
AB 20 TIC807_M8 14 3.918 1.95
0.521 2.79
5.
44 AB 1
TIC807_M
15 3.937 1.90
0.49
.88
4.
AB Negative 24 4.735 1.17
0.24
4.23
.2
A
indicates data missing or illegible when filed [0115] Std Dev=standard deviation [0116] SEM=Standard error on the mean [0117] Lo 95%=Lower limit at 95% confidence interval [0118] Up 95%=Upper limit at 95% confidence interval [0119] T grouping=using a least significant difference test. F value=101.1756, df=15.44, Pr<0.0001
[0120] 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 see cotton yield of the non-transgenic control, e.g. season-long square retention.
[0121] 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 See cotton yield from plants from three of the seven events was statistically higher than to seed cotton yield of the non-transgenic control.
[0122] In another example, cotton plants from thirty-four transgenic events expressing TIC807_M13 demonstrated growth chamber efficacy compared to the non-transgenic control Mesh bags were placed around the whole cotton plants at flowering stage (instead of just around single branches described earlier in this example). Five plants per event were evaluated and the average number of Lygus lineolaris insects recovered (nymphs to adults to 2.sup.nd generation Lygus) per plant was significantly lower than the average number of Lygus lineolaris insects per non-transgenic plant.
[0123] 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
[0124] 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.
[0125] Polynucleotide segment from SEQ ID NO:192 (encodes for TIC807_807. 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].
[0126] 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-US-00010 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 Std Lower Upper t Construct Sample Source fold Score 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 per 900 0.00 0.00 0.00 0.00 0.00 E [ES] construct 100 3.00 0.00 0.00 3.00
.00 A TIC807_M8 300 2.33 0.58 0.33 0.
0
BC 900 0.00 0.
0 0.00 0.00 0.00 E [ET] 100 2.67 0.
8 0.
3 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 E None Control 100 2.00 0.00 0.00
.00 2.00
Alfalfa 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 0 0.00 0.00 0.00 0.00 0.00 E in the diet
indicates data missing or illegible when filed
TABLE-US-00011 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 Std Lower Upper t Construct Sample Source fold mortality Dev SEM 95% 95% Grouping [ER] Pooled Alfalfa 100 4.17 7.22 4.1 −13.76 22.09 CD T
IC807_M8
300 0.00 0.
0 0.00 0.00 0.00 CD
construct 900 1
.10 1
.
.24 −18.06 44.
CD [ES] 100
.
.
.62
.
7
.12 AB
I
807_M8 300 41.
7 1
.0
11.
−5.
7 89.1
B 900 0.00 0.00 0.
0 0.00 0.00 CD [ET] 100 64.88 1
.
1 11.
0 15.42 114.
4 A TIC807_M8 300 16.6
1
.09 11.02
.77 6
.10
900 1
.50 12.
0 7.
−1
.
4
.
CD None Control 100 12.
0 12.
0 7.
−1
.
4
.
CD Alfalfa 300 0.00 0.00 0.00 0.00 0.00 CD
00 8.33 1
.43 8.
−2
.
2
.19 CD No Alfalfa incorporated 0 2.50
.01 1.81 −1.
8 6.
8 D in the diet
indicates data missing or illegible when filed
Example 8
Plants Co-Expressing an eHTP and a Second Insect Inhibitory Protein Exhibiting Lygus Species Inhibitory Activity
[0127] 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-US-00012 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.
[0128] 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-US-00013 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)