US 20140274885A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2014/0274885 A1 Cong et al. (43) Pub. Date: Sep. 18, 2014

(54) PHI-4 POLYPEPTIDES AND METHODS FOR (52) U.S. Cl. THEIR USE CPC ...... A0IN 43/40 (2013.01); C07K 14/195 (71) Applicant: PIONEER HI-BRED (2013.01) INTERNATIONAL, INC., Johnston, IA USPC ...... 514/4.5; 530/350; 536/23.7 (US) (57) ABSTRACT (72) Inventors: Ruth Cong, Palo Alto, CA (US); Jingtong Hou, San Pablo, CA (US); Compositions and methods for controlling pests are provided. Zhenglin Hou, Ankeny, IA (US); Phillip The methods involve transforming organisms with a nucleic Patten, Menlo Park, CA (US): Takashi acid sequence encoding an insecticidal protein. In particular, Yamamoto, Dublin, CA (US) the nucleic acid sequences are useful for preparing plants and (73) Assignee: PIONEER HI-BRED microorganisms that possess insecticidal activity. Thus, INTERNATIONAL, INC, Johnston, IA transformed bacteria, plants, plant cells, plant tissues and (US) seeds are provided. Compositions are insecticidal nucleic acids and proteins of bacterial species. The sequences find use (21) Appl. No.: 13/839,702 in the construction of expression vectors for Subsequent trans (22) Filed: Mar 15, 2013 formation into organisms of interest, as probes for the isola tion of other homologous (or partially homologous) genes. Publication Classification The insecticidal proteins find use in controlling, inhibiting (51) Int. Cl. growth or killing lepidopteran, coleopteran, dipteran, fungal, AOIN 43/40 (2006.01) hemipteran, and nematode pest populations and for produc C07K I4/95 (2006.01) ing compositions with insecticidal activity. Patent Application Publication Sep. 18, 2014 Sheet 1 of 5 US 2014/0274885 A1

100% SFR12-004 90% :

80% : : 70% 60% : & Ax205 w : ; : : : 8 50% : '-' half : s: : * Fu 40% : : redicted 30% : C50

20% : & 10% 0% -...... 5. 50 dose (ppm) 500 Patent Application Publication Sep. 18, 2014 Sheet 2 of 5 US 2014/0274885 A1

& Axmi2OS & SFR12-004

Patent Application Publication Sep. 18, 2014 Sheet 4 of 5 US 2014/0274885 A1

Fig. 4

1. 8

1. 6 & y = 0.544x + 3.5735 A. R = 0.106

2

8 A.O6 2

O ~~~~~~~~~~~~~~~~~~ O 2 3 4 5 6 7 8 9 O 11 2 3 4 15 1.6 17 18 Mean Fold improvement (EC50)

US 2014/0274885 A1 Sep. 18, 2014

PH-4 POLYPEPTIDES AND METHODS FOR important insect pests. In some cases, insects can develop THEIR USE resistance to different insecticidal compounds, which raises the need to identify alternative biological control agents for REFERENCE TO SEQUENCE LISTING pest control. SUBMITTED ELECTRONICALLY 0007 Accordingly, there remains a need for new pesti 0001. The official copy of the sequence listing is submitted cidal proteins with different ranges of insecticidal activity electronically via EFS-Web as an ASCII formatted sequence against insect pests, e.g., insecticidal proteins which are listing with a file named “5192 Sequence Listing created active against a variety of insects in the order Lepidoptera and on Mar. 15, 2013, and having a size of 3730 kilobytes and is the order Hemiptera including but not limited to species filed concurrently with the specification. The sequence listing belonging to the family Pentatomidae, the family Plataspidae contained in this ASCII formatted document is part of the and the family Cydnidae. In addition, there remains a need for specification and is herein incorporated by reference in its biopesticides having activity against a variety of insect pests entirety. that have developed resistance to existing pesticides. LENGTHY TABLES SUMMARY OF THE INVENTION 0002 The patent application contains a lengthy table sec 0008 Compositions and methods for conferring pesticidal activity to bacteria, plants, plant cells, tissues and seeds are tion as an ASCII formatted document, which is part of the provided. Compositions include nucleic acid molecules specification and is herein incorporated by reference in its encoding sequences for pesticidal and insecticidal polypep entirety. A copy of the tables are available in electronic form tides, vectors comprising those nucleic acid molecules, and from the USPTO web site (http://seqdata.uspto.gov/). An host cells comprising the vectors. Compositions also include electronic copy of the table will also be available from the the pesticidal polypeptide sequences and antibodies to those USPTO upon request and payment of the fee set forth in 37 polypeptides. The nucleic acid sequences can be used in DNA CFR 1.19(b)(3) constructs or expression cassettes for transformation and FIELD OF THE INVENTION expression in organisms, including microorganisms and plants. The nucleotide or amino acid sequences may be syn 0003. This disclosure relates to the field of molecular biol thetic sequences that have been designed for expression in an ogy. Provided are novel genes that encode pesticidal proteins. organism including, but not limited to, a microorganism or a These pesticidal proteins and the nucleic acid sequences plant. Compositions also comprise transformed bacteria, encoding them are useful in preparing pesticidal formulations plants, plant cells, tissues and seeds. and in the production of transgenic pest-resistant plants. 0009. In particular, isolated or recombinant nucleic acid molecules are provided encoding PHI-4 polypeptides includ BACKGROUND OF THE INVENTION ing amino acid substitutions, amino acid deletions, amino 0004 Biological control of insect pests of agricultural sig acid insertions, and fragments thereof, and combinations nificance using a microbial agent, Such as fungi, bacteria, or thereof. Additionally, amino acid sequences corresponding to another species of insect affords an environmentally friendly the PHI-4 polypeptides are encompassed. Nucleic acid and commercially attractive alternative to synthetic chemical sequences that are complementary to a nucleic acid sequence pesticides. Generally speaking, the use of biopesticides pre of the embodiments, or that hybridize to a sequence of the sents a lower risk of pollution and environmental hazards, and embodiments are also encompassed. biopesticides provide greater target specificity than is char 0010 Methods are provided for producing the polypep acteristic of traditional broad-spectrum chemical insecti tides and for using those polypeptides for controlling, inhib cides. In addition, biopesticides often costless to produce and iting growth or killing a Lepidopteran, Coleopteran, nema thus improve economic yield for a wide variety of crops. tode, fungi, Hemipteran and/or Dipteran pests. The 0005 Certain species of microorganisms of the genus transgenic plants of the embodiments express one or more of Bacillus are known to possess pesticidal activity against a the pesticidal sequences disclosed herein. In various embodi range of insect pests including Lepidoptera, Diptera, ments, the transgenic plant further comprises one or more Coleoptera, Hemiptera and others. Bacillus thuringiensis additional genes for insect resistance, for example, one or (Bt) and Bacillus popilliae are among the most Successful more additional genes for controlling coleopteran, lepi biocontrol agents discovered to date. Insect pathogenicity has dopteran, hemipteran or nematodepests. It will be understood also been attributed to strains of B. larvae, B. lentimorbus, B. by one of skill in the art that the transgenic plant may com sphaericus and B. cereus. Microbial insecticides, particularly prise any gene imparting an agronomic trait of interest. those obtained from Bacillus strains, have played an impor 0011 Methods for detecting the nucleic acids and tant role in agriculture as alternatives to chemical pest control. polypeptides of the embodiments in a sample are also 0006 Crop plants have been developed with enhanced included. Akit for detecting the presence of a PHI-4 polypep insect resistance by genetically engineering crop plants to tide or detecting the presence of a nucleotide sequence encod produce pesticidal proteins from Bacillus. For example, corn ing a PHI-4 polypeptide in a sample is provided. The kit is and cotton plants have been genetically engineered to pro provided along with all reagents and control samples neces duce pesticidal proteins isolated from strains of Bt. These sary for carrying out a method for detecting the intended genetically engineered crops are now widely used in agricul agent, as well as instructions for use. ture and have provided the farmer with an environmentally 0012. The compositions and methods of the embodiments friendly alternative to traditional insect-control methods. are useful for the production of organisms with enhanced pest While they have proven to be very successful commercially, resistance or tolerance. These organisms and compositions these genetically engineered, insect-resistant crop plants pro comprising the organisms are desirable for agricultural pur vide resistance to only a narrow range of the economically poses. The compositions of the embodiments are also useful US 2014/0274885 A1 Sep. 18, 2014 for generating altered or improved proteins that have pesti position 46 is Glu or ASn; the amino acid at position 52 is Ile cidal activity, or for detecting the presence of PHI-4 polypep or Val; the amino acid at position 97 is Arg, Asp, Glu or ASn; tides or nucleic acids in products or organisms. the amino acid at position 98 is Tyr or Phe; the amino acid at The following embodiments are encompassed by the present position 99 is Lys or Leu; the amino acid at position 145 is Leu disclosure. or Val; the amino acid at position 150 is Argor Gln; the amino 0013 Embodiment 1 is a PHI-4 polypeptide having acid at position 151 is Asp or Ser; the amino acid at position improved insecticidal activity compared to AXMI-205 (SEQ 153 is Leu or Ile; the amino acid at position 163 is Leu or Val; ID NO:35). the amino acid at position 171 is Tyr or Phe; the amino acid at 0014 Embodiment 2 is the PHI-4 polypeptide of embodi position 172 is Ile or Leu; the amino acid at position 182 is ment 1, wherein the insecticidal activity is increased about 1.5 Asp or Gln; the amino acid at position 196 is Glin or Asn; the fold or greater compared to AXMI-205 (SEQID NO: 35). amino acid at position 206 is Tyr or Phe; the amino acid at 0015 Embodiment 3 is the PHI-4 polypeptide of embodi position 210 is Val or Ile; the amino acid at position 216 is Glu ment 1, wherein the insecticidal activity is increased about 2 or Gln; the amino acid at position 220 is Glu, Gln, His or Asp; fold or greater compared to AXMI-205 (SEQID NO: 35). the amino acid at position 278 is Glu or Asn; the amino acid 0016 Embodiment 4 is the PHI-4 polypeptide of embodi at position 283 is Ile or Val; the amino acid at position 289 is ment 1, wherein the insecticidal activity is increased about 2.5 LyS, Gln or Leu; the amino acid at position 293 is Arg, Gln or fold or greater compared to AXMI-205 (SEQID NO: 35). Glu; the amino acid at position 328 is Lys or Glu; the amino 0017 Embodiment 5 is the PHI-4 polypeptide of embodi acid at position 333 is Ser, Lys or Val; the amino acid at ment 1, wherein the insecticidal activity is increased about 3 position 334 is Gly, Lys or Arg; the amino acid at position336 fold or greater compared to AXMI-205 (SEQID NO: 35). is Gly or Ala; the amino acid at position 338 is Seror Val; the 0018 Embodiment 6 is the PHI-4 polypeptide of embodi amino acid at position 339 is Glu, ASnor Gln; the amino acid ment 1, wherein the insecticidal activity is increased about 5 at position 342 is Ala or Ser; the amino acid at position 346 is fold or greater compared to AXMI-205 (SEQID NO: 35). Pro or Ala; the amino acid at position 354 is Met or Leu; the 0019 Embodiment 7 is the PHI-4 polypeptide of any one amino acid at position 355 is Val or Ile; the amino acid at of embodiments 1-6, wherein the improved insecticidal activ position 370 is His or Arg; the amino acid at position 389 is ity compared to AXMI-205 (SEQ ID NO: 35) is against Trp or Leu; the amino acid at position 393 is Trp or Leu; the Western Corn Root Worm (WCRW) larvae. amino acid at position 396 is Ala, Leu, Lys, Thr or Gly; the 0020 Embodiment 8 is the PHI-4 polypeptide of any one amino acid at position 401 is Ser. His, Gly, Lys or Pro; the of embodiments 1-7, wherein the improved insecticidal activ amino acid at position 402 is Lys, His, Gly or Trp; the amino ity compared to AXMI-205 (SEQID NO:35) is quantified as acid at position 403 is Asp or Tyr; the amino acid at position a Mean FAE Index. 410 is Ile or Val; the amino acid at position 412 is Pro or Ala; 0021 Embodiment 9 is the PHI-4 polypeptide of any one the amino acid at position 416 is Arg or Glu; the amino acid at of embodiments 1-7, wherein the improved insecticidal activ position 417 is Ala or Ser; the amino acid at position 426 is ity compared to AXMI-205 (SEQID NO:35) is quantified as Thr or Ser; the amino acid at position 442 is Glin or Glu; the an EC50 value. amino acid at position 447 is Asp or Lys; the amino acid at 0022. Embodiment 10 is the PHI-4 polypeptide of any one position 452 is Glin or Lys; the amino acid at position 454 is of embodiments 1-7, wherein the improved activity com Arg or Gln; the amino acid at position 455 is Val or Ile; the pared to AXMI-205 (SEQID NO:35) is quantified as a Mean amino acid at position 457 is Trp or ASn; the amino acid at Deviation Score. position 461 is Thr or Ser; the amino acid at position 462 is 0023 Embodiment 11 is the PHI-4 polypeptide of any one Gly or Ala; the amino acid at position 500 is Arg or Gln; the of embodiments 1-10, wherein the PHI-4 polypeptide com amino acid at position 509 is Lys or Gln; the amino acid at prises one or more amino acid Substitutions compared to the position 520 is Lys, Glu or Gln; and the amino acid at position native amino acid at position 40, 42, 43, 46, 52.97, 98, 99, 527 is Gln or Lys. 145, 150, 151,153, 163, 171, 172, 182, 196, 206, 210, 216, (0027 Embodiment 15 is the PHI-4 polypeptide of any one 220, 278, 283, 289, 293, 328,333,334, 336,338,339, 342, of embodiments 1-11 and 14, having 1 to 54 amino acid 346, 354, 355, 370, 389, 393, 396, 401, 402,403, 410, 412, substitutions compared to SEQID NO:35. 416, 417,426,442, 447, 452, 454, 455, 457, 461, 462, 500, 0028 Embodiment 16 is the PHI-4 polypeptide of any one 509, 520 or 527 of SEQID NO:35. of embodiments 1-11 and 14, having 1 to 27 amino acid 0024. Embodiment 12 is the PHI-4 polypeptide of substitutions compared to SEQID NO:35. embodiment 11, further comprising one or more amino acid (0029 Embodiment 17 is the PHI-4 polypeptide of any one substitutions at position 86,359,464, 465,466,467, 468,499 of embodiments 1-11 and 14, having 1 to 20 amino acid or 517. substitutions compared to SEQID NO:35. 0025 Embodiment 13 is the PHI-4 polypeptide of 0030 Embodiment 18 is the PHI-4 polypeptide of any one embodiment 11 or 12, wherein the amino acid at position 86 of embodiments 1-11 and 14, having 1 to 15 amino acid is Glu or Thr; the amino acid at position 359 is Gly or Ala; the substitutions compared to SEQID NO:35. amino acid at position 464 is Arg, Ala, Lys, Asp or ASn; the 0031 Embodiment 19 is the PHI-4 polypeptide of any one amino acid at position 465 is Lys or Met, the amino acid at of embodiments 12 or 13, comprising 2 to 54 amino acid position 467 is Val, Ala, Leu or Thr; the amino acid at position substitutions compared to SEQID NO:35. 468 is Seror Leu; the amino acid at position 499 is Glu or Ala, 0032 Embodiment 20 is the PHI-4 polypeptide of any one or the amino acid at position 517 is Glu or Arg. of embodiments 12 or 13, comprising 2 to 27 amino acid 0026. Embodiment 14 is the PHI-4 polypeptide of substitutions compared to SEQID NO:35. embodiment 11, 12 or 13, wherein the amino acid at position 0033 Embodiment 21 is the PHI-4 polypeptide of any one 40 is Leu or Ile; the amino acid at position 42 is Asp or ASn; of embodiments 12 or 13, comprising 2 to 20 amino acid the amino acid at position 43 is Phe or Glu; the amino acid at substitutions compared to SEQID NO:35. US 2014/0274885 A1 Sep. 18, 2014

0034 Embodiment 22 is the PHI-4 polypeptide of any one loop residue is selected from position 92,93, 94, 95, 96, 97. of embodiments 12 or 13, comprising 2 to 15 amino acid 98.99, 100, 101, 102, 103, 211,212, 213,214, 215, 216, 217, substitutions compared to SEQID NO:35. 218, 219, 220 of SEQID NO:35. 0035 Embodiment 23 is the PHI-4 polypeptide of any one 0047 Embodiment 31 is the PHI-4 polypeptide of any one of embodiments 1-22, wherein the PHI-4 polypeptide has at of embodiments 27-30, wherein the receptor binding loops least 80% identity to SEQID NO:35. are between about amino acid 332 (Asp) and 340 (Asp), 395 0036 Embodiment 24 is the PHI-4 polypeptide of any one (Asp) and 403 (Asp), 458 (Asp) and 466 (Asp) relative to of embodiments 1-22, wherein the PHI-4 polypeptide has at SEQID NO:35. least 90% identity to SEQID NO:35. 0048 Embodiment 32 is the PHI-4 polypeptide of 0037 Embodiment 25 is the PHI-4 polypeptide of any one embodiment 31, wherein the receptor binding loop residue is of embodiments 1-22, wherein the PHI-4 polypeptide has at selected from positions 332, 333,334, 335, 336, 337, 338, least 95% identity to SEQID NO:35. 339, 340,395, 396, 397, 398, 399, 400, 401, 402, 403, 458, 0038 Embodiment 26 is the PHI-4 polypeptide of any one 459, 460, 461, 462,463,464, 465, 466 of SEQID NO:35. of embodiments 1-22, wherein the PHI-4 polypeptide has at 0049 Embodiment 33 is the PHI-4 polypeptide of any one least 97% identity to SEQID NO:35. of embodiments 27-32, wherein the protease sensitive region 0039 Embodiment 27 is a PHI-4 polypeptide, comprising residue is selected from about amino acid residues between at least one amino acid Substitution at a residue relative to 305 (Lys) and 316 (Lys) and 500 (Arg) and 535 (Lys) relative SEQ ID NO:35 in structural domain selected from: to SEQID NO:35. 0040 a hydrophilic residue. 0050 Embodiment 34 is the PHI-4 polypeptide of 0041 a residue in a membrane insertion initiation loop: embodiment 27, wherein the protease is trypsin. 0042 a residue in a receptor binding loop; and 0051. Embodiment 35 is the PHI-4 polypeptide of any one 0043 a residue in a protease sensitive region, wherein of embodiments 27-34, wherein the PHI-4 polypeptide has at the PHI-4 polypeptide has increased insecticidal activity least 80% sequence identity to SEQID NO:35. compared to SEQID NO:35. 0052 Embodiment 36 is the PHI-4 polypeptide of any one 0044) Embodiment 28 is the PHI-4 polypeptide of of embodiments 27-34, wherein the PHI-4 polypeptide has at embodiment 27, wherein the hydrophilic residues are Asp, least 90% sequence identity to SEQID NO:35. Glu, Lys, Arg, His, Ser, Thr, Tyr, Trp, ASn, Gln, and Cys. 0053 Embodiment 37 is the PHI-4 polypeptide of any one 0045 Embodiment 29 is the PHI-4 polypeptide of of embodiments 27-34, wherein the PHI-4 polypeptide has at embodiment 27 or 28, wherein the membrane insertion loops least 95% sequence identity to SEQID NO:35. are between about amino acid at position 92 (Val) and 101 0054 Embodiment 38 is the PHI-4 polypeptide of any one (Ala) and at position 211 (Gly) and 220 (Glu) relative to SEQ of embodiments 27-34, wherein the PHI-4 polypeptide has at ID NO: 35. least 97% sequence identity to SEQID NO:35. 0046 Embodiment 30 is the PHI-4 polypeptide of 0055 Embodiment 39 is a PHI-4 polypeptide, comprising embodiment 29, wherein the membrane insertion initiation an amino acid sequence of the formula,

(SEQ ID NO : 3)

Met Xala Ser Ala Ala Asn Ala Gly Xaa Lieu. Gly Asn Lieu. Xaa Gly

Xaa. Thir Ser Xaa Gly Met Xaa Tyr Xaa Val Asn Gly Lieu. Tyr Ala

35 4 O 45 Ser Pro Glu Ser Lieu. Xaa Gly Glin Pro Lieu. Phe Xaa Xaa Gly Gly

SO 55 60 Xaa Lieu. Asp Ser Xaa Xaa Ile Glu Gly Xaa Xaa Xaa Xaa Phe Pro

65 70 7s Xaa Ser yet His Val His Thr Tyr Phe His Ser Asp Xaa Xaa Glin

8O 85 90 Xaa Val Ser Xaa Xaa Ile Xaa Xala Xala Arg Xaa Xaa Xaa Ser Xaa

95 1 OO 105 His Val Gly Xaa Ser Gly Xaa Xala Xala Lieu. Phe Ser Xaa Ser Xaa

11O 115 12O Ser Val Asp Xaa. Thir Thr Xala Xala Glin Glin Leu Xala Glu Ile Thr

125 13 O 135 Xaa Ser Ser Thr Arg Glu Xaa His Val Lieu. Trp Tyr Ile Ser Lieu.

14 O 145 150 ProGly Ala Ala Thir Lieu. Xaa Ser Met Lieu. Xaa Xala Xala Xaa Xaa

155 16 O 1.65 Xaa Asp Xaa Xaa Xaa Pro Asn Met Xaa Ala Met Xaa Lieu. Phe Xaa US 2014/0274885 A1 Sep. 18, 2014

- Continued

17O 17s 18O Xaa Xala Gly Pro Xaa Xaa Xaa Xala Xaa Ala Ala Val Gly Gly Arg

185 190 195 Lieu. Xaa Xala Xala Xaa Ala Ser Lys Xaa Lieu. Xaa Met Xaa Ser Ser

2 OO 2O5 21 O Xaa Ser Lieu. Ser Thir Thr Xaa Xala Xala Ser Xaa Xaa Ala Xala Xala

215 22O 225 Gly Glu Ile Xaa Ile Xaa His Gly Ser Xaa Met Glu Lys Glin Val

23 O 235 24 O Asn Ser Phe Xaa Xaa Xaa Ser Thr Ile Arg Xaa Thr Ala Thr Gly

245 250 255 Gly Llys Pro Gly Xaa Thr Xaa Arg Ile Lieu. His Gly Pro Asp Ser

26 O 265 27 O Xaa Xaa Ala Phe Ser Xaa Trp Ala Xala Ser Lieu Lleu Xaa Tyr Ala

27s 28O 285 Thir Lieu Met Asp Phe Xaa Thr Xaa Ser Lieu. Xaa Xaa Ile Xaa Ala

290 295 3OO Lieu. Xaa Asp Xaa Pro Xaa Xaa Xala Xala Glu Xaa Xaa Xaa Ala Xala

3. OS 310 315 Pro Xaa Xala Met Xaa Xaa Ser Glin Xaa Ser Ile Pro Xaa Val Asp

32O 3.25 33 O Xaa Val Lieu. Lieu Met Asp Ala Arg Pro Pro Met Val Xaa Ala Gly

335 34 O 345 Xaa Asp Xaa Xala Xaa Xaa Xaa Xala Xala Asp Xaa Xaa Xaa Xala Xala

350 355 360 Xaa Ser Thir Ser Xaa Xaa Tyr Lys Xaa Xala Gly Glin Phe Xaa Glin

365 37O 375 Arg Xaa His Xaa Ser Val Ala Asp Gly His Xaa Pro Ile Xaa Xala

38O 385 390 Asp Lieu. Phe Asp Xaa Gly Xaa Xala Xala Xala Pro Val Gly Xaa Glin

395 4 OO 405 Xaa Val Trp Asp Xaa Xaa Xaa Xala Gly Lys Xaa Xaa Xaa Tyr Xaa

410 415 42O Cys Trp Arg Xaa Xaa Xaa Xaa Glin Gly Tyr Xaa Xaa Xaa Gly Asp

425 43 O 435 Val Xala Met Lieu Ala Xaa Ser Gly Tyr Asn Pro Pro Asn Lieu Pro

4 4 O 445 450 Xaa Xala Xala Cys Xaa His Xaa Ser Lieu. Xaa Ala Xaa Xaa Xala Thr

45.5 460 465 Lieu. Xaa Xala Xala Xaa Trp Xaa Asp Xaa Xala Xaa Xaa Xaa Xala Xala

470 47s 48O Xaa Val Ser Lieu. Trp Xaa Pro Gly Ala Ala Gly Ala Val Ala Ser

485 490 495 Ser Cys Phe Ala Gly Val Pro Asn Tyr Asn Asn Pro Pro Asn Ser

5 OO 505 510 Gly Xaa Ile Xaa Xaa Lieu. Xaa Gly Ser Ile Ala Cys Val Xala Thr

515 52O 525 Ser Ala Ile Ala Ser Met Xaa Xala Met Xaa Ser Met Lieu. Ser Xaa

53 O 535 His Xaa Gly Met Glu Ala Met Met Ser Lys Lieu, US 2014/0274885 A1 Sep. 18, 2014 wherein Xaa at position 2 is Ala or Arg; Xaa at position 9 is Tyr. Thr, Asp, Phe, Ser, Met, Arg, Trp, Ile. His, Asn. Cys, Gly Gln, Lys or Glu; Xaa at position 14 is Pro or Ala; Xaa at or Ala; Xaa at position 291 is Glu or Glin; Xaa at position 292 position 16 is Val or Asp; Xaa at position 19 is Met or Leu: is Arg or Glin; Xaa at position 293 is Arg, Glu or Glin; Xaa at Xaaat position 22 is Gly or Ser; Xaaat position 24 is Asp, ASn position 294 is Val or Ala; Xaa at position 296 is Leu or Ile: or Glin; Xaa at position 36 is Leu or Met; Xaa at position 42 is Xaa at position 297 is Glu or Glin; Xaa at position 298 is Asp Asp, Asin or Glin; Xaa at position 43 is Phe or Glu; Xaa at or Glin; Xaa at position 300 is Phe or Tyr; Xaa at position 302 position 46 is Glu, Asp, Asn or Gly: Xaa at position 50 is Ile is Glu or Glin; Xaa at position 303 is Phe or Tyr; Xaa at or Val: Xaa at position 51 is Glu or Gln; Xaa at position 55 is position 305 is Lys or Glin; Xaa at position 306 is Gln or Lys: Argor Lys;Xaa at position 56 is Seror Thr; Xaa at position 57 Xaa at position 309 is Gln, Lys or Glu; Xaa at position 313 is is Tyr or Phe: Xaa at position 58 is Thr or Ser; Xaa at position LyS, Gln or Arg; Xaa at position 316 is Lys or Glin; Xaa at 61 is Arg, Lys or Glu; Xaa at position 73 is Phe or Tyr; Xaa at position 328 is Lys, Glu or Glin; Xaa at position 331 is Glu, position 74 is Lys, Glu, Gly, Arg, Met, Leu, His or Asp; Xaa ASnor Gln; Xaa at position333 is Ser, Arg, Gly, Lys, Val, Asn. at position 76 is Asp or Glin; Xaa at position 79 is Lys or Glu; Ala, His, Gln, Thr, Asp, Ile, Leu, Cys or Glu; Xaa at position Xaa at position 80 is Glu or Ser; Xaa at position 82 is Glu, Ile, 334 is Gly, Arg, Lys, Ile or Trp; Xaa at position 335 is Ser or Leu, Tyr or Glin; Xaa at position 83 is Glu or Glin; Xaa at Ala; Xaa at position 336 is Gly or Ala; Xaa at position 337 is position 84 is Tyror Phe, Xaa at position 86 is Glu or Glin; Xaa Ala, Val or Gly; Xaa at position 338 is Ser. His, Val, Lys, Ala, at position 87 is Lys or Gln; Xaa at position 88 is Met, Ile or Gly, Thr, Ile, Glu, Met, Arg, Pro, Asp, Asin or Leu; Xaa at Leu; Xaaat position 90 is Glin or Glu; Xaa at position 94 is Val position 339 is Glu, Asn., Gln, Ile, Pro, Met, Ser, Ala, Cys, or Ile: Xaa at position 97 is Arg, Asn., Asp, Glu, Gln, Gly or Phe, Val, Leu, Asp, Trp. His or Arg; Xaa at position 341 is Leu Ser; Xaa at position 98 is Tyr or Phe; Xaaat position 99 is Lys, or Val: Xaa at position 342 is Ala, Seror Val: Xaa at position Leu, Tyr, Ile, Met, Phe, Cys, Val or Asn; Xaa at position 103 343 is Val or Ile: Xaa at position 344 is Phe or Trp; Xaa at is Ala or Gly: Xaaat position 105 is Leu or Ile: Xaa at position position 345 is Asn or His; Xaa at position 346 is Pro or Ala; 109 is Phe, Lys, Gly, Met, Ser, Asp, Asn. Glu, Cys, Ala or Arg; Xaa at position 350 is Asn or Ser; Xaa at position 351 is Gly Xaa at position 112 is Thr or Ser; Xaa at position 113 is Asp, or Val: Xaa at position 354 is Met or Leu; Xaa at position 355 Glu or Met; Xaa at position 117 is Thror Ser; Xaa at position is Val, Ile or Leu; Xaa at position 359 is Gly or Ala; Xaa at 121 is Tyr or Phe: Xaa at position 127 is Ala or Thr; Xaa at position 362 is Asn or Ser; Xaa at position 364 is Ala or Ser; position 142 is Arg or Glu, Xaa at position 146 is Arg or Gln; Xaa at position 371 is Ala, Gly or Thr; Xaa at position 374 is Xaa at position 147 is Arg, Glu or Glin; Xaa at position 148 is Phe or Ile: Xaa at position 375 is Lys or Arg; Xaa at position Asp, Phe, Pro, Val, Glu, His, Trp, Ala, Arg, Leu, Ser, Gln or 380 is Leu or Gly:Xaa at position 382 is Val, Asp or Leu:Xaa Gly; Xaa at position 149 is Phe or Val: Xaa at position 150 is at position 383 is Leu, Ile or Val: Xaa at position 384 is Lys, Arg, Glnor Glu, Xaaat position 151 is Asp, Ser, Ala, ASn, Trp, Ala or Gly; Xaa at position 385 is Ala or Gly: Xaa at position Val, Gln, Cys, Met, Leu, Arg or Glu, Xaa at position 153 is 389 is Trp or Tyr; Xaa at position 391 is Arg, Leu, Glu, Gln or Leu or Ile; Xaa at position 154 is ASn or Asp; Xaa at position Asp; Xaa at position 395 is Asp or Cys; Xaa at position 396 is 155 is Asn or Lys; Xaa at position 159 is Pro or Asp; Xaa at Ala, Leu, Lys, ASn, Gly, Ile, Met, Arg, Tyr, Gln or His; Xaa at position 162 is Glu, Asp or Glin; Xaa at position 165 is Lys, position 397 is Gly, Argor Ala; Xaa at position 398 is Ser, Gln Glu, Gln, Pro, Thr, Ala, Leu, Gly, Asp, Val. His, Ile, Met, Trp, or Cys:Xaa at position 401 is Ser. His, Pro, Gly, Lys, Val, Arg, Phe, Tyr or Arg; Xaa at position 166 is Arg or Glin; Xaa at Ile, ASn, Phe, Thr, Ala, Asp, Met, Gln or Glu; Xaa at position position 167 is Tyr, Trp or Cys: Xaa at position 170 is Tyr or 402 is Lys, Phe, His, Arg, Trp, Gly, Asn. Leu, Tyr, Thr, Val, His; Xaa at position 171 is Tyr or Phe; Xaa at position 172 is Met, Pro or Ala; Xaa at position 403 is Asp, Tyr, Trp, Phe or Ile, Leu or Val; Xaa at position 173 is Ser or Ala; Xaa at Glu; Xaa at position 405 is Ala or Ser; Xaa at position 409 is position 174 is Glu or Glin; Xaa at position 182 is Asp or Gln; Ala or Pro; Xaa at position 410 is Ile or Val; Xaa at position Xaa at position 183 is Tyr or Val: Xaa at position 184 is Seror 411 is Pro or Ala; Xaa at position 412 is Pro or Ala; Xaa at Thr; Xaa at position 185 is Ala or Ser; Xaa at position 189 is position 416 is Arg, Glu or Glin; Xaaat position 417 is Ala, Ser Thr, Lys or Ile: Xaa at position 191 is Lys or Glin; Xaa at or Cys: Xaa at position 418 is Leu or Met; Xaaat position 422 position 193 is Asp or Asn; Xaa at position 196 is Gln, Lys, is Metor Val: Xaaat position 426 is Thror Ser; Xaa at position ASn, Asp, Glu, Ala, Ile or Arg; Xaa at position 202 is Ala or 436 is Asp or Lys; Xaa at position 437 is Tyr or Val: Xaa at Val: Xaa at position 203 is Glu, Thr or His: Xaa at position position 438 is Val or Arg; Xaa at position 440 is Val or Leu: 204 is Met or Ala; Xaa at position 206 is Tyr or Phe: Xaa at Xaa at position 442 is Gln, Lys or Glu, Xaa at position 445 is position 207 is Lys or Glin; Xaa at position 209 is Leu or Pro; Cys, Leu or Thr; Xaaat position 447 is Asp, Lys, Tyr, Ser, Glu, Xaa at position 210 is Val or Ile: Xaa at position 214 is Lys, Ile, Gly, Pro, Leu, Phe, Trp or Thr; Xaa at position 448 is Val Ser or Glin; Xaa at position 216 is Glu, Gln, Phe, Val, Tyr or or Ala; Xaa at position 449 is Glin or Glu; Xaa at position 452 Arg; Xaa at position 220 is Glu, His, Asp, Thr, Tyr, Val, Ser, is Gln, Lys or Glu; Xaa at position 453 is Asn or Asp; Xaa at Gln, Arg, Trp, Met, Ala, Phe, Ile, Leu, Cys or ASn; Xaa at position 454 is Arg, Tyr, Met, Ser, Val, Ile, Lys, Phe, Trp, Gln, position 229 is Arg or Glu; Xaa at position 230 is Seror Glu; Gly. His, Asp, Leu, Thr, Pro or ASn; Xaa at position 455 is Val Xaa at position 231 is Asn or Ser; Xaa at position 236 is Leu or Ile: Xaa at position 457 is Trp or ASn; Xaa at position 459 or Pro; Xaa at position 245 is Met or Leu; Xaa at position 247 is Lys, Met, Val, Trp, Gln, Ile, Thr, Ser. His, Cys, Tyr, Pro, is Asp or Tyr; Xaa at position 256 is Gln, Lys or Glu; Xaa at Asn, Ala, Arg or Glu, Xaa at position 460 is Gly or Ala; Xaa position 257 is Gln, Ile, Glu, Cys, Ser. His, Trp or Met; Xaa at at position 461 is Thror Ser; Xaa at position 462 is Gly or Ala; position 261 is Gln, Glu or Lys; Xaa at position 264 is Glu or Xaa at position 463 is Ala, Seror Gly; Xaa at position 464 is Gln; Xaa at position 268 is Asp or ASn; Xaa at position 276 is Arg, Gly, His, Gln, Thr or Phe; Xaa at position 465 is Lys, Ser or Ala; Xaa at position 278 is Glu, Asn or Glin; Xaa at ASn, Val, Met, Pro, Gly, Arg, Thr, His, Cys, Trp, Phe or Leu: position 281 is Gln, Lys or Glu; Xaa at position 282 is Pro or Xaa at position 466 is Asp or Arg, Xaa at position 471 is Gln, Gly; Xaa at position 284 is Trp or Arg; Xaa at position 287 is Lys or Glu, Xaa at position 497 is Asp or Glin; Xaa at position Ala or Cys;Xaaat position 289 is Lys, Leu, Val, Pro, Glu, Gln, 499 is Glu or Glin; Xaa at position 500 is Arg, Gln or Lys: Xaa US 2014/0274885 A1 Sep. 18, 2014

at position 502 is Arg, Glu or Glin; Xaa at position 509 is Lys, ID NO: 3 is an amino acid different from the corresponding Gln or Glu; Xaa at position 517 is Gln, Cys, Asn. Val or Pro; amino acid of SEQ ID NO: 35 and wherein the PHI-4 Xaa at position 518 is Glu or Glin; Xaa at position 520 is Lys, polypeptide has increased insecticidal activity compared to Gln or Glu; Xaa at position 525 is Glin or Lys; and Xaa at SEQID NO:35. position 527 is Gln, Lys, Pro, Cys, Glu, Ser. His, Phe or Trp; 0056. Embodiment 40 is a PHI-4 polypeptide, comprising wherein one or more amino acid(s) designated by Xaa in SEQ an amino acid sequence of the formula

(SEQ ID NO : 4) 10 15 Met Xaa Ser Ala Ala Asn Ala Gly Glin Luell Gly Asn Lell Pro Gly

2O 25 3O Wall. Thir Ser Met Gly Met Gly Tyr Xaa Wall Asn Gly Lell Tyr Ala

35 4 O 45 Ser Pro Glu Ser Lell Lell Gly Glin Pro Luell Phe Xaa Xaa Gly Gly

SO 55 60 Xala Luell Asp Ser Ile Glu Ile Glu Gly Arg Ser Thir Phe Pro

65 70 7s Arg Ser Met His Wall His Thir Phe His Ser Phe Xaa Glin

85 90 Asp Wall Ser Xaa Ile Xaa Glu Tyr Arg Glu Met Ser Glin

95 OO OS His Wall Gly Wall Ser Gly Xaa Xaa Xaa Lel Phe Ser Ala Ser Luell

15 Ser Wall Asp Xaa Thir Thir Asp Glin Glin Lell Thir Glu Ile

25 3O 35 Ser Ser Thir Glu Ala His Wall Lel Trp Ile Ser Luell

4 O 45 SO Pro Gly Ala Ala Lell Arg Ser Met Lel Arg Xaa Xaa Phe Xaa

55 60 65 Xaa Asp Xaa ASn Asn Pro Asn Met Pro Ala Met Xaa Lell Phe Xaa

Xaa Tyr Gly Pro Xaa Ile Ser Xaa Ala Wall Gly Gly

90 95 Luell Xala Ser Ala Ser Thir Lel Met Asp Ser Ser

2O5 21O Xala Ser Luell Ser Thir Ala Xaa Met Ser Xaa Ala Luell Wall

22O 225 Gly Glu Ile Xaa His Gly Ser Xaa Met Glu Glin Wall

23 O 235 24 O Asn. Ser Phe Arg Ser Asn Ser Thir Ile Arg Lell Thir Ala Thir Gly

245 250 255 Gly Pro Gly Met Thir Xaa Arg Ile Luell His Gly Pro Asp Ser

26 O 265 27 O Xaa Xala Ala Phe Ser Xaa Trp Ala Glu Ser Lell Lell Asp Ala

27s 28O 285 Thir Lieu. Met Asp Phe Ser Thir Xaa Ser Luell Xaa Pro Ile Trp Ala

290 295 3OO Lieu Ala Asp Xaa Pro Glu Arg Xaa Wall Glu Lell Glu Asp Ala Phe

3. OS 310 315 Pro Glu Phe Met Glin Ser Glin Glin Ser Ile Pro Xaa Wall Asp

3.25 330 Wall Luell Tell Met Asp Ala Arg Pro Pro Met Wall Xaa Ala Gly

335 34 O 345 Glu Asp Xaa Xaa Ser Xaa Ala Xaa Xaa Asp Lell Ala Xaa Phe Asn US 2014/0274885 A1 Sep. 18, 2014

- Continued 350 355 360 Xaa Ser Thr Ser Asn Gly Tyr Lys Met Gly Glin Phe Xaa Glin

365 375 Arg Asn His Ala Ser Val Ala Asp Gly His Ala Pro Ile Phe Lys

38O 385 390 Asp Lieu. Phe Asp Lell Gly Val Lieu Lys Ala Pro Val Gly Trp Glin

395 4 OO 405 Xaa Val Trp Asp Asp Xaa Gly Ser Gly Lys Xaa Xaa Xaa Ala

410 415 42O Trp Arg Ala Ile Xaa Xaa Glin Gly Tyr Xaa Xaa Xaa Gly Asp

425 43 O 435 Wal Met Met Lieu. Ala Xaa Ser Gly Tyr Asn Pro Pro Asn Luell Pro

4 4 O 445 450 Asp Tyr Val Xaa His Glin Ser Lieu. Cys Ala Xaa Val Glin Thir

45.5 460 465 Luell Xala Asn. Xala Xaa Trp Trp Asp Xaa Gly Xaa Xaa Xaa Xaa Xaa

470 48O Asp Wal Ser Lieu. Trp Xaa Pro Gly Ala Ala Gly Ala Val Ala Ser

485 490 495 Ser Phe Ala Gly Val Pro Asn Tyr Asn Asn Pro Pro Asn Ser

5 OO 505 510 Gly Asp Ile Glu Xaa Lieu. Arg Gly Ser Ile Ala Wall Xaa Thir

515 525 Ser Ala Ile Ala Ser Met Glin Glu Met Xala Ser Met Leu Ser Glin

53 O 535 His Xaa Gly Met Glu Ala Met Met Ser Lys Lieu, wherein Xaa at position 2 is Ala or Arg; Xaa at position 24 is Leu, Lys, ASnor Gly; Xaaat position 401 is Ser. His, Pro, Gly, Asp or ASn; Xaa at position 42 is Asp or ASn; Xaa at position LyS, Val or Arg; Xaa at position 402 is Lys, Phe, His, Arg, Gly, 43 is Phe or Glu, Xaa at position 46 is Glu or Asn; Xaa at Trp, Thr, Asn., Tyr or Met; Xaa at position 403 is Asp or Tyr; position 74 is Lys, Glu or Gly; Xaa at position 79 is Lys or Xaa at position 411 is Pro or Ala; Xaa at position 412 is Pro or Glu; Xaa at position 82 is Glu, Ile, Leu or Tyr; Xaa at position Ala; Xaa at position 416 is Arg or Glu, Xaa at position 417 is 97 is Arg, Asn., Asp, Glu, Gln or Gly: Xaa at position 98 is Tyr Ala or Ser; Xaa at position 418 is Leu or Met; Xaa at position or Phe, Xaa at position 99 is Lys, Leu, Tyr, Ile or Met; Xaa at 426 is Thr or Ser; Xaa at position 440 is Val or Leu; Xaa at position 109 is Phe, Lys, Gly, Met, Ser, Asp or Asn; Xaa at position 447 is Asp, Lys, Tyr, Ser, Glu or Ile: Xaa at position position 147 is Arg or Glu, Xaa at position 148 is Asp, Phe or 452 is Gln, Lys or Glu; Xaa at position 454 is Arg, Tyr, Met, Ser, Val, Ile, Lys, Phe, Trp or Gln; Xaa at position 455 is Val Pro; Xaa at position 150 is Arg or Glin; Xaa at position 151 is or Ile:Xaaat position 459 is Lys, Met, Val, Trp, Gln, Ile or Tyr; Asp, Ser, Ala or Asn; Xaa at position 153 is Leu or Ile: Xaa at Xaa at position 461 is Thr or Ser; Xaa at position 462 is Gly position 162 is Glu or Glin; Xaa at position 165 is Lys, Glu or or Ala; Xaa at position 463 is Ala or Ser; Xaa at position 464 Gln; Xaa at position 166 is Arg or Gln; Xaa at position 171 is is Arg, Gly or His; Xaa at position 465 is Lys, Asn. Val, Met, Tyr or Phe: Xaa at position 174 is Glu or Gln; Xaa at position Pro, Gly or Arg; Xaa at position 471 is Gln, Lys or Glu; Xaa 182 is Asp or Glin; Xaaat position 196 is Gln, Lys, ASnor Asp; at position 500 is Arg or Glin; Xaa at position 509 is Lys or Xaa at position 203 is Glu, Thr or His; Xaa at position 206 is Tyr or Phe: Xaa at position 216 is Glu or Gln; Xaa at position Gln; Xaa at position 520 is Lys, Gln or Glu; and Xaa at 220 is Glu, His, Asp, Thr, Tyr, Val, Seror Glin; Xaa at position position 527 is Gln, Lys, Pro, Cys or Glu; wherein one or more 247 is Asp or Tyr; Xaa at position 256 is Glin or Lys: Xaa at amino acid(s) designated by Xaa in SEQID NO. 4 is an amino position 257 is Glin or Ile: Xaa at position 261 is Glin or Glu; acid different from the corresponding amino acid of SEQID Xaa at position 278 is Glu or ASn; Xaa at position 281 is Gln, NO: 35 and wherein the PHI-4 polypeptide has increased Lys or Glu; Xaa at position 289 is Lys, Leu, Val, Pro, Glu, Gln, insecticidal activity compared to SEQID NO:35. Tyr, Thr or Asp; Xaa at position 293 is Arg, Glu or Glin; Xaa 0057 Embodiment 41 is the PHI-4 polypeptide of at position 313 is Lys or Glin; Xaa at position 328 is Lys, Glu embodiment 39 or 40, further comprising one or more amino or Glin; Xaa at position 333 is Ser, Gly, Lys, Val or Asn; Xaa acid substitutions at position 86,359,464, 465,466,467, 468, at position 334 is Gly, Arg, Lys or Ile: Xaa at position 336 is 499 or 517 of SEQID NO: 3 or SEQID NO: 4. Gly or Ala; Xaa at position 338 is Ser. His, Val, Lys or Ala; 0058 Embodiment 42 is the PHI-4 polypeptide of Xaaat position 339 is Glu, Asn. Ile or Pro; Xaaat position 343 embodiment 41, wherein the amino acid at position 86 is Glu is Val or Ile:Xaa at position 346 is Pro or Ala; Xaa at position or Thr; the amino acid at position 359 is Gly or Ala; the amino 355 is Val or Ile: Xaa at position 359 is Gly or Ala; Xaa at acid at position 464 is Arg, Ala, Lys, Asp or ASn; the amino position 391 is Arg, Glu or Glin; Xaa at position 396 is Ala, acid at position 465 is Lys or Met, the amino acid at position US 2014/0274885 A1 Sep. 18, 2014

467 is Val, Ala, Leu or Thr; the amino acid at position 468 is 0075 Embodiment 59 is a polynucleotide encoding a Seror Leu; the amino acid at position 499 is Glu or Ala, or the PHI-4 polypeptide, wherein the PHI-4 polypeptide has amino acid at position 517 is Glu or Arg. improved insecticidal activity compared to AXMI-205 (SEQ 0059 Embodiment 43 is the PHI-4 polypeptide of ID NO:35). embodiment 39-42, further comprising one or more conser 0076 Embodiment 60 is the polynucleotide of embodi Vative amino acid Substitution, insertion of one or more amino ment 59, wherein the insecticidal activity of the PHI-4 acids, deletion of one or more amino acids, and combinations polypeptide is increased about 1.5 fold or greater compared to thereof. AXMI-205 (SEQID NO:35). 0060 Embodiment 44 is the PHI-4 polypeptide of any one (0077. Embodiment 61 is the polynucleotide of embodi of embodiments 39-43, wherein the insecticidal activity is ment 59, wherein the insecticidal activity of the PHI-4 increased about 1.5 fold or greater compared to AXMI-205 polypeptide is increased about 2 fold or greater compared to (SEQ ID NO:35). AXMI-205 (SEQID NO:35). 0061 Embodiment 45 is the PHI-4 polypeptide of any one 0078 Embodiment 62 is the polynucleotide of embodi of embodiments 39-43, wherein the insecticidal activity is ment 59, wherein the insecticidal activity of the PHI-4 increased about 2 fold or greater compared to AXMI-205 polypeptide is increased about 2.5 fold or greater compared to (SEQ ID NO:35). AXMI-205 (SEQID NO:35). 0062 Embodiment 46 is the PHI-4 polypeptide of any one (0079 Embodiment 63 is the polynucleotide of embodi of embodiments 39-43, wherein the insecticidal activity is ment 59, wherein the insecticidal activity of the PHI-4 increased about 2.5 fold or greater compared to AXMI-205 polypeptide is increased about 3 fold or greater compared to (SEQ ID NO:35). AXMI-205 (SEQID NO:35). 0063 Embodiment 47 is the PHI-4 polypeptide of any one 0080 Embodiment 64 is the polynucleotide of embodi of embodiments 39-43, wherein the insecticidal activity is ment 59, wherein the insecticidal activity of the PHI-4 increased about 3 fold or greater compared to AXMI-205 polypeptide is increased about 5 fold or greater compared to (SEQ ID NO:35). AXMI-205 (SEQID NO:35). 0064. Embodiment 48 is the PHI-4 polypeptide of any one I0081 Embodiment 65 is the polynucleotide of any one of of embodiments 39-43, wherein the insecticidal activity is embodiments 59-64, wherein the improved insecticidal activ increased about 5 fold or greater compared to AXMI-205 ity compared to AXMI-205 (SEQ ID NO: 35) is against (SEQ ID NO:35). Western Corn Root Worm (WCRW) larvae. 0065 Embodiment 49 is the PHI-4 polypeptide of any one I0082 Embodiment 66 is the polynucleotide of any one of of embodiments 39-48, wherein the improved insecticidal embodiments 59-64, wherein the improved insecticidal activ activity compared to AXMI-205 (SEQID NO:35) is against ity compared to AXMI-205 (SEQID NO:35) is quantified as Western Corn Root Worm (WCRW) larvae. a Mean FAE Index. 0066 Embodiment 50 is the PHI-4 polypeptide of any one I0083. Embodiment 67 is the polynucleotide of any one of of embodiments 39-49, wherein the improved insecticidal embodiments 59-64, wherein the improved insecticidal activ activity compared to AXMI-205 (SEQID NO:35) is quanti ity compared to AXMI-205 (SEQID NO:35) is quantified as fied as a Mean FAE Index. an EC50 value. I0084 Embodiment 68 is the polynucleotide of any one of 0067. Embodiment 51 is the PHI-4 polypeptide of any one embodiments 59-64, wherein the improved activity com of embodiments 39-49, wherein the improved insecticidal pared to AXMI-205 (SEQID NO:35) is quantified as a Mean activity compared to AXMI-205 (SEQID NO:35) is quanti Deviation Score. fied as an EC50 value. I0085 Embodiment 69 is the polynucleotide of any one of 0068 Embodiment 52 is the PHI-4 polypeptide of any one embodiments 59-68, wherein the PHI-4 polypeptide com of embodiments 39-49, wherein the improved activity com prises one or more amino acid substitutions compared to the pared to AXMI-205 (SEQID NO:35) is quantified as a Mean native amino acid at position 40, 42, 43, 46, 52.97, 98, 99, Deviation Score. 145, 150, 151, 153, 163, 171, 172, 182, 196, 206, 210, 216, 0069. Embodiment 53 is the PHI-4 polypeptide of any one 220, 278, 283, 289, 293,328,333,334, 336,338,339, 342, of embodiments 39-49, having 1 to 54 amino acid substitu 346, 354, 355, 370, 389, 393, 396, 401, 402,403, 410, 412, tions at a position(s) designated as Xaa in SEQID NO: 3 or 4. 416, 417,426,442, 447, 452, 454, 455, 457, 461, 462, 500, 0070 Embodiment 54 is the PHI-4 polypeptide of any one 509, 520 or 527 of SEQID NO:35. of embodiments 39-49, having 1 to 27 amino acid substitu I0086 Embodiment 70 is the polynucleotide of embodi tions at a position(s) designated as Xaa in SEQID NO: 3 or 4. ment 69, wherein the PHI-4 polypeptide further comprises (0071 Embodiment 55 is the PHI-4 polypeptide of any one one or more amino acid Substitutions compared to the native of embodiments 39-49, having 1 to 20 amino acid substitu amino acid at position 86,359, 464, 465, 466, 467, 468, 499 tions at a position(s) designated as Xaa in SEQID NO: 3 or 4. or 517 of SEQID NO:35. 0072 Embodiment 56 is the PHI-4 polypeptide of any one I0087 Embodiment 71 is the polynucleotide of embodi of embodiments 39-49, having 1 to 15 amino acid substitu ment 70, wherein the amino acid at position 86 is Glu or Thr: tions at a position(s) designated as Xaa in SEQID NO: 3 or 4. the amino acid at position 359 is Gly or Ala; the amino acid at 0073 Embodiment 57 is the PHI-4 polypeptide of any one position 464 is Arg, Ala, LyS, Asp or ASn; the amino acid at of embodiments 1-56, wherein 1-25 amino acids are deleted position 465 is Lys or Met, the amino acid at position 467 is from the N-terminus of the PHI-4 polypeptide and/or C-ter Val, Ala, Leu or Thr; the amino acid at position 468 is Seror minus of the PHI-4 polypeptide. Leu; the amino acid at position 499 is Glu or Ala, or the amino 0074 Embodiment 58 is the PHI-4 polypeptide of any one acid at position 517 is Glu or Arg. of embodiments 1-53, wherein 1-20 amino acids are deleted I0088 Embodiment 72 is the polynucleotide of any one of from the C-terminus of the PHI-4 polypeptide. embodiments 69-71, wherein the amino acid at position 40 is US 2014/0274885 A1 Sep. 18, 2014

Leu or Ile; the amino acid at position 42 is Asp or ASn; the (0095 Embodiment 78 is the polynucleotide of embodi amino acid at position 43 is Phe or Glu; the amino acid at ments 70 or 71, wherein the PHI-4 polypeptide has 2 to 27 position 46 is Glu or Asn; the amino acid at position 52 is Ile amino acid substitutions compared to SEQID NO:35. or Val; the amino acid at position 97 is Arg, Asp, Glu or ASn; (0096 Embodiment 79 is the polynucleotide of embodi the amino acid at position 98 is Tyr or Phe; the amino acid at ments 70 or 71, wherein the PHI-4 polypeptide has 2 to 20 position 99 is Lys or Leu; the amino acid at position 145 is Leu amino acid substitutions compared to SEQID NO:35. or Val; the amino acid at position 150 is Argor Gln; the amino acid at position 151 is Asp or Ser; the amino acid at position (0097 Embodiment 80 is the polynucleotide of embodi 153 is Leu or Ile; the amino acid at position 163 is Leu or Val; ments 70 or 71, wherein the PHI-4 polypeptide has 2 to 15 the amino acid at position 171 is Tyr or Phe; the amino acid at amino acid substitutions compared to SEQID NO:35. position 172 is Ile or Leu; the amino acid at position 182 is 0.098 Embodiment 81 is the polynucleotide of any one of Asp or Gln; the amino acid at position 196 is Glin or ASn; the embodiments 59-80, wherein the PHI-4 polypeptide has at amino acid at position 206 is Tyr or least 80% identity to SEQID NO:35. I0089 Phe; the amino acid at position 210 is Val or Ile; the 0099 Embodiment 82 is the polynucleotide of any one of amino acid at position 216 is Glu or Gln; the amino acid at embodiments 59-80, wherein the PHI-4 polypeptide has at position 220 is Glu, Gln, His or Asp; the amino acid at posi least 90% identity to SEQID NO:35. tion 278 is Glu or Asn; the amino acid at position 283 is Ile or 0100 Embodiment 83 is the polynucleotide of any one of Val; the amino acid at position 289 is Lys, Gln or Leu; the embodiments 59-80, wherein the PHI-4 polypeptide has at amino acid at position 293 is Arg, Gln or Glu, the amino acid least 95% identity to SEQID NO:35. at position 328 is Lys or Glu; the amino acid at position333 is 0101 Embodiment 84 is the polynucleotide of any one of Ser, Lys or Val; the amino acid at position 334 is Gly, Lys or embodiments 59-80, wherein the PHI-4 polypeptide has at Arg; the amino acid at position 336 is Gly or Ala; the amino least 97% identity to SEQID NO:35. acid at position 338 is Ser or Val; the amino acid at position 0102 Embodiment 85 is a polynucleotide encoding a 339 is Glu, Asn or Gln; the amino acid at position 342 is Ala PHI-4 polypeptide, wherein the PHI-4 polypeptide has at or Ser; the amino acid at position 346 is Pro or Ala; the amino least one amino acid substitution at a residue relative to SEQ acid at position 354 is Met or Leu; the amino acid at position ID NO: 35 in structural domain selected from: 355 is Val or Ile; the amino acid at position 370 is His or Arg; the amino acid at position 389 is Trp or Leu; the amino acid at 0.103 a hydrophilic residue. position 393 is Trp or Leu; the amino acid at position 396 is 0.104 a residue in a membrane insertion initiation loop: Ala, Leu, Lys, Thr or Gly; the amino acid at position 401 is 0105 a residue in a receptor binding loop; and Ser. His, Gly, Lys or Pro; the amino acid at position 402 is Lys, 0106 a residue in a protease sensitive region, wherein His, Gly or Trp; the amino acid at position 403 is Asp or Tyr; the PHI-4 polypeptide has increased insecticidal activity the amino acid at position 410 is Ile or Val; the amino acid at compared to SEQID NO:35. position 412 is Pro or Ala; the amino acid at position 416 is Arg or Glu; the amino acid at position 417 is Ala or Ser; the 0107 Embodiment 86 is the polynucleotide of embodi amino acid at position 426 is Thr or Ser; the amino acid at ment 85, wherein the hydrophilic residues are Asp, Glu, Lys, position 442 is Glin or Glu; the amino acid at position 447 is Arg, His, Ser. Thr, Tyr, Trp, Asn., Gln, and Cys. Embodiment Asp or Lys; the amino acid at position 452 is Glin or Lys; the 87 is the polynucleotide of embodiment 85 or 86, wherein the amino acid at position 454 is Arg or Gln; the amino acid at membrane insertion loops are between about amino acid at position 455 is Val or Ile; the amino acid at position 457 is Trp position 92 (Val) and 101 (Ala) and at position 211 (Gly) and or ASn; the amino acid at position 461 is Thror Ser; the amino 220 (Glu) relative to SEQID NO:35. acid at position 462 is Gly or Ala; the amino acid at position 0.108 Embodiment 88 is the polynucleotide of embodi 500 is Argor Gln; the amino acid at position 509 is Lys or Gln; ment 87, wherein the PHI-4 polypeptide has one or more the amino acid at position 520 is Lys, Glu or Gln; and the amino Substitution compared to the native amino acid at posi amino acid at position 527 is Glin or Lys. tion 92,93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 211, 0090 Embodiment 73 is the polynucleotide of any one of 212, 213, 214, 215, 216, 217, 218, 219, and 220 of SEQ ID embodiments 59-69 and 72, wherein the PHI-4 polypeptide NO: 35. has 1 to 54 amino acid substitutions compared to SEQID NO: 0109 Embodiment 89 is the polynucleotide of any one of 35. embodiments 85, 86, 87 or 88, wherein the receptor binding 0091 Embodiment 74 is the polynucleotide of any one of loops are between aboutamino acid 332 (Asp) and 340 (Asp), embodiments 59-69 and 72, wherein the PHI-4 polypeptide 395 (Asp) and 403 (Asp), 458 (Asp) and 466 (Asp) relative to has 1 to 27 amino acid substitutions compared to SEQID NO: SEQID NO:35. 2 0110 Embodiment 90 is the polynucleotide of embodi 0092. Embodiment 75 is the polynucleotide of any one of ment 89, wherein the PHI-4 polypeptide has one or more embodiments 59-69 and 72, wherein the PHI-4 polypeptide amino Substitution compared to the native amino acid at posi has 1 to 20 amino acid substitutions compared to SEQID NO: tion 332,333,334, 335, 336,337, 338,339, 340,395,396, 35. 397, 398, 399, 400, 401, 402,403, 458, 459, 460, 461, 462, 0093 Embodiment 76 is the polynucleotide of any one of 463,464, 465,466 of SEQID NO:35. embodiments 59-69 and 72, wherein the PHI-4 polypeptide 0111 Embodiment 91 is the polynucleotide of any one of has 1 to 15 amino acid substitutions compared to SEQID NO: embodiments 85, 86, 87, 88, 89 or 90, wherein the protease 35. sensitive region residue is selected from about amino acid 0094) Embodiment 77 is the polynucleotide of embodi residues between position 305 (Lys) and position 316 (Lys) ments 70 or 71, wherein the PHI-4 polypeptide has 2 to 54 and/or position 500 (Arg) and position 535 (Lys) relative to amino acid substitutions compared to SEQID NO:35. SEQID NO:35. US 2014/0274885 A1 Sep. 18, 2014 10

0112 Embodiment 92 is the polynucleotide of any one of 0115 Embodiment 95 is the polynucleotide of any one of embodiments 85, 86, 87, 88, 89,90 or 91, wherein the pro embodiments 85-92, wherein the PHI-4 polypeptide has at tease is trypsin. least 95% sequence identity to SEQID NO:35. 0113 Embodiment 93 is the polynucleotide of any one of 0116 Embodiment 96 is the polynucleotide of any one of embodiments 85-92, wherein the PHI-4 polypeptide has at embodiments 85-92, wherein the PHI-4 polypeptide has at least 80% sequence identity to SEQID NO:35. least 97% sequence identity to SEQID NO:35. 0114 Embodiment 94 is the polynucleotide of any one of 0117 Embodiment 97 is a polynucleotide encoding a embodiments 85-92, wherein the PHI-4 polypeptide has at PHI-4 polypeptide, wherein the PHI-4 polypeptide comprises least 90% sequence identity to SEQID NO:35. an amino acid sequence of the formula,

(SEQ ID NO: 3) 15 Met Xala Ser Ala Ala Asn Ala Gly Xaa Lieu. Gly Asn Lieu. Xaa Gly

2O 25 Xaa. Thir Ser Xaa Gly Met Xaa Tyr Xaa Wall Asn Gly Lieu. Tyr Ala

35 4 O 45 Ser Pro Glu Ser Lell Xaa Gly Glin Pro Luell Phe Xaa Xaa Gly Gly

SO 55 60 Xaa Lieu. Asp Ser Xaa Xaa Ile Glu Gly Xaa Xaa Xaa Xaa Phe Pro

65 70 Xaa Ser yet His Wall His Thr Tyr Phe His Ser Asp Xaa Xala Glin

85 90 Xaa Val Ser Xaa Ile Xaa Xala Xala Arg Xaa Xala Xala Ser Xala

95 OO OS His Val Gly Xaa Ser Gly Xaa Xala Xala Lel Phe Ser Xala Ser Xaa

1O 15 Ser Val Asp Xaa Thr Xaa Xala Glin Glin Lieu. Xaa Glu Ile Thr

25 3O 35 Xaa Ser Ser Thir Glu Xaa His Wall Lel Trp Ile Ser Lieu

4 O 45 SO Pro Gly Ala Ala Thr Luell Xala Ser Met Lel Xaa Xala Xala Xala Xala

55 65 Xaa Asp Xaa Xaa Xaa Pro Asn. Met Xaa Met Xaa Lieu. Phe Xaa

70 Xaa Xala Gly Pro Xaa Xaa Xala Xala Xala Ala Val Gly Gly

90 95 Luell Xala Xala Xaa Ala Ser Xaa Lel Xaa Met Xala Ser Ser

2O5 Xala Ser Luell Ser Thr Xaa Xala Xala Ser Xaa Xaa Ala Xala Xala

225 Gly Glu Ile Xaa Xaa His Gly Ser Met Glu Glin Wall

23 O 235 24 O Asn. Ser Phe Xaa Xaa Xala Ser Thir Ile Arg Xaa Thr Ala Thr Gly

245 250 255 Gly Pro Gly Xaa Thr Xaa Arg Ile Luell His Gly Pro Asp Ser

26 O 265 27 O Xaa Xaa Ala Phe Ser Xaa Trp Ala Xala Ser Lieu. Luell Xala Ala

27s 28O 285 Thir Lieu Met Asp Phe Xala Thr Xala Ser Luell Xaa Xaa Ile Xaa Ala

290 295 Lieu. Xaa Asp Xaa Pro Xaa Xala Xala Xala Glu Xaa Xaa Xaa Ala Xala

3. OS 310 315 Pro Xala Xala Met Xaa Xaa Ser Glin Xaa Ser Ile Pro Xaa Val Asp

32O 3.25 330 Xaa Val Lieu. Tell Met Asp Ala Arg Pro Pro Met Val Xaa Ala Gly US 2014/0274885 A1 Sep. 18, 2014 11

- Continued

335 34 O 345 Xaa Asp Xaa Xala Xaa Xaa Xaa Xala Xala Asp Xaa Xaa Xaa Xaa Xaa

350 355 360 Xala Ser Thir Ser Xaa Xaa Tyr Xaa Xala Gly Glin Phe Xaa Glin

365 37O 375 Arg Xaa His Xaa Ser Val Ala Asp Gly His Xaa Pro Ile Xaa Xaa

385 390 Asp Lieu. Phe Asp Gly Xaa Xala Xala Xala Pro Val Gly Xaa Glin

395 4 OO 405 Xaa Val Trp Asp Xaa Xaa Xaa Xala Gly Lys Xaa Xaa Xaa Xaa

410 415 42O Trp Arg Xaa Xaa Xaa Xaa Glin Gly Tyr Xaa Xaa Xaa Gly Asp

425 43 O 435 Wall Xala Met Lieu. Ala Xaa Ser Gly Asn. Pro Pro Asn Luell Pro

4 4 O 445 450 Xaa Xala Xala Xaa His Xaa Ser Lieu. Xaa Ala Xaa Xaa Xaa Thir

45.5 460 465 Luell Xala Xala Xala Xaa Trp Xaa Asp Xaa Xala Xaa Xaa Xaa Xaa Xaa

470 48O Xaa Val Ser Lieu Trp Xaa Pro Gly Ala Ala Gly Ala Val Ala Ser

485 490 495 Ser Cys Phe Ala Gly Wall Pro Asn Asn. Asn. Pro Pro Asn Ser

5 OO 505 510 Gly Xaa Ile Xaa Xaa Lieu. Xaa Gly Ser Ile Ala Wall Xaa Thir

515 525 Ser Ala Ile Ala Ser Met Xala Xala Met Xala Ser Met Leu Ser Xaa

53 O 535 His Xaa Gly Met Glu Ala Met Met Ser Lys Lieu, wherein Xaa at position 2 is Ala or Arg; Xaa at position 9 is Asp, Phe, Pro, Val, Glu, His, Trp, Ala, Arg, Leu, Ser, Gln or Gln, Lys or Glu; Xaa at position 14 is Pro or Ala; Xaa at Gly: Xaa at position 149 is Phe or Val: Xaa at position 150 is position 16 is Val or Asp; Xaa at position 19 is Met or Leu: Arg, Glnor Glu, Xaaat position 151 is Asp, Ser, Ala, ASn, Trp, Xaaat position 22 is Gly or Ser; Xaaat position 24 is Asp, ASn Val, Gln, Cys, Met, Leu, Arg or Glu, Xaa at position 153 is or Glin; Xaa at position 36 is Leu or Met; Xaa at position 42 is Leu or Ile; Xaa at position 154 is ASnor Asp; Xaa at position Asp, Asin or Glin; Xaa at position 43 is Phe or Glu; Xaa at 155 is Asn or Lys; Xaa at position 159 is Pro or Asp; Xaa at position 46 is Glu, Asp, Asn or Gly: Xaa at position 50 is Ile position 162 is Glu, Asp or Glin; Xaa at position 165 is Lys, or Val: Xaa at position 51 is Glu or Gln; Xaa at position 55 is Glu, Gln, Pro, Thr, Ala, Leu, Gly, Asp, Val. His, Ile, Met, Trp, Argor Lys;Xaa at position 56 is Seror Thr; Xaa at position 57 Phe, Tyr or Arg; Xaa at position 166 is Arg or Glin; Xaa at is Tyr or Phe: Xaa at position 58 is Thr or Ser; Xaa at position position 167 is Tyr, Trp or Cys: Xaa at position 170 is Tyr or 61 is Arg, Lys or Glu; Xaa at position 73 is Phe or Tyr; Xaa at His; Xaa at position 171 is Tyr or Phe, Xaa at position 172 is position 74 is Lys, Glu, Gly, Arg, Met, Leu, His or Asp; Xaa Ile, Leu or Val; Xaa at position 173 is Ser or Ala; Xaa at at position 76 is Asp or Glin; Xaa at position 79 is Lys or Glu; position 174 is Glu or Glin; Xaa at position 182 is Asp or Gln; Xaa at position 80 is Glu or Ser; Xaa at position 82 is Glu, Ile, Xaa at position 183 is Tyr or Val: Xaa at position 184 is Seror Leu, Tyr or Glin; Xaa at position 83 is Glu or Glin; Xaa at Thr; Xaa at position 185 is Ala or Ser; Xaa at position 189 is position 84 is Tyror Phe, Xaa at position 86 is Glu or Glin; Xaa Thr, Lys or Ile: Xaa at position 191 is Lys or Glin; Xaa at at position 87 is Lys or Gln; Xaa at position 88 is Met, Ile or position 193 is Asp or Asn; Xaa at position 196 is Gln, Lys, Leu; Xaaat position 90 is Glin or Glu; Xaa at position 94 is Val ASn, Asp, Glu, Ala, Ile or Arg; Xaa at position 202 is Ala or or Ile: Xaa at position 97 is Arg, Asn., Asp, Glu, Gln, Gly or Val: Xaa at position 203 is Glu, Thr or His: Xaa at position Ser; Xaa at position 98 is Tyr or Phe; Xaaat position 99 is Lys, 204 is Met or Ala; Xaa at position 206 is Tyr or Phe: Xaa at Leu, Tyr, Ile, Met, Phe, Cys, Val or Asn; Xaa at position 103 position 207 is Lys or Glin; Xaa at position 209 is Leu or Pro; is Ala or Gly: Xaaat position 105 is Leu or Ile: Xaa at position Xaa at position 210 is Val or Ile: Xaa at position 214 is Lys, 109 is Phe, Lys, Gly, Met, Ser, Asp, Asn. Glu, Cys, Ala or Arg; Ser or Glin; Xaa at position 216 is Glu, Gln, Phe, Val, Tyr or Xaa at position 112 is Thr or Ser; Xaa at position 113 is Asp, Arg; Xaa at position 220 is Glu, His, Asp, Thr, Tyr, Val, Ser, Glu or Met; Xaa at position 117 is Thror Ser; Xaa at position Gln, Arg, Trp, Met, Ala, Phe, Ile, Leu, Cys or Asn; Xaa at 121 is Tyr or Phe: Xaa at position 127 is Ala or Thr; Xaa at position 229 is Arg or Glu; Xaa at position 230 is Seror Glu; position 142 is Arg or Glu, Xaa at position 146 is Arg or Gln; Xaa at position 231 is Asn or Ser; Xaa at position 236 is Leu Xaa at position 147 is Arg, Glu or Glin; Xaa at position 148 is or Pro; Xaa at position 245 is Met or Leu; Xaa at position 247 US 2014/0274885 A1 Sep. 18, 2014 is Asp or Tyr; Xaa at position 256 is Gln, Lys or Glu; Xaa at position 397 is Gly, Argor Ala; Xaa at position 398 is Ser, Gln position 257 is Gln, Ile, Glu, Cys, Ser. His, Trp or Met; Xaa at or Cys:Xaa at position 401 is Ser. His, Pro, Gly, Lys, Val, Arg, position 261 is Gln, Glu or Lys; Xaa at position 264 is Glu or Ile, ASn, Phe, Thr, Ala, Asp, Met, Gln or Glu; Xaa at position Gln; Xaa at position 268 is Asp or ASn; Xaa at position 276 is 402 is Lys, Phe, His, Arg, Trp, Gly, Asn. Leu, Tyr, Thr, Val, Met, Pro or Ala; Xaa at position 403 is Asp, Tyr, Trp, Phe or Ser or Ala; Xaa at position 278 is Glu, Asn or Glin; Xaa at Glu; Xaa at position 405 is Ala or Ser; Xaa at position 409 is position 281 is Gln, Lys or Glu; Xaa at position 282 is Pro or Ala or Pro; Xaa at position 410 is Ile or Val; Xaa at position Gly; Xaa at position 284 is Trp or Arg; Xaa at position 287 is 411 is Pro or Ala; Xaa at position 412 is Pro or Ala; Xaa at Ala or Cys;Xaaat position 289 is Lys, Leu, Val, Pro, Glu, Gln, position 416 is Arg, Glu or Glin; Xaaat position 417 is Ala, Ser Tyr, Thr, Asp, Phe, Ser, Met, Arg, Trp, Ile. His, Asn. Cys, Gly or Cys: Xaa at position 418 is Leu or Met; Xaaat position 422 or Ala; Xaa at position 291 is Glu or Glin; Xaa at position 292 is Metor Val: Xaaat position 426 is Thror Ser; Xaa at position is Arg or Glin; Xaa at position 293 is Arg, Glu or Glin; Xaa at 436 is Asp or Lys; Xaa at position 437 is Tyr or Val: Xaa at position 294 is Val or Ala; Xaa at position 296 is Leu or Ile: position 438 is Val or Arg; Xaa at position 440 is Val or Leu: Xaa at position 297 is Glu or Glin; Xaa at position 298 is Asp Xaa at position 442 is Gln, Lys or Glu, Xaa at position 445 is or Glin; Xaa at position 300 is Phe or Tyr; Xaa at position 302 Cys, Leu or Thr; Xaaat position 447 is Asp, Lys, Tyr, Ser, Glu, is Glu or Glin; Xaa at position 303 is Phe or Tyr; Xaa at Ile, Gly, Pro, Leu, Phe, Trp or Thr; Xaa at position 448 is Val position 305 is Lys or Glin; Xaa at position 306 is Gln or Lys: or Ala; Xaa at position 449 is Glin or Glu; Xaa at position 452 Xaa at position 309 is Gln, Lys or Glu; Xaa at position 313 is is Gln, Lys or Glu; Xaa at position 453 is Asn or Asp; Xaa at Lys, Gln or Arg; Xaa at position 316 is Lys or Glin; Xaa at position 454 is Arg, Tyr, Met, Ser, Val, Ile, Lys, Phe, Trp, Gln, position 328 is Lys, Glu or Glin; Xaa at position 331 is Glu, Gly. His, Asp, Leu, Thr, Pro or ASn; Xaa at position 455 is Val ASnor Gln; Xaaat position333 is Ser, Arg, Gly, Lys, Val, Asn. or Ile: Xaa at position 457 is Trp or ASn; Xaa at position 459 Ala, His, Gln, Thr, Asp, Ile, Leu, Cys or Glu; Xaa at position is Lys, Met, Val, Trp, Gln, Ile, Thr, Ser. His, Cys, Tyr, Pro, 334 is Gly, Arg, Lys, Ile or Trp; Xaa at position 335 is Ser or Asn, Ala, Arg or Glu, Xaa at position 460 is Gly or Ala; Xaa Ala; Xaa at position 336 is Gly or Ala; Xaa at position 337 is at position 461 is Thror Ser; Xaa at position 462 is Gly or Ala; Ala, Val or Gly; Xaa at position 338 is Ser. His, Val, Lys, Ala, Xaa at position 463 is Ala, Seror Gly; Xaa at position 464 is Gly, Thr, Ile, Glu, Met, Arg, Pro, Asp, Asn or Leu; Xaa at Arg, Gly, His, Gln, Thr or Phe; Xaa at position 465 is Lys, position 339 is Glu, Asn., Gln, Ile, Pro, Met, Ser, Ala, Cys, ASn, Val, Met, Pro, Gly, Arg, Thr, His, Cys, Trp, Phe or Leu: Phe, Val, Leu, Asp, Trp. His or Arg; Xaa at position 341 is Leu Xaa at position 466 is Asp or Arg, Xaa at position 471 is Gln, or Val: Xaa at position 342 is Ala, Seror Val: Xaa at position Lys or Glu, Xaa at position 497 is Asp or Glin; Xaa at position 343 is Val or Ile: Xaa at position 344 is Phe or Trp; Xaa at 499 is Glu or Glin; Xaa at position 500 is Arg, Gln or Lys: Xaa position 345 is Asn or His: Xaa at position 346 is Pro or Ala; at position 502 is Arg, Glu or Glin; Xaa at position 509 is Lys, Xaa at position 350 is Asn or Ser; Xaa at position 351 is Gly Gln or Glu; Xaa at position 517 is Gln, Cys, Asn. Val or Pro; or Val: Xaa at position 354 is Met or Leu; Xaa at position 355 Xaa at position 518 is Glu or Glin; Xaa at position 520 is Lys, is Val, Ile or Leu; Xaa at position 359 is Gly or Ala; Xaa at Gln or Glu; Xaa at position 525 is Glin or Lys; and Xaa at position 362 is Asn or Ser; Xaa at position 364 is Ala or Ser; position 527 is Gln, Lys, Pro, Cys, Glu, Ser. His, Phe or Trp; Xaa at position 371 is Ala, Gly or Thr; Xaa at position 374 is wherein one or more amino acid(s) designated by Xaa in SEQ Phe or Ile: Xaa at position 375 is Lys or Arg; Xaa at position ID NO: 3 is an amino acid different from the corresponding 380 is Leu or Gly; Xaa at position 382 is Val, Asp or Leu; Xaa amino acid of SEQ ID NO: 35 and wherein the PHI-4 at position 383 is Leu, Ile or Val: Xaa at position 384 is Lys, polypeptide has increased insecticidal activity compared to Ala or Gly; Xaa at position 385 is Ala or Gly: Xaa at position SEQID NO:35. 389 is Trp or Tyr; Xaa at position 391 is Arg, Leu, Glu, Gln or 0118 Embodiment 98 is a polynucleotide encoding a Asp; Xaa at position 395 is Asp or Cys; Xaa at position 396 is PHI-4 polypeptide, wherein the PHI-4 polypeptide comprises Ala, Leu, Lys, ASn, Gly, Ile, Met, Arg, Tyr, Gln or His; Xaa at an amino acid sequence of the formula,

(SEQ ID NO : 4)

Met Xala Ser Ala Ala Asn Ala Gly Glin Lieu. Gly Asn Lieu Pro Gly

Wall. Thir Ser Met Gly Met Gly Tyr Xaa Val Asn Gly Lieu. Tyr Ala

35 4 O 45 Ser Pro Glu Ser Lieu. Lieu. Gly Glin Pro Lieu. Phe Xaa Xaa Gly Gly

SO 55 60 Xaa Lieu. Asp Ser Ile Glu Ile Glu Gly Arg Ser Tyr Thr Phe Pro

65 70 7s Arg Ser Met His Val His Thr Tyr Phe His Ser Asp Phe Xaa Glin

8O 85 90 Asp Wal Ser Xaa Glu Ile Xaa Glu Tyr Arg Glu Lys Met Ser Glin

95 1 OO 105 His Val Gly Val Ser Gly Xaa Xala Xala Lieu. Phe Ser Ala Ser Lieu.

11O 115 12O Ser Val Asp Xaa Thr Thr Thr Asp Glin Gln Lieu. Thr Glu Ile Thr US 2014/0274885 A1 Sep. 18, 2014 13

- Continued

25 13 O 135 Ser Ser Thir Glu Ala His Wall Luell Trp Ile Ser Luell

4 O 145 15 O Pro Gly Ala Ala Lell Arg Ser Met Luell Arg Xaa Xaa Phe Xaa

55 160 1.65 Xaa Asp Xaa Asn Asn Pro Asn Met Pro Ala Met Xaa Lell Phe Xaa

17s 18O Xaa Gly Pro Xaa Ile Ser Xaa Ala Ala Wall Gly Gly Arg

190 195 Lell Xaa Ser Ala Ser Thir Luell Lys Met Asp Ser Ser

2O5 21 O Xaa Ser Luell Ser Thir Ala Xaa Met Ser Xaa Ala Luell Wall

22O 225 Gly Glu Ile Xaa His Gly Ser Xaa Met Glu Glin Wall

23 O 235 24 O Asn Ser Phe Arg Ser Asn Ser Thir Ile Arg Luell Thir Ala Thir Gly

245 250 255 Gly Pro Gly Met Thir Xaa Arg Ile Luell His Gly Pro Asp Ser

26 O 265 27 O Xaa Xaa Ala Phe Ser Xaa Trp Ala Glu Ser Luell Lell Asp Ala

27s 28O 285 Thir Luell Met Asp Phe Ser Thir Xaa Ser Luell Xaa Pro Ile Trp Ala

290 295 3OO Lell Ala Asp Xaa Pro Glu Arg Xaa Wall Glu Luell Glu Asp Ala Phe

3. OS 310 315 Pro Glu Phe Met Lys Glin Ser Glin Glin Ser Ile Pro Xaa Wall Asp

32O 3.25 33 O Wall Luell Luell Met Asp Ala Arg Pro Pro Met Wall Xaa Ala Gly

335 34 O 345 Glu Asp Xaa Xaa Ser Xaa Ala Xaa Xaa Asp Luell Ala Xaa Phe Asn

350 355 360 Xaa Ser Thir Ser Asn Gly Met Xaa Gly Glin Phe Xaa Glin

365 37O 375 Arg Asn His Ala Ser Wall Ala Asp Gly His Ala Pro Ile Phe

38O 385 390 Asp Luell Phe Asp Lell Gly Wall Luell Ala Pro Wall Gly Trp Glin

395 4 OO 405 Xaa Wall Trp Asp Asp Xaa Gly Ser Gly Xaa Xaa Xaa Ala

410 415 42O Trp Arg Ala Ile Xaa Xaa Glin Gly Tyr Xaa Xaa Xaa Gly Asp

425 43 O 435 Wall Met Met Luell Ala Xaa Ser Gly Asn Pro Pro Asn Luell Pro

4 4 O 445 450 Asp Wall Xaa His Glin Ser Luell Ala Xaa Wall Glin Thir

45.5 460 465 Lell Xaa Asn Xaa Xaa Trp Trp Asp Xaa Gly Xaa Xaa Xaa Xaa Xaa

470 47s 48O Asp Wall Ser Luell Trp Xaa Pro Gly Ala Ala Gly Ala Wall Ala Ser

485 490 495 Ser Phe Ala Gly Wall Pro Asn Asn ASn Pro Pro Asn Ser

5 OO 505 510 Gly Asp Ile Glu Xaa Lell Arg Gly Ser Ile Ala Wall Xaa Thir US 2014/0274885 A1 Sep. 18, 2014

- Continued

515 52O 525 Ser Ala Ile Ala Ser Met Glin Glu Met Xala Ser Met Leu Ser Glin

53 O 535 His Xaa Gly Met Glu Ala Met Met Ser Lys Lieu, wherein Xaa at position 2 is Ala or Arg; Xaa at position 24 is prises one or more amino acid Substitutions at position 86. Asp or ASn; Xaa at position 42 is Asp or ASn; Xaa at position 359,464, 465,466,467, 468,499 or 517 of SEQID NO:3 or 43 is Phe or Glu, Xaa at position 46 is Glu or Asn; Xaa at SEQID NO: 4. position 74 is Lys, Glu or Gly; Xaa at position 79 is Lys or I0120 Embodiment 100 is the polynucleotide of embodi Glu; Xaa at position 82 is Glu, Ile, Leu or Tyr; Xaa at position ment 99, wherein the amino acid at position 86 is Glu or Thr: 97 is Arg, Asn., Asp, Glu, Gln or Gly: Xaa at position 98 is Tyr the amino acid at position 359 is Gly or Ala; the amino acid at or Phe, Xaa at position 99 is Lys, Leu, Tyr, Ile or Met; Xaa at position 464 is Arg, Ala, LyS, Asp or ASn; the amino acid at position 109 is Phe, Lys, Gly, Met, Ser, Asp or Asn; Xaa at position 465 is Lys or Met, the amino acid at position 467 is position 147 is Arg or Glu, Xaa at position 148 is Asp, Phe or Val, Ala, Leu or Thr; the amino acid at position 468 is Seror Pro; Xaa at position 150 is Arg or Glin; Xaa at position 151 is Leu; the amino acid at position 499 is Glu or Ala, or the amino Asp, Ser, Ala or Asn; Xaa at position 153 is Leu or Ile: Xaa at acid at position 517 is Glu or Arg. position 162 is Glu or Glin; Xaa at position 165 is Lys, Glu or I0121 Embodiment 101 is the polynucleotide of any one of Gln; Xaa at position 166 is Arg or Gln; Xaa at position 171 is embodiments 97-100, wherein the PHI-4 polypeptide further comprises one or more conservative amino acid substitution, Tyr or Phe: Xaa at position 174 is Glu or Gln; Xaa at position insertion of one or more amino acids, deletion of one or more 182 is Asp or Glin; Xaaat position 196 is Gln, Lys, ASnor Asp; amino acids, and combinations thereof. Xaa at position 203 is Glu, Thr or His; Xaa at position 206 is 0.122 Embodiment 102 is the polynucleotide of any one of Tyr or Phe: Xaa at position 216 is Glu or Gln; Xaa at position embodiments 97-101, wherein the insecticidal activity of the 220 is Glu, His, Asp, Thr, Tyr, Val, Seror Glin; Xaa at position PHI-4 polypeptide is increased about 1.5 fold or greater com 247 is Asp or Tyr; Xaa at position 256 is Glin or Lys: Xaa at pared to AXMI-205 (SEQID NO:35). position 257 is Glin or Ile: Xaa at position 261 is Glin or Glu; I0123 Embodiment 103 is the polynucleotide of any one of Xaa at position 278 is Glu or ASn; Xaa at position 281 is Gln, embodiments 97-101, wherein the insecticidal activity of the Lys or Glu; Xaa at position 289 is Lys, Leu, Val, Pro, Glu, Gln, PHI-4 polypeptide is increased about 2 fold or greater com Tyr, Thr or Asp; Xaa at position 293 is Arg, Glu or Glin; Xaa pared to AXMI-205 (SEQID NO:35). at position 313 is Lys or Glin; Xaa at position 328 is Lys, Glu 0.124. Embodiment 104 is the polynucleotide of any one of or Glin; Xaa at position 333 is Ser, Gly, Lys, Val or Asn; Xaa embodiments 97-101, wherein the insecticidal activity of the at position 334 is Gly, Arg, Lys or Ile: Xaa at position 336 is PHI-4 polypeptide is increased about 2.5 fold or greater com Gly or Ala; Xaa at position 338 is Ser. His, Val, Lys or Ala; pared to AXMI-205 (SEQID NO:35). Xaaat position 339 is Glu, Asn. Ile or Pro; Xaaat position 343 0.125 Embodiment 105 is the polynucleotide of any one of is Val or Ile:Xaa at position 346 is Pro or Ala; Xaa at position embodiments 97-101, wherein the insecticidal activity of the 355 is Val or Ile: Xaa at position 359 is Gly or Ala; Xaa at PHI-4 polypeptide is increased about 3 fold or greater com position 391 is Arg, Glu or Glin; Xaa at position 396 is Ala, pared to AXMI-205 (SEQID NO:35). Leu, Lys, ASnor Gly; Xaa at position 401 is Ser. His, Pro, Gly, 0.126 Embodiment 106 is the polynucleotide of any one of LyS, Val or Arg; Xaa at position 402 is Lys, Phe, His, Arg, Gly, embodiments 97-101, wherein the insecticidal activity of the Trp, Thr, Asn., Tyr or Met; Xaa at position 403 is Asp or Tyr; PHI-4 polypeptide is increased about 5 fold or greater com Xaa at position 411 is Pro or Ala; Xaa at position 412 is Pro or pared to AXMI-205 (SEQID NO:35). Ala; Xaa at position 416 is Arg or Glu, Xaa at position 417 is I0127 Embodiment 107 is the polynucleotide of any one of Ala or Ser; Xaa at position 418 is Leu or Met; Xaa at position embodiments 97-106, wherein the improved insecticidal 426 is Thr or Ser; Xaa at position 440 is Val or Leu; Xaa at activity compared to AXMI-205 (SEQID NO:35) is against position 447 is Asp, Lys, Tyr, Ser, Glu or Ile: Xaa at position Western Corn Root Worm (WCRW) larvae. 452 is Gln, Lys or Glu; Xaa at position 454 is Arg, Tyr, Met, I0128 Embodiment 108 is the polynucleotide of any one of Ser, Val, Ile, Lys, Phe, Trp or Gln; Xaa at position 455 is Val embodiments 97-107, wherein the improved insecticidal or Ile:Xaaat position 459 is Lys, Met, Val, Trp, Gln, Ile or Tyr; activity compared to AXMI-205 (SEQID NO:35) is quanti Xaa at position 461 is Thr or Ser; Xaa at position 462 is Gly fied as a Mean FAE Index. or Ala; Xaa at position 463 is Ala or Ser; Xaa at position 464 I0129. Embodiment 109 is the polynucleotide of any one of is Arg, Gly or His; Xaa at position 465 is Lys, Asn. Val, Met, embodiments 97-107, wherein the improved insecticidal Pro, Gly or Arg; Xaa at position 471 is Gln, Lys or Glu; Xaa activity compared to AXMI-205 (SEQID NO:35) is quanti at position 500 is Arg or Glin; Xaa at position 509 is Lys or fied as an EC50 value. Gln; Xaa at position 520 is Lys, Gln or Glu; and Xaa at 0.130 Embodiment 110 is the polynucleotide of any one of position 527 is Gln, Lys, Pro, Cys or Glu; wherein one or more embodiments 97-107, wherein the improved activity com amino acid(s) designated by Xaa in SEQIDNO: 4 is an amino pared to AXMI-205 (SEQID NO:35) is quantified as a Mean acid different from the corresponding amino acid of SEQID Deviation Score. NO: 35 and wherein the PHI-4 polypeptide has increased I0131 Embodiment 111 is the polynucleotide of any one of insecticidal activity compared to SEQID NO:35. embodiments 97-110, wherein the PHI-4 polypeptide has 1 to 0119 Embodiment 99 is the polynucleotide of embodi 54 amino acid substitutions at a position(s) designated as Xaa ments 97 or 98, wherein the PHI-4 polypeptide further com in SEQID NO:3 or 4. US 2014/0274885 A1 Sep. 18, 2014

0132 Embodiment 112 is the polynucleotide of any one of ness, drought resistance or tolerance, cold resistance or tol embodiments 97-110, wherein the PHI-4 polypeptide has 1 to erance, Salt resistance or tolerance and increased yield under 27 amino acid Substitutions at a position(s) designated as Xaa StreSS. in SEQID NO:3 or 4. 0145 Embodiment 126 is seed, grain or processed product 0.133 Embodiment 113 is the polynucleotide of any one of thereof of the transgenic plant of any one of embodiments embodiments 97-110, wherein the PHI-4 polypeptide has 1 to 121-126, wherein the seed, grain, or processed product 20 amino acid Substitutions at a position(s) designated as Xaa thereof comprises the polynucleotide of any one of embodi in SEQID NO:3 or 4. ments 121-125. 0134 Embodiment 114 is the polynucleotide of any one of 0146 Embodiment 127 is an expression cassette, com embodiments 97-110, wherein the PHI-4 polypeptide has 1 to prising the polynucleotide of any one of embodiments 59-116 15 amino acid Substitutions at a position(s) designated as Xaa operably linked to one or more regulatory sequences directing in SEQID NO:3 or 4. expression of the PHI-4 polypeptide. 0135 Embodiment 115 is the polynucleotide of any one of 0147 Embodiment 128 is a transgenic plant or plant cell, embodiments 97-114, wherein 1-25 amino acids are deleted comprising the expression cassette of embodiment 127. from the N-terminus of the PHI-4 polypeptide and/or C-ter 0148 Embodiment 129 is seed, grain or processed product minus of the PHI-4 polypeptide. thereof of the transgenic plant of embodiment 128, wherein 0.136 Embodiment 116 is the polynucleotide of any one of the seed, grain, or processed product thereof comprises the embodiments 97-114, wherein 1-20 amino acids are deleted recombinant nucleic acid molecule of embodiment of 1 and from the C-terminus of the PHI-4 polypeptide. the recombinant nucleic acid molecule of 24. 0149 Embodiment 130 is the seed of embodiment 129, 0.137 Embodiment 117 is a composition, comprising an wherein one or more seed treatment has been applied to the insecticidally-effective amount of the PHI-4 polypeptide of seed. any one of embodiments 1-58. 0150 Embodiment 131 is a method for expressing in a 0138 Embodiment 118 is a method of inhibiting growth plant a polynucleotide encoding an insecticidal protein, com or killing an insect pest, comprising contacting the insect pest prising with the composition of embodiment 117. 0151 (a) inserting into a plant cell the polynucleotide of 0139 Embodiment 119 is a method for controlling an any one of embodiment 59-116 encoding the PHI-4 polypep insect pest population resistant to a pesticidal protein, com tide; prising contacting the resistant insect pest population with the 0152 (b) obtaining a transformed plant cell comprising composition of embodiment 117. the nucleic acid sequence of step (a); and 0140 Embodiment 120 is the method of controlling an 0153 (c) generating from the transformed plant cell a insect pest population resistant to an pesticidal protein of plant capable of expressing the polynucleotide encoding the embodiment 119, wherein the pesticidal protein is selected PHI-4 polypeptide. from Cry1Ac, Cry1Ab, Cry1A.105, Cry1Ac, Cry1F, 0154 Embodiment 132 is a method for protecting a plant Cry1 Fa2, Cry 1F, Cry2Ab, Cry3A, mCry3A, Cry3Bb1, from an insect pest, comprising expressing in the plant or cell Cry34Ab1, Cry35Ab1, Vip3A, Cry9c, eCry3.1Ab and CBI thereof, an insecticidally-effective amount of the PHI-4 Bt. polypeptide of any one of embodiments 1-58. 0141 Embodiment 121 is a transgenic plant or progeny 0155 Embodiment 133 is a method for controlling an thereof, comprising the polynucleotide of any one of embodi insect pest population, comprising contacting the insect pest ments 59-116. Embodiment 122 is the transgenic plant or population with an insecticidally-effective amount of the progeny thereof of embodiment 121, wherein the transgenic PHI-4 polypeptide of any one of embodiments 1-58. plant is a monocotyledon. 0156 Embodiment 134 is a method of inhibiting growth 0142 Embodiment 123 is the transgenic plant or progeny or killing an insect pest, comprising contacting the insect pest thereof of embodiment 122, wherein the plant is selected with a composition comprising an insecticidally-effective from barley, corn, oat, rice, rye, Sorghum, turfgrass, Sugar amount of the PHI-4 polypeptide of any one of embodiments cane, wheat, alfalfa, banana, broccoli, bean, cabbage, canola, 1-58. carrot, cassaya, cauliflower, celery, citrus, cotton, a cucurbit, 0157 Embodiment 135 is a method for controlling an eucalyptus, flax, garlic, grape, onion, lettuce, pea, peanut, insect pest population resistant to a pesticidal protein, com pepper, potato, poplar, pine, Sunflower, safflower, soybean, prising contacting the insect pest population with an insecti Strawberry, Sugar beet, Sweet potato, tobacco, tomato orna cidally-effective amount of the PHI-4 polypeptide of any one mental, shrub, nut, chickpea, pigeon pea, millets, hops and of embodiments 1-58. pasture grasses. 0158 Embodiment 136 is a fusion protein comprising the 0143 Embodiment 124 is the transgenic plant or progeny PHI-4 polypeptide of any one of embodiments 1-58. 0159 Embodiment 137 is a fusion protein comprising the thereof of embodiment 123, further comprising one or more PHI-4 polypeptide of any one of embodiments 1-58 and a additional transgenic traits. maltose binding protein. 0144. Embodiment 125 is the transgenic plant of embodi ment 124, wherein the one or more additional transgenic trait 0160 Embodiment 138 is the fusion protein of embodi is selected from insect resistance, herbicide resistance, fungal ment 137, wherein the maltose binding protein has an amino resistance, viral resistance, stress tolerance, disease resis acid sequence of SEQID NO: 830 or SEQID NO: 831. tance, male sterility, stalk strength, increased yield, modified starches, improved oil profile, balanced amino acids, high BRIEF DESCRIPTION OF THE FIGURES lysine or methionine, increased digestibility, improved fiber (0161 FIG. 1 shows FAE analysis of MPB::PHI-4-SFR12 quality, flowering, ear and seed development, enhancement of 004 (SEQID NO 31). The reference protein is MBP::PHI-4 nitrogen utilization efficiency, altered nitrogen responsive polypeptide of (SEQID NO: 6). The % Response is given on US 2014/0274885 A1 Sep. 18, 2014 they axis. The dose of the toxin fragment is given on the X axis 0.168. The present disclosure is drawn to compositions and in parts per million (ppm). Half dose (+) and full dose (*) methods for controlling pests. The methods involve trans indicate a mean response from six replicate wells at the indi forming organisms with a nucleic acid sequence encoding a cated concentration. All data are from a single experiment. PHI-4 polypeptide. In particular, the nucleic acid sequences (0162 FIG. 2 shows EC50 analysis of MBP::PHI-4 of the embodiments are useful for preparing plants and micro polypeptide of SEQID NO 6 and MPB::PHI-4-SFR12-004 organisms possessing pesticidal activity. Thus, transformed (SEQID NO 35). The % Response is given on they axis. The bacteria, plants, plant cells, plant tissues and seeds are pro dose of the toxin fragment is given on the X axis in parts per vided. Compositions are pesticidal nucleic acids and proteins million (ppm). The % Response is given on the y axis. The of bacterial species. The nucleic acid sequences find use in the dose of the toxin fragment is given on the X axis in parts per construction of expression vectors for Subsequent transfor million (ppm). The protein concentration (toxin portion of the mation into organisms of interest, as probes for the isolation protein only) is given on the X axis. Each symbol indicates a of other homologous (or partially homologous) genes, and for mean response from twenty-four replicate wells at the indi the generation of altered PHI-4 polypeptides by methods cated concentration. All data are from a single experiment. known in the art, Such as site directed mutagenesis, domain triangles: MBP::PHI-4 (SEQID NO: 6): circles: MBP::PHI swapping or DNA shuffling. The PHI-4 polypeptides find use 4-SFR12-004 (SEQ ID NO: 31). in controlling, inhibiting growth or killing Lepidopteran, 0163 FIG.3 shows the amino acids sequence of the C-ter Coleopteran, Dipteran, fungal, Hemipteran, and nematode minal portion of the PHI-4 polypeptide (SEQ ID NO: 2) is pest populations and for producing compositions with pesti given. Three stretches of sequence corresponding to nine cidal activity. Insect pests of interest include, but are not amino acid motifs that align to the putative Sugar binding loop limited to, the superfamily of stink bugs and other related motifD-X-G-(S/T)-G-X-D (SEQID NO:40) are highlighted insects including, but not limited to, species belonging to the in grey. family Pentatomidae (Nezara viridula, Halyomorpha halys, (0164 FIG. 4 shows EC50 data for SEQID NO: 610, SEQ Piezodorus guildini, Euschistus servus, Acrosternum hilare, IDNO:595, SEQIDNO:584, SEQID NO:591, SEQID NO: Euschistus heros, Euschistus tristigmus, Acrosternum hilare, 576, SEQID NO: 73, SEQID NO: 74, SEQID NO: 75, SEQ Dichelops fircatus, Dichelops melacanthus, and Bagrada ID NO: 79, SEQID NO: 81, SEQID NO: 150, SEQID NO: hilaris (Bagrada Bug)), the family Plataspidae (Megacopta 150, SEQID NO: 149, SEQ ID NO: 167, SEQID NO: 167, cribraria—Bean plataspid), and the family Cydnidae (Scap SEQID NO: 164, SEQID NO: 164, SEQID NO: 170, SEQ tocoris Castanea—Root Stink bug) and Lepidoptera species IDNO: 170, SEQIDNO:795, SEQID NO: 794, SEQID NO: including but not limited to: diamond-back moth, e.g., Heli 784, SEQID NO: 799, SEQID NO: 785, SEQID NO: 788, coverpa zea Boddie; soybean looper, e.g., Pseudoplusia SEQ ID NO: 786, SEQ ID NO: 796, SEQ ID NO: 787. The includens Walker and Velvet bean caterpillar e.g., Anticarsia x-axis corresponds to the mean fold improvement in EC50, gemmatalis Hübner. relative to MBP::PHI-4 (SEQID NO : 6) from at least three independent EC50 measurements. The y-axis corresponds to (0169. By “pesticidal toxin' or “pesticidal protein' is the mean fold improvement of the mean FAE Index from at intended a toxin that has toxic activity against one or more least three independent measurements. pests, including, but not limited to, members of the Lepi (0165 FIG.5. T0 seedlings in the V3-V4 growth stage were doptera, Diptera, Hemiptera and Coleoptera orders or the challenged as described (Oleson J. et al J. Economic Ento Nematoda phylum or a protein that has homology to Such a mology 98:1-8; 2005) and root nodal injury scores were protein. Pesticidal proteins have been isolated from organ recorded. The groups indicated along the X axis were either isms including, for example, Bacillus sp., Pseudomonas sp., controls or were transformed with a plant vector of Example Photorhabdus sp., Xenorhabdus sp., Clostridium bifermen 18: 1, untransformed control; 2. positive control transgenic tans and Paenibacillus popilliae. Pesticidal proteins include expressing positive control protein; 3. PHI-4 polypeptide but are not limited to: insecticidal proteins from Pseudomo mas sp. such as PSEEN3174 (Monalysin, (2011) PLOS (SEQ ID NO: 22): 4, PHI-4-B09 polypeptide (SEQID NO: Pathogens, 7:1-13) from Pseudomonas protegens Strain 23); 5, PHI-4-D09 polypeptide (SEQ ID NO: 24); 6 PHI-4- CHAO and Pf-5 (previously fluorescens) (Pechy–Tarr, (2008) H08 polypeptide (SEQID NO: 25). Environmental Microbiology 10:2368-2386: GenBank DETAILED DESCRIPTION Accession No. EU400157); from Pseudomonas Taiwanensis (Liu, et al., (2010).J. Agric. Food Chem. 58:12343-12349) 0166 It is to be understood that this invention is not lim and from Pseudomonas pseudoalcligenes (Zhang, et al., ited to the particular methodology, protocols, cell lines, gen (2009) Annals of Microbiology 59:45-50 and Li, et al., (2007) era, and reagents described, as such may vary. It is also to be Plant Cell Tiss. Organ Cult. 89:159-168); insecticidal pro understood that the terminology used herein is for the purpose teins from Photorhabdus sp. and Xenorhabdus sp. (Hinch of describing particular embodiments only, and is not liffe, et al., (2010) The Open Toxinology Journal 3:101-118 intended to limit the scope of the present invention. and Morgan, et al., (2001) Applied and Envir: Micro. 67:2062 0167 As used herein the singular forms “a”, “and”, and 2069), U.S. Pat. No. 6,048,838, and U.S. Pat. No. 6,379,946; “the include plural referents unless the context clearly dic and Ö-endotoxins including, but not limited to, the cry1, cry2, tates otherwise. Thus, for example, reference to “a cell cry3, cry4, crys, cry6, cry7, cry8, cry9, cry 10, cry 11, cry 12, includes a plurality of such cells and reference to “the pro cry 13, cry 14, cry 15, cry 16, cry 17, cry 18, cry 19, cry20, cry21, tein’ includes reference to one or more proteins and equiva cry 22, cry23, cry24, cry25, cry26, cry27, cry 28, cry 29, cry lents thereof knownto those skilled in the art, and so forth. All 30, cry31, cry32, cry33, cry34, cry35, cry36, cry37, cry38, technical and Scientific terms used herein have the same cry39, cry40, cry41, cry42, cry43, cry44, cry45, cry 46, meaning as commonly understood to one of ordinary skill in cry47, cry49, cry 51 and crys5 classes of 8-endotoxin genes the art to which this invention belongs unless clearly indi and the B. thuringiensis cytolytic cytl and cyt2 genes. Mem cated otherwise. bers of these classes of B. thuringiensis insecticidal proteins

US 2014/0274885 A1 Sep. 18, 2014

#BAD22577), Cry46Aa (Accession #BAC79010), 2011/0263488; AXMI-R1 and related proteins of US 2010/ Cry46Aa2 (Accession #BAG68906), Cry46Ab (Accession O197592: AXMI221Z, AXMI222Z, AXMI223Z, AXMI224Z #BAD35170), Cry47Aa (Accession #AY950229), Cry48Aa and AXMI225Z of WO 2011/103248: AXMI218, AXMI219, (Accession #AJ841948), Cry48Aa2 (Accession AXMI220, AXMI226, AXMI227, AXMI228, AXMI229, #AM237205), Cry48Aa3 (Accession #AM237206), AXMI230, and AXMI231 of WO1 1/103,247; AXMI-115, Cry48Ab (Accession #AM237207), Cry48Ab2 (Accession AXMI-113, AXMI-005, AXMI-163 and AXMI-184 of U.S. #AM237208), Cry49Aa (Accession #AJ841948), Cry49Aa2 Pat. No. 8,334431; AXMI-001, AXMI-002, AXMI-030, (Accession #AM237201), Cry49Aa3 (Accession AXMI-035, and AXMI-045 of US 2010/0298211: AXMI #AM237203), Cry49Aa4 (Accession #AM237204), 066 and AXMI-076 of US20090144852: AXMI 128, Cry49Ab1 (Accession #AM237202), Crys0Aa1 (Accession AXMI130, AXMI131, AXMI133, AXMI140, AXMI141, #AB253419), Crys1Aa1 (Accession #DQ836184), AXMI142, AXMI 143, AXMI144, AXMI146, AXMI148, Cry52Aa1 (Accession #EF613489), Crys3Aa1 (Accession AXMI149, AXMI152, AXMI153, AXMI154, AXMI155, #EF633476), Crys4Aa1 (Accession #EU339367), AXMI156, AXMI157, AXMI158, AXMI162, AXMI165, Crys5Aa1 (Accession #EU121521), Crys5Aa2 (Accession AXMI166, AXMI167, AXMI168, AXMI169, AXMI170, #AAE33526), Cyt1Aa (GenBank Accession #X03182), AXMI171, AXMI172, AXMI173, AXMI174, AXMI175, Cyt1 Ab (GenBank Accession #X98793), Cyt1B (GenBank AXMI176, AXMI177, AXMI178, AXMI179, AXMI 180, Accession #U37196), Cyt2A (GenBank Accession AXMI 181, AXMI 182, AXMI185, AXMI186, AXMI 187, #Z14147), Cyt2B (GenBank Accession #U52043). AXMI 188, AXMI 189 of U.S. Pat. No. 8,318,900; AXMI079, 0171 Examples of 8-endotoxins also include but are not AXMI080, AXMI081, AXMI082, AXMI091, AXMI092, limited to Cry1A proteins of U.S. Pat. Nos. 5,880,275 and AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, 7,858,849; a DIG-3 or DIG-11 toxin (N-terminal deletion of AXMI101, AXMI1 02, AXMI103, AXMI104, AXMI107, C.-helix 1 and/or C-helix 2 variants of cry proteins such as AXMI108, AXMI109, AXMI110, AXMI111, AXMI112, Cry1A, Cry3A) of U.S. Pat. Nos. 8.304,604 and 8,304,605, AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, Cry1B of U.S. patent application Ser. No. 10/525,318; Cry 1C AXMI120, AXMI121, AXMI122, AXMI123, AXMI124, of U.S. Pat. No. 6,033,874; Cry1F of U.S. Pat. Nos. 5,188, AXMI1257, AXMI1268, AXMI127, AXMI129, AXMI164, 960, 6,218, 188; Cry1A/F chimeras of U.S. Pat. Nos. 7,070, AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, 982; 6,962,705 and 6,713,063); a Cry2 protein such as AXMI137 of US 2010/0005543; cry proteins such as Cry1A Cry2Ab protein of U.S. Pat. No. 7,064.249); a Cry3A protein and Cry3A having modified proteolytic sites of U.S. Pat. No. including but not limited to an engineered hybrid insecticidal 8,319,019; a Cry1Ac, Cry2Aa and Cry1Catoxin protein from protein (eHIP) created by fusing unique combinations of Bacillus thuringiensis strain VBTS 2528 of US Patent Appli variable regions and conserved blocks of at least two different cation Publication Number 2011/0064710. Other Cry pro Cry proteins (US Patent Application Publication Number teins are well known to one skilled in the art (see, Crickmore, 2010/0017914); a Cry4 protein; a Crys protein; a Cry6 pro et al., “Bacillus thuringiensis toxin nomenclature' (2011), at tein; Cry8 proteins of U.S. Pat. Nos. 7,329,736, 7,449,552, lifesci. Sussex.ac.uk/home/Neil Crickmore/Bt? which can be 7,803,943, 7,476,781, 7,105,332, 7,378.499 and 7,462,760; a accessed on the world-wide web using the “www’ prefix). Cry9 protein such as such as members of the Cry9A, Cry9B, The insecticidal activity of Cry proteins is well known to one Cry9C, Cry9D, Cry9E, and Cry9F families; a Cry 15 protein skilled in the art (for review, see, van Frannkenhuyzen, (2009) of Naimov, et al., (2008) Applied and Environmental Micro J. Invert. Path. 101:1-16). The use of Cry proteins as trans biology 74:7145-7151; a Cry22, a Cry34Ab1 protein of U.S. genic plant traits is well known to one skilled in the art and Pat. Nos. 6,127,180, 6,624,145 and 6,340,593; a CryET33 Cry-transgenic plants including but not limited to Cry1Ac, and cryET34 protein of U.S. Pat. Nos. 6.248,535, 6,326,351, Cry1Ac+Cry2Ab, Cry1Ab, Cry1A.105, Cry1F, Cry1 Fa2, 6,399,330, 6,949,626, 7,385,107 and 7,504,229; a CryET33 Cry1F+Cry1Ac, Cry2Ab, Cry3A, mCry3A, Cry3Bb1, and CryET34 homologs of US Patent Publication Number Cry34Ab1, Cry35Ab1, Vip3A, mCry3A, Cry9c and CBI-Bt 2006/0191034, 2012/0278954, and PCT Publication Number have received regulatory approval (see, Sanahuja, (2011) WO 2012/139004: a Cry35Ab1 protein of U.S. Pat. Nos. Plant Biotech Journal 9:283-300 and the CERA (2010) GM 6,083,499, 6,548,291 and 6,340,593: a Cry46 protein, a Cry Crop Database Center for Environmental Risk Assessment 51 protein, a Cry binary toxin; a TIC901 or related toxin; (CERA), ILSI Research Foundation, Washington D.C. at TIC807 of US 2008/0295207; ET29, ET37, TIC809, TIC810, cera-gmc.org/index.php?action gm crop database which TIC812, TIC127, TIC128 of PCT US 2006/033867; AXMI can be accessed on the world-wide web using the “www.” 027, AXMI-036, and AXMI-038 of U.S. Pat. No. 8,236,757: prefix). More than one pesticidal proteins well known to one AXMI-031, AXMI-039, AXMI-040, AXMI-049 of U.S. Pat. skilled in the art can also be expressed in plants such as No. 7,923,602: AXMI-018, AXMI-020, and AXMI-021 of Vip3Ab & Cry1 Fa (US2012/0317682), Cry 1BE & Cry1F WO 2006/083891: AXMI-010 of WO 2005/038032: AXMI (US2012/0311746), Cry1CA & Cry1AB (US2012/ 003 of WO 2005/021585; AXMI-008 of US 2004/0250311; 0311745), Cry1F & CryCa (US2012/0317681), Cry1DA & AXMI-006 of US 2004/0216186: AXMI-007 of US 2004/ Cry 1BE (US2012/0331590), Cry1DA & Cry1 Fa (US2012/ 0210965; AXMI-009 of US 2004/0210964: AXMI-014 of US 0331589), Cry1AB & Cry1BE (US2012/0324606), and 2004/0197917: AXMI-004 of US 2004/0197916: AXMI-028 Cry1 Fa & Cry2Aa, Cry1 I or Cry1E (US2012/0324605). Pes and AXMI-029 of WO 2006/119457: AXMI-007, AXMI ticidal proteins also include insecticidal lipases including 008, AXMI-008Orf2, AXMI-009, AXMI-014 and AXMI-004 lipid acyl hydrolases of U.S. Pat. No. 7,491,869, and choles of WO 2004/074462: AXMI-150 of U.S. Pat. No. 8,084,416: terol oxidases such as from Streptomyces (Purcell et al. AXMI-205 of US2011 0023184: AXMI-011, AXMI-012, (1993) Biochem Biophy's Res Commun 15:1406-1413). Pes AXMI-013, AXMI-015, AXMI-019, AXMI-044, AXMI ticidal proteins also include VIP (vegetative insecticidal pro 037, AXMI-043, AXMI-033, AXMI-034, AXMI-022, teins) toxins of U.S. Pat. Nos. 5,877,012, 6,107.279 6,137, AXMI-023, AXMI-041, AXMI-063, and AXMI-064 of US 033, 7,244,820, 7,615,686, and 8,237,020 and the like. Other US 2014/0274885 A1 Sep. 18, 2014 20

VIP proteins are well known to one skilled in the art (see, nucleic acid is derived. For purposes of the disclosure, “iso lifesci. Sussex.ac.uk/home/Neil Crickmore/Bt/vip.html lated' or “recombinant' when used to refer to nucleic acid which can be accessed on the world-wide web using the molecules excludes isolated chromosomes. For example, in “www’ prefix). Pesticidal proteins also include toxin com various embodiments, the recombinant nucleic acid molecule plex (TC) proteins, obtainable from organisms such as encoding a PHI-4 polypeptide can contain less than about 5 Xenorhabdus, Photorhabdus and Paenibacillus (see, U.S. kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleic acid Pat. Nos. 7,491,698 and 8,084,418). Some TC proteins have sequences that naturally flank the nucleic acid molecule in “stand alone' insecticidal activity and other TC proteins genomic DNA of the cell from which the nucleic acid is enhance the activity of the stand-alone toxins produced by the derived. same given organism. The toxicity of a “stand-alone' TC 0176 A variety of polynucleotides encoding a PHI-4 protein (from Photorhabdus, Xenorhabdus or Paenibacillus, polypeptide(s) or related proteins are contemplated. Such for example) can be enhanced by one or more TC protein polynucleotides are useful for production of PHI-4 polypep “potentiators' derived from a source organism of a different tides in host cells when operably linked to suitable promoter, genus. There are three main types of TC proteins. As referred transcription termination and/or polyadenylation sequences. to herein, Class A proteins (“Protein A') are stand-alone Such polynucleotides are also useful as probes for isolating toxins. Class B proteins (“Protein B) and Class C proteins homologous or Substantially homologous polynucleotides (“Protein C) enhance the toxicity of Class A proteins. encoding PHI-4 polypeptides or related proteins. Examples of Class A proteins are TchA, TcdA, XptA1 and 0177. The present invention provides isolated or recombi XptA2. Examples of Class B proteins are TcaG, TcdB, nant polynucleotides that encode any of the PHI-4 polypep XptB1Xb and XptC1 Wi. Examples of Class C proteins are tides disclosed herein. Those having ordinary skill in the art TccC, XptC1Xb and XptB1 Wi. Pesticidal proteins also will readily appreciate that due to the degeneracy of the include spider, Snake and Scorpion venom proteins. Examples genetic code, a multitude of nucleotide sequences encoding of spider venom peptides include but not limited to lyc B-glucosidase polypeptides of the present invention exist. otoxin-1 peptides and mutants thereof (U.S. Pat. No. 8.334, Table 1 is a Codon Table that provides the synonymous 366). codons for each amino acid. For example, the codons AGA, 0172. In some embodiments the PHI-4 polypeptides AGG, CGA, CGC, CGG, and CGU all encode the amino acid include amino acid sequences deduced from the full-length arginine. Thus, at every position in the nucleic acids of the nucleic acid sequences disclosed herein, and amino acid invention where an arginine is specified by a codon, the codon sequences that are shorter than the full-length sequences, can be altered to any of the corresponding codons described either due to the use of an alternate downstream start site or above without altering the encoded polypeptide. It is under due to processing that produces a shorter protein having pes stood that U in an RNA sequence corresponds to T in a DNA ticidal activity. Processing may occur in the organism the Sequence. protein is expressed in, or in the pest after ingestion of the protein. TABLE 1. 0173 Thus, provided herein are novel isolated or recom binant nucleic acid sequences that confer pesticidal activity. Alanine Ala A. GCA GCC GCG GCU Also provided are the amino acid sequences of PHI-4 Cysteine Cys C UGC UGU polypeptides. The protein resulting from translation of these PHI-4 polypeptide genes allows cells to control or kill pests Aspartic acid Asp D GAC GAU that ingest it. Glutamic acid Glu E GAA GAG Nucleic Acid Molecules, and Variants and Fragments Thereof Phenylalanine Phe F UUC UUU 0.174. One aspect pertains to isolated or recombinant Glycine Gly G GGA. GGC GGG GGU nucleic acid molecules comprising nucleic acid sequences encoding PHI-4 polypeptides and polypeptides or biologi Histidine His H CAC CAU cally active portions thereof, as well as nucleic acid molecules Isoleucine Ile I AUA AUC AUU sufficient for use as hybridization probes to identify nucleic acid molecules encoding proteins with regions of sequence Lysine Lys K AAA AAG homology. As used herein, the term “nucleic acid molecule' is intended to include DNA molecules (e.g., recombinant Leucine Leu L UUA UUG. CUA CUC CUG. CUU DNA, cDNA, genomic DNA, plastid DNA, mitochondrial Methionine Met M AUG DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The Asparagine As n N AAC AAU nucleic acid molecule can be single-stranded or double Proline Pro P CCA CCC CCG CCU stranded, but preferably is double-stranded DNA. 0175. An "isolated” or “recombinant nucleic acid mol Glutamine Glin Q CAA CAG ecule (or DNA) is used herein to refer to a nucleic acid sequence (or DNA) that is no longer in its natural environ Arginine Arg R AGA AGG CGA CGC CGG CGU ment, for example in an in vitro or in a recombinant bacterial Serine Ser S AGC AGU UCA, UCC UCG UCU or plant host cell. In some embodiments, an "isolated” or “recombinant nucleic acid is free of sequences (preferably Threonine Thir T ACA ACC ACG ACU protein encoding sequences) that naturally flank the nucleic Waline Wall W. GUA GUC GUG GUU acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the US 2014/0274885 A1 Sep. 18, 2014

TABLE 1 - continued nucleic acid sequence such that it can hybridize to the given nucleic acid sequence to thereby form a stable duplex. In Tryptophan Trp W UGG Some embodiments the nucleic acid molecule encoding a Tyrosine Tyr Y UAC UAU PHI-4 polypeptide is a nucleic acid molecule having the sequence set forth in SEQID NO: 1, SEQID NO: 7, SEQID NO: 11, SEQID NOS: 24-30. The corresponding amino acid 0178. Such “silent variations” are one species of “conser sequences for the insecticidal protein encoded by these vative' variation. One of ordinary skill in the art will recog nucleic acid sequences are set forth in SEQID NO: 1, SEQID nize that each codon in a nucleic acid (except AUG, which is NO: 7, SEQID NO: 11, SEQID NOS: 24-30. ordinarily the only codon for methionine) can be modified by 0182. In some embodiments the nucleic acid molecule standard techniques to encode a functionally identical encoding a PHI-4 polypeptide is a polynucleotide having a polypeptide. Accordingly, each silent variation of a nucleic nucleotide sequence encoding a polypeptide comprising an acid which encodes a polypeptide is implicit in any described amino acid sequence having at least 80%, 81%, 82%, 83%, sequence. The invention contemplates and provides each and 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, every possible variation of nucleic acid sequence encoding a 94%.95%,96%.97%.98%,99% or greater sequence identity polypeptide of the invention that could be made by selecting to the amino acid sequence of SEQID NO:35, wherein the combinations based on possible codon choices. These com polypeptide has pesticidal activity. In some embodiments the binations are made in accordance with the standard triplet nucleic acid molecule encoding a PHI-4 polypeptide is a genetic code (set forthin Table 1), as applied to the polynucle polynucleotide having a nucleotide sequence encoding a otide sequences of the present invention. polypeptide comprising an amino acid sequence having 80% 0179 A group of two or more different codons that, when and 95% identity, to the amino acid sequence of SEQID NO: translated in the same context, all encode the same amino 35, wherein the polypeptide has pesticidal activity. acid, are referred to herein as “synonymous codons.” Poly 0183. In some embodiments the nucleic acid molecule nucleotides encoding PHI-4 polypeptides of the present dis encoding a PHI-4 polypeptide is a polynucleotide having a closure may be codon optimized for expression in a particular nucleotide sequence encoding a polypeptide comprising an host organism by modifying the polynucleotides to conform amino acid sequence having at least 80% identity, to the with the optimum codon usage of the desired host organism. amino acid sequence of SEQ ID NO: 51-819, wherein the Those having ordinary skill in the art will recognize that polypeptide has pesticidal activity. tables and other references providing preference information 0184. In some embodiments the nucleic acid molecule for a wide range of organisms are readily available. encoding a PHI-4 polypeptide is a polynucleotide having a 0180 Polynucleotides encoding a PHI-4 polypeptide can nucleotide sequence encoding a polypeptide comprising an also be synthesized de novo from a PHI-4 polypeptide amino acid sequence SEQID NO: 4, wherein Xaa at position sequence. The sequence of the polynucleotide gene can be 9 is Gln, Lys or Glu; Xaa at position 14 is Pro or Ala; Xaa at deduced from a PHI-4 polypeptide sequence through use of position 16 is Val or Asp; Xaa at position 19 is Met or Leu: the genetic code. Computer programs such as "BackTrans Xaaat position 22 is Gly or Ser; Xaaat position 24 is Asp, ASn late” (GCGTM Package. Acclerys, Inc. San Diego, Calif.) can or Glin; Xaa at position 36 is Leu or Met; Xaa at position 42 is be used to convert a peptide sequence to the corresponding Asp, Asn or Glin; Xaa at position 43 is Phe or Glu; Xaa at nucleotide sequence encoding the peptide. Examples of position 46 is Glu, Asp, Asn or Gly: Xaa at position 50 is Ile PHI-4 polypeptide sequences that can be used to obtain cor or Val: Xaa at position 51 is Glu or Gln; Xaa at position 55 is responding nucleotide encoding sequences include, but are Argor Lys;Xaa at position 56 is Seror Thr; Xaa at position 57 not limited to, the PHI-4 polypeptide sequence of SEQ ID is Tyr or Phe, Xaa at position 58 is Thr or Ser; Xaa at position NO:2, SEQID NO:3, SEQID NO:4, SEQID NOs: 51-819. 61 is Arg, Lys or Glu; Xaa at position 73 is Phe or Tyr; Xaa at Furthermore, synthetic PHI-4 polynucleotide sequences of position 74 is Lys, Glu, Gly, Arg, Met, Leu, His or Asp; Xaa the invention can be designed so that they will be expressed in at position 76 is Asp or Glin; Xaa at position 79 is Lys or Glu; plants. U.S. Pat. No. 5,500,365 describes a method for syn Xaa at position 80 is Glu or Ser; Xaa at position 82 is Glu, Ile, thesizing plant genes to improve the expression level of the Leu, Tyr or Glin; Xaa at position 83 is Glu or Glin; Xaa at protein encoded by the synthesized gene. This method relates position 84 is Tyror Phe, Xaa at position 86 is Glu or Glin; Xaa to the modification of the structural gene sequences of the at position 87 is Lys or Gln; Xaa at position 88 is Met, Ile or exogenous transgene, to cause them to be more efficiently Leu; Xaa at position 90 is Glin or Glu; Xaa at position 94 is Val transcribed, processed, translated and expressed by the plant. or Ile: Xaa at position 97 is Arg, Asn, Asp, Glu, Gln, Gly or Features of genes that are expressed well in plants include Ser; Xaa at position 98 is Tyr or Phe; Xaaat position 99 is Lys, elimination of sequences that can cause undesired intron Leu, Tyr, Ile, Met, Phe, Cys, Val or Asn; Xaa at position 103 splicing or polyadenylation in the coding region of a gene is Ala or Gly: Xaaat position 105 is Leu or Ile: Xaa at position transcript while retaining Substantially the amino acid 109 is Phe, Lys, Gly, Met, Ser, Asp, Asn. Glu, Cys, Ala or Arg; sequence of the toxic portion of the insecticidal protein. A Xaa at position 112 is Thr or Ser; Xaa at position 113 is Asp, similar method for obtaining enhanced expression of trans Glu or Met; Xaa at position 117 is Thror Ser; Xaa at position genes in monocotyledonous plants is disclosed in U.S. Pat. 121 is Tyr or Phe: Xaa at position 127 is Ala or Thr; Xaa at No. 5,689,052. position 142 is Arg or Glu, Xaa at position 146 is Arg or Gln; 0181. In some embodiments the nucleic acid molecule Xaa at position 147 is Arg, Glu or Glin; Xaa at position 148 is encoding a PHI-4 polypeptide is a polynucleotide having the Asp, Phe, Pro, Val, Glu, His, Trp, Ala, Arg, Leu, Ser, Gln or sequence set forth in SEQID NO: 1, SEQID NO: 7, SEQID Gly: Xaa at position 149 is Phe or Val: Xaa at position 150 is NO: 11, SEQ ID NOS: 24-30 and variants, fragments and Arg, Glnor Glu, Xaaat position 151 is Asp, Ser, Ala, ASn, Trp, complements thereof. By “complement' is intended a nucleic Val, Gln, Cys, Met, Leu, Arg or Glu, Xaa at position 153 is acid sequence that is Sufficiently complementary to a given Leu or Ile; Xaa at position 154 is ASnor Asp; Xaa at position US 2014/0274885 A1 Sep. 18, 2014 22

155 is Asn or Lys; Xaa at position 159 is Pro or Asp; Xaa at Ala, Leu, Lys, ASn, Gly, Ile, Met, Arg, Tyr, Gln or His; Xaa at position 162 is Glu, Asp or Glin; Xaa at position 165 is Lys, position 397 is Gly, Argor Ala; Xaa at position 398 is Ser, Gln Glu, Gln, Pro, Thr, Ala, Leu, Gly, Asp, Val. His, Ile, Met, Trp, or Cys:Xaa at position 401 is Ser. His, Pro, Gly, Lys, Val, Arg, Phe, Tyr or Arg; Xaa at position 166 is Arg or Glin; Xaa at Ile, ASn, Phe, Thr, Ala, Asp, Met, Gln or Glu; Xaa at position position 167 is Tyr, Trp or Cys: Xaa at position 170 is Tyr or 402 is Lys, Phe, His, Arg, Trp, Gly, Asn. Leu, Tyr, Thr, Val, His; Xaa at position 171 is Tyr or Phe; Xaa at position 172 is Met, Pro or Ala; Xaa at position 403 is Asp, Tyr, Trp, Phe or Ile, Leu or Val; Xaa at position 173 is Ser or Ala; Xaa at Glu; Xaa at position 405 is Ala or Ser; Xaa at position 409 is position 174 is Glu or Glin; Xaa at position 182 is Asp or Gln; Ala or Pro; Xaa at position 410 is Ile or Val; Xaa at position Xaa at position 183 is Tyr or Val: Xaa at position 184 is Seror 411 is Pro or Ala; Xaa at position 412 is Pro or Ala; Xaa at Thr; Xaa at position 185 is Ala or Ser; Xaa at position 189 is position 416 is Arg, Glu or Glin; Xaaat position 417 is Ala, Ser Thr, Lys or Ile: Xaa at position 191 is Lys or Glin; Xaa at or Cys: Xaa at position 418 is Leu or Met; Xaaat position 422 position 193 is Asp or Asn; Xaa at position 196 is Gln, Lys, is Metor Val: Xaaat position 426 is Thror Ser; Xaa at position ASn, Asp, Glu, Ala, Ile or Arg; Xaa at position 202 is Ala or 436 is Asp or Lys; Xaa at position 437 is Tyr or Val: Xaa at Val: Xaa at position 203 is Glu, Thr or His: Xaa at position position 438 is Val or Arg; Xaa at position 440 is Val or Leu: 204 is Met or Ala; Xaa at position 206 is Tyr or Phe: Xaa at Xaa at position 442 is Gln, Lys or Glu, Xaa at position 445 is position 207 is Lys or Glin; Xaa at position 209 is Leu or Pro; Cys, Leu or Thr; Xaaat position 447 is Asp, Lys, Tyr, Ser, Glu, Xaa at position 210 is Val or Ile: Xaa at position 214 is Lys, Ile, Gly, Pro, Leu, Phe, Trp or Thr; Xaa at position 448 is Val Ser or Glin; Xaa at position 216 is Glu, Gln, Phe, Val, Tyr or or Ala; Xaa at position 449 is Glin or Glu; Xaa at position 452 Arg; Xaa at position 220 is Glu, His, Asp, Thr, Tyr, Val, Ser, is Gln, Lys or Glu; Xaa at position 453 is Asn or Asp; Xaa at Gln, Arg, Trp, Met, Ala, Phe, Ile, Leu, Cys or ASn; Xaa at position 454 is Arg, Tyr, Met, Ser, Val, Ile, Lys, Phe, Trp, Gln, position 229 is Arg or Glu; Xaa at position 230 is Seror Glu; Gly. His, Asp, Leu, Thr, Pro or ASn; Xaa at position 455 is Val Xaa at position 231 is Asn or Ser; Xaa at position 236 is Leu or Ile: Xaa at position 457 is Trp or ASn; Xaa at position 459 or Pro; Xaa at position 245 is Met or Leu; Xaa at position 247 is Lys, Met, Val, Trp, Gln, Ile, Thr, Ser. His, Cys, Tyr, Pro, is Asp or Tyr; Xaa at position 256 is Gln, Lys or Glu; Xaa at Asn, Ala, Arg or Glu, Xaa at position 460 is Gly or Ala; Xaa position 257 is Gln, Ile, Glu, Cys, Ser. His, Trp or Met; Xaa at at position 461 is Thror Ser; Xaa at position 462 is Gly or Ala; position 261 is Gln, Glu or Lys; Xaa at position 264 is Glu or Xaa at position 463 is Ala, Seror Gly; Xaa at position 464 is Gln; Xaa at position 268 is Asp or ASn; Xaa at position 276 is Arg, Gly, His, Gln, Thr or Phe; Xaa at position 465 is Lys, Ser or Ala; Xaa at position 278 is Glu, Asn or Glin; Xaa at ASn, Val, Met, Pro, Gly, Arg, Thr, His, Cys, Trp, Phe or Leu: position 281 is Gln, Lys or Glu; Xaa at position 282 is Pro or Xaa at position 466 is Asp or Arg; Xaa at position 471 is Gln, Gly; Xaa at position 284 is Trp or Arg; Xaa at position 287 is Lys or Glu, Xaa at position 497 is Asp or Glin; Xaa at position Ala or Cys;Xaaat position 289 is Lys, Leu, Val, Pro, Glu, Gln, 499 is Glu or Glin; Xaa at position 500 is Arg, Gln or Lys: Xaa Tyr, Thr, Asp, Phe, Ser, Met, Arg, Trp, Ile. His, Asn. Cys, Gly at position 502 is Arg, Glu or Glin; Xaa at position 509 is Lys, or Ala; Xaa at position 291 is Glu or Glin; Xaa at position 292 Gln or Glu; Xaa at position 517 is Gln, Cys, Asn. Val or Pro; is Arg or Glin; Xaa at position 293 is Arg, Glu or Glin; Xaa at Xaa at position 518 is Glu or Glin; Xaa at position 520 is Lys, position 294 is Val or Ala; Xaa at position 296 is Leu or Ile: Gln or Glu; Xaa at position 525 is Glin or Lys; and Xaa at Xaa at position 297 is Glu or Glin; Xaa at position 298 is Asp position 527 is Gln, Lys, Pro, Cys, Glu, Ser. His, Phe or Trp; or Glin; Xaa at position 300 is Phe or Tyr; Xaa at position 302 and having one or more amino acid substitutions at positions is Glu or Glin; Xaa at position 303 is Phe or Tyr; Xaa at designated as Xaa in SEQID NO. 4 and wherein the PHI-4 position 305 is Lys or Glin; Xaa at position 306 is Gln or Lys: polypeptide has increased insecticidal activity compared to Xaa at position 309 is Gln, Lys or Glu; Xaa at position 313 is SEQID NO:35. Lys, Gln or Arg; Xaa at position 316 is Lys or Glin; Xaa at 0185. In some embodiments the nucleic acid molecule position 328 is Lys, Glu or Glin; Xaa at position 331 is Glu, encoding a PHI-4 polypeptide is a polynucleotide having a ASnor Gln; Xaaat position333 is Ser, Arg, Gly, Lys, Val, Asn. nucleotide sequence encoding a polypeptide comprising an Ala, His, Gln, Thr, Asp, Ile, Leu, Cys or Glu; Xaa at position amino acid sequence SEQID NO:3, wherein Xaa at position 334 is Gly, Arg, Lys, Ile or Trp; Xaa at position 335 is Ser or 24 is Asp or ASn; Xaa at position 42 is Asp or ASn; Xaa at Ala; Xaa at position 336 is Gly or Ala; Xaa at position 337 is position 43 is Phe or Glu; Xaa at position 46 is Glu or Asn; Ala, Val or Gly; Xaa at position 338 is Ser. His, Val, Lys, Ala, Xaaat position 74 is Lys, Glu or Gly: Xaaat position 79 is Lys Gly, Thr, Ile, Glu, Met, Arg, Pro, Asp, Asn or Leu; Xaa at or Glu; Xaa at position 82 is Glu, Ile, Leu or Tyr; Xaa at position 339 is Glu, Asn., Gln, Ile, Pro, Met, Ser, Ala, Cys, position 97 is Arg, ASn, Asp, Glu, Gln or Gly; Xaa at position Phe, Val, Leu, Asp, Trp. His or Arg; Xaa at position 341 is Leu 98 is Tyror Phe, Xaaat position 99 is Lys, Leu, Tyr, Ile or Met; or Val: Xaa at position 342 is Ala, Seror Val: Xaa at position Xaa at position 109 is Phe, Lys, Gly, Met, Ser, Asp or Asn; 343 is Val or Ile: Xaa at position 344 is Phe or Trp; Xaa at Xaa at position 147 is Arg or Glu, Xaa at position 148 is Asp, position 345 is Asn or His: Xaa at position 346 is Pro or Ala; Phe or Pro; Xaa at position 150 is Arg or Glin; Xaa at position Xaa at position 350 is Asn or Ser; Xaa at position 351 is Gly 151 is Asp, Ser, Ala or Asn; Xaa at position 153 is Leu or Ile: or Val: Xaa at position 354 is Met or Leu; Xaa at position 355 Xaa at position 162 is Glu or Glin; Xaa at position 165 is Lys, is Val, Ile or Leu; Xaa at position 359 is Gly or Ala; Xaa at Glu or Glin; Xaa at position 166 is Arg or Gln; Xaa at position position 362 is Asn or Ser; Xaa at position 364 is Ala or Ser; 171 is Tyr or Phe; Xaa at position 174 is Glu or Glin; Xaa at Xaa at position 371 is Ala, Gly or Thr; Xaa at position 374 is position 182 is Asp or Glin; Xaa at position 196 is Gln, Lys, Phe or Ile: Xaa at position 375 is Lys or Arg; Xaa at position Asin or Asp; Xaa at position 203 is Glu, Thr or His; Xaa at 380 is Leu or Gly; Xaa at position 382 is Val, Asp or Leu; Xaa position 206 is Tyr or Phe: Xaa at position 216 is Glu or Gln; at position 383 is Leu, Ile or Val: Xaa at position 384 is Lys, Xaa at position 220 is Glu, His, Asp, Thr, Tyr, Val, Seror Gln; Ala or Gly; Xaa at position 385 is Ala or Gly: Xaa at position Xaa at position 247 is Asp or Tyr; Xaa at position 256 is Gln 389 is Trp or Tyr; Xaa at position 391 is Arg, Leu, Glu, Gln or or Lys: Xaa at position 257 is Glin or Ile: Xaa at position 261 Asp; Xaa at position 395 is Asp or Cys; Xaa at position 396 is is Gln or Glu; Xaa at position 278 is Glu or Asn; Xaa at US 2014/0274885 A1 Sep. 18, 2014 position 281 is Gln, Lys or Glu, Xaa at position 289 is Lys, for example, in controlling expression of pesticidal activity, Leu, Val, Pro, Glu, Gln, Tyr, Thr or Asp; Xaa at position 293 since functional pesticidal polypeptide will only be expressed is Arg, Glu or Glin; Xaa at position 313 is Lys or Glin; Xaa at if all required fragments are expressed in an environment that position 328 is Lys, Glu or Glin; Xaa at position 333 is Ser, permits splicing processes to generate functional product. In Gly, Lys, Val or ASn; Xaa at position 334 is Gly, Arg, Lys or another example, introduction of one or more insertion Ile: Xaa at position 336 is Gly or Ala; Xaa at position 338 is sequences into a polynucleotide can facilitate recombination Ser. His, Val, Lys or Ala; Xaa at position 339 is Glu, Asn. Ile with a low homology polynucleotide; use of an intron or or Pro; Xaa at position 343 is Val or Ile:Xaa at position 346 is intein for the insertion sequence facilitates the removal of the Pro or Ala; Xaa at position 355 is Val or Ile: Xaa at position intervening sequence, thereby restoring function of the 359 is Gly or Ala; Xaa at position 391 is Arg, Glu or Gln; Xaa encoded variant. at position 396 is Ala, Leu, Lys, ASn or Gly, Xaa at position 0189 Nucleic acid molecules that are fragments of these 401 is Ser. His, Pro, Gly, Lys, Val or Arg; Xaa at position 402 nucleic acid sequences encoding PHI-4 are also encompassed is Lys, Phe, His, Arg, Gly, Trp, Thr, Asn., Tyr or Met; Xaa at by the embodiments. By “fragment' is intended a portion of position 403 is Asp or Tyr; Xaa at position 411 is Pro or Ala; the nucleic acid sequence encoding a PHI-4. A fragment of a Xaa at position 412 is Pro or Ala; Xaa at position 416 is Arg nucleic acid sequence may encode a biologically active por or Glu; Xaa at position 417 is Ala or Ser; Xaa at position 418 tion of a PHI-4 polypeptide or it may be a fragment that can be is Leu or Met; Xaa at position 426 is Thr or Ser; Xaa at used as a hybridization probe or PCR primer using methods position 440 is Val or Leu, Xaa at position 447 is Asp, LyS, disclosed below. Nucleic acid molecules that are fragments of Tyr, Ser, Glu or Ile: Xaa at position 452 is Gln, Lys or Glu; a nucleic acid sequence encoding a PHI-4 comprise at least Xaa at position 454 is Arg, Tyr, Met, Ser, Val, Ile, Lys, Phe, about 50, 100, 200, 300, 400, 500, 600 or 700, contiguous Trp or Glin; Xaa at position 455 is Val or Ile: Xaa at position nucleotides, or up to the number of nucleotides present in a 459 is Lys, Met, Val, Trp, Gln, Ile or Tyr; Xaa at position 461 full-length nucleic acid sequence encoding a PHI-4 disclosed is Thror Ser; Xaaat position 462 is Gly or Ala; Xaa at position herein, depending upon the intended use. By "contiguous 463 is Ala or Ser; Xaa at position 464 is Arg, Gly or His: Xaa nucleotides is intended nucleotide residues that are immedi at position 465 is Lys, Asn. Val, Met, Pro, Gly or Arg; Xaa at ately adjacent to one another. Fragments of the nucleic acid position 471 is Gln, Lys or Glu; Xaa at position 500 is Arg or sequences of the embodiments will encode protein fragments Gln; Xaa at position 509 is Lys or Gln; Xaa at position 520 is that retain the biological activity of the PHI-4 and, hence, Lys, Gln or Glu; and Xaa at position 527 is Gln, Lys, Pro, Cys retain insecticidal activity. By “retains activity” is intended or Glu, and having one or more amino acid Substitutions at that the fragment will encode a polypeptide having at least positions designated as Xaa in SEQID NO:3 and wherein the about 30%, at least about 50%, at least about 70%, 80%, 90%, PHI-4 polypeptide has increased insecticidal activity com 95% or higher of the insecticidal activity of the full-length pared to SEQID NO:35. PHI-4. In one embodiment, the insecticidal activity is Lep 0186. In some embodiments exemplary nucleic acid mol odoptera activity. In another embodiment, the insecticidal ecules encode a PHI-4 of SEQ ID NO: 51-819 as well as activity is Hemiptera activity. amino acid Substitutions, amino acid deletions, insertions and 0190. In some embodiments a fragment of a nucleic acid fragments thereof and combinations thereof. sequence encoding a PHI-4 encoding a biologically active 0187. In some embodiments the nucleic acid molecules portion of a protein will encode at least about 15, 25, 30, 50. encode a PHI-4 polypeptide of Table 3, Table 4, Table 5, Table 75, 100, 125, 150, 175, 200, or 250, contiguous amino acids, 6, Table 7, Megatable 1, and Megatable 2 and combinations of or up to the total number of amino acids present in a full the amino acid Substitutions thereof and amino acid deletions length PHI-4 of the embodiments. In some embodiments, the and/or insertions thereof. fragment is an N-terminal or a C-terminal truncation of at 0188 Also provided are nucleic acid molecules that least about 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, encode transcription and/or translation products that are Sub 18, 19, 20, 25 or more amino acids relative to SEQID NO:35, sequently spliced to ultimately produce functional PHI-4. SEQID NOs: 51-819 or variants thereof, e.g., by proteolysis, Splicing can be accomplished in vitro or in vivo, and can insertion of a start codon, deletion of the codons encoding the involve cis- or trans-splicing. The Substrate for splicing can be deleted amino acids with the concomitant insertion of a stop polynucleotides (e.g., RNA transcripts) or polypeptides. An codon or by insertion of a stop codon in the coding sequence. example of cis-splicing of a polynucleotide is where an intron In some embodiments, the fragments encompassed herein inserted into a coding sequence is removed and the two flank result from the removal of the N-terminal 1, 2, 3, 4, 5, 6, 7, 8, ing exon regions are spliced to generate a PHI-4 encoding 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, sequence. An example of trans splicing would be where a 26, 27, 28, 29, 30, 31, 32,33,34, or more amino acids relative polynucleotide is encrypted by separating the coding to SEQID NO:35, SEQID NOs: 51-819 or variants thereof, sequence into two or more fragments that can be separately e.g., by proteolysis or by insertion of a start codon in the transcribed and then spliced to form the full-length pesticidal coding sequence. encoding sequence. The use of a splicing enhancer sequence, 0191 In some embodiments the PHI-4 are encoded by a which can be introduced into a construct, can facilitate splic nucleic acid sequence Sufficiently identical to the nucleic acid ing either in cis or trans-splicing of polypeptides (U.S. Pat. sequence of SEQID NO: 1, SEQID NO: 7, SEQID NO: 11, Nos. 6,365.377 and 6,531,316). Thus, in some embodiments SEQ ID NOS: 24-30. By “sufficiently identical” is intended the polynucleotides do not directly encode a full-length PHI an amino acid or nucleic acid sequence that has at least about 4, but rather encode a fragment or fragments of a PHI-4. 60% or 65% sequence identity, about 70% or 75% sequence These polynucleotides can be used to express a functional identity, about 80%, 81%, 82%, 83%, 84%, 85%. 86%, 87%, PHI-4 through a mechanism involving splicing, where splic 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, ing can occurat the level of polynucleotide (e.g., intron/exon) 98%, to 99% or greater sequence identity compared to a and/or polypeptide (e.g., intein/extein). This can be useful, reference sequence using one of the alignment programs US 2014/0274885 A1 Sep. 18, 2014 24 described herein using standard parameters. In some embodi software packages, such as the ALIGNX(R) module of the ments the sequence homology identity is against the full Vector NTIR) Program Suite (Invitrogen Corporation, Carls length sequence of the polynucleotide encoding a PHI-4 or bad, Calif.). After alignment of amino acid sequences with against the full length sequence of a PHI-4 polypeptide. In ClustalW, the percent amino acid identity can be assessed. A some embodiments the polynucleotide encoding the PHI-4 non-limiting example of a Software program useful for analy has at least about 50%, 55%, 60%. 65%, 70%, 75%, 80%, sis of ClustalW alignments is GENEDOCTM. GENEDOCTM 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, (Karl Nicholas) allows assessment of amino acid (or DNA) 91%, 92%.93%, 94%, 95%, 96%,97%.98%, 99% or greater similarity and identity between multiple proteins. Another sequence identity compared to SEQID NO: 1, SEQID NO: non-limiting example of a mathematical algorithm utilized 7, SEQID NO: 11, SEQID NOS: 24-30. One of skill in the art for the comparison of sequences is the algorithm of Myers will recognize that these values can be appropriately adjusted and Miller, (1988) CABIOS 4:11-17. Such an algorithm is to determine corresponding identity of proteins encoded by incorporated into the ALIGN program (version 2.0), which is two nucleic acid sequences by taking into account codon part of the GCG Wisconsin Genetics Software Package, Ver degeneracy, amino acid similarity, reading frame positioning, sion 10 (available from Accelrys, Inc., 9685 Scranton Rd., and the like. San Diego, Calif., USA). When utilizing the ALIGN program 0.192 To determine the percent identity of two amino acid for comparing amino acid sequences, a PAM120 weight resi sequences or of two nucleic acids, the sequences are aligned due table, a gap length penalty of 12 and a gap penalty of 4 can for optimal comparison purposes. The percent identity be used. between the two sequences is a function of the number of 0196. Another non-limiting example of a mathematical identical positions shared by the sequences (i.e., percent algorithm utilized for the comparison of sequences is the identity=number of identical positions/total number of posi algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. tions (e.g., overlapping positions)x100). In one embodiment, 48(3):443-453, used GAP Version 10 software to determine the two sequences are the same length. In another embodi sequence identity or similarity using the following default ment, the comparison is across the entirety of the reference parameters: % identity and % similarity for a nucleic acid sequence (e.g., across the entirety of SEQIDNO:35 or across sequence using GAP Weight of 50 and Length Weight of 3 the entirety of one of SEQID NO: 1, SEQID NO: 7, SEQID and the nwsgapdna.cmpii scoring matrix; % identity or % NO: 11, SEQID NOS: 24-30). The percent identity between similarity for an amino acid sequence using GAP weight of 8 two sequences can be determined using techniques similar to and length weight of 2, and the BLOSUM62 scoring pro those described below, with or without allowing gaps. In gram. Equivalent programs may also be used. By "equivalent calculating percent identity, typically exact matches are program' is intended any sequence comparison program that, counted. for any two sequences in question, generates an alignment 0193 The determination of percent identity between two having identical nucleotide residue matches and an identical sequences can be accomplished using a mathematical algo percent sequence identity when compared to the correspond rithm. A non-limiting example of a mathematical algorithm ing alignment generated by GAPVersion 10. utilized for the comparison of two sequences is the algorithm 0197) The embodiments also encompass nucleic acid mol of Karlin and Altschul, (1990) Proc. Natl. Acad. Sci. USA ecules encoding variants of PHI-4 polypeptide. “Variants of 87:2264, modified as in Karlin and Altschul, (1993) Proc. the PHI-4 polypeptide encoding nucleic acid sequences Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is include those sequences that encode the PHI-4 polypeptides incorporated into the BLASTN and BLASTX programs of disclosed herein but that differ conservatively because of the Altschul, et al., (1990).J. Mol. Biol. 215:403. degeneracy of the genetic code as well as those that are 0194 BLAST nucleotide searches can be performed with Sufficiently identical as discussed above. Naturally occurring the BLASTN program, score=100, wordlength=12, to obtain allelic variants can be identified with the use of well-known nucleic acid sequences homologous to pesticidal-like nucleic molecular biology techniques, such as polymerase chain acid molecules. BLAST protein searches can be performed reaction (PCR) and hybridization techniques as outlined with the BLASTX program, score=50, wordlength=3, to below. Variant nucleic acid sequences also include syntheti obtain amino acid sequences homologous to pesticidal pro cally derived nucleic acid sequences that have been gener tein molecules. To obtain gapped alignments for comparison ated, for example, by using site-directed mutagenesis but purposes, Gapped BLAST (in BLAST 2.0) can be utilized as which still encode the PHI-4 polypeptides disclosed as dis described in Altschul, et al., (1997) Nucleic Acids Res. cussed below. 25:3389. Alternatively, PSI-Blast can be used to perform an 0198 The skilled artisan will further appreciate that iterated search that detects distant relationships between mol changes can be introduced by mutation of the nucleic acid ecules. See. Altschul, et al., (1997) supra. When utilizing sequences thereby leading to changes in the amino acid BLAST, Gapped BLAST, and PSI-Blast programs, the sequence of the encoded PHI-4 polypeptides, without alter default parameters of the respective programs (e.g., BLASTX ing the biological activity of the proteins. Thus, variant and BLASTN) can be used. Alignment may also be per nucleic acid molecules can be created by introducing one or formed manually by inspection. more nucleotide Substitutions, nucleotide additions and/or 0.195 Another non-limiting example of a mathematical nucleotide deletions into the corresponding nucleic acid algorithm utilized for the comparison of sequences is the sequence disclosed herein, Such that one or more amino acid ClustalW algorithm (Higgins, et al., (1994) Nucleic Acids Substitutions, amino acid additions or amino acid deletions Res. 22:4673-4680). ClustalW compares sequences and are introduced into the encoded protein. Mutations can be aligns the entirety of the amino acid or DNA sequence and introduced by Standard techniques, such as site-directed thus can provide data about the sequence conservation of the mutagenesis and PCR-mediated mutagenesis. Such variant entire amino acid sequence. The ClustalW algorithm is used nucleic acid sequences are also encompassed by the present in several commercially available DNA/amino acid analysis invention. US 2014/0274885 A1 Sep. 18, 2014

0199 Alternatively, variant nucleic acid sequences can be Genet. 25(4):436-439; Stemmer, et al., (1999) Tumor Target made by introducing mutations randomly along all or part of ing 4:1-4; Ness et al. (1999) Nat Biotechnol 17:893-896: the coding sequence, such as by Saturation mutagenesis and Changet al. (1999) Nat Biotechnol 17:793-797; Minshulland the resultant mutants can be screened for ability to confer Stemmer, (1999) Curr Opin Chem Biol 3:284-290; Chris pesticidal activity to identify mutants that retain activity. Fol tians, et al., (1999) Nat Biotechnol 17:259-264; Crameri, et lowing mutagenesis, the encoded protein can be expressed al., (1998) Nature 391:288-291; Crameri, et al., (1997) Nat recombinantly, and the activity of the protein can be deter Biotechnol 15:436-438: Zhang, et al., (1997) PNAS USA mined using standard assay techniques. 94:4504-4509: Patten, et al., (1997) Curr Opin Biotechnol 0200. The polynucleotides of the disclosure and frag 8:724-733; Crameri, et al., (1996) Nat Med 2:100-103; ments thereofare optionally used as substrates for a variety of Crameri, et al., (1996) Nat Biotechnol 14:315-319; Gates, et recombination and recursive recombination reactions, in al., (1996).J Mol Biol 255:373-386; Stemmer, (1996) “Sexual addition to standard cloning methods as set forth in, e.g., PCR and Assembly PCRIn: The Encyclopedia of Molecular Ausubel, Berger and Sambrook, i.e., to produce additional Biology. VCH Publishers, New York. pp. 447-457; Crameri pesticidal polypeptide homologues and fragments thereof and Stemmer, (1995) BioTechniques 18:194-195; Stemmer, with desired properties. A variety of such reactions are et al., (1995) Gene, 164:49-53: Stemmer, (1995) Science known, including those developed by the inventors and their 270:1510; Stemmer, (1995) Bio/Technology 13:549-553; co-workers. Methods for producing a variant of any nucleic Stemmer, (1994) Nature 370:389-391 and Stemmer, (1994) acid listed herein comprising recursively recombining Such PNAS USA 91:10747-10751. polynucleotide with a second (or more) polynucleotide, thus 0205 Mutational methods of generating diversity include, forming a library of variant polynucleotides are also embodi for example, site-directed mutagenesis (Ling, et al., (1997) ments of the disclosure, as are the libraries produced, the cells Anal Biochem 254(2):157-178; Dale, et al., (1996) Methods comprising the libraries, and any recombinant polynucleotide Mol Biol 57:369-374; Smith, (1985) Ann Rev. Genet. 19:423 produces by Such methods. Additionally, Such methods 462. Botstein and Shortle, (1985) Science 229:1193–1201; optionally comprise selecting a variant polynucleotide from Carter, (1986) Biochem J 237: 1-7 and Kunkel, (1987) “The Such libraries based on pesticidal activity, as is wherein Such efficiency of oligonucleotide directed mutagenesis' in recursive recombination is done in vitro or in vivo. Nucleic Acids & Molecular Biology (Eckstein and Lilley, 0201 A variety of diversity generating protocols, includ eds. Springer Verlag, Berlin)); mutagenesis using uracil con ing nucleic acid recursive recombination protocols are avail taining templates (Kunkel, (1985) PNAS USA 82:488-492; able and fully described in the art. The procedures can be used Kunkel, et al., (1987) Methods Enzymol 154:367-382 and separately, and/or in combination to produce one or more Bass, et al., (1988) Science 242:240-245); oligonucleotide variants of a nucleic acid or set of nucleic acids, as well as directed mutagenesis (Zoller and Smith, (1983) Methods variants of encoded proteins. Individually and collectively, Enzymol 100:468-500; Zoller and Smith, (1987) Methods these procedures provide robust, widely applicable ways of Enzymol 154:329-350; Zoller and Smith, (1982) Nucleic generating diversified nucleic acids and sets of nucleic acids Acids Res 10:6487-6500), phosphorothioate-modified DNA (including, e.g., nucleic acid libraries) useful, e.g., for the mutagenesis (Taylor, et al., (1985) NuclAcids Res 13:8749 engineering or rapid evolution of nucleic acids, proteins, 8764; Taylor, et al., (1985) Nucl Acids Res 13:8765-8787 pathways, cells and/or organisms with new and/or improved (1985); Nakamaye and Eckstein (1986) Nucl Acids Res characteristics. 14:9679–9698; Sayers, et al., (1988) Nucl Acids Res 16:791 0202 While distinctions and classifications are made in 802 and Sayers, et al., (1988) Nucl Acids Res 16:803-814); the course of the ensuing discussion for clarity, it will be mutagenesis using gapped duplex DNA (Kramer, et al., appreciated that the techniques are often not mutually exclu (1984) Nucl Acids Res 12:9441-9456; Kramer and Fritz, sive. Indeed, the various methods can be used singly or in (1987) Methods Enzymol 154:350-367; Kramer, et al., (1988) combination, in parallel or in series, to access diverse Nucl Acids Res 16:7207 and Fritz, et al., (1988) Nucl Acids sequence variants. Res 16:6987-6999). 0203 The result of any of the diversity generating proce 0206. Additional suitable methods include point mis dures described herein can be the generation of one or more match repair (Kramer, et al., (1984) Cell 38:879-887), nucleic acids, which can be selected or screened for nucleic mutagenesis using repair-deficient host strains (Carter, et al., acids with or which confer desirable properties or that encode (1985) Nucl Acids Res 13:4431-4443 and Carter, (1987) proteins with or which confer desirable properties. Following Methods in Enzymol 154:382-403), deletion mutagenesis diversification by one or more of the methods herein, or (Eghtedarzadeh and Henikoff, (1986) Nucl Acids Res otherwise available to one of skill, any nucleic acids that are 14:5115), restriction-selection and restriction-purification produced can be selected for a desired activity or property, (Wells, et al., (1986) Phil Trans RSoc Lond A317:415-423), e.g. pesticidal activity, or, such activity at a desired pH, etc. mutagenesis by total gene synthesis (Nambiar, et al., (1984) This can include identifying any activity that can be detected, Science 223:1299-1301: Sakamar and Khorana, (1988) Nucl for example, in an automated or automatable format, by any Acids Res 14:6361-6372; Wells, et al., (1985) Gene 34:315 of the assays in the art, see, e.g., discussion of Screening of 323 and Grundström, et al., (1985) Nucl Acids Res 13:3305 insecticidal activity, infra. A variety of related (or even unre 3316), double-strand break repair (Mandecki, (1986) PNAS lated) properties can be evaluated, in serial or in parallel, at USA, 83:7177-7181 and Arnold, (1993) Curr Opin Biotech the discretion of the practitioner. 4:450-455). Additional details on many of the above methods 0204. Descriptions of a variety of diversity generating pro can be found in Methods Enzymol Volume 154, which also cedures for generating modified nucleic acid sequences, e.g., describes useful controls for trouble-shooting problems with those coding for polypeptides having pesticidal activity, or various mutagenesis methods. fragments thereof, are found in the following publications 0207 Additional details regarding various diversity gen and the references cited therein: Soong, et al., (2000) Nat erating methods can be found in the following US patents, US 2014/0274885 A1 Sep. 18, 2014 26

PCT Publications and Applications and EPO Publications: associate the MS data of given cell lysate or desired molecular U.S. Pat. No. 5,723,323, U.S. Pat. No. 5,763,192, U.S. Pat. weight enriched samples (excised from SDS-PAGE gel of No. 5,814,476, U.S. Pat. No. 5,817,483, U.S. Pat. No. 5,824, relevant molecular weight bands to a PHI-4 polypeptide) with 514, U.S. Pat. No. 5,976,862, U.S. Pat. No. 5,605,793, U.S. sequence information of a PHI-4 polypeptide and its Pat. No. 5,811,238, U.S. Pat. No. 5,830,721, U.S. Pat. No. homologs. Any match in peptide sequences indicates the 5,834,252, U.S. Pat. No. 5,837,458, WO 1995/22625, WO potential of having the homologous proteins in the samples. 1996/33207, WO 1997/20078, WO 1997/35966, WO 1999/ Additional techniques (protein purification and molecular 41402, WO 1999/41383, WO 1999/41369, WO 1999/41368, biology) can be used to isolate the protein and identify the EP 752008, EP 0932670, WO 1999/23107, WO 1999/21979, sequences of the homologs. WO 1998/31837, WO 1998/27230, WO 1998/27230, WO 0212. In hybridization methods, all or part of the pesticidal 2000/00632, WO 2000/09679, WO 1998/42832, WO 1999/ nucleic acid sequence can be used to screen cDNA or 29902, WO 1998/41653, WO 1998/41622, WO 1998/42727, genomic libraries. Methods for construction of such cDNA WO 2000/18906, WO 2000/04190, WO 2000/42561, WO and genomic libraries are generally known in the art and are 2000/42559, WO 2000/42560, WO 2001/23401, and PCT/ disclosed in Sambrook and Russell. (2001), supra. The so USO1/06775. called hybridization probes may be genomic DNA fragments, 0208. The nucleotide sequences of the embodiments can cDNA fragments, RNA fragments or other oligonucleotides, also be used to isolate corresponding sequences from other and may be labeled with a detectable group such as P. or any organisms, particularly other bacteria. In this manner, meth other detectable marker, such as other radioisotopes, a fluo ods such as PCR, hybridization and the like can be used to rescent compound, an enzyme, or an enzyme co-factor. identify such sequences based on their sequence homology to Probes for hybridization can be made by labeling synthetic the sequences set forth herein. Sequences that are selected oligonucleotides based on the known PHI-4 polypeptide based on their sequence identity to the entire sequences set encoding nucleic acid sequence disclosed herein. Degenerate forth herein or to fragments thereof are encompassed by the primers designed on the basis of conserved nucleotides or embodiments. Such sequences include sequences that are amino acid residues in the nucleic acid sequence or encoded orthologs of the disclosed sequences. The term “orthologs' amino acid sequence can additionally be used. The probe refers to genes derived from a common ancestral gene and typically comprises a region of nucleic acid sequence that which are found in different species as a result of speciation. hybridizes under stringent conditions to at least about 12, at Genes found in different species are considered orthologs least about 25, at least about 50, 75, 100,125, 150, 175 or 200 when their nucleotide sequences and/or their encoded protein consecutive nucleotides of nucleic acid sequence encoding a sequences share substantial identity as defined elsewhere PHI-4 polypeptide of the disclosure or a fragment or variant herein. Functions of orthologs are often highly conserved thereof. Methods for the preparation of probes for hybridiza among species. tion are generally known in the art and are disclosed in Sam 0209. In a PCR approach, oligonucleotide primers can be brook and Russell, (2001), supra, herein incorporated by designed for use in PCR reactions to amplify corresponding reference. DNA sequences from cDNA or genomic DNA extracted from 0213 For example, an entire nucleic acid sequence, any organism of interest. Methods for designing PCR primers encoding a PHI-4 polypeptide, disclosed herein, or one or and PCR cloning are generally known in the art and are more portions thereof, may be used as a probe capable of disclosed in Sambrook, et al., (1989) Molecular Cloning. A specifically hybridizing to corresponding nucleic acid Laboratory Manual (2d ed., Cold Spring Harbor Laboratory sequences encoding PHI-4 polypeptide-like sequences and Press, Plainview, N.Y.), hereinafter “Sambrook”. See also, messenger RNAs. To achieve specific hybridization under a Innis, et al., eds. (1990) PCR Protocols: A Guide to Methods variety of conditions, such probes include sequences that are and Applications (Academic Press, New York); Innis and unique and are preferably at least about 10 nucleotides in Gelfand, eds. (1995) PCR Strategies (Academic Press, New length, or at least about 20 nucleotides in length. Such probes York); and Innis and Gelfand, eds. (1999) PCR Methods may be used to amplify corresponding pesticidal sequences Manual (Academic Press, New York). Known methods of from a chosen organism by PCR. This technique may be used PCR include, but are not limited to, methods using paired to isolate additional coding sequences from a desired organ primers, nested primers, single specific primers, degenerate ism or as a diagnostic assay to determine the presence of primers, gene-specific primers, Vector-specific primers, par coding sequences in an organism. Hybridization techniques tially-mismatched primers, and the like. include hybridization screening of plated DNA libraries (ei 0210. To identify potential PHI-4 polypeptides from bac ther plaques or colonies; see, for example, Sambrook, et al., terial collections, the bacterial cell lysates can be screened (1989) Molecular Cloning: A Laboratory Manual (2d ed., with antibodies generated against a PHI-4 polypeptide using Cold Spring Harbor Laboratory Press, Cold Spring Harbor, Western blotting and/or ELISA methods. This type of assays N.Y.). can be performed in a high throughput fashion. Positive 0214) Hybridization of such sequences may be carried out samples can be further analyzed by various techniques such under stringent conditions. By “stringent conditions' or as antibody based protein purification and identification. “stringent hybridization conditions' is intended conditions Methods of generating antibodies are well known in the art as under which a probe will hybridize to its target sequence to a discussed infra. detectably greater degree than to other sequences (e.g., at 0211 Alternatively, mass spectrometry based protein least 2-fold over background). Stringent conditions are identification method can be used to identify homologs of a sequence-dependent and will be different in different circum PHI-4 polypeptide using protocols in the literatures (Patter stances. By controlling the stringency of the hybridization son, (1998), 10(22):1-24, Current Protocol in Molecular and/or washing conditions, target sequences that are 100% Biology published by John Wiley & Son Inc). Specifically, complementary to the probe can be identified (homologous LC-MS/MS based protein identification method is used to probing). Alternatively, stringency conditions can be adjusted US 2014/0274885 A1 Sep. 18, 2014 27 to allow some mismatching in sequences so that lower Molecular Biology, Chapter 2 (Greene Publishing and Wiley degrees of similarity are detected (heterologous probing). Interscience, New York). See, Sambrook, et al., (1989) Generally, a probe is less than about 1000 nucleotides in Molecular Cloning: A Laboratory Manual (2d ed., Cold length, preferably less than 500 nucleotides in length. Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). 0215 Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 MNaion, Proteins and Variants and Fragments. Thereof typically about 0.01 to 1.0 M Na ion concentration (or other 0217 PHI-4 polypeptides are encompassed by the disclo salts) at pH 7.0 to 8.3 and the temperature is at least about 30° sure. By “PHI-4 polypeptide' or “PHI-4 protein” as used C. for short probes (e.g., 10 to 50 nucleotides) and at least herein interchangeably is intended a polypeptide that has about 60° C. for long probes (e.g., greater than 50 nucle increased insecticidal activity againstone or more insect pests otides). Stringent conditions may also be achieved with the of the Lepidoptera and/or Coleoptera orders compared to the addition of destabilizing agents such as formamide. Exem protein of SEQID NO:35, and is sufficiently identical to the plary low stringency conditions include hybridization with a protein of SEQID NO:35. A variety of PHI-4 polypeptides buffer solution of 30 to 35% formamide, 1 MNaCl, 1% SDS are contemplated. (sodium dodecyl sulphate) at 37° C., and a wash in 1x to 0218. The Western Corn Rootworm active protein AXMI 2xSSC (20xSSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 205 (SEQID NO:35) encoded by the polynucleotide of SEQ to 55° C. Exemplary moderate stringency conditions include ID NO: 34 was identified from the Chromobacterium Strain hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS ATX 2024 (US20110023184). Synthetic genes encoding at 37° C., and a wash in 0.5x to 1XSSC at 55 to 60° C. AXMI-205: AXMI-205 variants having a truncation of the Exemplary high Stringency conditions include hybridization last 10 and 20 amino acids from the C-terminus (SEQID NO: in 50% formamide, 1 MNaCl, 1% SDS at 37°C., and a wash 36 and SEQID NO:37); and alanine scanning at every other in 0.1xSSC at 60 to 65° C. Optionally, wash buffers may residue from residue 307-536 of AXMI-205 (SEQ ID NO: comprise about 0.1% to about 1% SDS. Duration of hybrid 35), with 5307A, D315A, V317A, S349A, G351A, K353A, ization is generally less than about 24 hours, usually about 4 V355A, D395A, G399A, W407A, G419A, P435A, S443A, to about 12 hours. K465A, V467A, F483A, P4.87A, S495A, D497A, E499A, 0216) Specificity is typically the function of post-hybrid K509A, and 1513A identified as having WCRW activity; are ization washes, the critical factors being the ionic strength disclosed in US2011 0023184. AXMI-205 variants evo 24 and temperature of the final wash solution. For DNA-DNA (E499A); evo25 (V467A SEQ ID NO: 41); evo30 hybrids, the Tm can be approximated from the equation of (V467L SEQ ID NO: 42); PMIibl PoolIG2 p2al 1 Meinkoth and Wahl, (1984) Anal. Biochem. 138:267-284: (5468L SEQID NO: 45); PMIibl Pool IG2 plcl (V467T T81.5° C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)- SEQID NO: 46), PMIibl Pool IG2 pla4 (R464N SEQ ID 500/L: where M is the molarity of monovalent cations, 96 GC NO: 47), evo34 (E86T SEQ ID NO: 43) and evo35 is the percentage of guanosine and cytosine nucleotides in the (Q517R SEQ ID NO. 44) having increased insecticidal DNA,% form is the percentage of formamide in the hybrid activity are disclosed in WO2013/016617. ization solution, and L is the length of the hybrid in base pairs. 0219. As used herein, the terms “protein.” “peptide mol The Tm is the temperature (under defined ionic strength and ecule' or “polypeptide' includes any molecule that com pH) at which 50% of a complementary target sequence prises five or more amino acids. It is well known in the art that hybridizes to a perfectly matched probe. Tm is reduced by protein, peptide or polypeptide molecules may undergo about 1° C. for each 1% of mismatching; thus, Tm, hybrid modification, including post-translational modifications, ization, and/or wash conditions can be adjusted to hybridize Such as, but not limited to, disulfide bond formation, glyco to sequences of the desired identity. For example, if Sylation, phosphorylation or oligomerization. Thus, as used sequences with 90% identity are sought, the Tm can be herein, the terms “protein.” “peptide molecule' or “polypep decreased 10° C. Generally, stringent conditions are selected tide' includes any protein that is modified by any biological to be about 5° C. lower than the thermal melting point (Tm) or non-biological process. The terms "amino acid' and for the specific sequence and its complement at a defined “amino acids’ refer to all naturally occurring L-amino acids. ionic strength and pH. However, severely stringent conditions 0220. A “recombinant protein’ is used to refer to a protein can utilize a hybridization and/or wash at 1, 2, 3 or 4°C. lower that is no longer in its natural environment, for example in than the thermal melting point (Tm); moderately stringent vitro or in a recombinant bacterial or plant host cell. A PHI-4 conditions can utilize a hybridization and/or wash at 6, 7, 8, 9 polypeptide that is substantially free of cellular material or 10° C. lower than the thermal melting point (Tm): low includes preparations of protein having less than about 30%, stringency conditions can utilize a hybridization and/or wash 20%, 10% or 5% (by dry weight) of non-pesticidal protein at 11, 12, 13, 14, 15, or 20°C. lower than the thermal melting (also referred to herein as a "contaminating protein'). point (Tm). Using the equation, hybridization and wash com 0221 By “improved activity” or “increased activity” is positions, and desired Tm, those of ordinary skill will under intended an increase of at least about 10%, at least about 15%, stand that variations in the stringency of hybridization and/or at least about 20%, at least about 25%, at least about 30%, at wash solutions are inherently described. If the desired degree least about 35%, at least about 40%, at least about 50%, at of mismatching results in a Tm of less than 45° C. (aqueous least about 60%, at least about 70%, at least about 80%, at solution) or 32° C. (formamide solution), it is preferred to least about 90%, at least about 100%, at least about 110%, at increase the SSC concentration so that a higher temperature least about 120%, at least about 130%, at least about 140%, at can be used. An extensive guide to the hybridization of least about 150%, at least about 160%, at least about 170%, at nucleic acids is found in Tijssen, (1993) Laboratory Tech least about 180%, at least about 190%, at least about 200%, at niques in Biochemistry and Molecular Biology Hybridiza least about 210% at least about 220%, at least about 230%, at tion with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, least about 240%, at least about 250%, at least about 260%, at N.Y.); and Ausubel, et al., eds. (1995) Current Protocols in least about 270%, at least about 280%, at least about 290%, at US 2014/0274885 A1 Sep. 18, 2014 28 least about 300%, at least about 3.10%, at least about 320%, at 350%, at least about 360%, at least about 370%, at least about least about 330%, at least about 340%, at least about 350%, at 380%, at least about 390%, at least about 400%, at least about least about 360%, at least about 370%, at least about 380%, at 410%, at least about 420%, at least about 430%, at least about least about 390%, at least about 400%, at least about 410%, at 440%, at least about 450%, at least about 460%, at least about least about 420%, at least about 430%, at least about 440%, at 470%, at least about 480%, at least about 490%, at least about least about 450%, at least about 460%, at least about 470%, at 500%, at least about 510%, at least about 520%, at least about least about 480%, at least about 490%, at least about 500%, at 530%, at least about 540%, at least about 550%, at least about least about 510%, at least about 520%, at least about 530%, at 560%, at least about 570%, at least about 580%, at least about least about 540%, at least about 550%, at least about 560%, at 590%, at least about 600%, at least about 650%, at least about least about 570%, at least about 580%, at least about 590%, at 700%, at least about 750%, at least about 800%, at least about least about 600%, at least about 650%, at least about 700%, at 850%, at least about 900%, at least about 950%, at least about least about 750%, at least about 800%, at least about 850%, at 1000% or higher, or at least about 1.1-fold, at least about least about 900%, at least about 950%, at least about 1000% 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, or at or higher, or at least about 1.1-fold, at least about 1.2-fold, at least about 1.5-fold, at least about 1.6-fold, at least about least about 1.3-fold, at least about 1.4-fold, or at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 2-fold, at least about 2.1-fold, at least about 2.2-fold, at about 1.8-fold, at least about 1.9-fold, at least about 2-fold, at least about 2.3-fold, at least about 2.4-fold, at least about least about 2.1-fold, at least about 2.2-fold, at least about 2.5-fold, at least about 2.6-fold, at least about 2.7-fold, at least 2.3-fold, at least about 2.4-fold, at least about 2.5-fold, at least about 2.8-fold, at least about 2.9-fold, at least about 3-fold, at about 2.6-fold, at least about 2.7-fold, at least about 2.8-fold, least about 3.1-fold, at least about 3.2-fold, at least about at least about 2.9-fold, at least about 3-fold, at least about 3.3-fold, at least about 3.4-fold, at least about 3.5-fold, at least 3.1-fold, at least about 3.2-fold, at least about 3.3-fold, at least about 3.6-fold, at least about 3.7-fold, at least about 3.8-fold, about 3.4-fold, at least about 3.5-fold, at least about 3.6-fold, at least about 3.9-fold, at least about 4-fold, at least about at least about 3.7-fold, at least about 3.8-fold, at least about 4.1-fold, at least about 4.2-fold, at least about 4.3-fold, at least 3.9-fold, at least about 4-fold, at least about 4.1-fold, at least about 4.4-fold, at least about 4.5-fold, at least about 4.6-fold, about 4.2-fold, at least about 4.3-fold, at least about 4.4-fold, at least about 4.7-fold, at least about 4.8-fold, at least about at least about 4.5-fold, at least about 4.6-fold, at least about 4.9-fold, at least about 5-fold, at least about 5.1-fold, at least 4.7-fold, at least about 4.8-fold, at least about 4.9-fold, at least about 5.2-fold, at least about 5.3-fold, at least about 5.4-fold, about 5-fold, at least about 5.1-fold, at least about 5.2-fold, at at least about 5.5-fold, at least about 5.6-fold, at least about least about 5.3-fold, at least about 5.4-fold, at least about 5.7-fold, at least about 5.8-fold, at least about 5.9-fold, at least 5.5-fold, at least about 5.6-fold, at least about 5.7-fold, at least about 6-fold, at least about 6.1-fold, at least about 6.2-fold, at about 5.8-fold, at least about 5.9-fold, at least about 6-fold, at least about 6.3-fold, at least about 6.4-fold, at least about least about 6.1-fold, at least about 6.2-fold, at least about 6.5-fold, at least about 6.6-fold, at least about 6.7-fold, at least 6.3-fold, at least about 6.4-fold, at least about 6.5-fold, at least about 6.8-fold, at least about 6.9-fold, at least about 7-fold, at about 6.6-fold, at least about 6.7-fold, at least about 6.8-fold, least about 7.1-fold, at least about 7.2-fold, at least about at least about 6.9-fold, at least about 7-fold, at least about 7.3-fold, at least about 7.4-fold, at least about 7.5-fold, at least 7.1-fold, at least about 7.2-fold, at least about 7.3-fold, at least about 7.6-fold, at least about 7.7-fold, at least about 7.8-fold, about 7.4-fold, at least about 7.5-fold, at least about 7.6-fold, at least about 7.9-fold, at least about 8-fold, at least about at least about 7.7-fold, at least about 7.8-fold, at least about 8.1-fold, at least about 8.2-fold, at least about 8.3-fold, at least 7.9-fold, at least about 8-fold, at least about 8.1-fold, at least about 8.4-fold, at least about 8.5-fold, at least about 8.6-fold, about 8.2-fold, at least about 8.3-fold, at least about 8.4-fold, at least about 8.7-fold, at least about 8.8-fold, at least about at least about 8.5-fold, at least about 8.6-fold, at least about 8.9-fold, at least about 9-fold, at least about 9.1-fold, at least 8.7-fold, at least about 8.8-fold, at least about 8.9-fold, at least about 9.2-fold, at least about 9.3-fold, at least about 9.4-fold, about 9-fold, at least about 9.1-fold, at least about 9.2-fold, at at least about 9.5-fold, at least about 9.6-fold, at least about least about 9.3-fold, at least about 9.4-fold, at least about 9.7-fold, at least about 9.8-fold, at least about 9.9-fold, at least 9.5-fold, at least about 9.6-fold, at least about 9.7-fold, at least about 10-fold, or greater reduction in the EC50 of the PHI-4 about 9.8-fold, at least about 9.9-fold, at least about 10-fold, polypeptide relative to the pesticidal activity of AXMI-205 or higher increase in the pesticidal activity of the variant (SEQ ID NO:35). protein relative to the pesticidal activity of AXMI-205 (SEQ 0223. In some embodiments the EC50 of the PHI-4 ID NO:35). polypeptide is <100 ppm, <90 ppm, <80 ppm, <70 ppm, <60 0222. In some embodiments, the improvement consists of ppm, <50 ppm, <45 ppm, <40 ppm, <35 ppm, <30 ppm, <25 a decrease in the EC50 of at least about 10%, at least about ppm, <20 ppm, <19 ppm, <18 ppm, <17 ppm, <16 ppm, <15 15%, at least about 20%, at least about 25%, at least about ppm, <14 ppm, <13 ppm, <12 ppm, <11 ppm, <10 ppm, <9 30%, at least about 35%, at least about 40%, at least about ppm, <8 ppm, <7 ppm, <6 ppm, <5 ppm, <4 ppm, <3 ppm, <2 50%, at least about 60%, at least about 70%, at least about ppm, <1 ppm, <0.9 ppm, <0.8 ppm, <0.7 ppm, <0.6 ppm, <0.5 80%, at least about 90%, at least about 100%, at least about ppm, <0.4 ppm, <0.3 ppm, <0.2 ppm, <0.1 ppm, 110%, at least about 120%, at least about 130%, at least about 0224. In some embodiments, the improvement consists of 14.0%, at least about 150%, at least about 160%, at least about an increase in the Mean FAE 170%, at least about 180%, at least about 190%, at least about 0225 Index of at least about 10%, at least about 15%, at 200%, at least about 210% at least about 220%, at least about least about 20%, at least about 25%, at least about 30%, at 23.0%, at least about 240%, at least about 250%, at least about least about 35%, at least about 40%, at least about 50%, at 260%, at least about 270%, at least about 280%, at least about least about 60%, at least about 70%, at least about 80%, at 290%, at least about 300%, at least about 3.10%, at least about least about 90%, at least about 100%, at least about 110%, at 320%, at least about 330%, at least about 340%, at least about least about 120%, at least about 130%, at least about 140%, at US 2014/0274885 A1 Sep. 18, 2014 29 least about 150%, at least about 160%, at least about 170%, at 190%, at least about 200%, at least about 210% at least about least about 180%, at least about 190%, at least about 200%, at 220%, at least about 230%, at least about 240%, at least about least about 210% at least about 220%, at least about 230%, at 250%, at least about 260%, at least about 270%, at least about least about 240%, at least about 250%, at least about 260%, at 280%, at least about 290%, at least about 300%, at least about least about 270%, at least about 280%, at least about 290%, at 3.10%, at least about 320%, at least about 330%, at least about least about 300%, at least about 3.10%, at least about 320%, at 340%, at least about 350%, at least about 360%, at least about least about 330%, at least about 340%, at least about 350%, at 370%, at least about 380%, at least about 390%, at least about least about 360%, at least about 370%, at least about 380%, at 400%, at least about 410%, at least about 420%, at least about least about 390%, at least about 400%, at least about 410%, at 430%, at least about 440%, at least about 450%, at least about least about 420%, at least about 430%, at least about 440%, at 460%, at least about 470%, at least about 480%, at least about least about 450%, at least about 460%, at least about 470%, at 490%, at least about 500%, at least about 510%, at least about least about 480%, at least about 490%, at least about 500%, at 520%, at least about 530%, at least about 540%, at least about least about 510%, at least about 520%, at least about 530%, at 550%, at least about 560%, at least about 570%, at least about least about 540%, at least about 550%, at least about 560%, at 580%, at least about 590%, at least about 600%, at least about least about 570%, at least about 580%, at least about 590%, at 650%, at least about 700%, at least about 750%, at least about least about 600%, at least about 650%, at least about 700%, at 800%, at least about 850%, at least about 900%, at least about least about 750%, at least about 800%, at least about 850%, at 950%, at least about 1000% or higher, or at least about 1.1- least about 900%, at least about 950%, at least about 1000% fold, at least about 1.2-fold, at least about 1.3-fold, at least or higher, or at least about 1.1-fold, at least about 1.2-fold, at about 1.4-fold, or at least about 1.5-fold, at least about 1.6- least about 1.3-fold, at least about 1.4-fold, or at least about fold, at least about 1.7-fold, at least about 1.8-fold, at least 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.9-fold, at least about 2-fold, at least about 2.1-fold, at about 1.8-fold, at least about 1.9-fold, at least about 2-fold, at least about 2.2-fold, at least about 2.3-fold, at least about least about 2.1-fold, at least about 2.2-fold, at least about 2.4-fold, at least about 2.5-fold, at least about 2.6-fold, at least 2.3-fold, at least about 2.4-fold, at least about 2.5-fold, at least about 2.7-fold, at least about 2.8-fold, at least about 2.9-fold, about 2.6-fold, at least about 2.7-fold, at least about 2.8-fold, at least about 3-fold, at least about 3.1-fold, at least about at least about 2.9-fold, at least about 3-fold, at least about 3.2-fold, at least about 3.3-fold, at least about 3.4-fold, at least 3.1-fold, at least about 3.2-fold, at least about 3.3-fold, at least about 3.5-fold, at least about 3.6-fold, at least about 3.7-fold, about 3.4-fold, at least about 3.5-fold, at least about 3.6-fold, at least about 3.8-fold, at least about 3.9-fold, at least about at least about 3.7-fold, at least about 3.8-fold, at least about 4-fold, at least about 4.1-fold, at least about 4.2-fold, at least 3.9-fold, at least about 4-fold, at least about 4.1-fold, at least about 4.3-fold, at least about 4.4-fold, at least about 4.5-fold, about 4.2-fold, at least about 4.3-fold, at least about 4.4-fold, at least about 4.6-fold, at least about 4.7-fold, at least about at least about 4.5-fold, at least about 4.6-fold, at least about 4.8-fold, at least about 4.9-fold, at least about 5-fold, at least 4.7-fold, at least about 4.8-fold, at least about 4.9-fold, at least about 5.1-fold, at least about 5.2-fold, at least about 5.3-fold, about 5-fold, at least about 5.1-fold, at least about 5.2-fold, at at least about 5.4-fold, at least about 5.5-fold, at least about least about 5.3-fold, at least about 5.4-fold, at least about 5.6-fold, at least about 5.7-fold, at least about 5.8-fold, at least 5.5-fold, at least about 5.6-fold, at least about 5.7-fold, at least about 5.9-fold, at least about 6-fold, at least about 6.1-fold, at about 5.8-fold, at least about 5.9-fold, at least about 6-fold, at least about 6.2-fold, at least about 6.3-fold, at least about least about 6.1-fold, at least about 6.2-fold, at least about 6.4-fold, at least about 6.5-fold, at least about 6.6-fold, at least 6.3-fold, at least about 6.4-fold, at least about 6.5-fold, at least about 6.7-fold, at least about 6.8-fold, at least about 6.9-fold, about 6.6-fold, at least about 6.7-fold, at least about 6.8-fold, at least about 7-fold, at least about 7.1-fold, at least about at least about 6.9-fold, at least about 7-fold, at least about 7.2-fold, at least about 7.3-fold, at least about 7.4-fold, at least 7.1-fold, at least about 7.2-fold, at least about 7.3-fold, at least about 7.5-fold, at least about 7.6-fold, at least about 7.7-fold, about 7.4-fold, at least about 7.5-fold, at least about 7.6-fold, at least about 7.8-fold, at least about 7.9-fold, at least about at least about 7.7-fold, at least about 7.8-fold, at least about 8-fold, at least about 8.1-fold, at least about 8.2-fold, at least 7.9-fold, at least about 8-fold, at least about 8.1-fold, at least about 8.3-fold, at least about 8.4-fold, at least about 8.5-fold, about 8.2-fold, at least about 8.3-fold, at least about 8.4-fold, at least about 8.6-fold, at least about 8.7-fold, at least about at least about 8.5-fold, at least about 8.6-fold, at least about 8.8-fold, at least about 8.9-fold, at least about 9-fold, at least 8.7-fold, at least about 8.8-fold, at least about 8.9-fold, at least about 9.1-fold, at least about 9.2-fold, at least about 9.3-fold, about 9-fold, at least about 9.1-fold, at least about 9.2-fold, at at least about 9.4-fold, at least about 9.5-fold, at least about least about 9.3-fold, at least about 9.4-fold, at least about 9.6-fold, at least about 9.7-fold, at least about 9.8-fold, at least 9.5-fold, at least about 9.6-fold, at least about 9.7-fold, at least about 9.9-fold, at least about 10-fold, or higher increase in the about 9.8-fold, at least about 9.9-fold, at least about 10-fold, Mean Deviation Score of the PHI-4 polypeptide relative to the or higher increase in the Mean FAE Index of the PHI-4 pesticidal activity of AXMI-205 (SEQ ID NO:35). polypeptide relative to the pesticidal activity of AXMI-205 0227. In some embodiments the improved activity of the (SEQ ID NO:35). PHI-4 polypeptide is relative to the pesticidal activity of 0226. In some embodiments, the improvement consists of AXMI-205(evo25) (SEQ ID NO: 41), AXMI-205(evo30) an increase in the Mean Deviation Score of at least about (SEQ ID NO: 42), Axmi205 PMIibl Pool IG2 p2al 1 (muta 10%, at least about 15%, at least about 20%, at least about tion S468L: SEQID NO: 45), Axmi205 PMIibl PoolIG2 plcl 25%, at least about 30%, at least about 35%, at least about (mutation V467T: SEQ ID NO: 46), Axmi205 PMIibl Poo 40%, at least about 50%, at least about 60%, at least about IIG2 pla4 (mutation R464N: SEQ ID NO: 47), AXMI-205 70%, at least about 80%, at least about 90%, at least about (evo34) (SEQ ID NO: 43) or AXMI-205(evo35) (SEQ ID 100%, at least about 110%, at least about 120%, at least about NO:44). 13.0%, at least about 140%, at least about 150%, at least about 0228 “Mean FAE Index” (MFI) refers to the mean of 160%, at least about 170%, at least about 180%, at least about multiple FAEGN an arithmetic mean of FAEGN. As used US 2014/0274885 A1 Sep. 18, 2014 30 herein, the "Mean Deviation Score” refers to the arithmetic to AXMI-205 (SEQID NO:35). In some embodiments PHI-4 mean of multiple Deviation Scores. polypeptides are provided having amino acid Substitutions at 0229. In some embodiments the PHI-4 polypeptides have Solvent exposed Surface residues to modify the protein char increased insecticidal activity against one or more insect pests acteristics of AXMI-205 (SEQID NO:35), including but not of the order Coleoptera. limited to the ionic polarity of the protein surface. In some 0230. In some embodiments the PHI-4 polypeptides have embodiments PHI-4 polypeptides are provided having amino increased insecticidal activity against Diabrotica virgifera acid Substitutions at hydrophilic residues such as Asp, Glu, larvae Western Corn Root Worm (WCRW). LyS, Arg, His, Ser, Thr, Tyr, Trp, ASn, Gln, and Cys. In some 0231 "Fragments’ or “biologically active portions' embodiments the PHI-4 polypeptides are provided having include polypeptide fragments comprising amino acid amino acid substitutions changing a Lysine or Arginine to a sequences sufficiently identical to a PHI-4 polypeptide and Glutamine, Glutamic Acid, Asparagine or Glutamic Acid; that exhibit insecticidal activity. "Fragments’ or “biologi changing a Glutamic Acid or Aspartic Acid to a Lysine, cally active portions' include polypeptide fragments com Asparagine or Glutamine; and changing a Glutamine to a prising amino acid sequences Sufficiently identical to the Asparagine or Lysine. amino acid sequence set forth in SEQID NO:35 and SEQID 0235. In some embodiments PHI-4 polypeptides are pro NOs: 51-819 and that exhibit insecticidal activity. A biologi vided having amino acid substitutions at residues in a mem cally active portion of a PHI-4 polypeptide can be a polypep brane insertion loop. In some embodiments PHI-4 polypep tide that is, for example, 10, 25, 50, 100, 150, 200, 250, 300, tides are provided having amino acid Substitutions in a 350, 400, 450, 500, 516, 517,518,519, 520,521, 522, 523, membrane insertion loop between about amino acid at posi 524,525,526,527, 528,529,530,531,532,533,534 or 535 tion 92 (Val) and 101 (Ala) and/or at position 211 (Gly) and amino acids in length. Such biologically active portions can 220 (Glu) relative to SEQID NO:35. be prepared by recombinant techniques and evaluated for 0236. In some embodiments PHI-4 polypeptides are pro insecticidal activity. As used here, a fragment comprises at vided having amino acid Substitutions at residues and recep least 8 contiguous amino acids of a PHI-4 polypeptide. In tor binding loops. In some embodiments PHI-4 polypeptides Some embodiments a fragment comprises at least 8 contigu are provided having amino acid Substitutions at residues and ous amino acids of SEQID NO: 2 or SEQID NOs: 51-819. In receptor binding loops between about amino acid 332 (Asp) Some embodiments a fragment comprises at least 8 contigu and 340 (Asp), 395 (Asp) and 403 (Asp), 458 (Asp) and 466 ous amino acids of SEQID NO: 2 or SEQID NOs: 51-819. (Asp) relative to SEQID NO:35. The embodiments encompass other fragments, however, such 0237. In some embodiments PHI-4 polypeptides are pro as any fragment in the protein greater than about 10, 20, 30, vided having amino acid substitutions at residues in a pro 50, 100, 150, 200, 250 or more amino acids. tease sensitive region. In some embodiments PHI-4 polypep 0232. In some embodiments, the fragment is an N-termi tides are provided having amino acid Substitutions at residues nal and/or a C-terminal truncation of at least about 1, 2, 3, 4, in a protease sensitive region from about amino acid residues 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or between 305 (Lys) and 316 (Lys) and 500 (Arg) and 535 (Lys) more amino acids relative to SEQID NO: 2, SEQID NO:35 relative to SEQID NO:35. or SEQID NOs: 51-819 or variants thereof e.g., by proteoly 0238. By variants is intended proteins or polypeptides sis, by insertion of a start codon, by deletion of the codons encoding the deleted amino acids and concomitant insertion having an amino acid sequence that is at least about 50%, of a start codon and/or insertion of a stop codon. In some 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, embodiments, the fragments encompassed herein result from 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, the removal of the C-terminal 1,2,3,4,5,6,7,8,9, 10, 11, 12, 95%, 96%, 97%, 98% or 99% identical to the parental amino 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or acid sequence. In some embodiments a PHI-4 polypeptide 29 amino acids relative to SEQ ID NO:35, SEQID NO: 2, has at least about 60%, 65%, about 70%, 75%, at least about SEQID NO:3, SEQID NO. 4 or SEQID NOs: 51-819, and 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, variants thereof by proteolysis or by insertion of a start codon, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%.98% or 99% or by deletion of the codons encoding the deleted amino acids greater identity across the entire length of the amino acid and concomitant insertion of a start codon. In particular sequence of SEQID NO: 2 or SEQID NOs: 51-819. In some embodiments the proteolytic cleavage site is between Lys at embodiments a PHI-4 polypeptide has at least about 80%, 520 and Ser at 521 Seror Lys at 313 and Val at 314 of SEQID 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, NO: 35 or variants thereof. It is well known in the art that 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or polynucleotide encoding the truncated PHI-4 polypeptide greater identity across the entire length of the amino acid can be engineered to add a start codon at the N-terminus Such sequence of SEQID NO: 2 or SEQID NOs: 51-819. as ATG encoding methionine or methionine followed by an 0239. In some embodiments a PHI-4 polypeptide com alanine. It is also well known in the art that depending on what prises an amino acid sequence having at least 80% identity, to host the PHI-4 polypeptide is expressed in the methionine the amino acid sequence of SEQID NO: 2, SEQ ID NO: 3, may be partially of completed processed off. SEQID NO. 4 or SEQID NOs: 51-819, wherein the polypep 0233. In some embodiments fragments, biologically tide has insecticidal activity. active portions, of SEQ ID NO: 2, SEQ ID NO:3, SEQ ID 0240. In some embodiments a PHI-4 polypeptide com NO. 4 or SEQID NOs: 51-819 as well as amino acid substi prises an amino acid sequence having at least 90% identity to tutions, amino acid deletions and/or insertions thereof are the amino acid sequence of SEQID NO: 2, SEQ ID NO: 3, also provided, and may be used to practice the methods of the SEQID NO. 4 or SEQID NOs: 51-819, wherein the polypep disclosure. tide has insecticidal activity. 0234. In some embodiments PHI-4 polypeptides are pro 0241. In some embodiments a PHI-4 polypeptide com vided having one or more amino acid Substitution compared prises an amino acid sequence having at least 95% identity to US 2014/0274885 A1 Sep. 18, 2014

the amino acid sequence of SEQID NO: 2, SEQ ID NO: 3, or Pro; Xaa at position 245 is Met or Leu; Xaa at position 247 SEQID NO. 4 or SEQID NOs: 51-819 wherein the polypep is Asp or Tyr; Xaa at position 256 is Gln, Lys or Glu; Xaa at tide has insecticidal activity. position 257 is Gln, Ile, Glu, Cys, Ser. His, Trp or Met; Xaa at 0242. In some embodiments a PHI-4 polypeptide com position 261 is Gln, Glu or Lys: Xaa at position 264 is Glu or prises an amino acid sequence having at least 97% identity to Gln; Xaa at position 268 is Asp or ASn; Xaa at position 276 is the amino acid sequence of SEQID NO: 2, SEQ ID NO: 3, Ser or Ala; Xaa at position 278 is Glu, Asin or Glin; Xaa at SEQID NO. 4 or SEQID NOs: 51-819, wherein the polypep position 281 is Gln, Lys or Glu; Xaa at position 282 is Pro or tide has insecticidal activity. Gly: Xaa at position 284 is Trp or Arg; Xaa at position 287 is 0243 In some embodiments a PHI-4 polypeptide com Ala or Cys;Xaaat position 289 is Lys, Leu, Val, Pro, Glu, Gln, prises an amino acid sequence of SEQID NO: 4, wherein Xaa Tyr. Thr, Asp, Phe, Ser, Met, Arg, Trp, Ile. His, Asn. Cys, Gly at position 9 is Gln, Lys or Glu; Xaa at position 14 is Pro or or Ala; Xaa at position 291 is Glu or Glin; Xaa at position 292 Ala; Xaa at position 16 is Valor Asp;Xaa at position 19 is Met is Arg or Glin; Xaa at position 293 is Arg, Glu or Glin; Xaa at or Leu; Xaa at position 22 is Gly or Ser; Xaa at position 24 is position 294 is Val or Ala; Xaa at position 296 is Leu or Ile: Asp, Asn or Glin; Xaa at position 36 is Leu or Met; Xaa at Xaa at position 297 is Glu or Glin; Xaa at position 298 is Asp position 42 is Asp, Asn or Glin; Xaa at position 43 is Phe or or Glin; Xaa at position 300 is Phe or Tyr; Xaa at position 302 Glu, Xaa at position 46 is Glu, Asp, ASn or Gly, Xaa at is Glu or Glin; Xaa at position 303 is Phe or Tyr; Xaa at position 50 is Ile or Val: Xaa at position 51 is Glu or Glin; Xaa position 305 is Lys or Glin; Xaa at position 306 is Gln or Lys: at position 55 is Arg or Lys; Xaa at position 56 is Ser or Thr: Xaa at position 309 is Gln, Lys or Glu; Xaa at position 313 is Xaa at position 57 is Tyr or Phe: Xaa at position 58 is Thr or LyS, Gln or Arg; Xaa at position 316 is Lys or Glin; Xaa at Ser; Xaa at position 61 is Arg, Lys or Glu; Xaa at position 73 position 328 is Lys, Glu or Glin; Xaa at position 331 is Glu, is Phe or Tyr; Xaa at position 74 is Lys, Glu, Gly, Arg, Met, ASnor Gln; Xaa at position333 is Ser, Arg, Gly, Lys, Val, Asn. Leu, His or Asp; Xaa at position 76 is Asp or Glin; Xaa at Ala, His, Gln, Thr, Asp, Ile, Leu, Cys or Glu; Xaa at position position 79 is Lys or Glu:Xaa at position 80 is Glu or Ser; Xaa 334 is Gly, Arg, Lys, Ile or Trp; Xaa at position 335 is Ser or at position 82 is Glu, Ile, Leu, Tyr or Glin; Xaa at position 83 Ala; Xaa at position 336 is Gly or Ala; Xaa at position 337 is is Glu or Glin; Xaa at position 84 is Tyr or Phe: Xaa at position Ala, Val or Gly; Xaa at position 338 is Ser. His, Val, Lys, Ala, 86 is Glu or Glin; Xaa at position 87 is Lys or Glin; Xaa at Gly, Thr, Ile, Glu, Met, Arg, Pro, Asp, Asin or Leu; Xaa at position 88 is Met, Ile or Leu:Xaaat position 90 is Glin or Glu; position 339 is Glu, Asn., Gln, Ile, Pro, Met, Ser, Ala, Cys, Xaa at position 94 is Val or Ile: Xaa at position 97 is Arg, Asn. Phe, Val, Leu, Asp, Trp. His or Arg; Xaa at position 341 is Leu Asp, Glu, Gln, Gly or Ser; Xaa at position 98 is Tyr or Phe: or Val: Xaa at position 342 is Ala, Ser or Val: Xaa at position Xaa at position 99 is Lys, Leu, Tyr, Ile, Met, Phe, Cys, Val or 343 is Val or Ile: Xaa at position 344 is Phe or Trp; Xaa at Asn; Xaa at position 103 is Ala or Gly; Xaa at position 105 is position 345 is Asn or His; Xaa at position 346 is Pro or Ala; Leu or Ile:Xaaat position 109 is Phe, Lys, Gly, Met, Ser, Asp, Xaa at position 350 is Asn or Ser; Xaa at position 351 is Gly ASn, Glu, Cys, Ala or Arg; Xaa at position 112 is Thr or Ser; or Val: Xaa at position 354 is Met or Leu; Xaa at position 355 Xaa at position 113 is Asp, Glu or Met; Xaa at position 117 is is Val, Ile or Leu; Xaa at position 359 is Gly or Ala; Xaa at Thror Ser; Xaa at position 121 is Tyr or Phe: Xaa at position position 362 is Asn or Ser; Xaa at position 364 is Ala or Ser; 127 is Ala or Thr; Xaa at position 142 is Arg or Glu; Xaa at Xaa at position 371 is Ala, Gly or Thr; Xaa at position 374 is position 146 is Arg or Glin; Xaa at position 147 is Arg, Glu or Phe or Ile: Xaa at position 375 is Lys or Arg; Xaa at position Gln; Xaa at position 148 is Asp, Phe, Pro, Val, Glu, His, Trp, 380 is Leu or Gly; Xaa at position 382 is Val, Asp or Leu; Xaa Ala, Arg, Leu, Ser, Gln or Gly; Xaa at position 149 is Phe or at position 383 is Leu, Ile or Val: Xaa at position 384 is Lys, Val: Xaa at position 150 is Arg, Gln or Glu; Xaa at position Ala or Gly; Xaa at position 385 is Ala or Gly: Xaa at position 151 is Asp, Ser, Ala, Asn., Trp, Val, Gln, Cys, Met, Leu, Arg or 389 is Trp or Tyr; Xaa at position 391 is Arg, Leu, Glu, Gln or Glu; Xaa at position 153 is Leu or Ile: Xaa at position 154 is Asp; Xaa at position 395 is Asp or Cys; Xaa at position 396 is ASnor Asp; Xaa at position 155 is ASn or LyS, Xaa at position Ala, Leu, Lys, ASn, Gly, Ile, Met, Arg, Tyr, Gln or His; Xaa at 159 is Pro or Asp; Xaa at position 162 is Glu, Asp or Glin; Xaa position 397 is Gly, Argor Ala; Xaa at position 398 is Ser, Gln at position 165 is Lys, Glu, Gln, Pro. Thr, Ala, Leu, Gly, Asp, or Cys:Xaa at position 401 is Ser. His, Pro, Gly, Lys, Val, Arg, Val. His, Ile, Met, Trp, Phe, Tyr or Arg; Xaa at position 166 is Ile, ASn, Phe, Thr, Ala, Asp, Met, Gln or Glu; Xaa at position Arg or Gln; Xaa at position 167 is Tyr, Trp or Cys; Xaa at 402 is Lys, Phe, His, Arg, Trp, Gly, Asn. Leu, Tyr, Thr, Val, position 170 is Tyr or His: Xaa at position 171 is Tyr or Phe: Met, Pro or Ala; Xaa at position 403 is Asp, Tyr, Trp, Phe or Xaa at position 172 is Ile, Leu or Val: Xaa at position 173 is Glu; Xaa at position 405 is Ala or Ser; Xaa at position 409 is Seror Ala; Xaa at position 174 is Glu or Glin; Xaa at position Ala or Pro; Xaa at position 410 is Ile or Val; Xaa at position 182 is Asp or Glin; Xaa at position 183 is Tyr or Val; Xaa at 411 is Pro or Ala; Xaa at position 412 is Pro or Ala; Xaa at position 184 is Ser or Thr; Xaa at position 185 is Ala or Ser; position 416 is Arg, Glu or Glin; Xaaat position 417 is Ala, Ser Xaa at position 189 is Thr, Lys or Ile: Xaa at position 191 is or Cys: Xaa at position 418 is Leu or Met; Xaaat position 422 Lys or Glin; Xaa at position 193 is Asp or ASn; Xaa at position is Metor Val: Xaaat position 426 is Thror Ser; Xaa at position 196 is Gln, Lys, ASn, Asp, Glu, Ala, Ile or Arg; Xaaat position 436 is Asp or Lys; Xaa at position 437 is Tyr or Val: Xaa at 202 is Ala or Val: Xaa at position 203 is Glu, Thr or His; Xaa position 438 is Val or Arg; Xaa at position 440 is Val or Leu: at position 204 is Metor Ala; Xaaat position 206 is Tyror Phe: Xaa at position 442 is Gln, Lys or Glu, Xaa at position 445 is Xaa at position 207 is Lys or Glin; Xaa at position 209 is Leu Cys, Leu or Thr; Xaaat position 447 is Asp, Lys, Tyr, Ser, Glu, or Pro; Xaa at position 210 is Val or Ile:Xaa at position 214 is Ile, Gly, Pro, Leu, Phe, Trp or Thr; Xaa at position 448 is Val Lys, Seror Glin; Xaa at position 216 is Glu, Gln, Phe, Val, Tyr or Ala; Xaa at position 449 is Glin or Glu; Xaa at position 452 or Arg; Xaa at position 220 is Glu, His, Asp, Thr, Tyr, Val, Ser, is Gln, Lys or Glu; Xaa at position 453 is Asn or Asp; Xaa at Gln, Arg, Trp, Met, Ala, Phe, Ile, Leu, Cys or ASn; Xaa at position 454 is Arg, Tyr, Met, Ser, Val, Ile, Lys, Phe, Trp, Gln, position 229 is Arg or Glu; Xaa at position 230 is Seror Glu; Gly. His, Asp, Leu, Thr, Pro or ASn; Xaa at position 455 is Val Xaa at position 231 is Asn or Ser; Xaa at position 236 is Leu or Ile: Xaa at position 457 is Trp or ASn; Xaa at position 459 US 2014/0274885 A1 Sep. 18, 2014 32 is Lys, Met, Val, Trp, Gln, Ile, Thr, Ser. His, Cys, Tyr, Pro, position 328 is Lys, Glu or Glin; Xaa at position 333 is Ser, Asn, Ala, Arg or Glu; Xaa at position 460 is Gly or Ala; Xaa Gly, Lys, Val or ASn; Xaa at position 334 is Gly, Arg, Lys or at position 461 is Thror Ser; Xaa at position 462 is Gly or Ala; Ile: Xaa at position 336 is Gly or Ala; Xaa at position 338 is Xaa at position 463 is Ala, Seror Gly; Xaa at position 464 is Ser. His, Val, Lys or Ala; Xaa at position 339 is Glu, Asn. Ile Arg, Gly, His, Gln, Thr or Phe: Xaa at position 465 is Lys, or Pro; Xaa at position 343 is Val or Ile:Xaa at position 346 is ASn, Val, Met, Pro, Gly, Arg, Thr, His, Cys, Trp, Phe or Leu: Pro or Ala; Xaa at position 355 is Val or Ile: Xaa at position Xaa at position 466 is Asp or Arg, Xaa at position 471 is Gln, 359 is Gly or Ala; Xaa at position 391 is Arg, Glu or Gln; Xaa Lys or Glu, Xaa at position 497 is Asp or Glin; Xaa at position at position 396 is Ala, Leu, Lys, ASn or Gly, Xaa at position 499 is Glu or Glin; Xaa at position 500 is Arg, Gln or Lys: Xaa 401 is Ser. His, Pro, Gly, Lys, Val or Arg; Xaa at position 402 at position 502 is Arg, Glu or Glin; Xaa at position 509 is Lys, is Lys, Phe, His, Arg, Gly, Trp, Thr, Asn., Tyr or Met; Xaa at Gln or Glu; Xaa at position 517 is Gln, Cys, Asn. Val or Pro; position 403 is Asp or Tyr; Xaa at position 411 is Pro or Ala; Xaa at position 518 is Glu or Glin; Xaa at position 520 is Lys, Xaa at position 412 is Pro or Ala; Xaa at position 416 is Arg Gln or Glu; Xaa at position 525 is Glin or Lys; and Xaa at or Glu; Xaa at position 417 is Ala or Ser; Xaa at position 418 position 527 is Gln, Lys, Pro, Cys, Glu, Ser. His, Phe or Trp; is Leu or Met; Xaa at position 426 is Thr or Ser; Xaa at and having one or more amino acid substitutions at positions position 440 is Val or Leu, Xaa at position 447 is Asp, Lys, designated as Xaa in SEQID NO. 4 and wherein the PHI-4 Tyr, Ser, Glu or Ile: Xaa at position 452 is Gln, Lys or Glu; polypeptide has increased insecticidal activity compared to Xaa at position 454 is Arg, Tyr, Met, Ser, Val, Ile, Lys, Phe, SEQID NO: 2; and amino acid substitutions, deletions, inser Trp or Glin; Xaa at position 455 is Val or Ile: Xaa at position tions, and fragments thereof, and combinations thereof. 459 is Lys, Met, Val, Trp, Gln, Ile or Tyr; Xaa at position 461 0244. In some embodiments a PHI-4 polypeptide com is Thror Ser; Xaaat position 462 is Gly or Ala; Xaa at position prises an amino acid sequence of SEQID NO: 4 having 1, 2, 463 is Ala or Ser; Xaa at position 464 is Arg, Gly or His: Xaa 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, at position 465 is Lys, Asn. Val, Met, Pro, Gly or Arg; Xaa at 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33,34, 35,36, 37,38, position 471 is Gln, Lys or Glu; Xaa at position 500 is Arg or 39, 40, 41,42, 43,44, 45,46, 47, 48,49, 50, 51, 52,53,54, 55, Gln; Xaa at position 509 is Lys or Gln; Xaa at position 520 is 56, 57, 58, 59, 60 or 61 amino acid substitutions, in any Lys, Gln or Glu; and Xaa at position 527 is Gln, Lys, Pro, Cys combination, at residues designated by Xaa in SEQID NO: 4 or Glu, and having one or more amino acid Substitutions at compared to the native amino acid at the corresponding posi positions designated as Xaa in SEQID NO:3 and wherein the tion of SEQID NO: 2. PHI-4 polypeptide has increased insecticidal activity com 0245. In some embodiments a PHI-4 polypeptide com pared to SEQID NO:35; and amino acid substitutions, dele prises an amino acid sequence of SEQID NO: 4 having 1, 2, tions, insertions, and fragments thereof, and combinations 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 thereof. amino acid Substitutions, in any combination, at residues 0247. In some embodiments a PHI-4 polypeptide com designated by Xaa in SEQID NO: 4 compared to the native prises an amino acid sequence of SEQID NO: 3 having 1, 2, amino acid at the corresponding position of SEQID NO: 2. In 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, Some embodiments a PHI-4 polypeptide comprises an amino 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33,34, 35,36, 37,38, acid sequence having at least 80%, 81%, 82%. 83%. 84%, 39, 40, 41,42, 43,44, 45,46, 47, 48,49, 50, 51, 52,53,54, 55, 85%. 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 56, 57,58, 59, 60, 61, 62,63, 64, 65, 66, 67,68, 69,70, 71,72, 95%,96%.97%.98%,99% or greater sequence identity to the 73, 74, 75 or 76 amino acid substitutions, in any combination, amino acid sequence of SEQID NO: 4. at residues designated by Xaa in SEQID NO:3 compared to 0246. In some embodiments a PHI-4 polypeptide com the native amino acid at the corresponding position of SEQID prises an amino acid sequence of SEQID NO:3, wherein Xaa NO: 2. at position 24 is Asp or ASn; Xaa at position 42 is Asp or ASn; 0248. In some embodiments PHI-4 polypeptide comprises Xaa at position 43 is Phe or Glu, Xaa at position 46 is Glu or an amino acid sequence of SEQID NO:3 having 1, 2, 3, 4, 5, Asn; Xaa at position 74 is Lys, Glu or Gly; Xaa at position 79 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, is Lys or Glu, Xaa at position 82 is Glu, Ile, Leu or Tyr; Xaa 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35,36, 37,38, 39, 40, at position 97 is Arg, ASn, Asp, Glu, Gln or Gly, Xaa at 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 or 54 amino position 98 is Tyr or Phe, Xaa at position 99 is Lys, Leu, Tyr, acid Substitutions, in any combination, at residues designated Ile or Met; Xaa at position 109 is Phe, Lys, Gly, Met, Ser, Asp by Xaa in SEQID NO:3 compared to the native amino acid or ASn; Xaa at position 147 is Arg or Glu, Xaa at position 148 at the corresponding position of SEQID NO: 2. is Asp, Phe or Pro; Xaa at position 150 is Arg or Glin; Xaa at 0249. In some embodiments PHI-4 polypeptide comprises position 151 is Asp, Ser, Ala or Asn; Xaa at position 153 is an amino acid sequence of SEQID NO:3 having 1, 2, 3, 4, 5, Leu or Ile: Xaa at position 162 is Glu or Glin; Xaa at position 6, 7, 8, 9, 10 11, 12, 13, 14 or 15 amino acid substitutions, in 165 is Lys, Glu or Glin; Xaaat position 166 is Argor Glin; Xaa any combination, at residues designated by Xaa in SEQ ID at position 171 is Tyror Phe, Xaaat position 174 is Glu or Gln; NO:3 compared to the native amino acid at the corresponding Xaa at position 182 is Asp or Glin; Xaa at position 196 is Gln, position of SEQID NO: 2. Lys, Asin or Asp; Xaa at position 203 is Glu, Thr or His: Xaa 0250 In some embodiments a PHI-4 polypeptide com at position 206 is Tyror Phe, Xaaat position 216 is Glu or Gln; prises an amino acid sequence having at least 80%, 81%, Xaa at position 220 is Glu, His, Asp, Thr, Tyr, Val, Seror Gln; 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, Xaa at position 247 is Asp or Tyr; Xaa at position 256 is Gln 92%, 93%, 94%. 95%, 96%, 97%, 98%, 99% or greater or Lys: Xaa at position 257 is Glin or Ile: Xaa at position 261 sequence identity to the amino acid sequence of SEQID NO: is Gln or Glu; Xaa at position 278 is Glu or ASn; Xaa at 3 position 281 is Gln, Lys or Glu, Xaa at position 289 is Lys, 0251. In some embodiments a PHI-4 polypeptide com Leu, Val, Pro, Glu, Gln, Tyr, Thr or Asp; Xaa at position 293 prises one or more amino acid substitutions compared to the is Arg, Glu or Glin; Xaa at position 313 is Lys or Glin; Xaa at native amino acid at position 40, 42, 43, 46, 52.97, 98, 99, US 2014/0274885 A1 Sep. 18, 2014

145, 150, 151,153, 163, 171, 172, 182, 196, 206, 210, 216 359, 399, 464, 465, 466, 467, 468, 499 or 517, wherein the 220, 278, 283, 289, 293, 328,333,334, 336,338,339, 342, amino acid at position 86 is Glu or Thr; the amino acid at 346, 354, 355, 370, 389, 393, 396, 401, 402,403, 410, 412, position 359 is Gly or Ala; the amino acid at position 399 is 416, 417,426,442, 447, 452, 454, 455, 457, 461, 462, 500, Gly or Ala; the amino acid at position 464 is Arg, Ala, LyS, 509, 520 or 527 of SEQID NO:35. Asp or ASn; the amino acid at position 465 is Lys or Met, the 0252. In some embodiments a PHI-4 polypeptide com amino acid at position 467 is Val, Ala, Leu or Thr; the amino prises one or more amino acid Substitutions compared to the acid at position 468 is Ser or Leu; the amino acid at position native amino acid at position 40, 42, 43, 46, 52.97, 98, 99, 499 is Glu or Ala, or the amino acid at position 517 is Glu or 145, 150, 151,153, 163, 171, 172, 182, 196, 206, 210, 216, Arg. 220, 278, 283, 289, 293, 328,333,334, 336,338,339, 342, 0255. In some embodiments exemplary PHI-4 polypep 346, 354, 355, 370, 389, 393, 396, 401, 402,403, 410, 412, tides are encoded by the polynucleotide sequence set forth in 416, 417,426,442, 447, 452, 454, 455, 457, 461, 462, 500, SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID 509, 520 or 527 of SEQID NO:35 wherein the amino acid at NOS: 24-30. position 40 is Leu or Ile; the amino acid at position 42 is Asp 0256 In some embodiments a PHI-4 polypeptide includes or Asn; the amino acid at position 43 is Phe or Glu; the amino variants where an amino acid that is part of a proteolytic acid at position 46 is Glu or ASn; the amino acid at position 52 cleavage site is changed to anotheramino acid to eliminate or is Ile or Val; the amino acid at position 97 is Arg, Asp, Glu or alter the proteolytic cleavage at that site. In some embodi Asn; the amino acid at position 98 is Tyr or Phe; the amino ments the proteolytic cleavage is by a protease in the insect acid at position 99 is Lys or Leu; the amino acid at position gut. In other embodiments the proteolytic cleavage is by a 145 is Leu or Val; the amino acid at position 150 is Argor Gln; plant protease in the transgenic plant. the amino acid at position 151 is Asp or Ser; the amino acid at 0257. In some embodiments exemplary PHI-4 polypep position 153 is Leu or Ile; the amino acid at position 163 is tides are the polypeptides shown in Table 3, Table 4, Table 5, Leu or Val; the amino acid at position 171 is Tyr or Phe; the Table 6, Table 7, Megatable 1, and Megatable 2 and combi amino acid at position 172 is Ile or Leu, the amino acid at nations of the amino Substitutions thereof as well as amino position 182 is Asp or Gln; the amino acid at position 196 is acid deletions, and or insertions and fragments thereof. Gln or ASn; the amino acid at position 206 is Tyr or Phe; the 0258. In some embodiments a PHI-4 polypeptide is amino acid at position 210 is Val or Ile; the amino acid at encoded by a nucleic acid molecule that hybridizes under position 216 is Glu or Gln; the amino acid at position 220 is stringent conditions to the nucleic acid molecule of SEQID Glu, Gln, His or Asp; the amino acid at position 278 is Glu or NO: 1, SEQID NO:7, SEQID NO: 11, SEQID NOS:24-30. Asn; the amino acid at position 283 is Ile or Val; the amino Variants include polypeptides that differ in amino acid acid at position 289 is Lys, Gln or Leu; the amino acid at sequence due to mutagenesis. Variant proteins encompassed position 293 is Arg, Gln or Glu; the amino acid at position 328 by the disclosure are biologically active, that is they continue is Lys or Glu; the amino acid at position333 is Ser, Lys or Val; to possess the desired biological activity (i.e. pesticidal activ the amino acid at position 334 is Gly, Lys or Arg; the amino ity) of the native protein. By “retains activity” is intended that acid at position 336 is Gly or Ala; the amino acid at position the variant will have at least about 30%, at least about 50%, at 338 is Seror Val; the amino acid at position 339 is Glu, Asn or least about 70% or at least about 80% of the insecticidal Gln; the amino acid at position 342 is Ala or Ser; the amino activity of the native protein. In some embodiments, the vari acid at position 346 is Pro or Ala; the amino acid at position ants may have improved activity over the native protein. 354 is Met or Leu; the amino acid at position 355 is Val or Ile: 0259 Bacterial genes quite often possess multiple the amino acid at position 370 is His or Arg; the amino acid at methionine initiation codons in proximity to the start of the position 389 is Trp or Leu; the amino acid at position 393 is open reading frame. Often, translation initiation at one or Trp or Leu; the amino acid at position 396 is Ala, Leu, LyS, more of these start codons will lead to generation of a func Thror Gly; the amino acid at position 401 is Ser. His, Gly, Lys tional protein. These start codons can include ATG codons. or Pro; the amino acid at position 402 is Lys, His, Gly or Trp; However, bacteria Such as Bacillus sp. also recognize the the amino acid at position 403 is Asp or Tyr; the amino acid at codon GTG as a start codon, and proteins that initiate trans position 410 is Ile or Val; the amino acid at position 412 is Pro lation at GTG codons contain a methionine at the first amino or Ala; the amino acid at position 416 is Argor Glu; the amino acid. On rare occasions, translation in bacterial systems can acid at position 417 is Ala or Ser; the amino acid at position initiate at a TTG codon, though in this event the TTG encodes 426 is Thror Ser; the amino acid at position 442 is Gln or Glu; a methionine. Furthermore, it is not often determined a priori the amino acid at position 447 is Asp or Lys; the amino acid at which of these codons are used naturally in the bacterium. position 452 is Glin or Lys; the amino acid at position 454 is Thus, it is understood that use of one of the alternate methion Arg or Gln; the amino acid at position 455 is Val or Ile; the ine codons may also lead to generation of pesticidal proteins. amino acid at position 457 is Trp or ASn; the amino acid at These pesticidal proteins are encompassed in the present position 461 is Thr or Ser; the amino acid at position 462 is disclosure and may be used in the methods of the present Gly or Ala; the amino acid at position 500 is Arg or Gln; the disclosure. It will be understood that, when expressed in amino acid at position 509 is Lys or Gln; the amino acid at plants, it will be necessary to alter the alternate start codon to position 520 is Lys, Glu or Gln; and the amino acid at position ATG for proper translation. 527 is Glin or LyS., and amino acid deletions, insertions and 0260. In another aspect the PHI-4 polypeptide may be fragments thereof, and combinations thereof. expressed as a precursor protein with an intervening sequence 0253) In some embodiments the PHI-4 polypeptide com that catalyzes multi-step, post translational protein splicing. prising one or more amino acid Substitutions at position 86. Protein splicing involves the excision of an intervening 359, 399, 464, 465,466,467, 468, 499 or 517. sequence from a polypeptide with the concomitant joining of 0254. In some embodiments the PHI-4 polypeptide com the flanking sequences to yield a new polypeptide (Chong, et prising one or more amino acid Substitutions at position 86. al., (1996).J. Biol. Chem. 271:22159-22168). This interven US 2014/0274885 A1 Sep. 18, 2014 34 ing sequence or protein splicing element, referred to as the 3' end of the first fragment coding for the N-terminal part inteins, which catalyze their own excision through three coor of the PHI-4 polypeptide and the 3' intein coding sequence is dinated reactions at the N-terminal and C-terminal splice linked to the 5' end of the second fragment coding for the junctions: an acyl rearrangement of the N-terminal cysteine C-terminal part of the PHI-4 polypeptide. or Serine; a transesterfication reaction between the two ter 0262. In general, the trans-splicing partners can be mini to form a branched ester or thioester intermediate and designed using any split intein, including any naturally-oc peptide bond cleavage coupled to cyclization of the intein curring or artificially-split split intein. Several naturally-oc C-terminal asparagine to free the intein (Evans, et al., (2000) curring split inteins are known, for example: the split intein of J. Biol. Chen. 275:9091-9094. The elucidation of the mecha the DnaB gene of Synechocystis sp. PCC6803 (see, Wu, et al., nism of protein splicing has led to a number of intein-based (1998) Proc Natl AcadSci USA 95(16): 9226-31 and Evans, et applications (Comb, et al., U.S. Pat. No. 5,496.714; Comb, et al., (2000).J. Biol Chem 275(13):9091-4 and of the DnaB gene al., U.S. Pat. No. 5,834,247; Camarero and Muir, (1999).J. from Nostoc punctiforme (see, Iwai, et al., (2006) FEBS Lett Amer: Chem. Soc. 121:5597-5598; Chong, et al., (1997) Gene 580(7):1853-8). Non-split inteins have been artificially split 192:271-281, Chong, et al., (1998) Nucleic Acids Res. in the laboratory to create new split inteins, for example: the 26:5109-5115; Chong, et al., (1998) J. Biol. Chem. 273: artificially split Ssp DnaB intein (see, Wu, et al., (1998) 10567-10577; Cotton, et al., (1999).J. Am. Chem. Soc. 121: Biochim Biophy's Acta 1387:422-32) and split Sce VMA 1100-1101: Evans, et al., (1999).J. Biol. Chem. 274:18359 intein (see, Brenzel, et al., (2006) Biochemistry 45(6): 1571 18363; Evans, et al., (1999).J. Biol. Chem. 274:3923-3926: 8) and an artificially split fungal mini-intein (see, Elleuche, et Evans, et al., (1998) Protein Sci. 7:2256-2264: Evans, et al., al., (2007) Biochem Biophy's Res Commun 355(3):830-4). (2000).J. Biol. Chem. 275:9091-9094; Iwai and Pluckthun, There are also intein databases available that catalogue (1999) FEBS Lett. 459:166-172: Mathys, et al., (1999) Gene known inteins (see, for example the online-database available 231:1-13; Mills, et al., (1998) Proc. Natl. Acad. Sci. USA at: bioinformatics. Weizmann.ac.il/pietro/inteins/Intein 95:3543-3548; Muir, et al., (1998) Proc. Natl. Acad. Sci. USA stable.html, which can be accessed on the world-wide web 95:6705-6710; Otomo, et al., (1999) Biochemistry 38:16040 using the “www’ prefix). 16044: Otomo, et al., (1999).J. Biolmol. NMR 14:105-114; 0263 Naturally-occurring non-split inteins may have Scott, et al., (1999) Proc. Natl. Acad. Sci. USA 96:13638 endonuclease or other enzymatic activities that can typically 13643: Severinov and Muir, (1998) J. Biol. Chem. 273: be removed when designing an artificially-split split intein. 16205-16209; Shingledecker, et al., (1998) Gene 207:187 Such mini-inteins or minimized split inteins are well known 195; Southworth, et al., (1998) EMBO J. 17:918-926; in the art and are typically less than 200 amino acid residues Southworth, et al., (1999) Biotechniques 27:110-120: Wood, long (see, Wu, et al., (1998) Biochim Biophys Acta 1387:422 et al., (1999) Nat. Biotechnol. 17:889-892; Wu, et al., (1998a) 32). Suitable split inteins may have other purification Proc. Natl. Acad. Sci. USA95:9226-9231; Wu, et al., (1998b) enabling polypeptide elements added to their structure, pro Biochim Biophy's Acta 1387:422-432; Xu, et al., (1999) Proc. vided that such elements do not inhibit the splicing of the split Natl. Acad. Sci. USA 96:388-393:Yamazaki, et al., (1998).J. intein or are added in a manner that allows them to be Am. Chem. Soc. 120:5591-5592). For the application of removed prior to splicing. Protein splicing has been reported inteins in plant transgenes see Yang, J, et al., (Transgene Res using proteins that comprise bacterial intein-like (BIL) 15:583-593 (2006)) and Evans, et al., (Annu. Rev. Plant Biol. domains (see, Amitai, et al., (2003) Mol Microbiol 47:61-73) 56:375-392, (2005)). and hedgehog (Hog) auto-processing domains (the latter is 0261. In another aspect the PHI-4 polypeptide may be combined with inteins when referred to as the Hog/intein encoded by two separate genes where the intein of the pre superfamily or HINT family (see, Dassa, et al., (2004).J. Biol. cursor protein comes from the two genes, referred to as a Chem. 27932001-7) and domains such as these may also be split-intein and the two portions of the precursor are joined by used to prepare artificially-split inteins. In particular, non a peptide bond formation. This peptide bond formation is splicing members of Such families may be modified by accomplished by intein-mediated trans-splicing. For this pur molecular biology methodologies to introduce or restore pose, a first and a second expression cassette comprising the splicing activity in Such related species. Recent studies dem two separate genes further code for inteins capable of medi onstrate that splicing can be observed when a N-terminal split ating protein trans-splicing. By trans-splicing, the proteins intein component is allowed to react with a C-terminal split and polypeptides encoded by the first and second fragments intein component not found in nature to be its “partner'; for may be linked by peptide bond formation. Trans-splicing example, splicing has been observed utilizing partners that inteins may be selected from the nucleolar and organellar have as little as 30 to 50% homology with the “natural genomes of different organisms including eukaryotes, splicing partner (see, Dassa, et al., (2007) Biochemistry archaebacteria and eubacteria. Inteins that may be used for 46(1):322-30). Other such mixtures of disparate split intein are listed at neb.com/neb/inteins.html, which can be accessed partners have been shown to be unreactive one with another on the world-wide web using the “www’ prefix). The nucle (see, Brenzel, et al., 2006 Biochemistry 45(6):1571-8). How otide sequence coding for an intein may be split into a 5' and ever, it is within the ability of a person skilled in the relevant a 3' part that code for the 5' and the 3' part of the intein, art to determine whether a particular pair of polypeptides is respectively. Sequence portions not necessary for intein splic able to associate with each other to provide a functional ing (e.g., homing endonuclease domain) may be deleted. The intein, using routine methods and without the exercise of intein coding sequence is split Such that the 5' and the 3' parts inventive skill. are capable of trans-splicing. For selecting a Suitable splitting 0264. In another aspect the PHI-4 polypeptide is a circular site of the intein coding sequence, the considerations pub permuted variant. In certain embodiments the PHI-4 polypep lished by Southworth, et al., (1998) EMBO.J. 17:918-926 tide is a circular permuted variant of the polypeptide of SEQ may be followed. In constructing the first and the second ID NO: 2, SEQID NO:3, SEQ ID NO. 4 or SEQID NOs: expression cassette, the 5' intein coding sequence is linked to 51-819. The development of recombinant DNA methods has US 2014/0274885 A1 Sep. 18, 2014 made it possible to study the effects of sequence transposition the linker required. From those residues whose positions are on protein folding, structure and function. The approach used well defined are selected two residues that are close in in creating new sequences resembles that of naturally occur sequence to the chain ends, and the distance between their ring pairs of proteins that are related by linear reorganization c-alpha carbons is used to calculate an approximate length for of their amino acid sequences (Cunningham, et al., (1979) a linker between them. Using the calculated length as a guide, Proc. Natl. Acad. Sci. U.S.A. 76:3218-3222; Teather and linkers with a range of number of residues (calculated using 2 Erfle, (1990).J. Bacteriol. 172:3837-3841; Schimming, et al., to 3.8 A per residue) are then selected. These linkers may be (1992) Eur: J. Biochem. 204:13-19: Yamiuchi and Minami composed of the original sequence, shortened or lengthened kawa, (1991) FEBS Lett. 260:127-130; MacGregor, et al., as necessary, and when lengthened the additional residues (1996) FEBS Lett. 378:263-266). The first in vitro application may be chosen to be flexible and hydrophilic as described of this type of rearrangement to proteins was described by above; or optionally the original sequence may be substituted Goldenberg and Creighton (J. Mol. Biol. 165:407-413, 1983). for using a series of linkers, one example being the Gly-Gly In creating a circular permuted variant a new N-terminus is Gly-Ser cassette approach mentioned above; or optionally a selected at an internal site (breakpoint) of the original combination of the original sequence and new sequence hav sequence, the new sequence having the same order of amino ing the appropriate total length may be used. Sequences of acids as the original from the breakpoint until it reaches an pesticidal polypeptides capable of folding to biologically amino acid that is at or near the original C-terminus. At this active states can be prepared by appropriate selection of the point the new sequence is joined, either directly or through an beginning (amino terminus) and ending (carboxyl terminus) additional portion of sequence (linker), to an amino acid that positions from within the original polypeptide chain while is at or near the original N-terminus and the new sequence using the linker sequence as described above. Amino and continues with the same sequence as the original until it carboxyl termini are selected from within a common stretch reaches a point that is at or near the amino acid that was of sequence, referred to as a breakpoint region, using the N-terminal to the breakpoint site of the original sequence, this guidelines described below. A novel amino acid sequence is residue forming the new C-terminus of the chain. The length thus generated by selecting amino and carboxyl termini from of the amino acid sequence of the linker can be selected within the same breakpoint region. In many cases the selec empirically or with guidance from structural information, or tion of the new termini will be such that the original position by using a combination of the two approaches. When no of the carboxyl terminus immediately preceded that of the structural information is available, a small series of linkers amino terminus. However, those skilled in the art will recog can be prepared for testing using a design whose length is nize that selections of termini anywhere within the region varied in order to span a range from 0 to 50 A and whose may function, and that these will effectively lead to either sequence is chosen in order to be consistent with Surface amino acid deletions or additions to the amino or carboxyl exposure (hydrophilicity, Hopp and Woods, (1983) Mol. portions of the new sequence. It is a central tenet of molecular Immunol. 20:483-489: Kyte and Doolittle, (1982) J. Mol. biology that the primary amino acid sequence of a protein Biol. 157:105-132; solvent exposed surface area, Lee and dictates folding to the three-dimensional structure necessary Richards, (1971).J. Mol. Biol. 55:379-400) and the ability to for expression of its biological function. Methods are known adopt the necessary conformation without deranging the con to those skilled in the art to obtain and interpret three-dimen figuration of the pesticidal polypeptide (conformationally sional structural information using X-ray diffraction of single flexible; Karplus and Schulz, (1985) Naturwissenschaften protein crystals or nuclear magnetic resonance spectroscopy 72:212-213. Assuming an average of translation of 2.0 to 3.8 of protein solutions. Examples of structural information that A per residue, this would mean the length to test would be are relevant to the identification of breakpoint regions include between 0 to 30 residues, with 0 to 15 residues being the the location and type of protein secondary structure (alpha preferred range. Exemplary of Such an empirical series would and 3-10 helices, parallel and anti-parallel beta sheets, chain be to construct linkers using a cassette sequence Such as reversals and turns, and loops; Kabsch and Sander, (1983) Gly-Gly-Gly-Ser repeated n times, where n is 1, 2, 3 or 4. Biopolymers 22:2577-2637; the degree of solvent exposure of Those skilled in the art will recognize that there are many amino acid residues, the extent and type of interactions of Such sequences that vary in length or composition that can residues with one another (Chothia, (1984) Ann. Rev. Bio serve as linkers with the primary consideration being that they chem. 53:537-572) and the static and dynamic distribution of be neither excessively long nor short (cf., Sandhu, (1992) conformations along the polypeptide chain (Alber and Critical Rev. Biotech. 12:437-462); if they are too long, Mathews, (1987) Methods Enzymol. 154:51 1-533). In some entropy effects will likely destabilize the three-dimensional cases additional information is known about solvent exposure fold, and may also make folding kinetically impractical, and of residues; one example is a site of post-translational attach if they are too short, they will likely destabilize the molecule ment of carbohydrate which is necessarily on the surface of because of torsional or steric strain. Those skilled in the the protein. When experimental structural information is not analysis of protein structural information will recognize that available, or is not feasible to obtain, methods are also avail using the distance between the chain ends, defined as the able to analyze the primary amino acid sequence in order to distance between the c-alpha carbons, can be used to define make predictions of protein tertiary and secondary structure, the length of the sequence to be used, or at least to limit the Solvent accessibility and the occurrence of turns and loops. number of possibilities that must be tested in an empirical Biochemical methods are also sometimes applicable for selection of linkers. They will also recognize that it is some empirically determining Surface exposure when direct struc times the case that the positions of the ends of the polypeptide tural methods are not feasible; for example, using the identi chain are ill-defined in structural models derived from X-ray fication of sites of chain Scission following limited proteoly diffraction or nuclear magnetic resonance spectroscopy data, sis in order to infer surface exposure (Gentile and Salvatore, and that when true, this situation will therefore need to be (1993) Eur: J. Biochem. 218:603-621). Thus using either the taken into account in order to properly estimate the length of experimentally derived structural information or predictive US 2014/0274885 A1 Sep. 18, 2014 36 methods (e.g., Srinivisan and Rose, (1995) Proteins. Struct., ment, the PHI-4 polypeptide may be fused to the pelB pectate Funct. & Genetics 22:81-99) the parental amino acid lyase signal sequence to increase the efficiency of expression sequence is inspected to classify regions according to and purification of Such polypeptides in Gram-negative bac whether or not they are integral to the maintenance of sec teria (see, U.S. Pat. Nos. 5,576,195 and 5,846,818). Plant ondary and tertiary structure. The occurrence of sequences plastid transit peptide/polypeptide fusions are well known in within regions that are known to be involved in periodic the art (see, U.S. Pat. No. 7,193,133). Apoplast transit pep secondary structure (alpha and 3-10 helices, parallel and anti tides such as rice or barley alpha-amylase secretion signal are parallel beta sheets) are regions that should be avoided. Simi also well known in the art. The plastid transit peptide is larly, regions of amino acid sequence that are observed or predicted to have a low degree of Solvent exposure are more generally fused N-terminal to the polypeptide to be targeted likely to be part of the so-called hydrophobic core of the (e.g., the fusion partner). In one embodiment, the fusion protein and should also be avoided for selection of amino and protein consists essentially of the peptide transit plastid and carboxyl termini. In contrast, those regions that are known or the PHI-4 polypeptide to be targeted. In another embodiment, predicted to be in Surface turns or loops, and especially those the fusion protein comprises the peptide transit plastid and the regions that are known not to be required for biological activ polypeptide to be targeted. In Such embodiments, the plastid ity, are the preferred sites for location of the extremes of the transit peptide is preferably at the N-terminus of the fusion polypeptide chain. Continuous stretches of amino acid protein. However, additional amino acid residues may be sequence that are preferred based on the above criteria are N-terminal to the plastid transit peptide providing that the referred to as a breakpoint region. Polynucleotides encoding fusion protein is at least partially targeted to a plastid. In a circular permuted PHI-4 polypeptides with new N-terminus/ specific embodiment, the plastid transit peptide is in the C-terminus which contain a linker region separating the origi N-terminal half, N-terminal third or N-terminal quarter of the nal C-terminus and N-terminus can be made essentially fol fusion protein. Most or all of the plastid transit peptide is lowing the method described in Mullins, et al., (1994).J. Am. generally cleaved from the fusion protein upon insertion into Chem. Soc. 116:5529-5533. Multiple steps of polymerase the plastid. The position of cleavage may vary slightly chain reaction (PCR) amplifications are used to rearrange the between plant species, at different plant developmental DNA sequence encoding the primary amino acid sequence of stages, as a result of specific intercellular conditions or the the protein. Polynucleotides encoding circular permuted particular combination of transit peptide/fusion partner used. PHI-4 polypeptides with new N-terminus/C-terminus which In one embodiment, the plastid transit peptide cleavage is contain a linker region separating the original C-terminus and homogenous such that the cleavage site is identical in a popu N-terminus can be made based on the tandem-duplication lation of fusion proteins. In another embodiment, the plastid method described in Horlick, et al., (1992) Protein Eng. 5:427-431. Polymerase chain reaction (PCR) amplification of transit peptide is not homogenous, Such that the cleavage site the new N-terminus/C-terminus genes is performed using a varies by 1-10 amino acids in a population of fusion proteins. tandemly duplicated template DNA. The plastid transit peptide can be recombinantly fused to a second protein in one of several ways. For example, a restric 0265. In another aspect fusion proteins are provided that tion endonuclease recognition site can be introduced into the include within its amino acid sequence an amino acid nucleotide sequence of the transit peptide at a position corre sequence comprising a PHI-4 polypeptide including but not sponding to its C-terminal end and the same or a compatible limited to the polypeptide of SEQID NO: 2, SEQID NO: 3, site can be engineered into the nucleotide sequence of the SEQID NO:4 or SEQID NOs: 51-819 and active fragments protein to be targeted at its N-terminal end. Care must be thereof. taken in designing these sites to ensure that the coding 0266. In another aspect fusion proteins are provided com sequences of the transit peptide and the second protein are prising a PHI-4 polypeptide and a second pesticidal polypep kept “in frame to allow the synthesis of the desired fusion tide Such a Cry protein. Methods for design and construction protein. In some cases, it may be preferable to remove the of fusion proteins (and polynucleotides encoding same) are initiator methionine codon of the second protein when the known to those of skill in the art. Polynucleotides encoding a new restriction site is introduced. The introduction of restric PHI-4 polypeptide may be fused to signal sequences which tion endonuclease recognition sites on both parent molecules will direct the localization of the PHI-4 polypeptide to par and their Subsequent joining through recombinant DNA tech ticular compartments of a prokaryotic or eukaryotic cell and/ niques may result in the addition of one or more extra amino or direct the secretion of the PHI-4 polypeptide of the acids between the transit peptide and the second protein. This embodiments from a prokaryotic or eukaryotic cell. For generally does not affect targeting activity as long as the example, in E. coli, one may wish to direct the expression of transit peptide cleavage site remains accessible and the func the protein to the periplasmic space. Examples of signal tion of the second protein is not altered by the addition of sequences or proteins (or fragments thereof) to which the these extra amino acids at its N-terminus. Alternatively, one PHI-4 polypeptide may be fused in order to direct the expres skilled in the art can create a precise cleavage site between the sion of the polypeptide to the periplasmic space of bacteria transit peptide and the second protein (with or without its include, but are not limited to, the pelB signal sequence, the initiator methionine) using gene synthesis (Stemmer, et al., maltose binding protein (MBP) signal sequence, MBP, the (1995) Gene 164:49-53) or similar methods. In addition, the omp A signal sequence, the signal sequence of the periplasmic transit peptide fusion can intentionally include amino acids E. coli heat-labile enterotoxin B-subunit, and the signal downstream of the cleavage site. The amino acids at the sequence of alkaline phosphatase. Several vectors are com N-terminus of the mature protein can affect the ability of the mercially available for the construction of fusion proteins transit peptide to target proteins to plastids and/or the effi which will direct the localization of a protein, such as the ciency of cleavage following protein import. This may be pMAL series of vectors (particularly the pMAL-p series) dependent on the protein to be targeted. See, e.g., Comai, et available from New England Biolabs. In a specific embodi al., (1988).J. Biol. Chem. 263(29): 15104-9. US 2014/0274885 A1 Sep. 18, 2014 37

0267 In some embodiments fusion proteins are provide from the hinge region of heavy chain immunoglobulins IgG, comprising a PHI-4 polypeptide, a pesticidal protein Such as IgA, IgM, Ig|D or IgE provide an angular relationship between a cry protein, and an amino acid linker. the attached polypeptides. Especially useful are those hinge 0268. In some embodiments fusion proteins are provided regions where the cysteines are replaced with serines. Pre represented by a formula selected from the group consisting ferred linkers of the present invention include sequences of derived from murine IgG gamma2bhinge region in which the cysteines have been changed to serines. The fusion proteins are not limited by the form, size or number of linker 0269 where R' is a PHI-4 polypeptide, R is a pesticidal sequences employed and the only requirement of the linker is protein with a different but complementary activity to the that functionally it does not interfere adversely with the fold PHI-4 polypeptide, including but not limited to cry proteins: ing and function of the individual molecules of the fusion. a polypeptide that increases the solubility and/or stability of 0271 In another aspect chimeric PHI-4 polypeptide are the PHI-4 polypeptide; or a transit peptide or leader sequence. provided that are created through joining two or more por The R' polypeptide is fused either directly or through a linker tions of genes, which originally encoded separate insecticidal segment to the R polypeptide. The term “directly defines proteins from different species, to create achimeric gene. The fusions in which the polypeptides are joined without a peptide translation of the chimeric gene results in a single chimeric linker. Thus L represents a chemical bound or polypeptide pesticidal polypeptide with regions, motifs or domains segment to which both R' and Rare fused in frame, most derived from each of the original polypeptides. In certain commonly L is a linear peptide to which R" and Rare bound embodiments the chimeric protein comprises portions, by amide bonds linking the carboxy terminus of R' to the motifs, or domains of PHI-4 polypeptides in any combina amino terminus of L and carboxy terminus of L to the amino tion. In certain embodiments the chimeric insecticidal terminus of R. By “fused in frame' is meant that there is no polypeptide includes but not limited to the polypeptides of translation termination or disruption between the reading SEQID NO: 2, SEQID NO:3, SEQ ID NO. 4 or SEQ ID frames of R' and R. The linking group (L) is generally a NOS: 51-819. polypeptide of between 1 and 500 amino acids in length. The 0272. It is recognized that DNA sequences may be altered linkers joining the two molecules are preferably designed to by various methods, and that these alterations may result in (1) allow the two molecules to fold and act independently of DNA sequences encoding proteins with amino acid each other, (2) not have a propensity for developing an sequences different than that encoded by the wild-type (or ordered secondary structure which could interfere with the native) pesticidal protein. These proteins may be altered in functional domains of the two proteins, (3) have minimal various ways including amino acid substitutions, amino acid hydrophobic or charged characteristic which could interact deletions, amino acid truncations, and insertions of one or with the functional protein domains and (4) provide steric more amino acids, including up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, separation of R' and R such that R' and R could interact 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85,90, 100, simultaneously with their corresponding receptors on a single 105,110, 115, 120, 125, 130, 135, 140,145,150, 155 or more cell. Typically surface amino acids in flexible protein regions amino acid Substitutions, amino acid deletions and/or inser include Gly, Asn and Ser. Virtually any permutation of amino tions or combinations thereof compared to SEQID NO:35 acid sequences containing Gly, ASn and Ser would be SEQID NO: 2, SEQID NO:3, SEQ ID NO. 4 or SEQ ID expected to satisfy the above criteria for a linker sequence. NOs: 51-819. In some embodiments a PHI-4 polypeptide Other neutral amino acids, such as Thr and Ala, may also be comprises the deletion of 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, used in the linker sequence. Additional amino acids may also 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or more be included in the linkers due to the addition of unique restric amino acids from the C-terminus of the PHI-4 polypeptide tion sites in the linker sequence to facilitate construction of relative to the amino acid position of SEQID NO: 2, SEQID the fusions. NO:3, SEQID NO. 4 or SEQID NOs: 51-819. Methods for 0270. In some embodiments the linkers comprise Such manipulations are generally known in the art. For sequences selected from the group of formulas: (Gly-Ser), example, amino acid sequence variants of a PHI-4 polypep (Gly-Ser), (Gly-Ser), (Gly, Ser), or (AlaGlySer), where n tide can be prepared by mutations in the DNA. This may also is an integer. One example of a highly-flexible linker is the be accomplished by one of several forms of mutagenesis (GlySer)-rich spacer region present within the pill protein of and/or in directed evolution. In some aspects, the changes the filamentous bacteriophages, e.g., bacteriophages M13 or encoded in the amino acid sequence will not substantially fa (Schaller, et al., 1975). This region provides along, flexible affect the function of the protein. Such variants will possess spacer region between two domains of the pill Surface protein. the desired pesticidal activity. However, it is understood that Also included are linkers in which an endopeptidase recog the ability of a PHI-4 polypeptide to confer pesticidal activity nition sequence is included. Such a cleavage site may be may be improved by the use of Such techniques upon the valuable to separate the individual components of the fusion compositions of this disclosure. to determine if they are properly folded and active in vitro. 0273 For example, conservative amino acid substitutions Examples of various endopeptidases include, but are not lim may be made at one or more, predicted, nonessential amino ited to, Plasmin, Enterokinase, Kallikerin, Urokinase, Tissue acid residues. A “nonessential amino acid residue is a resi Plasminogen activator, clostripain, Chymosin, Collagenase, due that can be altered from the wild-type sequence of a Russell's Viper Venom Protease, Postproline cleavage PHI-4 polypeptide without altering the biological activity. A enzyme, V8 protease, Thrombin and factor Xa. In some “conservative amino acid substitution' is one in which the embodiments the linker comprises the amino acids EEKKN amino acid residue is replaced with an amino acid residue from the multi-gene expression vehicle (MGEV), which is having a similar side chain. Families of amino acid residues cleaved by vacuolar proteases as disclosed in US 2007/ having similar side chains have been defined in the art. These 0277263. In other embodiments, peptide linker segments families include: amino acids with basic side chains (e.g., US 2014/0274885 A1 Sep. 18, 2014 lysine, arginine, histidine); acidic side chains (e.g., aspartic are within +1 are particularly preferred and those within +0.5 acid, glutamic acid); polar, negatively charged residues and are even more particularly preferred. their amides (e.g., aspartic acid, asparagine, glutamic, acid, (0277. It is also understood in the art that the substitution of glutamine; uncharged polar side chains (e.g., glycine, aspar like amino acids can be made effectively on the basis of agine, glutamine, serine, threonine, tyrosine, cysteine); Small hydrophilicity. U.S. Pat. No. 4,554,101, states that the great aliphatic, nonpolar or slightly polar residues (e.g., Alanine, est local average hydrophilicity of a protein, as governed by serine, threonine, proline, glycine); nonpolar side chains the hydrophilicity of its adjacentamino acids, correlates with (e.g., alanine, Valine, leucine, isoleucine, proline, phenylala a biological property of the protein. nine, methionine, tryptophan); large aliphatic, nonpolar resi dues (e.g., methionine, leucine, isoleucine, Valine, cystine); 0278. As detailed in U.S. Pat. No. 4,554,101, the follow beta-branched side chains (e.g., threonine, Valine, isoleu ing hydrophilicity values have been assigned to amino acid cine); aromatic side chains (e.g., tyrosine, phenylalanine, residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+0.1); tryptophan, histidine); large aromatic side chains (e.g., glutamate (+3.0.+0.1); serine (+0.3); asparagine (+0.2): tyrosine, phenylalanine, tryptophan). glutamine (+0.2); glycine (O); threonine (-0.4); proline (-0. 5.+0.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); 0274 Amino acid substitutions may be made in noncon methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine served regions that retain function. In general. Such substitu (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3. tions would not be made for conserved amino acid residues, or for amino acid residues residing within a conserved motif. 4). where such residues are essential for protein activity. 0279 Alternatively, alterations may be made to the protein Examples of residues that are conserved and that may be sequence of many proteins at the amino or carboxy terminus essential for protein activity include, for example, residues without substantially affecting activity. This can include that are identical between all proteins contained in an align amino acid insertions, amino acid deletions, or amino acid ment of similar or related toxins to the sequences of the alterations introduced by modern molecular methods, such as embodiments (e.g., residues that are identical in an alignment PCR, including PCR amplifications that alter or extend the of homologous proteins). Examples of residues that are con protein coding sequence by virtue of inclusion of amino acid served but that may allow conservative amino acid substitu encoding sequences in the oligonucleotides utilized in the tions and still retain activity include, for example, residues PCR amplification. Alternatively, the protein sequences that have only conservative substitutions between all proteins added can include entire protein-coding sequences, such as contained in an alignment of similar or related toxins to the those used commonly in the art to generate protein fusions. sequences of the embodiments (e.g., residues that have only Such fusion proteins are often used to (1) increase expression conservative Substitutions between all proteins contained in of a protein of interest (2) introduce a binding domain, enzy the alignment homologous proteins). However, one of skill in matic activity, or epitope to facilitate either protein purifica the art would understand that functional variants may have tion, protein detection, or other experimental uses known in minor conserved or nonconserved alterations in the con the art (3) target Secretion or translation of a protein to a served residues. Guidance as to appropriate amino acid Sub Subcellular organelle, such as the periplasmic space of Gram stitutions that do not affect biological activity of the protein of negative bacteria, mitochondria or chloroplasts of plants or interest may be found in the model of Dayhoff, et al., (1978) the endoplasmic reticulum of eukaryotic cells, the latter of Atlas of Protein Sequence and Structure (Natl. Biomed. Res. which often results in glycosylation of the protein. Found. Washington, D.C.), herein incorporated by reference. 0280. In specific embodiments, the substitution is an ala 0275. In making such changes, the hydropathic index of nine for the native amino acid at the recited position(s). Also amino acids may be considered. The importance of the hydro encompassed are the nucleic acid sequence(s) encoding the pathic amino acid index in conferring interactive biologic variant protein or polypeptide. function on a protein is generally understood in the art (Kyte 0281 Variant nucleotide and amino acid sequences of the and Doolittle, (1982) J Mol Biol. 157(1):105-32). It is disclosure also encompass sequences derived from accepted that the relative hydropathic character of the amino mutagenic and recombinogenic procedures such as DNA acid contributes to the secondary structure of the resultant shuffling. With such a procedure, one or more different PHI-4 protein, which in turn defines the interaction of the protein polypeptide coding regions can be used to create a new PHI-4 with other molecules, for example, enzymes, Substrates, polypeptide possessing the desired properties. In this manner, receptors, DNA, antibodies, antigens and the like. libraries of recombinant polynucleotides are generated from a 0276. It is known in the art that certain amino acids may be population of related sequence polynucleotides comprising Substituted by otheramino acids having a similar hydropathic sequence regions that have Substantial sequence identity and index or score and still result in a protein with similar bio can be homologously recombined in vitro or in vivo. For logical activity, i.e., still obtain a biological functionally example, using this approach, sequence motifs encoding a equivalent protein. Each amino acid has been assigned a domain of interest may be shuffled between a pesticidal gene hydropathic index on the basis of its hydrophobicity and and other known pesticidal genes to obtain a new gene coding charge characteristics (Kyte and Doolittle, ibid). These are: for a protein with an improved property of interest, such as an isoleucine (+4.5); Valine (+4.2); leucine (+3.8); phenylala increased insecticidal activity. Strategies for such DNA shuf nine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); ala fling are known in the art. See, for example, Stemmer, (1994) nine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); Proc. Natl. Acad. Sci. USA 91:10747-10751; Stemmer, tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (1994) Nature 370:389-391; Crameri, et al., (1997) Nature (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); Biotech. 15:436-438; Moore, et al., (1997).J. Mol. Biol. 272: asparagine (-3.5); lysine (-3.9) and arginine (-4.5). In mak 336-347; Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA ing Such changes, the Substitution of amino acids whose 94:4504-4509: Crameri, et al., (1998) Nature 391:288-291 hydropathic indices are within +2 is preferred, those which and U.S. Pat. Nos. 5,605,793 and 5,837,458. US 2014/0274885 A1 Sep. 18, 2014 39

0282 Domain Swapping or shuffling is another mecha Nucleotide Constructs, Expression Cassettes and Vectors nism for generating altered PHI-4 polypeptides. Domains 0286 The use of the term “nucleotide constructs' herein is may be swapped between PHI-4 polypeptides, resulting in not intended to limit the embodiments to nucleotide con hybrid or chimeric toxins with improved pesticidal activity or structs comprising DNA. Those of ordinary skill in the art will target spectrum. Methods for generating recombinant pro recognize that nucleotide constructs particularly polynucle teins and testing them for pesticidal activity are well known in otides and oligonucleotides composed of ribonucleotides and the art (see, for example, Naimov, et al., (2001) Appl. Environ. combinations of ribonucleotides and deoxyribonucleotides Microbiol. 67:5328-5330; de Maagd, et al., (1996) Appl. may also be employed in the methods disclosed herein. The Environ. Microbiol. 62:1537-1543; Ge, et al., (1991).J. Biol. nucleotide constructs, nucleic acids, and nucleotide Chem. 266:17954-17958; Schnepf, et al., (1990) J. Biol. sequences of the embodiments additionally encompass all Chem. 265:20923-20930; Rang, et al., 91999) Appl. Environ. complementary forms of Such constructs, molecules and Microbiol. 65:2918-2925). sequences. Further, the nucleotide constructs, nucleotide molecules and nucleotide sequences of the embodiments Antibodies encompass all nucleotide constructs, molecules and 0283 Antibodies to a PHI-4 polypeptide of the embodi sequences which can be employed in the methods of the ments, or to variants or fragments thereof, are also encom embodiments for transforming plants including, but not lim passed. Methods for producing antibodies are well known in ited to, those comprised of deoxyribonucleotides, ribonucle the art (see, for example, Harlow and Lane, (1988) Antibod otides and combinations thereof. Such deoxyribonucleotides ies: A Laboratory Manual, Cold Spring Harbor Laboratory, and ribonucleotides include both naturally occurring mol Cold Spring Harbor, N.Y.; U.S. Pat. No. 4,196,265). ecules and synthetic analogues. The nucleotide constructs, 0284 Akit for detecting the presence of a PHI-4 polypep nucleic acids, and nucleotide sequences of the embodiments tide, or detecting the presence of a nucleotide sequence also encompass all forms of nucleotide constructs including, encoding a PHI-4 polypeptide, in a sample is provided. In one but not limited to, single-stranded forms, double-stranded embodiment, the kit provides antibody-based reagents for forms, hairpins, stem-and-loop structures and the like. detecting the presence of a PHI-4 polypeptide in a tissue 0287. A further embodiment relates to a transformed sample. In another embodiment, the kit provides labeled organism Such as an organism selected from plant and insect nucleic acid probes useful for detecting the presence of one or cells, bacteria, yeast, baculovirus, protozoa, nematodes and more polynucleotides encoding PHI-4 polypeptide(s). The algae. The transformed organism comprises a DNA molecule kit is provided along with appropriate reagents and controls of the embodiments, an expression cassette comprising the for carrying out a detection method, as well as instructions for DNA molecule or a vector comprising the expression cas use of the kit.” sette, which may be stably incorporated into the genome of the transformed organism. 0288 The sequences of the embodiments are provided in Receptor Identification and Isolation DNA constructs for expression in the organism of interest. 0285 Receptors to the PHI-4 polypeptide of the embodi The construct will include 5' and 3' regulatory sequences ments, or to variants or fragments thereof, are also encom operably linked to a sequence of the embodiments. The term passed. Methods for identifying receptors are well known in “operably linked as used herein refers to a functional linkage the art (see, Hofmann, et. al., (1988) Eur: J. Biochem. 173: between a promoter and a second sequence, wherein the 85-91; Gill, et al., (1995).J. Biol. Chem. 27277-27282) can be promoter sequence initiates and mediates transcription of the employed to identify and isolate the receptor that recognizes DNA sequence corresponding to the second sequence. Gen the PHI-4 polypeptides using the brush-border membrane erally, operably linked means that the nucleic acid sequences vesicles from Susceptible insects. In addition to the radioac being linked are contiguous and where necessary to join two tive labeling method listed in the cited literatures, PHI-4 protein coding regions in the same reading frame. The con polypeptide can be labeled with fluorescent dye and other struct may additionally contain at least one additional gene to common labels such as streptavidin. Brush-border membrane be cotransformed into the organism. Alternatively, the addi vesicles (BBMV) of susceptible insects such as soybean tional gene(s) can be provided on multiple DNA constructs. looper and Stink bugs can be prepared according to the pro 0289 Such a DNA construct is provided with a plurality of tocols listed in the references and separated on SDS-PAGE restriction sites for insertion of the PHI-4 polypeptide gene gel and blotted on suitable membrane. Labeled PHI-4 sequence to be under the transcriptional regulation of the polypeptides can be incubated with blotted membrane of regulatory regions. The DNA construct may additionally con BBMV and labeled the PHI-4 polypeptides can be identified tain selectable marker genes. with the labeled reporters. Identification of protein band(s) 0290. The DNA construct will generally include in the 5' that interact with the PHI-4 polypeptides can be detected by to 3' direction of transcription: a transcriptional and transla N-terminal amino acid gas phase sequencing or mass spec tional initiation region (i.e., a promoter), a DNA sequence of trometry based protein identification method (Patterson, the embodiments and a transcriptional and translational ter (1998) 10(22):1-24, Current Protocol in Molecular Biology mination region (i.e., termination region) functional in the published by John Wiley & Son Inc). Once the protein is organism serving as a host. The transcriptional initiation identified, the corresponding gene can be cloned from region (i.e., the promoter) may be native, analogous, foreign genomic DNA or cDNA library of the susceptible insects and or heterologous to the host organism and/or to the sequence of binding affinity can be measured directly with the PHI-4 the embodiments. Additionally, the promoter may be the polypeptides. Receptor function for insecticidal activity by natural sequence or alternatively a synthetic sequence. The the PHI-4 polypeptides can be verified by accomplished by term “foreign' as used herein indicates that the promoter is RNAi type of gene knock out method (Rajagopal, et al., not found in the native organism into which the promoter is (2002).J. Biol. Chem. 277:46849-46851). introduced. Where the promoter is “foreign” or "heterolo US 2014/0274885 A1 Sep. 18, 2014 40 gous' to the sequence of the embodiments, it is intended that 0295 Additional sequence modifications are known to the promoter is not the native or naturally occurring promoter enhance gene expression in a cellular host. These include for the operably linked sequence of the embodiments. As used elimination of sequences encoding spurious polyadenylation herein, a chimeric gene comprises a coding sequence oper signals, exon-intron splice site signals, transposon-like ably linked to a transcription initiation region that is heter repeats, and other well-characterized sequences that may be ologous to the coding sequence. Where the promoter is a deleterious to gene expression. The GC content of the native or natural sequence, the expression of the operably sequence may be adjusted to levels average for a given cellu linked sequence is altered from the wild-type expression, lar host, as calculated by reference to known genes expressed which results in an alteration in phenotype. in the host cell. The term "host cell as used herein refers to a 0291. In some embodiments the DNA construct may also cell which contains a vector and Supports the replication include a transcriptional enhancer sequence. As used herein, and/or expression of the expression vector is intended. Host the term an "enhancer refers to a DNA sequence which can cells may be prokaryotic cells Such as E. coli, or eukaryotic stimulate promoter activity and may be an innate element of cells such as yeast, insect, amphibian or mammalian cells or the promoter or a heterologous element inserted to enhance monocotyledonous or dicotyledonous plant cells. An the level or tissue-specificity of a promoter. Various enhanc example of a monocotyledonous host cell is a maize host cell. ers are known in the art including for example, introns with When possible, the sequence is modified to avoid predicted gene expression enhancing properties in plants (US Patent hairpin secondary mRNA structures. Application Publication Number 2009/0144863, the ubiq 0296. The expression cassettes may additionally contain 5' uitin intron (i.e., the maize ubiquitin intron 1 (see, for leader sequences. Such leader sequences can act to enhance example, NCBI sequence S94464)), the omega enhancer or translation. Translation leaders are known in the art and the omega prime enhancer (Gallie, et al., (1989) Molecular include: picornavirus leaders, for example, EMCV leader Biology of RNA ed. Cech (Liss, New York) 237-256 and (Encephalomyocarditis 5' noncoding region) (Elroy-Stein, et Gallie, et al., (1987) Gene 60:217-25), the CaMV 35S al., (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130); potyvi enhancer (see, e.g., Benfey, et al., (1990) EMBO.J. 9:1685 rus leaders, for example, TEV leader (Tobacco Etch Virus) 96) and the enhancers of U.S. Pat. No. 7,803,992 may also be (Gallie, et al., (1995) Gene 165(2):233-238), MDMV leader used, each of which is incorporated by reference. The above (Maize Dwarf Mosaic Virus), human immunoglobulin heavy list of transcriptional enhancers is not meant to be limiting. chain binding protein (BiP) (Macejak, et al., (1991) Nature Any appropriate transcriptional enhancer can be used in the 353:90-94); untranslated leader from the coat protein mRNA embodiments. of alfalfa mosaic virus (AMV RNA 4) (Jobling, et al., (1987) 0292. The termination region may be native with the tran Nature 325:622-625); tobacco mosaic virus leader (TMV) Scriptional initiation region, may be native with the operably (Gallie, et al., (1989) in Molecular Biology of RNA, ed. Cech linked DNA sequence of interest, may be native with the plant (Liss, New York), pp. 237-256) and maize chlorotic mottle host, or may be derived from another source (i.e., foreign or virus leader (MCMV) (Lommel, et al., (1991) Virology heterologous to the promoter, the sequence of interest, the 81:382-385). See also, Della-Cioppa, et al., (1987) Plant plant host or any combination thereof). Physiol. 84:965-968. Such constructs may also contain a 0293 Convenient termination regions are available from “signal sequence' or "leader sequence' to facilitate co-trans the Ti-plasmid of A. tumefaciens, such as the octopine Syn lational or post-translational transport of the peptide to cer thase and nopaline synthase termination regions. See also, tain intracellular structures such as the chloroplast (or other Guerineau, et al., (1991) Mol. Gen. Genet. 262:141-144: plastid), endoplasmic reticulum, or Golgi apparatus. Proudfoot, (1991) Cell 64:671-674; Sanfacon, et al., (1991) 0297. By “signal sequence' is intended a sequence that is Genes Dev. 5:141-149; Mogen, et al., (1990) Plant Cell known or Suspected to result in cotranslational or post-trans 2:1261-1272; Munroe, et al., (1990) Gene 91:151-158; Bal lational peptide transport across the cell membrane. In las, et al., (1989) Nucleic Acids Res. 17:7891-7903 and Joshi, eukaryotes, this typically involves Secretion into the Golgi et al., (1987) Nucleic Acid Res. 15:9627-9639. apparatus, with some resulting glycosylation. Insecticidal 0294. Where appropriate, a nucleic acid may be optimized toxins of bacteria are often synthesized as protoxins, which for increased expression in the host organism. Thus, where are protolytically activated in the gut of the target pest the host organism is a plant, the synthetic nucleic acids can be (Chang, (1987) Methods Enzymol. 153:507-516). In some synthesized using plant-preferred codons for improved embodiments, the signal sequence is located in the native expression. See, for example, Campbell and Gowri, (1990) sequence, or may be derived from a sequence of the embodi Plant Physiol. 92: 1-11 for a discussion of host-preferred ments. By “leader sequence' is intended any sequence that codon usage. For example, although nucleic acid sequences when translated, results in an amino acid sequence Sufficient of the embodiments may be expressed in both monocotyle to trigger co-translational transport of the peptide chain to a donous and dicotyledonous plant species, sequences can be Subcellular organelle. Thus, this includes leader sequences modified to account for the specific codon preferences and targeting transport and/or glycosylation by passage into the GC content preferences of monocotyledons or dicotyledons endoplasmic reticulum, passage to vacuoles, plastids includ as these preferences have been shown to differ (Murray et al. ing chloroplasts, mitochondria and the like. Nuclear-encoded (1989) Nucleic Acids Res. 17:477-498). Thus, the maize proteins targeted to the chloroplast thylakoid lumen compart preferred codon for a particular amino acid may be derived ment have a characteristic bipartite transit peptide, composed from known gene sequences from maize. Maize codon usage of a stromal targeting signal peptide and a lumen targeting for 28 genes from maize plants is listed in Table 4 of Murray, signal peptide. The stromal targeting information is in the et al., Supra. Methods are available in the art for synthesizing amino-proximal portion of the transit peptide. The lumen plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380, targeting signal peptide is in the carboxyl-proximal portion of 831, and 5,436,391 and Murray, et al., (1989) Nucleic Acids the transit peptide, and contains all the information for tar Res. 17:477-498, herein incorporated by reference. geting to the lumen. Recent research in proteomics of the US 2014/0274885 A1 Sep. 18, 2014

higher plant chloroplast has achieved in the identification of those discussed in U.S. Pat. Nos. 5,608,149; 5,608,144: numerous nuclear-encoded lumen proteins (Kieselbach et al. 5,604,121 : 5,569,597; 5,466,785; 5,399,680: 5,268,463: FEBS LETT 480:271-276, 2000; Peltier et al. Plant Cell 5,608,142 and 6,177,611. 12:319-341, 2000; Brickeretal. Biochim. Biophy's Acta 1503: 0301 Depending on the desired outcome, it may be ben 350-356, 2001), the lumen targeting signal peptide of which eficial to express the gene from an inducible promoter. Of can potentially be used in accordance with the present inven particular interest for regulating the expression of the nucle tion. About 80 proteins from Arabidopsis, as well as homolo otide sequences of the embodiments in plants are wound gous proteins from spinach and garden pea, are reported by inducible promoters. Such wound-inducible promoters, may Kieselbach et al., Photosynthesis Research, 78:249-264, respond to damage caused by insect feeding, and include 2003. In particular, table 2 of this publication, which is incor potato proteinase inhibitor (pin II) gene (Ryan, (1990) Ann. porated into the description herewith by reference, discloses Rev. Phytopath. 28:425-449: Duan, et al., (1996) Nature Bio 85 proteins from the chloroplast lumen, identified by their technology 14:494-498); wun1 and wun2, U.S. Pat. No. accession number (see also US Patent Application Publica 5,428,148; win1 and win2 (Stanford, et al., (1989) Mol. Gen. tion 2009/09044298). In addition, the recently published Genet. 215:200-208); system in (McGurl, et al., (1992) Sci draft version of the rice genome (Goffetal, Science 296:92 ence 225:1570-1573); WIP1 (Rohmeier, et al., (1993) Plant 100, 2002) is a suitable source for lumen targeting signal Mol. Biol. 22:783-792: Eckelkamp, et al., (1993) FEBS Let peptide which may be used in accordance with the present ters 323:73-76); MPI gene (Corderok, et al., (1994) Plant J. invention. Suitable chloroplast transit peptides (CTP) are 6(2): 141-150) and the like, herein incorporated by reference. well known to one skilled in the art also include chimeric CTPs comprising but not limited to, an N-terminal domain, a 0302 Additionally, pathogen-inducible promoters may be central domain or a C-terminal domain from a CTP from employed in the methods and nucleotide constructs of the Oryza sativa 1-deoxy-D xylulose-5-Phosphate Synthase, embodiments. Such pathogen-inducible promoters include Oryza sativa-Superoxide dismutase, Oryza sativa-soluble those from pathogenesis-related proteins (PR proteins), starch synthase, Oryza sativa-NADP-dependent Malic acid which are induced following infection by a pathogen; e.g., PR enzyme, Oryza sativa-Phospho-2-dehydro-3-deoxyhepto proteins, SAR proteins, beta-1,3-glucanase, chitinase, etc. nate Aldolase 2, Oryza sativa-L-AScorbate peroxidase 5. See, for example, Redolfi, et al., (1983) Neth, J. Plant Pathol. Oryza sativa-Phosphoglucan water dikinase, Zea May's 89:245-254; Uknes, et al., (1992) Plant Cell 4:645-656 and ssRUBISCO, Zea Mays-beta-glucosidase, Zea Mays-Malate Van Loon, (1985) Plant Mol. Virol. 4:111-116. See also, WO dehydrogenase, Zea May's Thioredoxin M-type US Patent 1999/43819, herein incorporated by reference. Application Publication 2012/0304336). 0303 Of interest are promoters that are expressed locally 0298. The PHI-4 polypeptide gene to be targeted to the at or near the site of pathogen infection. See, for example, chloroplast may be optimized for expression in the chloro Marineau, et al., (1987) Plant Mol. Biol. 9:335-342; Matton, plast to account for differences in codon usage between the et al., (1989) Molecular Plant-Microbe Interactions 2:325 plant nucleus and this organelle. In this manner, the nucleic 331; Somsisch, et al., (1986) Proc. Natl. Acad. Sci. USA acids of interest may be synthesized using chloroplast-pre 83:2427-2430; Somsisch, et al., (1988) Mol. Gen. Genet. ferred codons. See, for example, U.S. Pat. No. 5,380,831, 2:93-98 and Yang, (1996) Proc. Natl. Acad. Sci. USA herein incorporated by reference. 93:14972-14977. See also, Chen, et al., (1996) Plant J. 10:955-966: Zhang, et al., (1994) Proc. Natl. Acad. Sci. USA 0299. In preparing the expression cassette, the various 91:2507-2511; Warner, et al., (1993) Plant J. 3:191-201: Sie DNA fragments may be manipulated so as to provide for the bertz, et al., (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750, DNA sequences in the proper orientation and, as appropriate, 386 (nematode-inducible) and the references cited therein. Of in the proper reading frame. Toward this end, adapters or particular interest is the inducible promoter for the maize linkers may be employed to join the DNA fragments or other PRms gene, whose expression is induced by the pathogen manipulations may be involved to provide for convenient Fusarium moniliforme (see, for example, Cordero, et al., restriction sites, removal of superfluous DNA, removal of (1992) Physiol. Mol. Plant. Path. 41.189-200). restriction sites, or the like. For this purpose, in vitro 0304 Chemical-regulated promoters can be used to mutagenesis, primer repair, restriction, annealing, resubstitu modulate the expression of a gene in a plant through the tions, e.g., transitions and transversions, may be involved. application of an exogenous chemical regulator. Depending 0300. A number of promoters can be used in the practice of upon the objective, the promoter may be a chemical-inducible the embodiments. The promoters can be selected based on the promoter, where application of the chemical induces gene desired outcome. The nucleic acids can be combined with expression, or a chemical-repressible promoter, where appli constitutive, tissue-preferred, inducible, or other promoters cation of the chemical represses gene expression. Chemical for expression in the host organism. Suitable constitutive inducible promoters are known in the art and include, but are promoters for use in a plant host cell include, for example, the not limited to, the maize In2-2 promoter, which is activated by core promoter of the Rsyn? promoter and other constitutive benzenesulfonamide herbicide safeners, the maize GST pro promoters disclosed in WO 1999/43838 and U.S. Pat. No. moter, which is activated by hydrophobic electrophilic com 6,072,050; the core CaMV35S promoter (Odell, et al., (1985) pounds that are used as pre-emergent herbicides, and the Nature 313:810-812); rice actin (McElroy, et al., (1990) Plant tobacco PR-1a promoter, which is activated by salicylic acid. Cell 2:163-171); ubiquitin (Christensen, et al., (1989) Plant Other chemical-regulated promoters of interest include ste Mol. Biol. 12:619-632 and Christensen, et al., (1992) Plant roid-responsive promoters (see, for example, the glucocorti Mol. Biol. 18:675-689); pEMU (Last, et al., (1991) Theor: coid-inducible promoter in Schena, et al., (1991) Proc. Natl. Appl. Genet. 81:581–588); MAS (Velten, et al., (1984) EMBO Acad. Sci. USA 88:10421-10425 and McNellis, et al., (1998) J.3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026) and Plant J. 14(2):247-257) and tetracycline-inducible and tetra the like. Other constitutive promoters include, for example, cycline-repressible promoters (see, for example, Gatz, et al., US 2014/0274885 A1 Sep. 18, 2014 42

(1991) Mol. Gen. Genet. 227:229-237 and U.S. Pat. Nos. 681-691. See also, U.S. Pat. Nos. 5,837,876; 5,750,386; 5,814,618 and 5,789,156), herein incorporated by reference. 5,633,363; 5,459,252: 5,401,836; 5,110,732 and 5,023,179. (0308 “Seed-preferred” promoters include both “seed 0305 Tissue-preferred promoters can be utilized to target specific' promoters (those promoters active during seed enhanced PHI-4 polypeptide expression within a particular development such as promoters of seed storage proteins) as plant tissue. Tissue-preferred promoters include those dis well as “seed-germinating promoters (those promoters cussed in Yamamoto, et al., (1997) Plant J. 12(2)255-265; active during seed germination). See, Thompson, et al., Kawamata, et al., (1997) Plant Cell Physiol. 38(7):792-803; (1989) BioEssays 10:108, herein incorporated by reference. Hansen, et al., (1997) Mol. Gen Genet. 254(3):337-343; Rus Such seed-preferred promoters include, but are not limited to, sell, et al., (1997) Transgenic Res. 6(2): 157-168; Rinehart, et Cim1 (cytokinin-induced message); cz 19B 1 (maize 19 kDa al., (1996) Plant Physiol. 112(3):1331-1341; Van Camp, et Zein); and milps (myo-inositol-1-phosphate synthase) (see, al., (1996) Plant Physiol. 112(2):525-535; Canevascini, et al., U.S. Pat. No. 6.225,529, herein incorporated by reference). (1996) Plant Physiol. 112(2):513-524; Yamamoto, et al., Gamma-Zein and Glb-1 are endosperm-specific promoters. (1994) Plant Cell Physiol. 35(5):773-778; Lam, (1994) For dicots, seed-specific promoters include, but are not lim Results Probl. Cell Differ. 20:181-196; Orozco, et al., (1993) ited to, Kunitz trypsin inhibitor 3 (KTi3) (Jofuku, K. D. and Plant Mol. Biol. 23(6):1129-1138; Matsuoka, et al., (1993) Goldberg, R. B. Plant Cell 1:1079-1093, 1989), bean Proc Natl. Acad. Sci. USA 90(20):9586-9590 and Guevara B-phaseolin, napin, B-conglycinin, glycinin 1, soybean lectin, Garcia, et al., (1993) Plant J. 4(3):495-505. Such promoters cruciferin, and the like. For monocots, seed-specific promot can be modified, if necessary, for weak expression. ers include, but are not limited to, maize 15 kDa Zein, 22 kDa 0306 Leaf-preferred promoters are known in the art. See, Zein, 27 kDa Zein, g-Zein, waxy, shrunken 1, shrunken 2, for example, Yamamoto, et al., (1997) Plant J. 12(2):255-265; globulin 1, etc. See also, WO 2000/12733, where seed-pre Kwon, et al., (1994) Plant Physiol. 105:357-67: Yamamoto, ferred promoters from endl and end2 genes are disclosed; et al., (1994) Plant Cell Physiol. 35(5):773-778; Gotor, et al., herein incorporated by reference. In dicots, seed specific pro (1993) Plant J. 3:509-18; Orozco, et al., (1993) Plant Mol. moters include but are not limited to seed coat promoter from Biol. 23(6):1129-1138 and Matsuoka, et al., (1993) Proc. Arabidopsis, pFBAN; and the early seed promoters from Ara Natl. Acad. Sci. USA 90(20):9586-9590. bidopsis, p26, p63, and p63tr (U.S. Pat. Nos. 7.294,760 and 0307 Root-preferred or root-specific promoters are 7,847,153). A promoter that has “preferred expression in a known and can be selected from the many available from the particular tissue is expressed in that tissue to a greater degree literature or isolated de novo from various compatible spe than in at least one other plant tissue. Some tissue-preferred cies. See, for example, Hire, et al., (1992) Plant Mol. Biol. promoters show expression almost exclusively in the particu 2002): 207-218 (soybean root-specific glutamine synthetase lar tissue. gene); Keller and Baumgartner, (1991) Plant Cell 3 (10): 0309 Where low level expression is desired, weak pro 1051-1061 (root-specific control element in the GRP 1.8 gene moters will be used. Generally, the term “weak promoter as of French bean); Sanger, et al., (1990) Plant Mol. Biol. 14(3): used herein refers to a promoter that drives expression of a 433-443 (root-specific promoter of the mannopine synthase coding sequence at a low level. By low level expression at (MAS) gene of Agrobacterium tumefaciens) and Miao, et al., levels of about /1000 transcripts to about/100,000 transcripts to (1991) Plant Cell3(1):11-22 (full-length cDNA clone encod about /500,000 transcripts is intended. Alternatively, it is rec ing cytosolic glutamine synthetase (GS), which is expressed ognized that the term “weak promoters' also encompasses in roots and root nodules of soybean). See also, Bogusz, et al., promoters that drive expression in only a few cells and not in (1990) Plant Ce112(7):633-641, where two root-specific pro others to give a total low level of expression. Where a pro moters isolated from hemoglobin genes from the nitrogen moter drives expression at unacceptably high levels, portions fixing nonlegume Parasponia andersonii and the related non of the promoter sequence can be deleted or modified to nitrogen-fixing nonlegume Trema tomentosa are described. decrease expression levels. The promoters of these genes were linked to a B-glucu 0310. Such weak constitutive promoters include, for ronidase reporter gene and introduced into both the nonle example the core promoter of the Rsyn7 promoter (WO 1999/ gume Nicotiana tabacum and the legume Lotus corniculatus, 43838 and U.S. Pat. No. 6,072,050), the core 35S CaMV and in both instances root-specific promoteractivity was pre promoter, and the like. Other constitutive promoters include, served. Leach and Aoyagi, (1991) describe their analysis of for example, those disclosed in U.S. Pat. Nos. 5,608, 149: the promoters of the highly expressed rolC and roll D root 5,608,144; 5,604,121 : 5,569,597; 5,466,785; 5,399,680; inducing genes of Agrobacterium rhizogenes (see, Plant Sci 5,268,463; 5,608,142 and 6,177,611, herein incorporated by ence (Limerick) 79(1):69-76). They concluded that enhancer reference. and tissue-preferred DNA determinants are dissociated in 0311. The above list of promoters is not meant to be lim those promoters. Teen, et al., (1989) used gene fusion to lacz iting. Any appropriate promoter can be used in the embodi to show that the Agrobacterium T-DNA gene encoding mentS. octopine synthase is especially active in the epidermis of the 0312 Generally, the expression cassette will comprise a root tip and that the TR2 gene is root specific in the intact selectable marker gene for the selection of transformed cells. plant and stimulated by wounding in leaf tissue, an especially Selectable marker genes are utilized for the selection of trans desirable combination of characteristics foruse with an insec formed cells or tissues. Marker genes include genes encoding ticidal or larvicidal gene (see, EMBO.J. 8(2):343-350). The antibiotic resistance, such as those encoding neomycin phos TR1 gene fused to mptII (neomycin phosphotransferase II) photransferase II (NEO) and hygromycin phosphotransferase showed similar characteristics. Additional root-preferred (HPT), as well as genes conferring resistance to herbicidal promoters include the VfBNOD-GRP3 gene promoter compounds, such as glufosinate ammonium, bromoxynil. (Kuster, et al., (1995) Plant Mol. Biol. 29(4):759-772) and imidazolinones and 2,4-dichlorophenoxyacetate (2,4-D). rolB promoter (Capana, et al., (1994) Plant Mol. Biol. 25(4): Additional examples of Suitable selectable marker genes US 2014/0274885 A1 Sep. 18, 2014

include, but are not limited to, genes encoding resistance to plants, plant organs (e.g., leaves, Sterns, roots, etc.), seeds, chloramphenicol (Herrera Estrella, et al., (1983) EMBO.J. plant cells, propagules, embryos and progeny of the same. 2:987-992); methotrexate (Herrera Estrella, et al., (1983) Plant cells can be differentiated or undifferentiated (e.g. cal Nature 303:209-213 and Meijer, et al., (1991) Plant Mol. lus, Suspension culture cells, protoplasts, leaf cells, root cells, Biol. 16:807-820); streptomycin (Jones, et al., (1987) Mol. phloem cells, and pollen). Gen. Genet. 210:86-91); spectinomycin (Bretagne-Sagnard, 0316 Transformation protocols as well as protocols for et al., (1996) Transgenic Res. 5:131-137); bleomycin (Hille, introducing nucleotide sequences into plants may vary et al., (1990) Plant Mol. Biol. 7:171-176); sulfonamide depending on the type of plant or plant cell, i.e., monocot or (Guerineau, et al., (1990) Plant Mol. Biol. 15:127-136); bro dicot, targeted for transformation. Suitable methods of intro moxynil (Stalker, et al., (1988) Science 242:419–423); gly ducing nucleotide sequences into plant cells and Subsequent phosate (Shaw, et al., (1986) Science 233:478-481 and U.S. insertion into the plant genome include microinjection patent application Ser. Nos. 10/004.357 and 10/427,692); (Crossway, et al., (1986) Biotechniques 4:320-334), elec phosphinothricin (DeBlock, et al., (1987) EMBO.J. 6:2513 troporation (Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA 2518). See generally, Yarranton, (1992) Curr. Opin. Biotech. 83:5602-5606), Agrobacterium-mediated transformation 3:506-511; Christopherson, et al., (1992) Proc. Natl. Acad. (U.S. Pat. Nos. 5,563,055 and 5,981,840), direct genetransfer Sci. USA 89:6314-6318; Yao, et al., (1992) Cell 71:63-72; (Paszkowski, et al., (1984) EMBO.J. 3:2717-2722) and bal Reznikoff, (1992) Mol. Microbiol. 6:2419-2422: Barkley, et listic particle acceleration (see, for example, U.S. Pat. Nos. al., (1980) in The Operon, pp. 177-220; Hu, et al., (1987) Cell 4,945,050; 5,879,918; 5,886,244 and 5,932,782; Tomes, et 48:555-566; Brown, et al., (1987) Cell 49:603-612: Figge, et al., (1995) in Plant Cell, Tissue, and Organ Culture. Funda al., (1988) Cell 52:713-722: Deuschle, et al., (1989) Proc. mental Methods, ed. Gamborg and Phillips, (Springer-Verlag, Natl. Acad. Sci. USA 86:5400-5404: Fuerst, et al., (1989) Berlin) and McCabe, et al., (1988) Biotechnology 6:923-926) Proc. Natl. Acad. Sci. USA 86:2549-2553: Deuschle, et al., and Lecl transformation (WO 2000/28058). For potato trans (1990) Science 248:480–483; Gossen, (1993) Ph.D. Thesis, formation see, Tu, et al., (1998) Plant Molecular Biology University of Heidelberg; Reines, et al., (1993) Proc. Natl. 37:829-838 and Chong, et al., (2000) Transgenic Research Acad. Sci. USA 90:1917-1921; Labow, et al., (1990) Mol. 9:71-78. Additional transformation procedures can be found Cell. Biol. 10:3343-3356: Zambretti, et al., (1992) Proc. Natl. in Weissinger, et al., (1988) Ann. Rev. Genet. 22:421-477; Acad. Sci. USA 89:3952-3956: Bairn, et al., (1991) Proc. Sanford, et al., (1987) Particulate Science and Technology Natl. Acad. Sci. USA 88:5072-5076; Wyborski, et al., (1991) 5:27-37 (onion); Christou, et al., (1988) Plant Physiol. Nucleic Acids Res. 19:4647-4653; Hillenand-Wissman, 87:671-674 (soybean); McCabe, et al., (1988) Bio/Technol (1989) Topics Mol. Struc. Biol. 10:143-162: Degenkolb, et ogy 6:923-926 (soybean): Finer and McMullen, (1991) In al., (1991) Antimicrob. Agents Chemother: 35:1591-1595: Vitro Cell Dev. Biol. 27P:175-182 (soybean): Singh, et al., Kleinschnidt, et al., (1988) Biochemistry 27:1094-1104; (1998) Theor: Appl. Genet. 96.319-324 (soybean); Datta, et Bonin, (1993) Ph.D. Thesis, University of Heidelberg: Gos al., (1990) Biotechnology 8:736-740 (rice); Klein, et al., sen, et al., (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize): Oliva, et al., (1992) Antimicrob. Agents Chemother: 36:913 Klein, et al., (1988) Biotechnology 6:559-563 (maize): U.S. 919; Hlavka, et al., (1985) Handbook of Experimental Phar Pat. Nos. 5,240,855; 5,322,783 and 5,324,646; Klein, et al., macology, Vol. 78 (Springer-Verlag, Berlin) and Gill, et al., (1988) Plant Physiol. 91:440-444 (maize): Fromm, et al., (1988) Nature 334:721-724. Such disclosures are herein (1990) Biotechnology 8:833-839 (maize): Hooykaas-Van incorporated by reference. Slogteren, et al., (1984) Nature (London) 311:763-764; U.S. 0313 The above list of selectable marker genes is not Pat. No. 5,736,369 (cereals); Bytebier, et al., (1987) Proc. meant to be limiting. Any selectable marker gene can be used Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet, et in the embodiments. al., (1985) in The Experimental Manipulation of Ovule Tis sues, ed. Chapman, et al., (Longman, N.Y.), pp. 197-209 Plant Transformation (pollen); Kaeppler, et al., (1990) Plant Cell Reports 9:415 0314. The methods of the embodiments involve introduc 418 and Kaeppler, et al., (1992) Theor: Appl. Genet. 84:560 ing a polypeptide or polynucleotide into a plant. “Introduc 566 (whisker-mediated transformation); D Halluin, et al., ing is intended to mean presenting to the plant the polynucle (1992) Plant Cell 4:1495-1505 (electroporation); Li, et al., otide or polypeptide in Such a manner that the sequence gains (1993) Plant Cell Reports 12:250-255 and Christou and Ford, access to the interior of a cell of the plant. The methods of the (1995) Annals of Botany 75:407-413 (rice); Osjoda, et al., embodiments do not depend on a particular method for intro (1996) Nature Biotechnology 14:745-750 (maize via Agro ducing a polynucleotide or polypeptide into a plant, only that bacterium tumefaciens); all of which are herein incorporated the polynucleotide or polypeptides gains access to the interior by reference. of at least one cell of the plant. Methods for introducing 0317. In specific embodiments, the sequences of the polynucleotide or polypeptides into plants are known in the embodiments can be provided to a plant using a variety of art including, but not limited to, stable transformation meth transient transformation methods. Such transient transforma ods, transient transformation methods and virus-mediated tion methods include, but are not limited to, the introduction methods. of the PHI-4 polypeptide or variants and fragments thereof 0315 “Stable transformation' is intended to mean that the directly into the plant or the introduction of the PHI-4 nucleotide construct introduced into a plant integrates into the polypeptide transcript into the plant. Such methods include, genome of the plant and is capable of being inherited by the for example, microinjection or particle bombardment. See, progeny thereof. “Transient transformation' is intended to for example, Crossway, et al., (1986) Mol. Gen. Genet. 202: mean that a polynucleotide is introduced into the plant and 179-185: Nomura, et al., (1986) Plant Sci. 44:53-58; Hepler, does not integrate into the genome of the plant or a polypep et al., (1994) Proc. Natl. Acad. Sci. 91:2176-2180 and Hush, tide is introduced into a plant. By “plant' is intended whole et al., (1994) The Journal of Cell Science 107: 775-784, all of US 2014/0274885 A1 Sep. 18, 2014 44 which are herein incorporated by reference. Alternatively, the immature or mature embryos, Suspension cultures, undiffer PHI-4 polypeptide polynucleotide can be transiently trans entiated callus, protoplasts, etc.), followed by applying a formed into the plant using techniques known in the art. Such maximum threshold level of appropriate selection (depend techniques include viral vector system and the precipitation ing on the selectable marker gene) to recover the transformed of the polynucleotide in a manner that precludes Subsequent plant cells from a group of untransformed cell mass. Follow release of the DNA. Thus, transcription from the particle ing integration of heterologous foreign DNA into plant cells, bound DNA can occur, but the frequency with which it is one then applies a maximum threshold level of appropriate released to become integrated into the genome is greatly selection in the medium to kill the untransformed cells and reduced. Such methods include the use of particles coated separate and proliferate the putatively transformed cells that with polyethylimine (PEI: Sigma #P3143). Survive from this selection treatment by transferring regularly 0318 Methods are known in the art for the targeted inser to a fresh medium. By continuous passage and challenge with tion of a polynucleotide at a specific location in the plant appropriate selection, one identifies and proliferates the cells genome. In one embodiment, the insertion of the polynucle that are transformed with the plasmid vector. Molecular and otide at a desired genomic location is achieved using a site biochemical methods can then be used to confirm the pres specific recombination system. See, for example, WO 1999/ ence of the integrated heterologous gene of interest into the 25821, WO 1999/25854, WO 1999/25840, WO 1999/25855 genome of the transgenic plant. and WO 1999/25853, all of which are herein incorporated by 0321 Explants are typically transferred to a fresh supply reference. Briefly, the polynucleotide of the embodiments can of the same medium and cultured routinely. Subsequently, the be contained in transfer cassette flanked by two non-identical transformed cells are differentiated into shoots after placing recombination sites. The transfer cassette is introduced into a on regeneration medium Supplemented with a maximum plant have stably incorporated into its genome a target site threshold level of selecting agent. The shoots are then trans which is flanked by two non-identical recombination sites ferred to a selective rooting medium for recovering rooted that correspond to the sites of the transfer cassette. An appro shoot or plantlet. The transgenic plantlet then grows into a priate recombinase is provided and the transfer cassette is mature plant and produces fertile seeds (e.g., Hiei, et al., integrated at the target site. The polynucleotide of interest is (1994) The Plant Journal 6:271-282; Ishida, et al., (1996) thereby integrated at a specific chromosomal position in the Nature Biotechnology 14:745-750). Explants are typically plant genome. transferred to a fresh supply of the same medium and cultured 0319 Plant transformation vectors may be comprised of routinely. A general description of the techniques and meth one or more DNA vectors needed for achieving plant trans ods for generating transgenic plants are found in Ayres and formation. For example, it is a common practice in the art to Park, (1994) Critical Reviews in Plant Science 13:219-239 utilize plant transformation vectors that are comprised of and Bommineni and Jauhar, (1997) Maydica 42:107-120. more than one contiguous DNA segment. These vectors are Since the transformed material contains many cells; both often referred to in the art as “binary vectors'. Binary vectors transformed and non-transformed cells are present in any as well as vectors with helperplasmids are most often used for piece of subjected target callus or tissue or group of cells. The Agrobacterium-mediated transformation, where the size and ability to kill non-transformed cells and allow transformed complexity of DNA segments needed to achieve efficient cells to proliferate results in transformed plant cultures. transformation is quite large, and it is advantageous to sepa Often, the ability to remove non-transformed cells is a limi rate functions onto separate DNA molecules. Binary vectors tation to rapid recovery of transformed plant cells and Suc typically contain a plasmid vector that contains the cis-acting cessful generation of transgenic plants. sequences required for T-DNA transfer (such as left border 0322 The cells that have been transformed may be grown and right border), a selectable marker that is engineered to be into plants in accordance with conventional ways. See, for capable of expression in a plant cell, and a "gene of interest' example, McCormick, et al., (1986) Plant Cell Reports 5:81– (a gene engineered to be capable of expression in a plant cell 84. These plants may then be grown, and either pollinated for which generation of transgenic plants is desired). Also with the same transformed strain or different strains and the present on this plasmid vector are sequences required for resulting hybrid having constitutive or inducible expression bacterial replication. The cis-acting sequences are arranged in of the desired phenotypic characteristic identified. Two or a fashion to allow efficient transfer into plant cells and expres more generations may be grown to ensure that expression of sion therein. For example, the selectable marker gene and the the desired phenotypic characteristic is stably maintained and pesticidal gene are located between the left and right borders. inherited and then seeds harvested to ensure that expression Often a second plasmid vector contains the trans-acting fac of the desired phenotypic characteristic has been achieved. tors that mediate T-DNA transfer from Agrobacterium to 0323. The nucleotide sequences of the embodiments may plant cells. This plasmid often contains the virulence func be provided to the plant by contacting the plant with a virus or tions (Vir genes) that allow infection of plant cells by Agro viral nucleic acids. Generally, such methods involve incorpo bacterium, and transfer of DNA by cleavage at border rating the nucleotide construct of interest within a viral DNA sequences and Vir-mediated DNA transfer, as is understood in or RNA molecule. It is recognized that the recombinant pro the art (Hellens and Mullineaux, (2000) Trends in Plant Sci teins of the embodiments may be initially synthesized as part ence 5:446-451). Several types of Agrobacterium strains (e.g. of a viral polyprotein, which later may be processed by pro LBA4404, GV3101, EHA101, EHA105, etc.) can be used for teolysis in vivo or in vitro to produce the desired PHI-4 plant transformation. The second plasmid vector is not nec polypeptide. It is also recognized that Such a viral polypro essary for transforming the plants by other methods such as tein, comprising at least a portion of the amino acid sequence microprojection, microinjection, electroporation, polyethyl of a PHI-4 polypeptide of the embodiments, may have the ene glycol, etc. desired pesticidal activity. Such viral polyproteins and the 0320 In general, plant transformation methods involve nucleotide sequences that encode for them are encompassed transferring heterologous DNA into target plant cells (e.g., by the embodiments. Methods for providing plants with US 2014/0274885 A1 Sep. 18, 2014 nucleotide constructs and producing the encoded proteins in (Sequoia sempervirens); true firs such as silver fir (Abies the plants, which involve viral DNA or RNA molecules are amabilis) and balsam fir (Abies balsamea); and cedars such as known in the art. See, for example, U.S. Pat. Nos. 5,889, 191; Western red cedar (Thuja plicata) and Alaska yellow-cedar 5,889, 190; 5,866,785: 5,589,367 and 5,316,931, herein (Chamaecyparis nootkatensis). Plants of the embodiments incorporated by reference. include crop plants (for example, corn, alfalfa, Sunflower, 0324 Methods for transformation of chloroplasts are Brassica, Soybean, cotton, safflower, peanut, Sorghum, known in the art. See, for example, Svab, et al., (1990) Proc. wheat, millet, tobacco, etc.). Such as corn and soybean plants. Natl. Acad. Sci. USA 87:8526-8530; Svab and Maliga, (1993) 0328 Turfgrasses include, but are not limited to: annual Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga, bluegrass (Poa annua); annual ryegrass (Lolium multiflo (1993) EMBO.J. 12:601-606. The method relies on particle rum); Canada bluegrass (Poa compressa); Chewings fescue gun delivery of DNA containing a selectable marker and (Festuca rubra); colonial bentgrass (Agrostis tenuis); creep targeting of the DNA to the plastid genome through homolo ing bentgrass (Agrostis palustris); crested wheatgrass (Agro gous recombination. Additionally, plastid transformation can pyron desertorum); fairway wheatgrass (Agropyron Cris be accomplished by transactivation of a silent plastid-borne tatum); hard fescue (Festuca longifolia); Kentucky bluegrass transgene by tissue-preferred expression of a nuclear-en (Poa pratensis); orchardgrass (Dactylis glomerata); peren coded and plastid-directed RNA polymerase. Such a system nial ryegrass (Lolium perenne); red fescue (Festuca rubra); has been reported in McBride, et al., (1994) Proc. Natl. Acad. redtop (Agrostis alba); rough bluegrass (Poa trivialis); sheep Sci., USA 91:7301-73.05. fescue (Festuca ovina); Smooth bromegrass (Bromus iner 0325 The embodiments further relate to plant-propagat mis); tall fescue (Festuca arundinacea); timothy (Phleum ing material of a transformed plant of the embodiments pratense); Velvet bentgrass (Agrostis canina); weeping alka including, but not limited to, seeds, tubers, corms, bulbs, ligrass (Puccinelia distans); western wheatgrass (Agropyron leaves, and cuttings of roots and shoots. Smithii); Bermuda grass (Cynodon spp.); St. Augustine grass 0326. The embodiments may be used for transformation (Stenotaphrum secundatum); Zoysia grass (Zoysia spp.); of any plant species, including, but not limited to, monocots Bahia grass (Paspalum notatum); carpet grass (Axonopus afi and dicots. Examples of plants of interest include, but are not nis); centipede grass (Eremochloa Ophiuroides): kikuyu grass limited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B. (Pennisetum clandesinum); seashore paspalum (Paspalum rapa, B. juncea), particularly those Brassica species useful as vaginatum); bluegramma (Bouteloua gracilis); buffalo grass Sources of seed oil, alfalfa (Medicago sativa), rice (Oryza (Buchloe dactyloids); Sideoats gramma (Bouteloua curtipen sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sor dula). ghum vulgare), millet (e.g., pearl millet (Pennisetum glau 0329 Plants of interest include grain plants that provide cum), proso millet (Panicum miliaceum), foxtail millet (Se seeds of interest, oil-seed plants, and leguminous plants. taria italica), finger millet (Eleusine coracana)), Sunflower Seeds of interest include grain seeds, such as corn, wheat, (Helianthus annuus), Safflower (Carthamus tinctorius), barley, rice, Sorghum, rye, millet, etc. Oil-seed plants include wheat (Triticum aestivum), soybean (Glycine max), tobacco cotton, soybean, Safflower, Sunflower, Brassica, maize, (Nicotiana tabacum), potato (Solanum tuberosum), peanuts alfalfa, palm, coconut, flax, castor, olive etc. Leguminous (Arachis hypogaea), cotton (Gossypium barbadense, Gos plants include beans and peas. Beans include guar, locust sypium hirsutum), Sweet potato (Ipomoea batatus), cassaya bean, fenugreek, soybean, garden beans, cowpea, mungbean, (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos lima bean, fava bean, lentils, chickpea, etc. nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma Cacao), tea (Camellia sinensis), Evaluation of Plant Transformation banana (Musa spp.), avocado (Persea americana), fig (Ficus Casica), guava (Psidium guajava), mango (Mangifera 0330. Following introduction of heterologous foreign indica), olive (Olea europaea), papaya (Carica papaya), DNA into plant cells, the transformation or integration of cashew (Anacardium Occidentale), macadamia (Macadamia heterologous gene in the plant genome is confirmed by vari integrifolia), almond (Prunus amygdalus), Sugar beets (Beta ous methods such as analysis of nucleic acids, proteins and vulgaris), Sugarcane (Saccharum spp.), oats, barley, Veg metabolites associated with the integrated gene. etables, ornamentals and conifers. 0331 PCR analysis is a rapid method to screen trans 0327 Vegetables include tomatoes (Lycopersicon escu formed cells, tissue or shoots for the presence of incorporated lentum), lettuce (e.g., Lactuca sativa), green beans (Phaseo gene at the earlier stage before transplanting into the soil lus vulgaris), lima beans (Phaseolus limensis), peas (Sambrook and Russell, (2001) Molecular Cloning: A Labo (Lathyrus spp.), and members of the genus Cucumis such as ratory Manual. Cold Spring Harbor Laboratory Press, Cold cucumber (C. sativus), cantaloupe (C. cantalupensis), and Spring Harbor, N.Y.). PCR is carried out using oligonucle muskmelon (C. melo). Ornamentals include azalea (Rhodo otide primers specific to the gene of interest or Agrobacterium dendron spp.), hydrangea (Macrophylla hydrangea), hibiscus vector background, etc. (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa 0332 Plant transformation may be confirmed by Southern spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), blot analysis of genomic DNA (Sambrook and Russell, carnation (Dianthus caryophyllus), poinsettia (Euphorbia (2001) supra). In general, total DNA is extracted from the pulcherrima), and chrysanthemum. Conifers that may be transformant, digested with appropriate restriction enzymes, employed in practicing the embodiments include, for fractionated in an agarose gel and transferred to a nitrocellu example, pines such as loblolly pine (Pinus taeda), slash pine lose or nylon membrane. The membrane or “blot' is then (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole probed with, for example, radiolabeled 32P target DNA frag pine (Pinus contorta) and Monterey pine (Pinus radiata); ment to confirm the integration of introduced gene into the Douglas-fir (Pseudotsuga menziesii); Western hemlock plant genome according to standard techniques (Sambrook (Tsuga Canadensis); Sitka spruce (Picea glauca); redwood and Russell. (2001) supra). US 2014/0274885 A1 Sep. 18, 2014 46

0333. In Northern blot analysis, RNA is isolated from stacked with one or more additional input traits (e.g., herbi specific tissues of transformant, fractionated in a formalde cide resistance, fungal resistance, virus resistance or stress hyde agarose gel, and blotted onto a nylon filter according to tolerance, disease resistance, male Sterility, Stalk strength, standard procedures that are routinely used in the art (Sam and the like) or output traits (e.g., increased yield, modified brook and Russell. (2001) supra). Expression of RNA starches, improved oil profile, balanced amino acids, high encoded by the pesticidal gene is then tested by hybridizing lysine or methionine, increased digestibility, improved fiber the filter to a radioactive probe derived from a pesticidal gene, quality, drought resistance, and the like). Thus, the polynucle by methods known in the art (Sambrook and Russell. (2001) otide embodiments can be used to provide a complete agro Supra). nomic package of improved crop quality with the ability to 0334 Western blot, biochemical assays and the like may flexibly and cost effectively control any number of agronomic be carried out on the transgenic plants to confirm the presence pests. of protein encoded by the pesticidal gene by standard proce dures (Sambrook and Russell, 2001, supra) using antibodies Transgenes Useful for Stacking Include but are not Limited that bind to one or more epitopes present on the PHI-4 tO: polypeptide. 0337 1. Transgenes that Confer Resistance to Insects or Disease and that Encode: Stacking of Traits in Transgenic Plant 0338 (A) Plant disease resistance genes. Plant defenses 0335 Transgenic plants may comprise a stack of one or are often activated by specific interaction between the product more insecticidal polynucleotides disclosed herein with one of a disease resistance gene (R) in the plant and the product of or more additional polynucleotides resulting in the produc a corresponding avirulence (AVr) gene in the pathogen. A tion or Suppression of multiple polypeptide sequences. Trans plant variety can be transformed with cloned resistance gene genic plants comprising stacks of polynucleotide sequences to engineer plants that are resistant to specific pathogen can be obtained by either or both of traditional breeding strains. See, for example, Jones, et al., (1994) Science 266: methods or through genetic engineering methods. These 789 (cloning of the tomato Cf-9 gene for resistance to Cla methods include, but are not limited to, breeding individual dosporium fillvum); Martin, et al., (1993) Science 262:1432 lines each comprising a polynucleotide of interest, transform (tomato Pto gene for resistance to Pseudomonas syringae pv. ing a transgenic plant comprising a gene disclosed herein tomato encodes a protein kinase); Mindrinos, et al., (1994) with a Subsequent gene, and co-transformation of genes into Cell 78: 1089 (Arabidopsis RSP2 gene for resistance to a single plant cell. As used herein, the term "stacked' includes Pseudomonas syringae), McDowell and Woffenden, (2003) having the multiple traits present in the same plant (i.e., both Trends Biotechnol. 21 (4):178-83 and Toyoda, et al., (2002) traits are incorporated into the nuclear genome, one trait is Transgenic Res. 11(6):567-82. A plant resistant to a disease is incorporated into the nuclear genome and one trait is incor one that is more resistant to a pathogen as compared to the porated into the genome of a plastid, or both traits are incor wild type plant. porated into the genome of a plastid). In one non-limiting 0339 (B) Genes encoding a Bacillus thuringiensis pro example, 'stacked traits’ comprise a molecular stack where tein, a derivative thereof or a synthetic polypeptide modeled the sequences are physically adjacent to each other. A trait, as thereon. See, for example, Geiser, et al., (1986) Gene 48: 109, used herein, refers to the phenotype derived from a particular who disclose the cloning and nucleotide sequence of a Bt sequence or groups of sequences. Co-transformation of genes delta-endotoxin gene. Moreover, DNA molecules encoding can be carried out using single transformation vectors com delta-endotoxin genes can be purchased from AmericanType prising multiple genes or genes carried separately on multiple Culture Collection (Rockville, Md.), for example, under vectors. If the sequences are stacked by genetically trans ATCC Accession Numbers 40098, 67136, 31995 and 31998. forming the plants, the polynucleotide sequences of interest Other non-limiting examples of Bacillus thuringiensis trans can be combined at any time and in any order. The traits can genes being genetically engineered are given in the following be introduced simultaneously in a co-transformation protocol patents and patent applications and hereby are incorporated with the polynucleotides of interest provided by any combi by reference for this purpose: U.S. Pat. Nos. 5,188,960; nation of transformation cassettes. For example, if two 5,689,052; 5,880,275; 5,986,177; 6,023,013, 6,060,594, sequences will be introduced, the two sequences can be con 6,063,597, 6,077,824, 6,620,988, 6,642,030, 6,713,259, tained in separate transformation cassettes (trans) or con 6,893,826, 7,105,332; 7,179,965, 7,208.474; 7,227,056, tained on the same transformation cassette (cis). Expression 7,288,643, 7,323,556, 7,329,736, 7,449,552, 7,468,278, of the sequences can be driven by the same promoter or by 7,510,878, 7,521,235, 7,544,862, 7,605,304, 7,696,412, different promoters. In certain cases, it may be desirable to 7,629,504, 7,705,216, 7,772,465, 7,790,846, 7,858,849 and introduce a transformation cassette that will Suppress the WO 1991/14778; WO 1999/31248: WO 2001/12731; WO expression of the polynucleotide of interest. This may be 1999/24581 and WO 1997/40162. combined with any combination of other suppression cas 0340 (C) A polynucleotide encoding an insect-specific settes or overexpression cassettes to generate the desired hormone or pheromone such as an ecdysteroid and juvenile combination of traits in the plant. It is further recognized that hormone, a variant thereof, a mimetic based thereon or an polynucleotide sequences can be stacked at a desired antagonistoragonist thereof. See, for example, the disclosure genomic location using a site-specific recombination system. by Hammock, et al., (1990) Nature 344:458, of baculovirus See, for example, WO 1999/25821, WO 1999/25854, WO expression of cloned juvenile hormone esterase, an inactiva 1999/25840, WO 1999/25855 and WO 1999/25853, all of tor of juvenile hormone. which are herein incorporated by reference. 0341 (D) A polynucleotide encoding an insect-specific 0336. In some embodiments the polynucleotides encoding peptide which, upon expression, disrupts the physiology of the PHI-4 polypeptides disclosed herein, alone or stacked the affected pest. For example, see the disclosures of Regan, with one or more additional insect resistance traits can be (1994).J. Biol. Chem. 269:9 (expression cloning yields DNA US 2014/0274885 A1 Sep. 18, 2014 47 coding for insect diuretic hormone receptor); Pratt, et al., 0348 (K) A gene encoding an insect-specific antibody or (1989) Biochem. Biophys. Res. Comm. 163:1243 (an allosta an immunotoxin derived therefrom. Thus, an antibody tar tin is identified in Diploptera puntata); Chattopadhyay, et al., geted to a critical metabolic function in the insect gut would (2004) Critical Reviews in Microbiology 30(1):33-54, Zjaw inactivate an affected enzyme, killing the insect. Cf. Taylor, et iony, (2004).J Nat Prod 67(2):300-310; Carlini and Grossi al., Abstract #497, SEVENTH INTL SYMPOSIUM ON de-Sa, (2002) Toxicon 40(11):1515-1539; Ussuf, et al., MOLECULAR PLANT-MICROBE INTERACTIONS (Ed (2001) Curr Sci. 80(7):847-853 and Vasconcelos and inburgh, Scotland, 1994) (enzymatic inactivation in trans Oliveira, (2004) Toxicon 44(4):385-403. See also, U.S. Pat. genic tobacco via production of single-chain antibody frag No. 5.266,317 to Tomalski, et al., who disclose genes encod ments). ing insect-specific toxins. 0349 (L) A gene encoding a virus-specific antibody. See, 0342 (E) A polynucleotide encoding an enzyme respon for example, Tavladoraki, et al., (1993) Nature 366:469, who sible for a hyperaccumulation of a monoterpene, a sesquiter show that transgenic plants expressing recombinant antibody pene, a steroid, hydroxamic acid, a phenylpropanoid deriva genes are protected from virus attack. tive or another non-protein molecule with insecticidal 0350 (M) A polynucleotide encoding a developmental activity. arrestive protein produced in nature by a pathogen or a para 0343 (F) A polynucleotide encoding an enzyme involved site. Thus, fungal endo alpha-1,4-D-polygalacturonases in the modification, including the post-translational modifi facilitate fungal colonization and plant nutrient release by cation, of a biologically active molecule; for example, a gly solubilizing plant cell wall homo-alpha-1,4-D-galacturonase. colytic enzyme, a proteolytic enzyme, a lipolytic enzyme, a See, Lamb, et al., (1992) Bio/Technology 10:1436. The clon nuclease, a cyclase, a transaminase, an esterase, a hydrolase, ing and characterization of a gene which encodes a bean a phosphatase, a kinase, a phosphorylase, a polymerase, an endopolygalacturonase-inhibiting protein is described by elastase, a chitinase and a glucanase, whether natural or Syn Toubart, et al., (1992) Plant J. 2:367. thetic. See, PCT Application WO 1993/02197 in the name of 0351 (N) A polynucleotide encoding a developmental Scott, et al., which discloses the nucleotide sequence of a arrestive protein produced in nature by a plant. For example, callase gene. DNA molecules which contain chitinase-encod Logemann, et al., (1992) Bio/Technology 10:305, have shown ing sequences can be obtained, for example, from the ATCC that transgenic plants expressing the barley ribosome-inacti under Accession Numbers 39637 and 67152. See also, Vating gene have an increased resistance to fungal disease. Kramer, et al., (1993) Insect Biochem. Molec. Biol. 23:691, 0352 (O) Genes involved in the Systemic Acquired Resis who teach the nucleotide sequence of a cDNA encoding tance (SAR) Response and/or the pathogenesis related genes. tobacco hookworm chitinase and Kawalleck, et al., (1993) Briggs, (1995) Current Biology 5(2), Pieterse and Van Loon, Plant Molec. Biol. 21:673, who provide the nucleotide (2004) Curr. Opin. Plant Bio. 7(4):456-64 and Somssich, sequence of the parsley ubi4-2 polyubiquitin gene, and U.S. (2003) Cell 113(7):815-6. Pat. Nos. 6,563,020; 7,145,060 and 7,087,810. 0353 (P) Antifungal genes (Cornelissen and Melchers, 0344 (G) A polynucleotide encoding a molecule that (1993) Pl. Physiol. 101:709-712 and Parijs, et al., (1991) stimulates signal transduction. For example, see the disclo Planta 183:258-264 and Bushnell, et al., (1998) Can. J. of sure by Botella, et al., (1994) Plant Molec. Biol. 24:757, of Plant Path. 2002): 137-149. Also see, U.S. patent application nucleotide sequences for mung bean calmodulin cDNA Ser. Nos. 09/950,933; 11/619,645; 11/657,710; 1 1/748,994; clones, and Griess, et al., (1994) Plant Physiol. 104:1467, 11/774,121 and U.S. Pat. Nos. 6,891,085 and 7,306,946. who provide the nucleotide sequence of a maize calmodulin LysM Receptor-like kinases for the perception of chitin frag cDNA clone. ments as a first step in plant defense response against fungal 0345 (H) A polynucleotide encoding a hydrophobic pathogens (US 2012/0110696). moment peptide. See, PCT Application WO 1995/16776 and U.S. Pat. No. 5,580,852 disclosure of peptide derivatives of 0354 (Q) Detoxification genes, such as for fumonisin, Tachyplesin which inhibit fungal plant pathogens) and PCT beauvericin, moniliformin and Zearalenone and their struc Application WO 1995/18855 and U.S. Pat. No. 5,607.914 turally related derivatives. For example, see, U.S. Pat. Nos. (teaches synthetic antimicrobial peptides that confer disease 5,716,820; 5,792,931; 5,798.255; 5,846,812: 6,083,736; resistance). 6,538,177; 6,388,171 and 6,812,380. 0346 (I) A polynucleotide encoding a membrane per 0355 (R) A polynucleotide encoding a Cystatin and cys mease, a channel former or a channel blocker. For example, teine proteinase inhibitors. See, U.S. Pat. No. 7,205.453. see the disclosure by Jaynes, et al., (1993) Plant Sci. 89:43, of 0356 (S) Defensingenes. See, WO 2003/0008.63 and U.S. heterologous expression of a cecropin-beta lytic peptide ana Pat. Nos. 6,911,577; 6,855,865; 6,777,592 and 7,238,781. log to render transgenic tobacco plants resistant to 0357 (T) Genes conferring resistance to nematodes. See, Pseudomonas Solanacearum. e.g., PCT Application WO 1996/30517: PCT Application 0347 (J) A gene encoding a viral-invasive protein or a WO 1993/19181, WO 2003/033651 and Urwin, et al., (1998) complex toxin derived therefrom. For example, the accumu Planta 204:472-479, Williamson, (1999) Curr Opin Plant lation of viral coat proteins in transformed plant cells imparts Bio.2(4):327-31; U.S. Pat. Nos. 6.284.948 and 7.301,069 and resistance to viral infection and/or disease development miR164 genes (WO 2012/058266). effected by the virus from which the coat protein gene is 0358 (U) Genes that confer resistance to Phytophthora derived, as well as by related viruses. See, Beachy, et al., Root Rot, such as the Rps 1. Rps 1-a, Rps 1-b, Rps 1-c. Rps (1990) Ann. Rev. Phytopathol. 28:451. Coat protein-mediated 1-d, Rps 1-e, Rps 1-k, Rps 2, Rps 3-a, Rps 3-b, Rps 3-c. Rps resistance has been conferred upon transformed plants 4. Rps 5, Rps 6. Rps 7 and other Rps genes. See, for example, against alfalfa mosaic virus, cucumber mosaic virus, tobacco Shoemaker, et al., Phytophthora Root Rot Resistance Gene streak virus, potato virus X, potato virus Y, tobacco etch virus, Mapping in Soybean, Plant Genome IV Conference, San tobacco rattle virus and tobacco mosaic virus. Id. Diego, Calif. (1995). US 2014/0274885 A1 Sep. 18, 2014 48

0359 (V) Genes that conferresistance to Brown Stem Rot, rated herein by reference for this purpose. Exemplary genes such as described in U.S. Pat. No. 5.689,035 and incorporated conferring resistance to phenoxy proprionic acids and cyclo by reference for this purpose. hexones, such as Sethoxydimand haloxyfop, are the Acc1-S1, 0360 (W) Genes that confer resistance to Colletotrichum, Acc1-52 and Acc1-53 genes described by Marshall, et al., such as described in US Patent Application Publication US (1992) Theor: Appl. Genet. 83:435. 2009/0035765 and incorporated by reference for this pur 0363 (C) A polynucleotide encoding a protein for resis pose. This includes the Rcg locus that may be utilized as a tance to herbicide that inhibits photosynthesis, such as a single locus conversion. triazine (psbA and gS+genes) and a benzonitrile (nitrilase 2. Transgenes that Confer Resistance to a Herbicide, for gene). Przibilla, et al., (1991) Plant Cell 3:169, describe the Example: transformation of Chlamydomonas with plasmids encoding 0361 (A) A polynucleotide encoding resistance to a her mutant psbA genes. Nucleotide sequences for nitrilase genes bicide that inhibits the growing point or meristem, Such as an are disclosed in U.S. Pat. No. 4,810,648 to Stalker and DNA imidazolinone or a Sulfonylurea. Exemplary genes in this molecules containing these genes are available under ATCC category code for mutant ALS and AHAS enzyme as Accession Numbers 53435, 67441 and 67442. Cloning and described, for example, by Lee, et al., (1988) EMBO.J. 7:1241 expression of DNA coding for a glutathione S-transferase is and Miki, et al., (1990) Theor: Appl. Genet. 80:449, respec described by Hayes, et al., (1992) Biochem. J. 285:173. tively. See also, U.S. Pat. Nos. 5,605,011; 5,013,659; 5,141, 0364 (D) A polynucleotide encoding a protein for resis 870; 5,767,361; 5,731, 180; 5,304.732; 4,761,373; 5,331,107; tance to Acetohydroxy acid synthase, which has been found 5,928,937 and 5,378,824; U.S. patent application Ser. No. to make plants that express this enzyme resistant to multiple 1 1/683,737 and International Publication WO 1996/33270. types of herbicides, has been introduced into a variety of 0362 (B) A polynucleotide encoding a protein for resis plants (see, e.g., Hattori, et al., (1995) Mol Gen Genet. 246: tance to Glyphosate (resistance imparted by mutant 419). Other genes that confer resistance to herbicides include: 5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA a gene encoding a chimeric protein of rat cytochrome genes, respectively) and other phosphono compounds such as P4507A1 and yeast NADPH-cytochrome P450 oxidoreduc glufosinate (phosphinothricin acetyl transferase (PAT) and tase (Shiota, et al., (1994) Plant Physiol 106:17), genes for Streptomyces hygroscopicus phosphinothricin acetyl trans glutathione reductase and Superoxide dismutase (Aono, et al., ferase (bar) genes), and pyridinoxy or phenoxy proprionic (1995) Plant Cell Physiol 36:1687) and genes for various acids and cyclohexones (ACCase inhibitor-encoding genes). phosphotransferases (Datta, et al., (1992) Plant Mol Biol See, for example, U.S. Pat. No. 4,940,835 to Shah, et al., 20:619). which discloses the nucleotide sequence of a form of EPSPS 0365 (E) A polynucleotide encoding resistance to a her which can confer glyphosate resistance. U.S. Pat. No. 5,627, bicide targeting Protoporphyrinogen oxidase (protox) which 061 to Barry, et al., also describes genes encoding EPSPS is necessary for the production of chlorophyll. The protox enzymes. See also, U.S. Pat. Nos. 6,566,587; 6,338,961: enzyme serves as the target for a variety of herbicidal com 6,248,876 B1; 6,040,497; 5,804,425; 5,633,435; 5,145,783: pounds. These herbicides also inhibit growth of all the differ 4,971,908: 5,312,910; 5,188,642; 5,094,945, 4,940,835; ent species of plants present, causing their total destruction. 5,866,775; 6,225,114 B1; 6,130,366; 5,310,667; 4,535,060; The development of plants containing altered protox activity 4,769,061; 5,633,448; 5,510,471; Re. 36,449; RE 37,287 E which are resistant to these herbicides are described in U.S. and U.S. Pat. No. 5,491,288 and International Publications Pat. Nos. 6,288,306 B1; 6,282,837 B1 and 5,767,373 and EP 1173580; WO 2001/66704; EP 1173581 and EP 1173582, International Publication WO 2001/12825. which are incorporated herein by reference for this purpose. 0366 (F) The aad-1 gene (originally from Sphingobium Glyphosate resistance is also imparted to plants that express a herbicidovorans) encodes the aryloxyalkanoate dioxygenase gene encoding a glyphosate oxido-reductase enzyme as (AAD-1) protein. The trait confers tolerance to 2,4-dichlo described more fully in U.S. Pat. Nos. 5,776,760 and 5,463, rophenoxyacetic acid and aryloxyphenoxypropionate (com 175, which are incorporated herein by reference for this pur monly referred to as “fop herbicides such as quizalofop) pose. In addition glyphosate resistance can be imparted to herbicides. The aad-1 gene, itself, for herbicide tolerance in plants by the over expression of genes encoding glyphosate plants was first disclosed in WO 2005/107437 (see also, US N-acetyltransferase. See, for example, U.S. Pat. Nos. 7.462, 2009/0093366). The aad-12 gene, derived from Delftia aci 481; 7,405,074 and US Patent Application Publication Num dovorans, which encodes the aryloxyalkanoate dioxygenase ber US 2008/0234130. A DNA molecule encoding a mutant (AAD-12) protein that confers tolerance to 2,4-dichlorophe aroA gene can be obtained under ATCC Accession Number noxyacetic acid and pyridyloxyacetate herbicides by deacti 39256, and the nucleotide sequence of the mutant gene is Vating several herbicides with an aryloxyalkanoate moiety, disclosed in U.S. Pat. No. 4,769,061 to Comai. EP Applica including phenoxy auxin (e.g., 2,4-D, MCPA), as well as tion Number 0 333 033 to Kumada, et al., and U.S. Pat. No. pyridyloxy auxins (e.g., fluoroxypyr, triclopyr). 4.975,374 to Goodman, et al., disclose nucleotide sequences of glutamine synthetase genes which confer resistance to 0367 (G) A polynucleotide encoding a herbicide resistant herbicides such as L-phosphinothricin. The nucleotide dicamba monooxygenase disclosed in US Patent Application sequence of a phosphinothricin-acetyl-transferase gene is Publication 2003/0135879 for imparting dicamba tolerance; provided in EP Application Numbers 0242 246 and 0242 236 0368 (H) A polynucleotide molecule encoding bromoxy to Leemans, et al.; De Greef, et al., (1989) Bio/Technology nil nitrilase (Bxn) disclosed in U.S. Pat. No. 4,810,648 for 7:61, describe the production of transgenic plants that express imparting bromoxynil tolerance; chimeric bar genes coding for phosphinothricin acetyl trans 0369 (I) A polynucleotide molecule encoding phytoene ferase activity. See also, U.S. Pat. Nos. 5,969,213; 5.489.520; (crt1) described in Misawa, et al., (1993) Plant J. 4:833-840 5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477; and in Misawa, et al., (1994) Plant J. 6:481-489 for norflu 5,646,024; 6,177,616 B1 and 5,879,903, which are incorpo razon tolerance. US 2014/0274885 A1 Sep. 18, 2014 49

3. Transgenes that Confer or Contribute to an Altered Grain Number 2003/0009011, WO 2003/027243, US Patent Appli Characteristic cation Publication Number 2003/0079247, WO 1999/05298, U.S. Pat. No. 6,197,561, U.S. Pat. No. 6,291,224, U.S. Pat. Such as: No. 6,391,348, WO 2002/059324, US Patent Application 0370 (A) Altered fatty acids, for example, by Publication Number 2003/007.9247, WO 1998/45448, WO 0371 (1) Down-regulation of stearoyl-ACP to increase 1999/55882, WO 2001/04147. stearic acid content of the plant. See, KnultZon, et al., (1992) 0383 (C) Altered carbohydrates affected, for example, by Proc. Natl. Acad. Sci. USA 89:2624 and WO 1999/64579 altering a gene for an enzyme that affects the branching (Genes to Alter Lipid Profiles in Corn). pattern of starch or, a gene altering thioredoxin Such as NTR 0372 (2) Elevating oleic acid via FAD-2 gene modifica and/or TRX (see, U.S. Pat. No. 6,531,648. which is incorpo tion and/or decreasing linolenic acid via FAD-3 gene modi rated by reference for this purpose) and/or a gamma Zein fication (see, U.S. Pat. Nos. 6,063,947; 6,323,392; 6,372,965 knock out or mutant such as cs27 or TUSC27 or en27 (see, and WO 1993/11245). U.S. Pat. No. 6,858,778 and US Patent Application Publica 0373 (3) Altering conjugated linolenic or linoleic acid tion Number 2005/0160488, US Patent Application Publica content, such as in WO 2001/12800. tion Number 2005/0204418, which are incorporated by ref 0374 (4) Altering LEC1, AGP, Dek1, Superal 1, mill ps, erence for this purpose). See, Shiroza, et al., (1988) J. various Ipagenes such as Ipal, Ipa3, hptorhggt. For example, Bacteriol. 170:810 (nucleotide sequence of Streptococcus see, WO 2002/42424, WO 1998/22604, WO 2003/011015, mutant fructosyltransferase gene), Steinmetz, et al., (1985) WO 2002/057439, WO 2003/011015, U.S. Pat. Nos. 6,423, Mol. Gen. Genet. 200:220 (nucleotide sequence of Bacillus 886, 6,197.561, 6,825,397 and US Patent Application Publi subtilis levansucrase gene), Pen, et al., (1992) Bio/Technol cation Numbers US 2003/0079247, US 2003/0204870 and ogy 10:292 (production of transgenic plants that express Rivera-Madrid, et al., (1995) Proc. Natl. Acad. Sci. 92:5620 Bacillus licheniformis alpha-amylase), Elliot, et al., (1993) 5624. Plant Molec. Biol. 21:515 (nucleotide sequences of tomato 0375 (5) Genes encoding delta-8 desaturase for making invertase genes), Søgaard, et al., (1993).J. Biol. Chem. 268: long-chain polyunsaturated fatty acids (U.S. Pat. Nos. 8,058, 22480 (site-directed mutagenesis of barley alpha-amylase 571 and 8.338,152), delta-9 desaturase for lowering saturated gene) and Fisher, et al., (1993) Plant Physiol. 102:1045 fats (U.S. Pat. No. 8,063,269), Primula A6-desaturase for (maize endosperm starch branching enzyme II), WO 1999/ improving omega-3 fatty acid profiles. 10498 (improved digestibility and/or starch extraction 0376 (6) Isolated nucleic acids and proteins associated through modification of UDP-D-xylose 4-epimerase, Fragile with lipid and Sugar metabolism regulation, in particular, 1 and 2, Refl, HCHL., C4H), U.S. Pat. No. 6.232,529 (method lipid metabolism protein (LMP) used in methods of produc of producing high oil seed by modification of starch levels ing transgenic plants and modulating levels of seed storage (AGP)). The fatty acid modification genes mentioned herein compounds including lipids, fatty acids, starches or seed Stor may also be used to affect starch content and/or composition age proteins and use in methods of modulating the seed size, through the interrelationship of the starch and oil pathways. seed number, seed weights, root length and leaf size of plants 0384 (D) Altered antioxidant content or composition, (EP2404499). Such as alteration of tocopherol or tocotrienols. For example, 0377 (7) Altering expression of a High-Level Expression see, U.S. Pat. No. 6,787,683, US Patent Application Publica of Sugar-Inducible 2 (HSI2) protein in the plant to increase or tion Number 2004/0034886 and WO 2000/68393 involving decrease expression of HSI2 in the plant. Increasing expres the manipulation of antioxidant levels and WO 2003/082899 sion of HSI2 increases oil content while decreasing expres through alteration of a homogentisate geranylgeranyl trans sion of HSI2 decreases abscisic acid sensitivity and/or ferase (hggt). increases drought resistance (US Patent Application Publica 0385 (E) Altered essential seed amino acids. For example, tion Number 2012/0066794). see, U.S. Pat. No. 6,127.600 (method of increasing accumu 0378 (8) Expression of cytochrome b5 (Cb5) alone or lation of essential amino acids in seeds), U.S. Pat. No. 6,080, with FAD2 to modulate oil content in plant seed, particular to 913 (binary methods of increasing accumulation of essential increase the levels of omega-3 fatty acids and improve the amino acids in seeds), U.S. Pat. No. 5,990,389 (high lysine), ratio of omega-6 to omega-3 fatty acids (US Patent Applica WO 1999/40209 (alteration of amino acid compositions in tion Publication Number 2011/019 1904). seeds), WO 1999/29882 (methods for altering amino acid 0379 (9) Nucleic acid molecules encoding wrinkled 1-like content of proteins), U.S. Pat. No. 5,850,016 (alteration of polypeptides for modulating sugar metabolism (U.S. Pat. No. amino acid compositions in seeds), WO 1998/20133 (pro 8,217,223). teins with enhanced levels of essential amino acids), U.S. Pat. 0380 (B) Altered phosphorus content, for example, by the No. 5,885,802 (high methionine), U.S. Pat. No. 5,885,801 0381 (1) Introduction of a phytase-encoding gene would (high threonine), U.S. Pat. No. 6,664,445 (plantamino acid enhance breakdown of phytate, adding more free phosphate biosynthetic enzymes), U.S. Pat. No. 6,459,019 (increased to the transformed plant. For example, see, Van Hartingsveldt, lysine and threonine), U.S. Pat. No. 6,441.274 (plant tryp et al., (1993) Gene 127:87, for a disclosure of the nucleotide tophan synthase beta subunit), U.S. Pat. No. 6,346,403 (me sequence of an Aspergillus niger phytase gene. thionine metabolic enzymes), U.S. Pat. No. 5,939,599 (high 0382 (2) Modulating a gene that reduces phytate content. sulfur), U.S. Pat. No. 5,912,414 (increased methionine), WO In maize, this, for example, could be accomplished, by clon 1998/56935 (plant amino acid biosynthetic enzymes), WO ing and then re-introducing DNA associated with one or more 1998/45458 (engineered seed protein having higher percent of the alleles, such as the LPA alleles, identified in maize age of essential amino acids), WO 1998/42831 (increased mutants characterized by low levels of phytic acid, such as in lysine), U.S. Pat. No. 5,633,436 (increasing sulfur amino acid WO 2005/113778 and/or by altering inositol kinase activity content), U.S. Pat. No. 5,559,223 (synthetic storage proteins as in WO 2002/059324, US Patent Application Publication with defined structure containing programmable levels of US 2014/0274885 A1 Sep. 18, 2014 50 essential amino acids for improvement of the nutritional 0394 (B) WO 199938977 describing genes, including value of plants), WO 1996/01905 (increased threonine), WO CBF genes and transcription factors effective in mitigating 1995/15392 (increased lysine), US Patent Application Publi the negative effects of freezing, high salinity and drought on cation Number 2003/0163838, US Patent Application Publi plants, as well as conferring other positive effects on plant cation Number 2003/0150014, US Patent Application Publi phenotype. cation Number 2004/0068767, U.S. Pat. No. 6,803,498, WO 0395 (C) US Patent Application Publication Number 2001 (79.516. 2004/0148,654 and WO 2001/36596 where abscisic acid is 4. Genes that Control Male-Sterility: altered in plants resulting in improved plant phenotype Such 0386 There are several methods of conferring genetic as increased yield and/or increased tolerance to abiotic stress. male sterility available. Such as multiple mutant genes at 0396 (D) WO 2000/006341, WO 2004/090143, U.S. Pat. separate locations within the genome that confer male steril Nos. 7,531,723 and 6,992.237 where cytokinin expression is ity, as disclosed in U.S. Pat. Nos. 4,654,465 and 4,727.219 to modified resulting in plants with increased stress tolerance, Brar, et al., and chromosomal translocations as described by Such as drought tolerance, and/or increased yield. Also see, Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. In addi WO 2002/02776, WO 2003/052063, JP 2002/281975, U.S. tion to these methods, Albertsen, et al., U.S. Pat. No. 5,432, Pat. No. 6,084, 153, WO 2001/64898, U.S. Pat. No. 6,177,275 068, describe a system of nuclear male sterility which and U.S. Pat. No. 6,107,547 (enhancement of nitrogen utili includes: identifying a gene which is critical to male fertility; Zation and altered nitrogen responsiveness). silencing this native gene which is critical to male fertility; 0397 (E) For ethylene alteration, see, US Patent Applica removing the native promoter from the essential male fertility tion Publication Number 2004/0128719, US Patent Applica gene and replacing it with an inducible promoter, inserting tion Publication Number 2003/0166197 and WO 2000/ this genetically engineered gene back into the plant; and thus 32761. creating a plant that is male sterile because the inducible 0398 (F) For plant transcription factors or transcriptional promoter is not “on” resulting in the male fertility gene not regulators of abiotic stress, see, e.g., US Patent Application being transcribed. Fertility is restored by inducing or turning Publication Number 2004/0098764 or US Patent Application “on”, the promoter, which in turn allows the gene that confers Publication Number 2004/0078852. male fertility to be transcribed. 0399 (G) Genes that increase expression of vacuolar 0387 (A) Introduction of a deacetylase gene under the pyrophosphatase such as AVP1 (U.S. Pat. No. 8,058,515) for control of a tapetum-specific promoter and with the applica increased yield; nucleic acid encoding a HSFA4 or a HSFA5 tion of the chemical N-Ac-PPT (WO 2001/29237). (Heat Shock Factor of the class A4 or A5) polypeptides, an 0388 (B) Introduction of various stamen-specific promot oligopeptide transporter protein (OPT4-like) polypeptide; a ers (WO 1992/13956, WO 1992/13957). plastochron2-like (PLA2-like) polypeptide or a Wuschel 0389 (C) Introduction of the barnase and the barstar gene related homeobox 1-like (WOX1-like) polypeptide (U. Patent (Paul, et al., (1992) Plant Mol. Biol. 19:611-622). Application Publication Number US 2011/0283420). 0390 For additional examples of nuclear male and female 04.00 (H) Down regulation of polynucleotides encoding sterility systems and genes, see also, U.S. Pat. Nos. 5,859, poly (ADP-ribose) polymerase (PARP) proteins to modulate 341; 6,297,426; 5,478,369; 5,824,524; 5,850,014 and 6,265, programmed cell death (U.S. Pat. No. 8,058,510) for 640, all of which are hereby incorporated by reference. increased vigor. 5. Genes that Create a Site for Site Specific DNA Integration. 04.01 (I) Polynucleotide encoding DTP21 polypeptides 0391 This includes the introduction of FRT sites that may for conferring drought resistance (US Patent Application be used in the FLP/FRT system and/or LOX sites that may be Publication Number US 2011/0277181). used in the Cre/LOXp system. For example, see, Lyznik, et al., 0402 (J) Nucleotide sequences encoding ACC Synthase 3 (2003) Plant Cell Rep. 21:925-932 and WO 1999/25821, (ACS3) proteins for modulating development, modulating which are hereby incorporated by reference. Other systems response to stress, and modulating stress tolerance (US Patent that may be used include the Gin recombinase of phage Mu Application Publication Number US 2010/0287669). (Maeser, et al., (1991) Vicki Chandler, The Maize Handbook 0403 (K) Polynucleotides that encode proteins that confer ch. 118 (Springer-Verlag 1994), the Pin recombinase of E. a drought tolerance phenotype (DTP) for conferring drought coli (Enomoto, et al., 1983) and the R/RS system of the pSRi resistance (WO 2012/058528). plasmid (Araki, et al., 1992). 0404 (L) Tocopherol cyclase (TC) genes for conferring 6. Genes that Affect Abiotic Stress Resistance drought and salt tolerance (US Patent Application Publication 0392 Including but not limited to flowering, ear and seed Number 2012/0272352). development, enhancement of nitrogen utilization efficiency, 04.05 (M) CAAX amino terminal family proteins for altered nitrogen responsiveness, drought resistance or toler stress tolerance (U.S. Pat. No. 8.338,661). ance, cold resistance or tolerance and salt resistance or toler 0406 (N) Mutations in the SAL1 encoding gene have ance and increased yield under stress. increased stress tolerance, including increased drought resis 0393 (A) For example, see: WO 2000/73475 where water tant (US Patent Application Publication Number 2010/ use efficiency is altered through alteration of malate: U.S. Pat. 0257633). Nos. 5,892,009, 5,965,705, 5,929,305, 5,891,859, 6,417,428, 0407 (O) Expression of a nucleic acid sequence encoding 6,664,446, 6,706,866, 6,717,034, 6,801,104, WO 2000/ a polypeptide selected from the group consisting of GRF 060089, WO 2001/026459, WO 2001/035725, WO 2001/ polypeptide, RAA1-like polypeptide, SYR polypeptide, 034726, WO 2001/035727, WO 2001/036444, WO 2001/ ARKL polypeptide, and YTP polypeptide increasing yield 036597, WO 2001/036598, WO 2002/015675, WO 2002/ related traits (US Patent Application Publication Number 0.17430, WO 2002/077185, WO 2002/079403, WO 2003/ 2011/0061133). 013227, WO 2003/013228, WO 2003/014327, WO 2004/ 0408 (P) Modulating expression in a plant of a nucleic 031349, WO 2004/076638, WO 1998.09521. acid encoding a Class III Trehalose Phosphate Phosphatase US 2014/0274885 A1 Sep. 18, 2014

(TPP) polypeptide for enhancing yield-related traits in plants, 0411 (C) Constitutive over-expression of maize lateral particularly increasing seed yield (US Patent Application organ boundaries (LOB) domain protein (Zm-LOBDP1) has Publication Number 2010/0024067). been shown to increase kernel number and total kernel weight 04.09. Other genes and transcription factors that affect per plant (US Patent Application Publication Number 2012/ plant growth and agronomic traits such as yield, flowering, 0079622). plant growth and/or plant structure, can be introduced or 0412 (D) Enhancing yield-related traits in plants by introgressed into plants, see e.g., WO 1997/49811 (LHY), modulating expression in a plant of a nucleic acid encoding a WO 1998/56918 (ESD4), WO 1997/10339 and U.S. Pat. No. VIM1 (Variant in Methylation 1)-like polypeptide or a VTC2 6,573,430 (TFL), U.S. Pat. No. 6,713,663 (FT), WO 1996/ like (GDP-L-galactose phosphorylase) polypeptide or a 14414 (CON), WO 1996/38560, WO 2001/21822 (VRN1), DUF1685 polypeptide or an ARF6-like (Auxin Responsive WO 2000/44918 (VRN2), WO 1999/49064 (GI), WO 2000/ Factor) polypeptide (WO 2012/038893). 46358 (FR1), WO 1997/29123, U.S. Pat. No. 6,794,560, U.S. Pat. No. 6,307,126 (GAI), WO 1999/09174 (D8 and Rht) and 0413 (E) Modulating expression in a plant of a nucleic WO 2004/076638 and WO 2004/031349 (transcription fac acid encoding a Step 20-like polypeptide or a homologue tors). thereof gives plants having increased yield relative to control 7. Genes that Confer Increased Yield plants (EP2431472). 0410 (A) A transgenic crop plant transformed by a 0414 (F) Genes encoding nucleoside diphosphatase 1-AminoCyclopropane-1-Carboxylate Deaminase-like kinase (NDK) polypeptides and homologs thereof for modi Polypeptide (ACCDP) coding nucleic acid, wherein expres fying the plant's root architecture (US Patent Application sion of the nucleic acid sequence in the crop plant results in Publication Number 2009/0064373). the plants increased root growth, and/or increased yield, 8. Genes that Confer Plant Digestibility. and/or increased tolerance to environmental stress as com pared to a wild type variety of the plant (U.S. Pat. No. 8,097. 0415 (A) Altering the level of xylan present in the cell 769). (B) Over-expression of maize zinc finger protein gene wall of a plant by modulating expression of Xylan synthase (Zm-ZFP1) using a seed preferred promoter has been shown (U.S. Pat. No. 8,173.866). to enhance plant growth, increase kernel number and total 0416) In some embodiment the stacked trait may be a trait kernel weight per plant (US Patent Application Publication or event that has received regulatory approval including but Number 2012/0079623). not limited to the events in Table 2A-1F. TABLE 2A

Triticum aestivum Wheat

Event Company Description AP2OSCL BASF Inc. Selection for a mutagenized version of the enzyme acetohydroxyacid synthase (AHAS), also known as acetolactate synthase (ALS) or acetolactate pyruvate-lyase. AP602CL BASF Inc. Selection for a mutagenized version of the enzyme acetohydroxyacid synthase (AHAS), also known as acetolactate synthase (ALS) or acetolactate pyruvate-lyase. BW255-2, BW238-3 BASF Inc. Selection for a mutagenized version of the enzyme acetohydroxyacid synthase (AHAS), also known as acetolactate synthase (ALS) or acetolactate pyruvate-lyase. BWT BASF Inc. Tolerance to imidazolinone herbicides induced by chemical mutagenesis of the acetohydroxyacid synthase (AHAS) gene using Sodium azide. MONT1800 Monsanto Company Glyphosate tolerant wheat variety produced by inserting a modified 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) encoding gene from the soil bacterium Agrobacterium tumefaciens, strain CP4. Cyanamid Crop Selection for a mutagenized version of the Protection enzyme acetohydroxyacid synthase (AHAS), also known as acetolactate synthase (ALS) or acetolactate pyruvate-lyase. Teal 11A BASF Inc. Selection for a mutagenized version of the enzyme acetohydroxyacid synthase (AHAS), also known as acetolactate synthase (ALS) or acetolactate pyruvate-lyase. A2704-12, A2704-21, Bayer CropScience Glufosinate ammonium herbicide tolerant ASS47-35 (Aventis CropScience Soybean produced by inserting a modified (AgrEvo)) phosphinothricin acetyltransferase (PAT) encoding gene from the soil bacterium Streptomyces viridochromogenes. US 2014/0274885 A1 Sep. 18, 2014 52

TABLE 2B Glycine max L. Soybean

Event Company Description

ASS47-127 Bayer CropScience Glufosinate ammonium herbicide tolerant (Aventis CropScience soybean produced by inserting a modified (AgrEvo)) phosphinothricin acetyltransferase (PAT) encoding gene from the soil bacterium Streptomyces viridochromogenes. BPS-CV127-9 BASF Inc. The introduced csir1-2 gene from Arabidopsis thaliana encodes an acetohydroxyacid synthase protein that confers tolerance to imidazolinone herbicides due to a point mutation that results in a single amino acid Substitution in which the serine residue at position 653 is replaced by asparagine (S653N). DP-3 OS423 Pioneer Hi-Bred High oleic acid soybean produced by inserting International Inc. additional copies of a portion of the omega-6 desaturase encoding gene, gm-fad2-1 resulting in silencing of the endogenous omega-6 desaturase gene (FAD2-1). DP356O43 Pioneer Hi-Bred Soybean event with two herbicide tolerance International Inc. genes: glyphosate N-acetlytransferase, which detoxifies glyphosate, and a modified acetolactate synthase (ALS) gene which is tolerant to ALS-inhibiting herbicides. G94-1, G94-19, G168 DuPont Canada High oleic acid soybean produced by inserting a Agricultural Products second copy of the fatty acid desaturase (GmFad2-1) encoding gene from soybean, which resulted in silencing of the endogenous host gene. GTS 40-3-2 Monsanto Company Glyphosate tolerant soybean variety produced by inserting a modified 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) encoding gene from the soil bacterium Agrobacterium timefaciens. GU262 Bayer CropScience Glufosinate ammonium herbicide tolerant (Aventis soybean produced by inserting a modified CropScience(AgrEvo)) phosphinothricin acetyltransferase (PAT) encoding gene from the soil bacterium Streptomyces viridochromogenes. MON877O1 Monsanto Company Resistance to lepidopteran pests of soybean including velvetbean caterpillar (Anticarsia gemmataiis) and soybean looper (Pseudoplitsia includens). MON877O1 x Monsanto Company Glyphosate herbicide tolerance through MON89788 expression of the EPSPS encoding gene from A. tumefaciens strain CP4, and resistance to lepidopteran pests of soybean including velvetbean caterpillar (Anticarsia gemmatais) and soybean looper (Pseudoplitsia includens) via expression of the Cry1Ac encoding gene from B. thiringiensis. MON89788 Monsanto Company Glyphosate-tolerant soybean produced by inserting a modified 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) encoding aroA (epsps) gene from Agrobacterium timefaciens CP4. OT96-15 Agriculture & Agri-Food Low linolenic acid soybean produced through Canada traditional cross-breeding to incorporate the novel trait from a naturally occurring fan1 gene mutant that was selected for low linolenic acid. W62, W98 Bayer CropScience Glufosinate ammonium herbicide tolerant (Aventis soybean produced by inserting a modified CropScience(AgrEvo)) phosphinothricin acetyltransferase (PAT) encoding gene from the soil bacterium Streptomyces hygroscopicals. US 2014/0274885 A1 Sep. 18, 2014 53

TABLE 2C TABLE 2D Medicago Saiva Alfalfa Heianthus annuus Sunflower Event Company Description Event Company Description J101, Monsanto Glyphosate herbicide tolerant alfalfa (lucerne) J163 Company and produced by inserting a gene encoding the X81359 BASF Inc. Tolerance to imidazolinone herbicides by Forage enzyme 5-enolypyruvylshikimate-3-phosphate Genetics synthase (EPSPS) from the CP4 strain of selection of a naturally occurring mutant. International Agrobacterium timefaciens.

TABLE 2E Oryza saiva Rice Event Company Description CL121, CL141, CFX51 BASF Inc. Tolerance to the imidazolinone herbicide, imazethalpyr, induced by chemical mutagenesis of the acetolactate synthase (ALS) enzyme using ethyl methanesulfonate (EMS). IMINTA-1, IMINTA-4 BASF Inc. Tolerance to imidazolinone herbicides induced by chemical mutagenesis of the acetolactate synthase (ALS) enzyme using sodium azide. LLRICEO6, LLRICE62 Aventis CropScience Glufosinate ammonium herbicide tolerant rice produced by inserting a modified phosphinothricin acetyltransferase (PAT) encoding gene from the soil bacterium Streptomyces hygroscopicus). LLRICE6O1 Bayer CropScience Glufosinate ammonium herbicide tolerant rice (Aventis produced by inserting a modified CropScience (AgrEvo)) phosphinothricin acetyltransferase (PAT) encoding gene from the soil bacterium Streptomyces hygroscopicus). PWC16 BASF Inc. Tolerance to the imidazolinone herbicide, imazethalpyr, induced by chemical mutagenesis of the acetolactate synthase (ALS) enzyme using ethyl methanesulfonate (EMS).

TABLE 2F Zea mass L. Maize Event Company Description 176 Syngenta Seeds, Inc. Insect-resistant maize produced by inserting the cry1Ab gene from Bacilius thiringiensis subsp. kunstaki. The genetic modification affords resistance to attack by the European corn borer (ECB). 3751IR Pioneer Hi-Bred Selection of somaclonal variants by culture of International Inc. embryos on imidazolinone containing media. 676,678,680 Pioneer Hi-Bred Male-sterile and glufosinate ammonium herbicide International Inc. tolerant maize produced by inserting genes encoding DNA adenine methylase and phosphinothricin acetyltransferase (PAT) from Escherichia coli and Streptomyces viridochromogenes, respectively. B16 (DLL25) Dekalb Genetics Glufosinate ammonium herbicide tolerant maize Corporation produced by inserting the gene encoding phosphinothricin acetyltransferase (PAT) from Streptomyces hygroscopicnis. Syngenta Seeds, Inc. Insect-resistant and herbicide tolerant maize produced by inserting the cry1Abgene from Bacillus thuringiensis subsp. kurstaki, and the phosphinothricin N-acetyltransferase (PAT) encoding gene from S. viridochromogenes. BT11 x GA21 Syngenta Seeds, Inc. Stacked insect resistant and herbicide tolerant maize produced by conventional crossbreeding of parental lines BT11 (OECD unique identifier: SYN-BTO11-1) and GA21 (OECD unique identifier: MON-OOO21-9). US 2014/0274885 A1 Sep. 18, 2014 54

TABLE 2F-continued Zea maps L. Maize Event Company Description BT11 XMIR162 Syngenta Seeds, Inc. Stacked insect resistant and herbicide tolerant maize produced by conventional crossbreeding of parental lines BT11 (OECD unique identifier: SYN-BTO11-1) and MIR162 (OECD unique identifier: SYN-IR162-4). Resistance to the European Corn Borer and tolerance to the herbicide glufosinate ammonium (Liberty) is derived from BT11, which contains the cry1Ab gene from Bacilius thairingiensis Subsp. ikirstaki, and the phosphinothricin N-acetyltransferase (PAT) encoding gene from S. viridochromogenes. Resistance to other lepidopteran pests, including H. zea, S.frugiperda, A. ipsilon, and S. albicosta, is derived from MIR162, which contains the vip3Aa gene from Bacilius thuringiensis strain AB88. BT11 x MIR162 x Syngenta Seeds, Inc. Bacilius thuringiensis Cry1Ab delta-endotoxin MIR604 protein and the genetic material necessary for its production (via elements of vector pZO1502) in Event Bt11 corn (OECD Unique Identifier: SYN BTO11-1) x Bacilius thuringiensis Vip3Aa20 insecticidal protein and the genetic material necessary for its production (via elements of vector pNOV1300) in Event MIR162 maize (OECD Unique Identifier: SYN-IR162-4) x modified Cry3A protein and the genetic material necessary for its production (via elements of vector pZM26) in Event MIR604 corn (OECD Unique Identifier: SYN-IR6O4-5). BT11 x Syngenta Seeds, Resistance to coleopteran pests, particularly corn rootworm pests MIR162 x Inc. (Diabroica spp.) and several lepidopteran pests of corn, including MIR604 x European corn borer (ECB, Ostrinia nubialis), corn earworm GA21 (CEW, Helicoverpa zea), fall army worm (FAW. Spodoptera fugiperda), and black cutworm (BCW, Agrotis ipsilon); tolerance to glyphosate and glufosinate-ammonium containing herbicides. BT11 x Syngenta Seeds, Stacked insect resistant and herbicide tolerant maize produced MIR604 Inc. by conventional cross breeding of parental lines BT11 (OECD unique identifier: SYN-BTO11-1) and MIR604 (OECD unique identifier: SYN-IR6O5-5). Resistance to the European Corn Borer and tolerance to the herbicide glufosinate ammonium (Liberty) is derived from BT11, which contains the cry1Ab gene from Bacilius thiringiensis Subsp. ikairs taki, and the phosphinothricin N acetyltransferase (PAT) encoding gene from S. viridochromogenes. Corn rootworm-resistance is derived from MIR604 which contains the mcry3A gene from Bacilius thiringiensis. BT11 x Syngenta Seeds, Stacked insect resistant and herbicide tolerant maize produced MIR604 x Inc. by conventional cross breeding of parental lines BT11 (OECD GA21 unique identifier: SYN-BTO11-1), MIR604 (OECD unique identifier: SYN-IR6O5-5) and GA21 (OECD unique identifier: MON-OOO21-9). Resistance to the European Corn Borer and tolerance to the herbicide glufosinate ammonium (Liberty) is derived from BT11, which contains the cry1Ab gene from Bacilius thiringiensis Subsp. ikairs taki, and the phosphinothricin N acetyltransferase (PAT) encoding gene from S. viridochromogenes. Corn rootworm-resistance is derived from MIR604 which contains the mcry3A gene from Bacillus thuringiensis. Tolerance to glyphosate herbicide is derived from GA21 which contains a modified EPSPS gene from maize. CBH-351 Aventis Insect-resistant and glufosinate ammonium herbicide tolerant CropScience maize developed by inserting genes encoding Cry9C protein from Bacilius thuringiensis subsp. tolworthi and phosphinothricin acetyltransferase (PAT) from Streptomyces hygroscopicus. DAS DOW AgroSciences Lepidopteran insect resistant and glufosinate ammonium O6275-8 LLC herbicide-tolerant maize variety produced by inserting the cry1F gene from Bacilius thairingiensis varaizawai and the phosphinothricin acetyltransferase (PAT) from Streptomyces hygroscopicals. DAS DOW AgroSciences Corn rootworm-resistant maize produced by inserting the 591.22-7 LLC and Pioneer Hi cry34Ab1 and cry35Ab1 genes from Bacilius thuringiensis strain Bred International PS149B1. The PAT encoding gene from Streptomyces Inc. viridochromogenes was introduced as a selectable marker. US 2014/0274885 A1 Sep. 18, 2014 55

TABLE 2F-continued Zea maps L. Maize Event Company Description DAS- DOW AgroSciences Stacked insect resistant and herbicide tolerant maize produced S9122-7 x LLC and Pioneer Hi- by conventional cross breeding of parental lines DAS-59122-7 NK603 Bred International (OECD unique identifier: DAS-59122-7) with NK603 (OECD Inc. unique identifier: MON-OO6O3-6). Corn rootworm-resistance is derived from DAS-59122-7 which contains the cry34Ab1 and cry35Ab1 genes from Bacilius thuringiensis strain PS149B1. Tolerance to glyphosate herbicide is derived from NK603. DAS-591.22-7 x DOW AgroSciences Stacked insect resistant and herbicide tolerant maize produced TC1507 x LLC and Pioneer by conventional cross breeding of parental lines DAS-59122-7 NK603 Hi-Bred (OECD unique identifier: DAS-59122-7) and TC1507 (OECD International Inc. unique identifier: DAS-O15O7-1) with NK603 (OECD unique identifier: MON-OO6O3-6). Corn rootworm-resistance is derived from DAS-59122-7 which contains the cry34Ab1 and cry35Ab1 genes from Bacilius thuringiensis strain PS149B1. Lepidopteran resistance and tolerance to glufosinate ammonium herbicide is derived from TC1507. Tolerance to glyphosate herbicide is derived from NK603. DBT418 Dekalb Genetics Insect-resistant and glufosinate ammonium herbicide tolerant Corporation maize developed by inserting genes encoding Cry1AC protein from Bacilius thiringiensis subsp kistaki and phosphinothricin acetyltransferase (PAT) from Streptomyces hygroscopicus DK4O4SR BASF Inc. Somaclonal variants with a modified acetyl-CoA-carboxylase (ACCase) were selected by culture of embryos on sethoxydim enriched medium. Event 3272 Syngenta Seeds, Maize line expressing a heat stable alpha-amylase gene Inc. amy797E for use in the dry-grind ethanol process. The phosphomannose isomerase gene from E. coli was used as a selectable marker. Event 98.140 Pioneer Hi-Bred Maize event expressing tolerance to glyphosate herbicide, via International Inc. expression of a modified bacterial glyphosate N acetlytransferase, and ALS-inhibiting herbicides, vial expression of a modified form of the maize acetolactate synthase enzyme. EXP191OIT Syngenta Seeds, Tolerance to the imidazolinone herbicide, imazethalpyr, Inc. (formerly induced by chemical mutagenesis of the acetolactate synthase Zeneca Seeds) (ALS) enzyme using ethyl methanesulfonate (EMS). GA21 Syngenta Seeds, Introduction, by particle bombardment, of a modified 5 Inc. (formerly enolpyruvylshikimate-3-phosphate synthase (EPSPS), an Zeneca Seeds) enzyme involved in the shikimate biochemical pathway for the production of the aromatic amino acids. GA21 x Monsanto Company Stacked insect resistant and herbicide tolerant corn hybrid MON810 derived from conventional cross-breeding of the parental lines GA21 (OECD identifier: MON-OOO21-9) and MON810 (OECD identifier: MON-OO81O-6). IT Pioneer Hi-Bred Tolerance to the imidazolinone herbicide, imazethalpyr, was International Inc. obtained by in vitro selection of Somaclonal variants. LYO38 Monsanto Company Altered amino acid composition, specifically elevated levels of lysine, through the introduction of the cordap A gene, derived from Corynebacterium glutamictim, encoding the enzyme dihydrodipicolinate synthase (cDHDPS). MIR162 Syngenta Seeds, Insect-resistant maize event expressing a Vip3A protein from Inc. Bacilius thuringiensis and the Escherichia coli PMI selectable marker MIR604 Syngenta Seeds, Corn rootworm resistant maize produced by transformation Inc. with a modified cry3A gene. The phosphomannose isomerase gene from E. coli was used as a selectable marker. MIR604 x Syngenta Stacked insect resistant and herbicide tolerant maize produced by GA21 Seeds, Inc. conventional cross breeding of parental lines MIR604 (OECD unique identifier: SYN-IR6O5-5) and GA21 (OECD unique identifier: MON OOO21-9). Corn rootworm-resistance is derived from MIR604 which contains the mcry3A gene from Bacilius thiringiensis. Tolerance to glyphosate herbicide is derived from GA21. MON801 OO Monsanto Insect-resistant maize produced by inserting the cry1Ab gene from Company Bacilius thiringiensis subsp. ikirstaki. The genetic modification affords resistance to attack by the European corn borer (ECB). MON802 Monsanto Insect-resistant and glyphosate herbicide tolerant maize produced Company by inserting the genes encoding the Cry1Ab protein from Bacilius thiringiensis and the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from A. tumefaciens strain CP4. MON809 Pioneer Hi- Resistance to European corn borer (Ostrinia nubialis) by Bred introduction of a synthetic cry1Ab gene. Glyphosate resistance via International introduction of the bacterial version of a plant enzyme, 5-enolpyruvyl Inc. shikimate-3-phosphate synthase (EPSPS). US 2014/0274885 A1 Sep 18, 2014 56

TABLE 2F-continued Zea maps L. Maize Event Company Description MON810 Monsanto Insect-resistant maize produced by inserting a truncated form of the Company cry1Ab gene from Bacilius thuringiensis subsp. kunstaki HD-1. The genetic modification affords resistance to attack by the European corn borer (ECB). MON810 x Monsanto Stacked insect resistant and enhanced lysine content maize derived LYO38 Company from conventional cross-breeding of the parental lines MON810 (OECD identifier: MON-OO81O-6) and LYO38 (OECD identifier: REN-OOO38-3). MON810 x Monsanto Stacked insect resistant and glyphosate tolerant maize derived from MON88O17 Company conventional cross-breeding of the parental lines MON810 (OECD identifier: MON-OO81O-6) and MON88017 (OECD identifier: MON 88O17-3). European corn borer (ECB) resistance is derived from a truncated form of the cry1Ab gene from Bacillus thuringiensis subsp. kurstaki HD-1 present in MON810. Corn rootworm resistance is derived from the cry3Bb1 gene from Bacilius thuringiensis Subspecies kumamotoensis strain EG4691 present in MON88017. Glyphosate tolerance is derived from a 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) encoding gene from Agrobacterium tumefaciens strain CP4 present in MON88017. MON832 Monsanto Introduction, by particle bombardment, of glyphosate oxidase (GOX) Company and a modified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), an enzyme involved in the shikimate biochemical pathway for the production of the aromatic amino acids. MON863 Monsanto Corn root worm resistant maize produced by inserting the cry3Bb1 Company gene from Bacilius thairingiensis Subsp. iki imamotoensis. MON863 x Monsanto Stacked insect resistant corn hybrid derived from conventional MON810 Company cross-breeding of the parental lines MON863 (OECD identifier: MON-OO863-5) and MON810 (OECD identifier: MON-OO81O-6) MON863 x Monsanto Stacked insect resistant and herbicide tolerant corn hybrid derived MON810 x Company from conventional cross-breeding of the stacked hybrid MON NK603 OO863-5 x MON-OO81O-6 and NK603 (OECD identifier: MON OO6O3-6). MON863 x Monsanto Company Stacked insect resistant and herbicide tolerant NK603 corn hybrid derived from conventional cross breeding of the parental lines MON863 (OECD identifier: MON-OO863-5) and NK603 (OECD identifier: MON-OO6O3-6). MON87460 Monsanto Company MON 87460 was developed to provide reduced yield loss under water-limited conditions compared to conventional maize. Efficacy in MON 87460 is derived by expression of the inserted Bacilius subtilis cold shock protein B (CspB). MON88O17 Monsanto Company Corn rootworm-resistant maize produced by inserting the cry3Bb1 gene from Bacilius thiringiensis Subspecies kinamotoensis strain EG4691. Glyphosate tolerance derived by inserting a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene from Agrobacterium tumefaciens strain CP4. MON89034 Monsanto Company Maize event expressing two different insecticidal proteins from Bacilius thiringiensis providing resistance to number of lepidopteran pests. MON89034 x Monsanto Company Stacked insect resistant and glyphosate tolerant MON88O17 maize derived from conventional cross-breeding of the parental lines MON89034 (OECD identifier: MON-89O34-3) and MON88017 (OECD identifier: MON-88O17-3). Resistance to Lepidopteran insects is derived from two cry genes present in MON89043. Corn rootworm resistance is derived from a single cry genes and glyphosate tolerance is derived from the 5 enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene from Agrobacterium tumefaciens present in MON88017. MON89034 x Monsanto Company Stacked insect resistant and herbicide tolerant NK603 maize produced by conventional crossbreeding of parental lines MON89034 (OECD identifier: MON-89O34-3) with NK603 (OECD unique identifier: MON-OO6O3-6). Resistance to Lepidopteran insects is derived from two cry enes present in MON89043. Tolerance to yphosate herbicide is derived from NK603. US 2014/0274885 A1 Sep. 18, 2014 57

TABLE 2F-continued Zea maps L. Maize Event Company Description MON89034 x Monsanto Company and Mycogen Stacked insect resistant and herbicide tolerant TC1507 x Seeds clo Dow AgroSciences LLC maize produced by conventional crossbreeding MON88O17 x of parental lines: MON89034, TC1507, DAS MON88017, and DAS-59122. Resistance to the 591.22-7 above-ground and below-ground insect pests and tolerance to glyphosate and glufosinate ammonium containing herbicides. MS3 Bayer CropScience (Aventis Malesterility caused by expression of the barnase CropScience(AgrEvo)) ribonuclease gene from Bacilitis amyloiquefaciens; PPT resistance was via PPT acetyltransferase (PAT). MS6 Bayer CropScience (Aventis Malesterility caused by expression of the barnase CropScience(AgrEvo)) ribonuclease gene from Bacilitis amyloiquefaciens; PPT resistance was via PPT acetyltransferase (PAT). NK603 Monsanto Company Introduction, by particle bombardment, of a modified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), an enzyme involved in the shikimate biochemical pathway for the production of the aromatic amino acids. NK6O3 x Monsanto Company Stacked insect resistant and herbicide tolerant MON810 corn hybrid derived from conventional cross breeding of the parental lines NK603 (OECD identifier: MON-OO6O3-6) and MON810 (OECD identifier: MON-OO81O-6). Monsanto Company Stacked glufosinate ammonium and glyphosate herbicide tolerant maize hybrid derived from conventional cross-breeding of the parental lines NK603 (OECD identifier: MON-OO6O3-6) and T25 (OECD identifier: ACS-ZM003-2). T14, T25 Bayer CropScience (Aventis Glufosinate herbicide tolerant maize produced by CropScience(AgrEvo)) inserting the phosphinothricin N-acetyltransferase (PAT) encoding gene from the aerobic actinomycete Streptomyces viridochromogenes. T2S X Bayer CropScience (Aventis Stacked insect resistant and herbicide tolerant MON810 CropScience(AgrEvo)) corn hybrid derived from conventional cross breeding of the parental lines T25 (OECD identifier: ACS-ZMOO3-2) and MON810 (OECD identifier: MON-OO81O-6). TC1507 Mycogen (co Dow AgroSciences); Insect-resistant and glufosinate ammonium Pioneer (cio DuPont) herbicide tolerant maize produced by inserting the cry1F gene from Bacilius thuringiensis var. aizawai and the phosphinothricin N acetyltransferase encoding gene from Streptomyces viridochromogenes. TC1507 x DOW AgroSciences LLC and Stacked insect resistant and herbicide tolerant DAS- Pioneer Hi-Bred International Inc. maize produced by conventional crossbreeding of 591.22-7 parental lines TC1507 (OECD unique identifier: DAS-O15O7-1) with DAS-59122-7 (OECD unique identifier: DAS-59122-7). Resistance to lepidopteran insects is derived from TC1507 due the presence of the cry1F gene from Bacillus thiringiensis var. aizawai. Corn rootworm resistance is derived from DAS-59122-7 which contains the cry34Ab1 and cry35Ab1 genes from Bacillus thuringiensis strain PS149B1. Tolerance to glufosinate ammonium herbicide is derived from TC1507 from the phosphinothricin N acetyltransferase encoding gene from Streptomyces viridochromogenes. TC1507 x DOW AgroSciences LLC Stacked insect resistant and herbicide tolerant NK603 corn hybrid derived from conventional cross breeding of the parental lines 1507 (OECD identifier: DAS-O15O7-1) and NK603 (OECD identifier: MON-OO6O3-6).

0417. Other events with regulatory approval are well Gene Silencing known to one skilled in the art and can be found at the Center for Environmental Risk Assessment (cera-gmc.org/ 0418. In some embodiments the stacked trait may be in the '?action gm crop database, which can be accessed using the form of silencing of one or more polynucleotides of interest www prefix). resulting in Suppression of one or more target pest polypep US 2014/0274885 A1 Sep. 18, 2014

tides. In some embodiments the silencing is achieved through 0427. Another variation describes the use of plant viral the use of a suppression DNA construct. sequences to direct the Suppression of proximal mRNA 0419. In some embodiments one or more of the PHI-4 encoding sequences (PCT Publication WO 1998/36083). polypeptides or fragments or variants thereof may be stacked 0428 Recent work has described the use of “hairpin' with one or more polynucleotides encoding one or more structures that incorporate all, or part, of an mRNA encoding polypeptides having insecticidal activity or agronomic traits sequence in a complementary orientation that results in a as set forth Supra and optionally may further include one or potential “stem-loop' structure for the expressed RNA (PCT more polynucleotides providing for gene silencing of one or Publication Number WO 1999/53050). In this case the stem is more target polynucleotides as discussed infra. formed by polynucleotides corresponding to the gene of 0420 “Suppression DNA construct” is a recombinant interest inserted in either sense or anti-sense orientation with DNA construct which when transformed or stably integrated respect to the promoter and the loop is formed by some into the genome of the plant, results in “silencing of a target polynucleotides of the gene of interest, which do not have a gene in the plant. The target gene may be endogenous or complement in the construct. This increases the frequency of transgenic to the plant. coSuppression or silencing in the recovered transgenic plants. 0421 “Silencing,” as used herein with respect to the target For review of hairpin suppression see, Wesley, et al., (2003) gene, refers generally to the suppression of levels of mRNA or Methods in Molecular Biology, Plant Functional Genomics: protein/enzyme expressed by the target gene, and/or the level Methods and Protocols 236:273-286. of the enzyme activity or protein functionality. The term 0429. A construct where the stem is formed by at least 30 “Suppression' includes lower, reduce, decline, decrease, nucleotides from a gene to be suppressed and the loop is inhibit, eliminate and prevent. “Silencing or “gene silenc formed by a random nucleotide sequence has also effectively ing does not specify mechanism and is inclusive, and not been used for suppression (WO 1999/61632). limited to, anti-sense, coSuppression, viral-suppression, hair 0430. The use of poly-Tand poly-A sequences to generate pin Suppression, stem-loop Suppression, RNAi-based the stem in the stem-loop structure has also been described approaches and Small RNA-based approaches. (WO 2002/00894). 0422. A suppression DNA construct may comprise a 0431 Yet another variation includes using synthetic region derived from a target gene of interest and may com repeats to promote formation of a stem in the stem-loop prise all or part of the nucleic acid sequence of the sense structure. Transgenic organisms prepared with Such recom Strand (or antisense Strand) of the target gene of interest. binant DNA fragments have been shown to have reduced Depending upon the approach to be utilized, the region may levels of the protein encoded by the nucleotide fragment be 100% identical or less than 100% identical (e.g., at least forming the loop as described in PCT Publication Number 50% or any integer between 51% and 100% identical) to all or WO 2002/00904. part of the sense Strand (or antisense Strand) of the gene of 0432 RNA interference refers to the process of sequence interest. specific post-transcriptional gene silencing in animals medi ated by short interfering RNAs (siRNAs) (Fire, et al., (1998) 0423 Suppression DNA constructs are well-known in the Nature 391:806). The corresponding process in plants is com art, are readily constructed once the target gene of interest is monly referred to as post-transcriptional gene silencing selected, and include, without limitation, coSuppression con (PTGS) or RNA silencing and is also referred to as quelling in structs, antisense constructs, viral-suppression constructs, fungi. The process of post-transcriptional gene silencing is hairpin Suppression constructs, stem-loop Suppression con thought to be an evolutionarily-conserved cellular defense structs, double-stranded RNA-producing constructs, and mechanism used to prevent the expression of foreign genes more generally, RNAi (RNA interference) constructs and and is commonly shared by diverse flora and phyla (Fire, et small RNA constructs such as siRNA (short interfering RNA) al., (1999) Trends Genet. 15:358). Such protection from for constructs and miRNA (microRNA) constructs. eign gene expression may have evolved in response to the 0424) “Antisense inhibition” refers to the production of production of double-stranded RNAs (dsRNAs) derived from antisense RNA transcripts capable of suppressing the expres viral infection or from the random integration of transposon sion of the target protein. elements into a host genome via a cellular response that 0425 “Antisense RNA” refers to an RNA transcript that is specifically destroys homologous single-stranded RNA of complementary to all or part of a target primary transcript or viral genomic RNA. The presence of dsRNA in cells triggers mRNA and that blocks the expression of a target isolated the RNAi response through a mechanism that has yet to be nucleic acid fragment (U.S. Pat. No. 5,107,065). The comple fully characterized. mentarity of an antisense RNA may be with any part of the 0433. The presence of long dsRNAs in cells stimulates the specific gene transcript, i.e., at the 5' non-coding sequence, 3' activity of a ribonuclease III enzyme referred to as dicer. non-coding sequence, introns or the coding sequence. Dicer is involved in the processing of the dsRNA into short 0426 “Cosuppression” refers to the production of sense pieces of dsRNA known as short interfering RNAs (siRNAs) RNA transcripts capable of suppressing the expression of the (Berstein, et al., (2001) Nature 409:363). Short interfering target protein. “Sense' RNA refers to RNA transcript that RNAs derived from dicer activity are typically about 21 to includes the mRNA and can be translated into protein within about 23 nucleotides in length and comprise about 19 base a cellor in vitro. CoSuppression constructs in plants have been pair duplexes (Elbashir, et al., (2001) Genes Dev. 15:188). previously designed by focusing on overexpression of a Dicer has also been implicated in the excision of 21- and nucleic acid sequence having homology to a native mRNA, in 22-nucleotide small temporal RNAs (stRNAs) from precur the sense orientation, which results in the reduction of all sor RNA of conserved structure that are implicated in trans RNA having homology to the overexpressed sequence (see, lational control (Hutvagner, et al., (2001) Science 293:834). Vaucheret, et al. (1998) Plant J. 16:651-659 and Gura, (2000) The RNAi response also features an endonuclease complex, Nature 404:804-808). commonly referred to as an RNA-induced silencing complex US 2014/0274885 A1 Sep. 18, 2014 59

(RISC), which mediates cleavage of single-stranded RNA target sequences, or from different regions of the same target having sequence complementarity to the antisense strand of sequence can be employed. For example, the Suppressor the siRNA duplex. Cleavage of the target RNA takes place in enhancer elements employed can comprise fragments of the the middle of the region complementary to the antisense target sequence derived from different region of the target strand of the siRNA duplex (Elbashir, et al., (2001) Genes sequence (i.e., from the 3'UTR, coding sequence, intron, and/ Dev. 15:188). In addition, RNA interference can also involve or 5' UTR). Further, the suppressor enhancer element can be Small RNA (e.g., miRNA) mediated gene silencing, presum contained in an expression cassette, as described elsewhere ably through cellular mechanisms that regulate chromatin herein, and in specific embodiments, the Suppressor enhancer structure and thereby prevent transcription of target gene element is on the same or on a different DNA vector or sequences (see, e.g., Allshire, (2002) Science 297: 1818 construct as the silencing element. The Suppressor enhancer 1819; Volpe, et al., (2002) Science 297:1833-1837; Jenuwein, element can be operably linked to a promoter as disclosed (2002) Science 297:2215-2218; and Hall, et al., (2002) Sci herein. It is recognized that the Suppressor enhancer element ence 297:2232–2237). As such, miRNA molecules of the can be expressed constitutively or alternatively, it may be invention can be used to mediate gene silencing via interac produced in a stage-specific manner employing the various tion with RNA transcripts or alternately by interaction with inducible or tissue-preferred or developmentally regulated particular gene sequences, wherein Such interaction results in promoters that are discussed elsewhere herein. gene silencing either at the transcriptional or post-transcrip 0437. In specific embodiments, employing both a silenc tional level. ing element and the Suppressor enhancer element the sys 0434 Methods and compositions are further provided temic production of RNAi occurs throughout the entire plant. which allow for an increase in RNAi produced from the In further embodiments, the plant or plant parts of the inven silencing element. In such embodiments, the methods and tion have an improved loading of RNAi into the phloem of the compositions employ a first polynucleotide comprising a plant than would be observed with the expression of the silencing element for a target pest sequence operably linked silencing element construct alone and, thus provide better to a promoteractive in the plant cell; and, a second polynucle control of phloem feeding insects by an RNAi approach. In otide comprising a Suppressor enhancer element comprising specific embodiments, the plants, plant parts, and plant cells the target pest sequence or an active variant or fragment of the invention can further be characterized as allowing for thereof operably linked to a promoter active in the plant cell. the production of a diversity of RNAi species that can The combined expression of the silencing element with Sup enhance the effectiveness of disrupting target gene expres pressor enhancer element leads to an increased amplification S1O. of the inhibitory RNA produced from the silencing element 0438. In specific embodiments, the combined expression over that achievable with only the expression of the silencing of the silencing element and the Suppressor enhancer element element alone. In addition to the increased amplification of increases the concentration of the inhibitory RNA in the plant the specific RNAi species itself, the methods and composi cell, plant, plant part, plant tissue orphloem over the level that tions further allow for the production of a diverse population is achieved when the silencing element is expressed alone. of RNAi species that can enhance the effectiveness of dis 0439. As used herein, an “increased level of inhibitory rupting target gene expression. As such, when the Suppressor RNA' comprises any statistically significant increase in the enhancer element is expressed in a plant cell in combination level of RNAi produced in a plant having the combined with the silencing element, the methods and composition can expression when compared to an appropriate control plant. allow for the systemic production of RNAi throughout the For example, an increase in the level of RNAi in the plant, plant; the production of greater amounts of RNAi than would plant part or the plant cell can comprise at least about a 1%, be observed with just the silencing element construct alone; about a 1%-5%, about a 5%-10%, about a 10%-20%, about a and, the improved loading of RNAi into the phloem of the 20%-30%, about a 30%-40%, about a 40%-50%, about a plant, thus providing better control of phloemfeeding insects 50%-60%, about 60-70%, about 70%–80%, about a 80%- by an RNAi approach. Thus, the various methods and com 90%, about a 90%-100% or greater increase in the level of positions provide improved methods for the delivery of RNAi in the plant, plant part, plant cell, or phloem when inhibitory RNA to the target organism. See, for example, US compared to an appropriate control. In other embodiments, 2009/O1880O8. the increase in the level of RNAi in the plant, plant part, plant 0435. As used herein, a “suppressor enhancer element' cell, or phloem can comprise at least about a 1 fold, about a 1 comprises a polynucleotide comprising the target sequence to fold-5 fold, about a 5 fold.-10 fold, about a 10 fold-20 fold, be suppressed or an active fragment or variant thereof. It is about a 20 fold-30 fold, about a 30 fold-40 fold, about a 40 recognize that the Suppressor enhancer element need not be fold-50 fold, about a 50 fold-60 fold, about 60 fold-70 fold, identical to the target sequence, but rather, the Suppressor about 70 fold-80 fold, about a 80 fold-90 fold, about a 90 enhancer element can comprise a variant of the target fold-100 fold or greater increase in the level of RNAi in the sequence, so long as the Suppressor enhancer element has plant, plant part, plant cell or phloem when compared to an Sufficient sequence identity to the target sequence to allow for appropriate control. Examples of combined expression of the an increased level of the RNAi produced by the silencing silencing element with Suppressor enhancer element for the element over that achievable with only the expression of the control of Stinkbugs and Lygus can be found in US 2011/ silencing element. Similarly, the Suppressor enhancer ele O3O1223 and US 2009/O1921.17. ment can comprise afragment of the target sequence, wherein 0440 Some embodiments relate to down-regulation of the fragment is of Sufficient length to allow for an increased expression of target genes in insect pest species by interfering level of the RNAi produced by the silencing element over that ribonucleic acid (RNA) molecules. WO 2007/074405 achievable with only the expression of the silencing element. describes methods of inhibiting expression of target genes in 0436. It is recognized that multiple suppressor enhancer invertebrate pests including Colorado potato beetle. WO elements from the same target sequence or from different 2005/110068 describes methods of inhibiting expression of US 2014/0274885 A1 Sep. 18, 2014 60 target genes in invertebrate pests including in particular West 0442 Microorganism hosts that are known to occupy the ern corn rootworm as a means to control insect infestation. “phytosphere' (phylloplane, phyllosphere, rhizosphere, and/ Furthermore, WO 2009/091864 describes compositions and or rhizoplana) of one or more crops of interest may be methods for the Suppression of target genes from insect pest selected. These microorganisms are selected so as to be species including pests from the Lygus genus. Nucleic acid capable of Successfully competing in the particular environ molecules including RNAi for targeting the vacuolar ATPase ment with the wild-type microorganisms, provide for stable H subunit, useful for controlling a coleopteran pest popula maintenance and expression of the gene expressing the PHI-4 tion and infestation as described in US Patent Application polypeptide, and desirably, provide for improved protection Publication 2012/O198586. WO 2012/055982 describes ribo of the pesticide from environmental degradation and inacti nucleic acid (RNA or double stranded RNA) that inhibits or Vation. down regulates the expression of a target gene that encodes: 0443 Such microorganisms include bacteria, algae, and an insect ribosomal protein Such as the ribosomal protein fungi. Of particular interest are microorganisms such as bac L19, the ribosomal protein L40 or the ribosomal protein teria, e.g., Pseudomonas, Erwinia, Serratia, Klebsiella, Xan 527A, an insect proteasome subunit Such as the Rpnó protein, thomonas, Streptomyces, Rhizobium, Rhodopseudomonas, the Pros 25, the Rpn2 protein, the proteasome beta 1 subunit Methylius, Agrobacterium, Acetobacter; Lactobacillus, protein or the Pros beta 2 protein; an insect B-coatomer of the Arthrobacter, Azotobacter; Leuconostoc, and Alcaligenes, COP1 vesicle, the y-coatomer of the COP1 vesicle, the fungi, particularly yeast, e.g., Saccharomyces, Cryptococcus, B'-coatomer protein or the -coatomer of the COP1 vesicle: Kluyveromyces, Sporobolomyces, Rhodotorula, and Aure an insect Tetraspanine 2 A protein which is a putative trans Obasidium. Of particular interest are such phytosphere bac membrane domain protein; an insect protein belonging to the terial species as Pseudomonas Syringae, Pseudomonas fluo actin family Such as Actin 5C, an insect ubiquitin-5E protein; rescens, Pseudomonas chlororaphis, Serratia marcescens, an insect Sec23 protein which is a GTPase activator involved Acetobacter xylinum, Agrobacteria, Rhodopseudomonas in intracellular protein transport; an insect crinkled protein spheroides, Xanthomonas campestris, Rhizobium melioti, which is an unconventional myosin which is involved in Alcaligenes entrophus, Clavibacter xyli and Azotobacter motor activity; an insect crooked neck protein which is vinelandii and phytosphere yeast species such as Rhodot involved in the regulation of nuclear alternative mRNA splic orula rubra, R. glutinis, R. marina, R. aurantiaca, Crypto ing; an insect vacuolar H+-ATPase G-subunit protein; and an coccus albidus, C. difluens, C. laurentii, Saccharomyces insect Tbp-1 such as Tat-binding protein. US Patent Applica rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, tion Publications 2012/029750, US 20120297501, and 2012/ S. odorus, Kluyveromyces veronae, and Aureobasidium pol 0322660 describe interfering ribonucleic acids (RNA or lulans. Of particular interest are the pigmented microorgan double stranded RNA) that functions upon uptake by an isms. Host organisms of particularinterest include yeast, Such insect pest species to down-regulate expression of a target as Rhodotorula spp., Aureobasidium spp., Saccharomyces gene in said insect pest, wherein the RNA comprises at least spp. (Such as S. cerevisiae), Sporobolomyces spp., phyl one silencing element wherein the silencing element is a loplane organisms such as Pseudomonas spp. (Such as P region of double-stranded RNA comprising annealed aeruginosa, P. fluorescens, P. chlororaphis), Erwinia spp., complementary strands, one strand of which comprises or and Flavobacterium spp., and other Such organisms, includ consists of a sequence of nucleotides which is at least partially ing Agrobacterium tumefaciens, E. coli, Bacillus subtilis, complementary to a target nucleotide sequence within the Bacillus cereus and the like. target gene. US Patent Application Publication 2012/ 0444 Genes encoding the PHI-4 polypeptides of the 0.164205 describe potential targets for interfering double embodiments can be introduced into microorganisms that stranded ribonucleic acids for inhibiting invertebrate pests multiply on plants (epiphytes) to deliver PHI-4 polypeptides including: a Chd3 Homologous Sequence, a Beta-Tubulin to potential target pests. Epiphytes, for example, can be gram Homologous Sequence, a 40 kDa V-ATPase Homologous positive or gram-negative bacteria. Sequence, a EF1C. Homologous Sequence, a 26S Proteosome Subunit p28 Homologous Sequence, a Juvenile Hormone 0445 Root-colonizing bacteria, for example, can be iso Epoxide Hydrolase Homologous Sequence, a Swelling lated from the plant of interest by methods known in the art. Dependent Chloride Channel Protein Homologous Specifically, a Bacillus cereus Strain that colonizes roots can Sequence, a Glucose-6-Phosphate 1-Dehydrogenase Protein be isolated from roots of a plant (see, for example, Handels Homologous Sequence, an Act 42A Protein Homologous man, et al., (1991) Appl. Environ. Microbiol. 56:713-718). Sequence, a ADP-Ribosylation Factor 1 Homologous Genes encoding the PHI-4 polypeptides of the embodiments Sequence, a Transcription Factor IIB Protein Homologous can be introduced into a root-colonizing Bacillus cereus by Sequence, a Chitinase Homologous Sequences, a Ubiquitin standard methods known in the art. Conjugating Enzyme Homologous Sequence, a Glyceralde 0446 Genes encoding PHI-4 polypeptides can be intro hyde-3-Phosphate Dehydrogenase Homologous Sequence, duced, for example, into the root-colonizing Bacillus by an Ubiquitin B Homologous Sequence, a Juvenile Hormone means of electro transformation. Specifically, genes encoding Esterase Homolog, and an Alpha Tubuliln Homologous the PHI-4 polypeptides can be cloned into a shuttle vector, for Sequence. example, pHT3101 (Lerecius, et al., (1989) FEMS Microbiol. Letts. 60:211-218. The shuttle vector pHT3101 containing Use in Pesticidal Control the coding sequence for the particular PHI-4 polypeptide gene can, for example, be transformed into the root-coloniz 0441 General methods for employing strains comprising ing Bacillus by means of electroporation (Lerecius, et al., a nucleic acid sequence of the embodiments, or a variant (1989) FEMS Microbiol. Letts. 60:211-218). Expression sys thereof, in pesticide control or in engineering other organisms tems can be designed so that PHI-4 polypeptides are secreted as pesticidal agents are known in the art. See, for example outside the cytoplasm of gram-negative bacteria, Such as E. U.S. Pat. No. 5,039,523 and EPO480762A2. coli, for example. Advantages of having PHI-4 polypeptides US 2014/0274885 A1 Sep. 18, 2014 secreted are: (1) avoidance of potential cytotoxic effects of adjuvants can be solid or liquid and correspond to the Sub the PHI-4 polypeptide expressed; and (2) improvement in the stances ordinarily employed in formulation technology, e.g. efficiency of purification of the PHI-4 polypeptide, including, natural or regenerated mineral Substances, solvents, dispers but not limited to, increased efficiency in the recovery and ants, wetting agents, tackifiers, binders or fertilizers. Like purification of the protein per volume cell broth and wise the formulations may be prepared into edible “baits” or decreased time and/or costs of recovery and purification per fashioned into pest “traps' to permit feeding or ingestion by unit protein. a target pest of the pesticidal formulation. 0447 PHI-4 polypeptides can be made to be secreted in E. 0451 Methods of applying an active ingredient oran agro coli, for example, by fusing an appropriate E. coli signal chemical composition that contains at least one of the PHI-4 peptide to the amino-terminal end of the PHI-4 polypeptide. polypeptides produced by the bacterial strains include leaf Signal peptides recognized by E. coli can be found in proteins application, seed coating and Soil application. The number of already known to be secreted in E. coli, for example the applications and the rate of application depend on the inten OmpA protein (Ghrayeb, et al., (1984) EMBO J, 3:2437 sity of infestation by the corresponding pest. 2442). Omp A is a major protein of the E. coli outer mem 0452. The composition may be formulated as a powder, brane, and thus its signal peptide is thought to be efficient in dust, pellet, granule, spray, emulsion, colloid, Solution, or the translocation process. Also, the OmpA signal peptide Such like, and may be prepared by Such conventional means does not need to be modified before processing as may be the as desiccation, lyophilization, homogenation, extraction, fil case for other signal peptides, for example lipoprotein signal tration, centrifugation, sedimentation, or concentration of a peptide (Duffaud, et al., (1987) Meth. Enzymol. 153:492). culture of cells comprising the polypeptide. In all Such com 0448 PHI-4 polypeptides of the embodiments can be fer positions that contain at least one Such pesticidal polypeptide, mented in a bacterial host and the resulting bacteria processed the polypeptide may be present in a concentration of from and used as a microbial spray in the same manner that Bt about 1% to about 99% by weight. strains have been used as insecticidal sprays. In the case of a 0453 Lepidopteran, dipteran, heteropteran, nematode, PHI-4 polypeptide that is secreted from Bacillus, the secre hemiptera, or coleopteran pests may be killed or reduced in tion signal is removed or mutated using procedures known in numbers in a given area by the methods of the disclosure, or the art. Such mutations and/or deletions prevent secretion of may be prophylactically applied to an environmental area to the PHI-4 polypeptide into the growth medium during the prevent infestation by a susceptible pest. Preferably the pest fermentation process. The PHI-4 polypeptides are retained ingests, or is contacted with, a pesticidally-effective amount within the cell, and the cells are then processed to yield the of the polypeptide. By "pesticidally-effective amount” is encapsulated PHI-4 polypeptides. Any Suitable microorgan intended an amount of the pesticide that is able to bring about ism can be used for this purpose. Pseudomonas has been used death to at least one pest, or to noticeably reduce pest growth, to express Bt toxins as encapsulated proteins and the resulting feeding, or normal physiological development. This amount cells processed and sprayed as an insecticide (Gaertner, et al., will vary depending on Such factors as, for example, the (1993), in: Advanced Engineered Pesticides, ed. Kim). specific target pests to be controlled, the specific environ 0449 Alternatively, the PHI-4 polypeptides are produced ment, location, plant, crop or agricultural site to be treated, the by introducing a heterologous gene into a cellular host. environmental conditions, and the method, rate, concentra Expression of the heterologous gene results, directly or indi tion, stability, and quantity of application of the pesticidally rectly, in the intracellular production and maintenance of the effective polypeptide composition. The formulations may pesticide. These cells are then treated under conditions that also vary with respect to climatic conditions, environmental prolong the activity of the toxin produced in the cell when the considerations, and/or frequency of application and/or sever cell is applied to the environment of target pest(s). The result ity of pest infestation. ing product retains the toxicity of the toxin. These naturally 0454. The pesticide compositions described may be made encapsulated PHI-4 polypeptides may then be formulated in by formulating either the bacterial cell, crystal and/or spore accordance with conventional techniques for application to Suspension, or isolated protein component with the desired the environment hosting a target pest, e.g., soil, water, and agriculturally-acceptable carrier. The compositions may be foliage of plants. See, for example EPA 0192319, and the formulated prior to administration in an appropriate means references cited therein. Such as lyophilized, freeze-dried, desiccated, or in an aqueous carrier, medium or Suitable diluent, such as Saline or other Pesticidal Compositions buffer. The formulated compositions may be in the form of a 0450. In some embodiments the active ingredients can be dust or granular material, or a Suspension in oil (vegetable or applied in the form of compositions and can be applied to the mineral), or water or oil/water emulsions, or as a wettable crop area or plant to be treated, simultaneously or in Succes powder, or in combination with any other carrier material Sion, with other compounds. These compounds can be fertil Suitable for agricultural application. Suitable agricultural car izers, weed killers, cryoprotectants, Surfactants, detergents, riers can be solid or liquid and are well known in the art. The pesticidal soaps, dormant oils, polymers, and/or time-release term 'agriculturally-acceptable carrier covers all adjuvants, or biodegradable carrier formulations that permit long-term inert components, dispersants, Surfactants, tackifiers, bind dosing of a target area following a single application of the ers, etc. that are ordinarily used in pesticide formulation tech formulation. They can also be selective herbicides, chemical nology; these are well known to those skilled in pesticide insecticides, Virucides, microbicides, amoebicides, pesti formulation. The formulations may be mixed with one or cides, fungicides, bacteriocides, nematocides, molluscicides more Solidor liquid adjuvants and prepared by various means, or mixtures of several of these preparations, if desired, e.g., by homogeneously mixing, blending and/or grinding the together with further agriculturally acceptable carriers, Sur pesticidal composition with Suitable adjuvants using conven factants or application-promoting adjuvants customarily tional formulation techniques. Suitable formulations and employed in the art of formulation. Suitable carriers and application methods are described in U.S. Pat. No. 6,468.523, US 2014/0274885 A1 Sep. 18, 2014 62 herein incorporated by reference. The plants can also be traZamide, Imazosulfuron, Mefenacet, Oxaziclomefone, treated with one or more chemical compositions, including Pyrazosulfuron, Pyributicarb, Quinclorac. Thiobencarb, one or more herbicide, insecticides, or fungicides. Exemplary Indanofan, Flufenacet, FentraZamide, Halosulfuron, Oxazi chemical compositions include: Fruits/Vegetables Herbi clomefone, Benzobicyclon, Pyriftalid, Penoxsulam, Bispyri cides: Atrazine, Bromacil, Diuron, Glyphosate, Linuron, bac, Oxadiargyl, Ethoxysulfuron, Pretilachlor, Mesotrione, Metribuzin, Simazine, Trifluralin, Fluazifop, Glufosinate, Tefuryltrione, OxadiaZone, Fenoxaprop, Pyrimisulfan; Rice Halo sulfuron Gowan, Paraquat, PropyZamide, Sethoxydim, Insecticides: Diazinon, Fenitrothion, Fenobucarb, Monocro Butafenacil, Halosulfuron, Indaziflam: Fruits/Vegetables tophos, Benfuracarb, Buprofezin, Dinotefuran, Fipronil, Imi Insecticides: Aldicarb, Bacillus thuriengiensis, Carbaryl, dacloprid, Isoprocarb. Thiacloprid, Chromafenozide. Thia Carbofuran, Chlorpyrifos, Cypermethrin, Deltamethrin, cloprid, Dinotefuran, Clothianidin, Ethiprole, Diazinon, Malathion, Abamectin, Cyfluthrin/beta-cyfluthrin, Flubendiamide, Rynaxypyr, Deltamethrin, Acetamiprid, Esfenvalerate, Lambda-cyhalothrin, Acequinocyl. Thiamethoxam, Cyazypyr, Spinosad, Spinotoram, Emamec Bifenazate, Methoxyfenozide, Novaluron, Chromafenozide, tin-Benzoate, Cypermethrin, Chlorpyriphos, Cartap, Metha Thiacloprid, Dinotefuran, Fluacrypyrim, Tolfenpyrad, midophos, Etofenprox. Triazophos, 4-(6-Chlorpyridin-3- Clothianidin, Spirodiclofen, Gamma-cyhalothrin, yl)methyl(2,2-difluorethyl)aminofuran-2(5H)-on, Spiromesifen, Spinosad, Rynaxypyr, CyaZypyr, Spinoteram, Carbofuran, Benfuracarb; Rice Fungicides: Thiophanate-me Triflumuron, Spirotetramat, Imidacloprid, Flubendiamide, thyl, AZOxystrobin, Carpropamid, Edifenphos, FerimZone, Thiodicarb. Metaflumizone, Sulfoxaflor, Cyflumetofen, Iprobenfos, Isoprothiolane, Pencycuron, Probenazole, Pyro Cyanopyrafen, Imidacloprid, Clothianidin, Thiamethoxam, quilon, Tricyclazole, Trifloxystrobin, Diclocymet, Fenoxaniil, Spinotoram, Thiodicarb, Flonicamid, Methiocarb, Emamec Simeconazole, Tiadinil; Cotton Herbicides: Diuron, Flu tin-benzoate, lindoxacarb. Forthiazate, Fenamiphos, Cadusa ometuron, MSMA, Oxyfluorfen, Prometryn, Trifluralin, phos, Pyriproxifen, Fenbutatin-oxid, Hexthiazox, Methomyl, CarfentraZone, Clethodim, Fluazifop-butyl, Glyphosate, 4-(6-Chlorpyridin-3-yl)methyl(2,2-difluorethyl)amino Norflurazon, Pendimethalin, Pyrithiobac-sodium, Triflox furan-2(5H)-on; Fruits/Vegetables Fungicides: Carbenda ysulfuron, Tepraloxydim, Glufosinate, Flumioxazin. Thidi Zim, Chlorothalonil, EBDCs, Sulphur. Thiophanate-methyl, aZuron; Cotton Insecticides: Acephate, Aldicarb, Chlorpyri AZOxystrobin, Cymoxanil, FluaZinam, Fosetyl, Iprodione, fos, Cypermethrin, Deltamethrin, Malathion, Kresoxim-methyl, Metalaxyl/mefenoxam, Trifloxystrobin, Monocrotophos, Abamectin, Acetamiprid, Emamectin Ben Ethaboxam, Iprovalicarb, Trifloxystrobin, Fenhexamid, Zoate, Imidacloprid, Indoxacarb, Lambda-Cyhalothrin, Spi Oxpoconazole fumarate, Cyazofamid, Fenamidone, Zoxam nosad. Thiodicarb, Gamma-Cyhalothrin, Spiromesifen, ide, Picoxystrobin, Pyraclostrobin, Cyflufenamid, Boscalid: Pyridalyl, Flonicamid, Flubendiamide, Triflumuron, Ryn Cereals Herbicides: Isoproturon, Bromoxynil, loxynil, Phe axypyr, Beta-Cyfluthrin, Spirotetramat, Clothianidin, Thia noxies, Chlorsulfuron, Clodinafop, Diclofop, Diflufenican, methoxam, Thiacloprid, Dinetofuran, Flubendiamide, Fenoxaprop, Florasulam, Fluoroxypyr. Metsulfuron, Triasul CyaZypyr, Spinosad, Spinotoram, gamma Cyhalothrin, furon, Flucarbazone, Iodosulfuron, Propoxycarbazone, 4-(6-Chlorpyridin-3-yl)methyl(2,2-difluorethyl)amino Picolinafen, Mesosulfuron, Beflubutamid, Pinoxaden, Ami furan-2(5H)-on, Thiodicarb, Avermectin, Flonicamid, dosulfuron, Thifensulfuron Methyl, Tribenuron, Flupyrsulfu Pyridalyl, Spiromesifen, Sulfoxaflor, Profenophos, Thriazo ron, Sulfosulfuron, Pyrasulfotole, PyroxSulam, Flufenacet, phos, Endosulfan; Cotton Fungicides: Etridiazole, Metal Tralkoxydim, Pyroxasulfon; Cereals Fungicides: Carbenda axyl, Quintozene: Soybean Herbicides: Alachlor, Bentazone, Zim, Chlorothalonil, AZOxystrobin, Cyproconazole, Cyprodi Trifluralin, Chlorimuron-Ethyl, Cloransulam-Methyl, nil, Fenpropimorph, Epoxiconazole, Kresoxim-methyl, Qui Fenoxaprop, Fomesafen, Fluazifop, Glyphosate, Imazamox, noxyfen, Tebuconazole, Trifloxystrobin, Simeconazole, Imazaquin, Imazethapyr. (S-)Metolachlor, Metribuzin, Pen Picoxystrobin, Pyraclostrobin, Dimoxystrobin, Prothiocona dimethalin, Tepraloxydim, Glufosinate: Soybean Insecti Zole, Fluoxastrobin: Cereals Insecticides: Dimethoate, cides: Lambda-cyhalothrin, Methomyl, Parathion, Thiocarb, Lambda-cyhalthrin, Deltamethrin, alpha-Cypermethrin, Imidacloprid, Clothianidin. Thiamethoxam, Thiacloprid, B-cyfluthrin, Bifenthrin, Imidacloprid, Clothianidin, Thia Acetamiprid, Dinetofuran, Flubendiamide, Rynaxypyr, methoxam, Thiacloprid, Acetamiprid, Dinetofuran, Clorphy CyaZypyr, Spinosad, Spinotoram, Emamectin-Benzoate, riphos, Metamidophos, Oxidemethon-methyl, Pirimicarb, Fipronil, Ethiprole, Deltamethrin, B-Cyfluthrin, gamma and Methiocarb; Maize Herbicides: Atrazine, Alachlor, Bro lambda Cyhalothrin, 4-(6-Chlorpyridin-3-yl)methyl(2.2- moxynil, Acetochlor, Dicamba, Clopyralid, (S-) Dimethena difluorethyl)aminofuran-2(5H)-on, Spirotetramat, Spinodi mid, Glufosinate, Glyphosate, Isoxaflutole, (S-)Metolachlor, clofen, Triflumuron, Flonicamid. Thiodicarb, beta-Cy Mesotrione, Nicosulfuron, Primisulfuron, Rimsulfuron, Sul fluthrin, Soybean Fungicides: AZOxystrobin, Cyproconazole, cotrione, Foramsulfuron, ToprameZone, Tembotrione, Epoxiconazole, Flutriafol, Pyraclostrobin, Tebuconazole, Saflufenacil. Thiencarbazone, Flufenacet, Pyroxasulfon; Trifloxystrobin, Prothioconazole, Tetraconazole; Sugarbeet Maize Insecticides: Carbofuran, Chlorpyrifos, Bifenthrin, Herbicides: Chloridazon, Desmedipham, Ethiofumesate, Fipronil, Imidacloprid, Lambda-Cyhalothrin, Tefluthrin, Ter Phenmedipham, Triallate, Clopyralid, Fluazifop, Lenacil, bufos. Thiamethoxam, Clothianidin, Spiromesifen, Fluben Metamitron, Quinmerac, Cycloxydim, Triflusulfuron, diamide, Triflumuron, Rynaxypyr, Deltamethrin, Thiodicarb, Tepraloxydim, Quizalofop; Sugarbeet Insecticides: Imida B-Cyfluthrin, Cypermethrin, Bifenthrin, Lufenuron, Triflu cloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetami moron, Tefluthrin, Tebupirimphos, Ethiprole, Cyazypyr. prid, Dinetofuran, Deltamethrin, B-Cyfluthrin, gamma/ Thiacloprid, Acetamiprid, Dinetofuran, Avermectin, Methio lambda Cyhalothrin, 4-(6-Chlorpyridin-3-yl)methyl(2.2- carb, Spirodiclofen, Spirotetramat; Maize Fungicides: Feni difluorethyl)aminofuran-2(5H)-on, Tefluthrin, Rynaxypyr. tropan, Thiram, Prothioconazole, Tebuconazole, Triflox Cyaxypyr. Fipronil, Carbofuran; Canola Herbicides: Clopy ystrobin; Rice Herbicides: Butachlor, Propanil, ralid, Diclofop, Fluazifop, Glufosinate, Glyphosate, Metaza AZimsulfuron, Bensulfuron, Cyhalofop, Daimuron, Fen chlor, Trifluralin Ethametsulfuron, Quinmerac, Quizalofop, US 2014/0274885 A1 Sep. 18, 2014

Clethodim, Tepraloxydim; Canola Fungicides: AZOX medinalis Guenée (rice leafroller); Desmia funeralis Hübner ystrobin, Carbendazim, Fludioxonil, Iprodione, Prochloraz, (grape leaffolder); Diaphania hyalinata Linnaeus (melon Vinclozolin; Canola Insecticides: Carbofuran, Organophos worm); D. nitidalis Stoll (pickleworm); Diatraea grandi phates, Pyrethroids. Thiacloprid, Deltamethrin, Imidaclo Osella Dyar (southwestern corn borer), D. Saccharalis Fabri prid, Clothianidin, Thiamethoxam, Acetamiprid, Dinetofu cius (Surgarcane borer); Eoreuma loftini Dyar (Mexican rice ran, B-Cyfluthrin, gamma and lambda Cyhalothrin, tau borer); Ephestia elutella Hübner (tobacco (cacao) moth); Fluvaleriate, Ethiprole, Spinosad, Spinotoram, Galleria mellonella Linnaeus (greater wax moth); Herpeto Flubendiamide, Rynaxypyr, Cyazypyr, 4-(6-Chlorpyridin gramma licarsisalis Walker (Sod webworm); Homoeosoma 3-yl)methyl(2,2-difluorethyl)aminofuran-2(5H)-on. electellum Hulst (Sunflower moth); Elasmopalpus lignosellus 0455. In some embodiments the herbicide is Atrazine, Zeller (lesser cornstalk borer); Achroia grisella Fabricius Bromacil, Diuron, Chlorsulfuron, Metsulfuron, Thifensulfu (lesser wax moth); Loxostege Sticticalis Linnaeus (beet web ron Methyl, Tribenuron, Acetochlor, Dicamba, Isoxaflutole, worm); Orthaga thyrisalis Walker (tea tree web moth); Nicosulfuron, Rimsulfuron, Pyrithiobac-sodium, Flumiox Maruca testulalis Geyer (bean pod borer); Plodia interpunc azin, Chlorimuron-Ethyl, Metribuzin, Quizalofop, S-meto tella Hübner (Indian meal moth); Scirpophaga incertulas lachlor, Hexazinne or combinations thereof. Walker (yellow stem borer); Udea rubigalis Guenée (celery 0456. In some embodiments the insecticide is Esfenvaler leaftier); and leafrollers, budworms, seed worms, and fruit ate, Chlorantraniliprole, Methomyl, Indoxacarb, Oxamyl, or worms in the family Tortricidae Acleris gloverana Walsing combinations thereof. ham (Western blackheaded budworm); A. variana Fernald (Eastern blackheaded budworm): Archips argyrospila Walker Pesticidal and Insecticidal Activity (fruit tree leaf roller); A. rosana Linnaeus (European leaf 0457 "Pest includes but is not limited to, insects, fungi, roller); and other Archips species, Adoxophyes Orana Fischer bacteria, nematodes, mites, ticks, and the like. Insect pests von Rösslerstamm (summer fruit tortrix moth); Cochylis hos include insects selected from the orders Coleoptera, Diptera, pes Walsingham (banded sunflower moth): Cydia latifer Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemi reana Walsingham (filbertworm): C. pomonella Linnaeus ptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, (coding moth); Platynota flavedana Clemens (variegated lea Anoplura, Siphonaptera, Trichoptera, etc., particularly Lepi froller); P Stultana Walsingham (omnivorous leafroller); doptera, and Hemiptera. Lobesia botrana Denis & Schiffermüller (European grape 0458. Those skilled in the art will recognize that not all vine moth); Spilonota ocellana Denis & Schiffermüller (eye compounds are equally effective against all pests. Com spotted bud moth); Endopiza viteana Clemens (grape berry pounds of the embodiments display activity against insect moth); Eupoecilia ambiguella Hübner (vine moth); Bonagota pests, which may include economically important agro salubricola Meyrick (Brazilian apple leafroller); Grapholita nomic, forest, greenhouse, nursery, ornamentals, food and molesta Busck (oriental fruit moth); Suleima helianthana fiber, public and animal health, domestic and commercial Riley (Sunflower bud moth); Argyrotaenia spp., Choristo structure, household and stored product pests. neura spp. 0459 Larvae of the order Lepidoptera include, but are not 0460 Selected other agronomic pests in the order Lepi limited to, armyworms, cutworms, loopers, and heliothines in doptera include, but are not limited to. Alsophila pometaria the family Noctuidae Spodoptera frugiperda J E Smith (fall Harris (fall cankerworm); Anarsia lineatella Zeller (peach armyworm): S. exigua Hübner (beet armyworm): S. litura twig borer); Anisota Senatoria J. E. Smith (orange striped Fabricius (tobacco cutworm, cluster caterpillar); Mamestra oakworm); Antheraea pernyi Guérin-Méneville (Chinese configurata Walker (bertha armyworm); M. brassicae Lin Oak Tussah Moth); Bombyx mori Linnaeus (Silkworm); Buc naeus (cabbage moth); Agrotis ipsilon Hufnagel (black cut culatrix thurberiella Busck (cotton leaf perforator); Collas worm); A. Orthogonia Morrison (western cutworm); A. sub eurytheme Boisduval (alfalfa caterpillar); Datana integer terranea Fabricius (granulate cutworm); Alabama argillacea rima Grote & Robinson (walnut caterpillar); Dendrolimus Hübner (cotton leaf worm); Trichoplusia ni Hübner (cabbage Sibiricus Tschetwerikov (Siberian silk moth), Ennomos sub looper); Pseudoplusia includens Walker (soybean looper); signaria Hübner (elm spanworm): Erannis tiliaria Harris Anticarsia gemmatalis Hübner (velvetbean caterpillar); (linden looper); Euproctis chrysorrhoea Linnaeus (browntail Hypena scabra Fabricius (green cloverworm); Heliothis vire moth); Harrisina americana Guérin-Méneville (grapeleaf scens Fabricius (tobacco budworm); Pseudaletia unipuncta skeletonizer); Hemileuca Oliviae Cockrell (range caterpillar); Haworth (armyworm); Athetis mindara Barnes and Mcdun Hyphantria cunea Drury (fall webworm); Keiferia lycoper nough (rough skinned cutworm); Euxoa messoria Harris Sicella Walsingham (tomato pinworm), Lambdina fiscellaria (darksided cutworm); Earias insulana Boisduval (spiny boll fiscellaria Hulst (Eastern hemlock looper); L. fiscellaria lugu worm); E. vittella Fabricius (spotted bollworm); Helicoverpa brosa Hulst (Western hemlock looper); Leucoma salicis Lin armigera Hübner (American bollworm); H. zea Boddie (corn naeus (satin moth); Lymantria dispar Linnaeus (gypsy moth); earworm or cotton bollworm); Melanchra picta Harris (Zebra Manduca quinquemaculata Haworth (five spotted hawk caterpillar); Egira (Xylomyges) curialis Grote (citrus cut moth, tomato hornworm); M. sexta Haworth (tomato horn worm); borers, casebearers, webworms, coneworms, and worm, tobacco hornworm); Operophtera brumata Linnaeus skeletonizers from the family Pyralidae Ostrinia nubilalis (winter moth); Paleacrita venata Peck (spring cankerworm): Hübner (European corn borer); Amyelois transitella Walker Papilio Cresphontes Cramer (giant Swallowtail, orange dog); (naval orangeworm); Anagasta kuehniella Zeller (Mediterra Phryganidia Californica Packard (California oakworm): nean flour moth); Cadra cautella Walker (almond moth); Phyllocnistis citrella Stainton (citrus leafminer); Phyllono Chilo suppressalis Walker (rice stem borer); C. partellus, rycter blancardella Fabricius (spotted tentiform leafminer); (Sorghum borer); Corcyra cephalonica Stainton (rice moth); Pieris brassicae Linnaeus (large white butterfly); P rapae Crambus caliginosellus Clemens (corn root webworm): C. Linnaeus (Small white butterfly); P inapi Linnaeus (green teterrellus Zincken (bluegrass webworm): Cnaphalocrocis veined white butterfly): Platyptilia carduidactyla Riley (arti US 2014/0274885 A1 Sep. 18, 2014 64 choke plume moth); Plutella xylostella LinnaeuS (diamond ovinus Linnaeus (keds); and other Brachycera, mosquitoes back moth); Pectinophora gossypiella Saunders (pink boll Aedes spp.; Anopheles spp., Culex spp., black flies Prosimu worm); Pontia protodice Boisduval & Leconte (Southern lium spp., Simulium spp.; biting midges, sand flies, Sciarids, cabbageworm); Sabulodes aegrotata Guenee (omnivorous and other Nematocera. looper); Schizura concinna J. E. Smith (red humped caterpil 0463 Included as insects of interest are adults and nymphs lar); Sitotroga cerealella Olivier (Angoumois grain moth); of the orders Hemiptera and Homoptera such as, but not Thaumetopoea pitvocampa Schiffermuller (pine procession limited to, adelgids from the family Adelgidae, plant bugs ary caterpillar); Tineola bisselliella Hummel (webbing from the family Miridae, cicadas from the family Cicadidae, clothesmoth); Tuta absolute Meyrick (tomato leafminer); leafhoppers, Empoasca spp.; from the family Cicadellidae, Yponomeuta padella Linnaeus (ermine moth); Heliothis sub planthoppers from the families Cixiidae, Flatidae, Fulgoroi flexia Guenee; Malacosoma spp. and Orgvia spp. dea, Issidae and Delphacidae, treehoppers from the family 0461 Of interest are larvae and adults of the order Membracidae, psyllids from the family Psyllidae, whiteflies Coleoptera including weevils from the families Anthribidae, from the family Aleyrodidae, aphids from the family Aphid Bruchidae, and Curculionidae (including, but not limited to: idae, phylloxera from the family Phylloxeridae, mealybugs Anthonomus grandis Boheman (boll weevil); Lissorhoptrus from the family Pseudococcidae, scales from the families Oryzophilus Kuschel (rice water weevil); Sitophilus grana Asterolecanidae, Coccidae, Dactylopiidae, Diaspididae, rius Linnaeus (granary weevil); S. Oryzae Linnaeus (rice wee Eriococcidae, Ortheziidae, Phoenicococcidae and Margar vil); Hypera punctata Fabricius (clover leaf weevil); Cylin odidae, lace bugs from the family Tingidae, Stink bugs from drocopturus adspersus LeConte (Sunflower stem weevil); the family Pentatomidae, cinch bugs, Blissus spp.; and other Smicronyx filvus LeConte (red sunflower seed weevil); S. seed bugs from the family Lygaeidae, Spittlebugs from the SOrdidus LeConte (gray Sunflower seed weevil); Sphenopho family Cercopidae squash bugs from the family Coreidae, rus maidis Chittenden (maize billbug)); flea beetles, cucum and red bugs and cotton stainers from the family Pyrrhoc ber beetles, rootworms, leaf beetles, potato beetles, and leaf oridae. miners in the family Chrysomelidae (including, but not 0464 Agronomically important members from the order limited to: Leptinotarsa decemlineata Say (Colorado potato Homoptera further include, but are not limited to: Acyrthisi beetle); Diabrotica virgifera virgifera LeConte (western corn phon pisum Harris (pea aphid): Aphis craccivora Koch (cow rootworm): D. barberi Smith & Lawrence (northern corn pea aphid); A. fabae Scopoli (black bean aphid); A. gossypii rootworm): D. undecimpunctata howardi Barber (southern Glover (cotton aphid, melon aphid); A. maidiradicis Forbes corn rootworm); Chaetocnema pulicaria Melsheimer (corn (corn root aphid); A. pomi De Geer (apple aphid); A. spirae flea beetle); Phyllotreta cruciferae Goeze (corn flea beetle); cola Patch (spirea aphid); Aulacorthum Solani Kaltenbach Colaspis brunnea Fabricius (grape colaspis); Oulema mel (foxglove aphid); Chaetosiphon fragaefolii Cockerell (Straw anopus Linnaeus (cereal leaf beetle); Zygogramma exclama berry aphid); Diuraphis noxia Kurdumov/Mordvilko (Rus tionis Fabricius (sunflower beetle)); beetles from the family sian wheat aphid); Dysaphis plantaginea Paaserini (rosy Coccinellidae (including, but not limited to: Epilachna apple aphid); Eriosoma lanigerum Hausmann (woolly apple varivestis Mulsant (Mexican bean beetle)); chafers and other aphid); Brevicoryne brassicae Linnaeus (cabbage aphid); beetles from the family Scarabaeidae (including, but not lim Hyalopterus pruni Geoffroy (mealy plum aphid); Lipaphis ited to: Popillia japonica Newman (Japanese beetle); Cyclo erysimi Kaltenbach (turnip aphid); Metopolophium dirrho cephala borealis Arrow (northern masked chafer, white dum Walker (cereal aphid); Macrosiphum euphorbiae Tho grub); C. immaculate Olivier (southern masked chafer, white mas (potato aphid); Myzus persicae Sulzer (peach-potato grub); Rhizotrogus maialis Razoumowsky (European cha aphid, greenpeach aphid); Nasonovia ribisnigri Mosley (let fer); Phyllophaga crimita Burmeister (white grub): Ligyrus tuce aphid); Pemphigus spp. (root aphids and gall aphids); gibbosus De Geer (carrot beetle)); carpet beetles from the Rhopalosiphum maidis Fitch (corn leaf aphid); R. padi Lin family Dermestidae; wireworms from the family Elateridae, naeus (bird cherry-oat aphid); Schizaphis graminum Rondani Eleodes spp., Melanotus spp.; Conoderus spp., Limonius (greenbug); Sipha flava Forbes (yellow Sugarcane aphid); spp.; Agriotes spp., Ctenicera spp., Aeolus spp., bark beetles Sitobion avenae Fabricius (English grain aphid); Therioaphis from the family Scolytidae and beetles from the family Tene maculate Buckton (spotted alfalfa aphid); Toxoptera aurantii brionidae. Boyer de Fonscolombe (black citrus aphid); and T. citricida 0462 Adults and immatures of the order Diptera are of Kirkaldy (brown citrus aphid); Adelges spp. (adelgids); Phyl interest, including leafminers Agromyza parvicornis Loew loxera devastatrix Pergande (pecan phylloxera); Bemisia (cornblotch leafminer); midges (including, but not limited to: tabaci Gennadius (tobacco whitefly, sweetpotato whitefly): Contarinia sorghicola Coquillett (Sorghum midge); May B. argentifolii Bellows & Perring (silverleaf whitefly): etiola destructor Say (Hessian fly); Sitodiplosis mosellana Dialeurodes citri Ashmead (citrus whitefly): Trialeurodes Géhin (wheat midge); Neolasioptera murtfeldtiana Felt, abutiloneus (bandedwinged whitefly) and T vaporariorum (sunflower seed midge)); fruit flies (Tephritidae), Oscinella Westwood (greenhouse whitefly); Empoasca fabae Harris frit Linnaeus (fruit flies); maggots (including, but not limited (potato leafhopper); Laodelphax striatellus Fallen (Smaller to: Delia platura Meigen (seedcorn maggot); D. coarctata brown planthopper); Macrolestes quadrilineatus Forbes (as Fallen (wheat bulb fly); and other Delia spp., Meromyza ter leafhopper); Nephotettix cinticeps Uhler (green leafhop americana Fitch (wheat stem maggot); Musca domestica per); N. nigropictus Stal (rice leafhopper); Nilaparvata Linnaeus (house flies); Fannia canicularis Linnaeus, Ffemo lugens Stal (brown planthopper); Peregrinus maidis Ash ralis Stein (lesser house flies); Stomoxys calcitrans Linnaeus mead (corn planthopper); Sogatella furcifera Horvath (white (stable flies)); face flies, horn flies, blow flies, Chrysomya backed planthopper); Sogatodes Orizicola Muir (rice delpha spp., Phormia spp.; and other muscoid fly pests, horse flies cid), Tiphlocyba pomaria McAtee (white apple leafhopper); Tabanus spp., botflies Gastrophilus spp., Oestrus spp., cattle Erythroneoura spp. (grape leafhoppers); Magicicada Septen grubs Hypoderma spp.; deerflies Chrysops spp.; Mellophagus decim Linnaeus (periodical cicada); Icerya purchasi Maskell US 2014/0274885 A1 Sep. 18, 2014

(cottony cushion scale); Ouadraspidiotus perniciosus Com Fabricius (black widow spider); and centipedes in the order stock (San Jose scale); Planococcus citri Risso (citrus mea Scutigeromorpha Such as Scutigera Coleoptrata Linnaeus lybug); Pseudococcus spp. (other mealybug complex); (house centipede). Cacopsylla pyricola Foerster (pear psylla); Trioza diospyri 0470 Insect pest of interest include the Superfamily of Ashmead (persimmon psylla). Stink bugs and other related insects including but not limited 0465 Agronomically important species of interest from to species belonging to the family Pentatomidae (Nezara the order Hemiptera include, but are not limited to: Acroster viridula, Halyomorpha haly's, Piezodorus guildini, Euschis nun hilare Say (green Stink bug); Anasa tristis De Geer tus servus, Acrosternum hilare, Euschistus heros, Euschistus (squash bug); Blissus leucopterus leucopterus Say (chinch tristigmus, Acrosternum hilare, Dichelops fircatus, Dich bug); Corythuca gossypii Fabricius (cotton lace bug); Cyr elops melacanthus, and Bagrada hilaris (Bagrada Bug)), the topeltis modesta Distant (tomato bug); Dysdercus suturellus family Plataspidae (Megacopta cribraria—Bean plataspid), Herrich-Schaffer (cotton stainer); Euschistus servus Say and the family Cydnidae (Scaptocoris Castanea—Root Stink (brown stink bug); E. variolarius Palisot de Beauvois (one bug); and Lepidoptera species including but not limited to: spotted Stink bug); Graptostethus spp. (complex of seed diamond-back moth, e.g., Helicoverpa zea Boddie; soybean bugs); Leptoglossus Corculus Say (leaf-footed pine seedbug); looper, e.g., Pseudoplusia includens Walker, and Velvet bean Lygus lineolaris Palisot de Beauvois (tarnished plant bug), L. caterpillar e.g., Anticarsia gemmatalis Hübner. Hesperus Knight (Western tarnished plant bug); L. pratensis 0471 Methods for measuring pesticidal activity are well Linnaeus (common meadow bug); L. rugulipennis Poppius known in the art. See, for example, Czapla and Lang, (1990) (European tarnished plant bug); Lygocoris pabulinus Lin J. Econ. Entomol. 83:2480-2485; Andrews, et al., (1988) naeus (common green capsid); Nezara viridula Linnaeus Biochem. J. 252: 199-206; Marrone, et al., (1985).J. of Eco (Southern green Stink bug); Oebalus pugnax Fabricius (rice nomic Entomology 78:290–293 and U.S. Pat. No. 5,743,477, Stink bug). Oncopeltus fasciatus Dallas (large milkweed all of which are herein incorporated by reference in their bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper). entirety. Generally, the protein is mixed and used in feeding assays. See, for example Marrone, et al., (1985).J. of Eco 0466 Furthermore, embodiments may be effective against nomic Entomology 78:290–293. Such assays can include con Hemiptera such, Calocoris norvegicus Gmelin (strawberry tacting plants with one or more pests and determining the bug); Orthops campestris Linnaeus; Plesiocoris rugicollis plant’s ability to survive and/or cause the death of the pests. Fallen (apple capsid), Cyrtopeltis modestus Distant (tomato 0472. Nematodes include parasitic nematodes such as bug); Cyrtopeltis notatus Distant (Suckfly); Spanagonicus root-knot, cyst, and lesion nematodes, including Heterodera albofasciatus Reuter (whitemarked fleahopper); Diaphno spp., Meloidogyne spp., and Globodera spp.; particularly coris chlorionis Say (honeylocust plant bug); Labopidicola members of the cyst nematodes, including, but not limited to, alli Knight (onion plant bug); Pseudatomoscelis seriatus Heterodera glycines (soybean cyst nematode); Heterodera Reuter (cotton fleahopper); Adelphocoris rapidus Say (rapid Schachtii (beet cyst nematode); Heterodera avenae (cereal plant bug); Poecilocapsus lineatus Fabricius (four-lined plant cyst nematode); and Globodera rostochiensis and Globodera bug); Nysius ericae Schilling (false chinch bug); Nysius pailida (potato cyst nematodes). Lesion nematodes include raphanus Howard (false chinch bug); Nezara viridula Lin Pratylenchus spp. naeus (Southern green Stink bug); Eurygaster spp.; Coreidae spp., Pyrrhocoridae spp.; Timidae spp., Blostomatidae spp.; Seed Treatment Reduviidae spp.; and Cimicidae spp. 0467 Also included are adults and larvae of the order 0473. To protect and to enhance yield production and trait Acari (mites) such as Aceria to Sichella Keifer (wheat curl technologies, seed treatment options can provide additional mite); Petrobia latens Müller (brown wheat mite); spider crop plan flexibility and cost effective control against insects, mites and red mites in the family Tetranychidae, Panonychus weeds and diseases. Seed material can be treated, typically ulmi Koch (European red mite); Tetranychus urticae Koch Surface treated, with a composition comprising combinations (two spotted spider mite); (T. mcdanieli McGregor of chemical or biological herbicides, herbicide safeners, (McDaniel mite); T cinnabarinus Boisduval (carmine spider insecticides, fungicides, germination inhibitors and enhanc mite); T turkestani Ugarov & Nikolski (strawberry spider ers, nutrients, plant growth regulators and activators, bacteri mite); flat mites in the family Tenuipalpidae, Brevipalpus cides, nematocides, avicides and/or molluscicides. These lewisi McGregor (citrus flat mite); rust and bud mites in the compounds are typically formulated together with further family Eriophyidae and other foliar feeding mites and mites carriers, Surfactants or application-promoting adjuvants cus important in human and animal health, i.e. dust mites in the tomarily employed in the art of formulation. The coatings family Epidermoptidae, follicle mites in the family Demodi may be applied by impregnating propagation material with a cidae, grain mites in the family Glycyphagidae, ticks in the liquid formulation or by coating with a combined wet or dry order Ixodidae. Ixodes Scapularis Say (deer tick); I. holocy formulation. Examples of the various types of compounds clus Neumann (Australian paralysis tick); Dermacentor vari that may be used as seed treatments are provided in The abilis Say (American dog tick); Amblyomma americanum Pesticide Manual: A World Compendium, C. D. S. Tomlin Linnaeus (lone star tick); and scab and itch mites in the Ed., Published by the British Crop Production Council, which families Psoroptidae, Pyemotidae, and Sarcoptidae. is hereby incorporated by reference. 0474 Some seed treatments that may be used on crop seed 0468. Insect pests of the order Thysanura are of interest, include, but are not limited to, one or more of abscisic acid, Such as Lepisma saccharina Linnaeus (silverfish); Thermo acilbenzolar-5-methyl, avermectin, amitrol, azaconazole, bia domestica Packard (firebrat). aZospirillum, azadirachtin, azoxystrobin, Bacillus spp. (in 0469 Additional arthropod pests covered include: spiders cluding one or more of cereus, firmus, megaterium, pumilis, in the order Araneae Such as Loxosceles reclusa Gertsch & sphaericus, Subtilis and/or thuringiensis species), Mulaik (brown recluse spider); and the Latrodectus mactans bradyrhizobium spp. (including one or more of betae, canar US 2014/0274885 A1 Sep. 18, 2014 66 iense, elkanii, iriomotense, japonicum, liaonigense, decreasing the number of offspring produced, producing less pachyrhizi and/or yuanmingense), captan, carboxin, chito fit pests, producing pests more Susceptible to predator attack san, clothianidin, copper, cyazypyr, difenoconazole, etidiaz or deterring the pests from eating the plant. ole, fipronil, fludioxonil, fluoxastrobin, fluguinconazole, flu 0478. In some embodiments methods are provided for razole, fluxofenim, harpin protein, imazalil, imidacloprid, controlling an insect pest population resistant to a pesticidal ipconazole, isoflavenoids, lipo-chitooligosaccharide, manco protein, comprising contacting the insect pest population Zeb, manganese, maneb, mefenoxam, metalaxyl, metcona with an insecticidally-effective amount of a recombinant Zole, myclobutanil, PCNB. penflufen, penicillium, penthi PHI-4 polypeptide. In some embodiments methods are pro opyriad, permethrine, picoxystrobin, prothioconazole, vided for controlling an insect pest population resistant to a pyraclostrobin, rynaxypyr, S-metolachlor, Saponin, sedax pesticidal protein, comprising contacting the insect pest ane, TCMTB, tebuconazole, thiabendazole, thiamethoxam, population with an insecticidally-effective amount of a thiocarb, thiram, tolclofos-methyl, triadimenol, trichoderma, recombinant pesticidal protein of SEQ ID NO: 2, SEQ ID trifloxystrobin, triticonazole and/or zinc. PCNB seed coat NO:3, SEQID NO:4, and SEQID NOs: 51-819 or a variant refers to EPA registration number 002935.00419, containing thereof. quintozen and terrazole. TCMTB refers to 2-(thiocyanometh 0479. In some embodiments methods are provided for ylthio) benzothiazole. protecting a plant from an insect pest, comprising expressing 0475 Seed varieties and seeds with specific transgenic in the plant or cell thereof a recombinant PHI-4 polypeptide. traits may be tested to determine which seed treatment In some embodiments methods are provided for protecting a options and application rates may complement such varieties plant from an insect pest, comprising expressing in the plant and transgenic traits in order to enhance yield. For example, a or cell thereof a recombinant pesticidal protein of SEQ ID variety with good yield potential but head Smut Susceptibility NO: 2, SEQ ID NO:3, SEQ ID NO: 4, and SEQ ID NOs: may benefit from the use of a seed treatment that provides 51-819 or variants thereof. protection against head Smut, a variety with good yield poten tial but cyst nematode susceptibility may benefit from the use Insect Resistance Management (IRM) Strategies of a seed treatment that provides protection against cyst 0480 Expression of B. thuringiensis 8-endotoxins in nematode, and so on. Likewise, a variety encompassing a transgenic corn plants has proven to be an effective means of transgenic trait conferring insect resistance may benefit from controlling agriculturally important insect pests (Perlak, et the second mode of action conferred by the seed treatment, a al., 1990; 1993). However, insects have evolved that are resis variety encompassing a transgenic trait conferring herbicide tant to B. thuringiensis 5-endotoxins expressed in transgenic resistance may benefit from a seed treatment with a safener plants. Such resistance, should it become widespread, would that enhances the plants resistance to that herbicide, etc. Fur clearly limit the commercial value of germplasm containing ther, the good root establishment and early emergence that genes encoding Such B. thuringiensis Ö-endotoxins. results from the proper use of a seed treatment may result in 0481. One way to increasing the effectiveness of the trans more efficient nitrogen use, a better ability to withstand genic insecticides against target pests and contemporane drought and an overall increase in yield potential of a variety ously reducing the development of insecticide-resistant pests or varieties containing a certain trait when combined with a is to use provide non-transgenic (i.e., non-insecticidal pro seed treatment. tein) refuges (a section of non-insecticidal crops/corn) for use with transgenic crops producing a single insecticidal protein Methods for Inhibiting Growth or Killing an Insect Pest and active against target pests. The United States Environmental Controlling an Insect Population Protection Agency (epa.gov/oppbppdl/biopesticides/pipS/bt 0476. In some embodiments methods are provided for corn refuge 2006.htm, which can be accessed using the inhibiting growth or killing an insect pest, comprising con www prefix) publishes the requirements for use with trans tacting the insect pest with an insecticidally-effective amount genic crops producing a single Bt protein active against target of a recombinant PHI-4 polypeptide. In some embodiments pests. In addition, the National Corn Growers Association, on methods are provided for inhibiting growth or killing an their website: (incga.com/insect-resistance-management insect pest, comprising contacting the insect pest with an fact-sheet-bt-corn, which can be accessed using the www insecticidally-effective amount of a recombinant pesticidal prefix) also provides similar guidance regarding refuge protein of SEQID NO: 2, SEQID NO:3, SEQID NO:4, and requirements. Due to losses to insects within the refuge area, SEQ ID NOs: 51-819 or a variant thereof. larger refuges may reduce overall yield. 0477. In some embodiments methods are provided for 0482 Another way of increasing the effectiveness of the controlling an insect pest population, comprising contacting transgenic insecticides against target pests and contempora the insect pest population with an insecticidally-effective neously reducing the development of insecticide-resistant amount of a recombinant PHI-4 polypeptide. In some pests would be to have a repository of insecticidal genes that embodiments methods are provided for controlling an insect are effective against groups of insect pests and which mani pest population, comprising contacting the insect pest popu fest their effects through different modes of action. lation with an insecticidally-effective amount of a recombi 0483 Expression in a plant of two or more insecticidal nant pesticidal protein of SEQID NO: 2, SEQID NO:3, SEQ compositions toxic to the same insect species, each insecti ID NO: 4, and SEQID NOs: 51-819 or a variant thereof. As cide being expressed at efficacious levels would be another used herein, by “controlling a pest population' or "controls a way to achieve control of the development of resistance. This pest' is intended any effect on a pest that results in limiting the is based on the principle that evolution of resistance against damage that the pest causes. Controlling a pest includes, but two separate modes of action is far more unlikely than only is not limited to, killing the pest, inhibiting development of one. Roush for example, outlines two-toxin strategies, also the pest, altering fertility or growth of the pest in Such a called "pyramiding or 'stacking for management of insec manner that the pest provides less damage to the plant, ticidal transgenic crops. (The Royal Society. Phil. Trans. R. US 2014/0274885 A1 Sep. 18, 2014 67

Soc. Lond. B. (1998)353:777-1786). Stacking or pyramiding insecticidal proteins toxic to Lepidoptera and/or Hemiptera of two different proteins each effective against the target pests insects but each exhibiting a different mode of effectuating its and with little or no cross-resistance can allow for use of a inhibiting growth or killing activity, wherein the two or more smaller refuge. The U.S. Environmental Protection Agency insecticidal proteins comprise a PHI-4 polypeptide and a cry requires significantly less (generally 5%) structured refuge of protein. Also provided are means for effective Lepidoptera non-Bt corn be planted than for single trait products (gener and/or Hemiptera insect resistance management of transgenic ally 20%). There are various ways of providing the IRM plants, comprising co-expressing at high levels in the plants effects of a refuge, including various geometric planting pat two or more insecticidal proteins toxic to Lepidoptera and/or terns in the fields and in-bag seed mixtures, as discussed Hemiptera insects but each exhibiting a different mode of further by Roush. effectuating its inhibiting growth or activity, wherein the two 0484. In some embodiments the PHI-4 polypeptides of the or more insecticidal proteins comprise a protein of SEQ ID disclosure are useful as an insect resistance management NO: 2, SEQID NO:3, SEQID NO: 4, SEQID NO: 51-819 strategy in combination (i.e., pyramided) with other pesti or variants thereof and a cry protein. cidal proteins include but are not limited to Bt toxins, 0493. In addition, methods are provided for obtaining Xenorhabdus sp. or Photorhabdus sp. insecticidal proteins, regulatory approval for planting or commercialization of and the like. plants expressing proteins insecticidal to insects in the order 0485 Provided are methods of controlling Lepidoptera Coleoptera, comprising the step of referring to, Submitting or and/or Hemiptera insect infestation(s) in a transgenic plant relying on insect assay binding data showing that the PHI-4 that promote insect resistance management, comprising polypeptide does not compete with binding sites for cry pro expressing in the plant at least two different insecticidal pro teins in Such insects. In addition, methods are provided for teins having different modes of action. obtaining regulatory approval for planting or commercializa 0486 In some embodiments the methods of controlling tion of plants expressing proteins insecticidal to insects in the Lepidoptera and/or Hemiptera insect infestation in a trans order Coleoptera, comprising the step of referring to, Submit genic plant and promoting insect resistance management the ting or relying on insect assay binding data showing that the at least one of the insecticidal proteins comprise a PHI-4 protein of SEQID NO: 2, SEQID NO:3, SEQID NO:4, SEQ polypeptide insecticidal to insects in the order Coleoptera. ID NO: 51-819 or variant thereof does not compete with 0487. In some embodiments the methods of controlling binding sites for cry proteins in Such insects. Lepidoptera and/or Hemiptera insect infestation in a trans genic plant and promoting insect resistance management the Methods for Increasing Plant Yield at least one of the insecticidal proteins comprises a protein of SEQID NO: 2, SEQID NO:3, SEQID NO: 4, SEQID NO: 0494 Methods for increasing plant yield are provided. 51-819 or variants thereof, insecticidal to insects in the order The methods comprise providing a plant or plant cell express Coleoptera. ing a polynucleotide encoding the pesticidal polypeptide 0488. In some embodiments the methods of controlling sequence disclosed herein and growing the plant or a seed Lepidoptera and/or Hemiptera insect infestation in a trans thereof in a field infested with a pest against which the genic plant and promoting insect resistance management polypeptide has pesticidal activity. In some embodiments, the comprise expressing in the transgenic planta PHI-4 polypep polypeptide has pesticidal activity against a lepidopteran, tide and a cry protein insecticidal to insects in the order coleopteran, dipteran, hemipteran or nematode pest, and the Coleoptera having different modes of action. field is infested with a lepidopteran, hemipteran, coleopteran, 0489. In some embodiments the methods of controlling dipteran or nematode pest. Coleoptera insect infestation in a transgenic plant and pro 0495. As defined herein, the "yield” of the plant refers to moting insect resistance management comprise in the trans the quality and/or quantity of biomass produced by the plant. genic plant a protein of SEQID NO: 2, SEQID NO:3, SEQ By “biomass” is intended any measured plant product. An ID NO: 4, SEQID NO: 51-819 or variants thereof and a cry increase in biomass production is any improvement in the protein insecticidal to insects in the order Lepidoptera and/or yield of the measured plant product. Increasing plant yield Hemiptera having different modes of action. has several commercial applications. For example, increasing 0490 Also provided are methods of reducing likelihood of plant leafbiomass may increase the yield of leafy vegetables emergence of Lepidoptera and/or Hemiptera insect resistance for human or animal consumption. Additionally, increasing to transgenic plants expressing in the plants insecticidal pro leaf biomass can be used to increase production of plant teins to control the insect species, comprising expression of a derived pharmaceutical or industrial products. An increase in PHI-4 polypeptide insecticidal to the insect species in com yield can comprise any statistically significant increase bination with a second insecticidal protein to the insect spe including, but not limited to, at least a 1% increase, at least a cies having different modes of action. 3% increase, at least a 5% increase, at least a 10% increase, at 0491 Also provided are methods of reducing likelihood of least a 20% increase, at least a 30%, at least a 50%, at least a emergence of Coleoptera insect resistance to transgenic 70%, at least a 100% or a greater increase in yield compared plants expressing in the plants insecticidal proteins to control to a plant not expressing the pesticidal sequence. the insect species, comprising expression of a protein of SEQ 0496. In specific methods, plant yield is increased as a IDNO: 2, SEQIDNO:3, SEQIDNO:4, SEQIDNO:51-819 result of improved pest resistance of a plant expressing a or variants thereof, insecticidal to the insect species in com PHI-4 polypeptide disclosed herein. Expression of the PHI-4 bination with a second insecticidal protein to the insect spe polypeptide results in a reduced ability of a pest to infest or cies having different modes of action. feed on the plant, thus improving plant yield. 0492. Also provided are means for effective Coleoptera 0497. These and other changes may be made to the inven insect resistance management of transgenic plants, compris tion in light of the above detailed description. In general, in ing co-expressing at high levels in the plants two or more the following claims, the terms used should not be construed US 2014/0274885 A1 Sep. 18, 2014

to limit the invention to the specific embodiments disclosed in sequencing primer followed by full length sequence confir the specification and the claims. mation with multiple sequencing primers. 0498. The entire disclosure of each document cited (in cluding patents, patent applications, journal articles, Example 2 abstracts, manuals, books, or other disclosures) in the Back ground of the Invention, Detailed Description, and Examples is herein incorporated by reference in their entireties. Purification of MBP::PHI-4 Fusion Polypeptides 0499. The following examples are put forth so as to pro vide those of ordinary skill in the art with a complete disclo 0503 Polynucleotides encoding PHI-4 polypeptides were Sure and description of how to make and use the Subject expressed in a modified pMAL vector (New England Bio invention, and are not intended to limit the scope of what is Lab) as a fusion (i.e. MBP::PHI-4: SEQID NO: 6) with MBP regarded as the invention. Efforts have been made to ensure (maltose binding protein). The pMAL vector was modified to accuracy with respect to the numbers used (e.g. amounts, attacha 6xHis tag to the N-terminus of MBP. In order to clone temperature, concentrations, etc.) but some experimental the polynucleotide encoding the MBP::PHI-4 fusion protein errors and deviations should be allowed for. Unless otherwise (SEQID NO: 6), Sph1 and BamH1 sites were engineered in indicated, parts are parts by weight, molecular weight is aver the vector at the cloning site. The polynucleotide (SEQ ID age molecular weight; temperature is in degrees centigrade; NO: 1) encoding the PHI-4 polypeptide (SEQID NO: 2) was and pressure is at or near atmospheric. amplified with a forward primer (SEQ ID NO:32) overlap ping the Sph1 site and a reverse primer (SEQ ID NO: 33) EXPERIMENTALS overlapping the BamH1 site. This PCR product was digested with Sph1 and BamH1 and cloned into pMAL that was precut Example 1 with the same enzymes. The forward primer was designed such that polynucleotides encoding both MBP and PHI-4 Generating PHI-4 Genes polypeptide (SEQ ID NO: 2) within the MBP::PHI-4 gene 0500 Polynucleotides having single codon substitutions (SEQID NO:5) were ligated in frame. The plasmid contain compared to the PHI-4 polypeptide of SEQ ID NO: 1 were ing the polynucleotide (SEQID NO: 5) encoding the MBP:: generated. As described in the examples below, the corre PHI-4 polypeptide (SEQ ID NO: 6) was transformed into E. sponding PHI-4 polypeptides were expressed, purified and coli BL21 (DE3) cells. The BL21 (DE3) cells were grown in assayed for WCRW insecticidal activity in order to assess the MagicMediaTM (Life Technologies) in either 96 deep well corresponding activity diversity. A reverse mutagenesis plates or flasks in a shaker running at 250 rpm at 37°C. for 8 primer and a complementary forward mutagenesis primer hrs. followed by 16° C. for 48-60 hrs. During the 16° C. were designed to create the desired amino acid substitution(s) incubation, the MBP::PHI-4 polypeptide fusion proteinaccu at the site(s) of interest. Typically the mutagenesis primer was mulated in the BL21 (DE3) cells as a soluble protein. between 30 to 45 bases in length with two or more bases, (0504. In order to purify the MBP::PHI-4 fusion protein usually 10 to 15, on both sides of the site of interest. In the (SEQID NO: 6), the E. coli cells were harvested by centrifu case of Saturation mutagenesis, degenerated primers that gation and treated in a lysozyme solution consisting of 2 cover all possible amino acid residues were used. mg/ml lysozyme in 50 ml sodium phosphate buffer at pH 8.0 0501 Unless otherwise noted, the mutagenic reactions containing 300 mM NaCl, 2 um endonuclease (Epicentre) were carried out using Agilent's QuikChangeTM Lightening and 5 mM MgCl, for 3 hrs. at 37°C. with gentle shaking. The Site-Directed Mutagenesis kit. Materials provided in the kit lysozyme treated E. coli cells were then disrupted with 1% are QuikChangeTM Lightening Enzyme, 10x QuikChangeTM Triton X100 and clear lysate containing the MBP::PHI-4 Lightning Buffer, dNTP mix, QuikSolution reagent and Dpn1 proteins were prepared by centrifugation at 4000 rpm, 30 min restriction enzyme according to the manufactures directions. (96 well plates) or 9000 rpm (flask produced samples). His 0502 PCR amplifications were typically carried out with tagged MBP-PHI-4 polypeptide fusion proteins were purified ExpandTM High Fidelity PCR system (Roche, Switzerland) in from the clear lysates by affinity chromatography using 50 ul containing 50-100 ng templates, 0.4-2 uM primer pair, NiNTA agarose (catalog #30450; Qiagen) following the 200 uM dNTPs and 2 Units of DNA polymerase. The manufacturer's standard procedure. For high throughput pro mutagenesis reaction was initiated by pre-heating the reac tein purification, Pall 96 deep well filter plates (Pall Corpo tion mixture to 94°C. for 3 min, followed by 16 cycles of the ration: Catalogue #5051) were used for the affinity chroma following cycling program: 94°C. for 1 min, 52°C. for 1 min tography. The purified MPB::PHI-4 polypeptide fusion and 68°C. for 8, 12, 16 or 24 min according to the length of protein was eluted from NiNTA agarose and passed through template. The mutagenesis reaction was completed by incu Sephadex G25 to change the phosphate buffer to 25 mM bation at 68°C. for 1 h. The PCR-amplification products were HEPES-NaOH, pH 8 and used in insect bioassays for deter evaluated by agarose gel electrophoresis. The PCR products mining the insecticidal activity against Western Corn Root were purified by QIAquickTM PCR purification kit (Qiagen, worm (WCRW). Calipar GXII capillary electrophoresis with Germany) and further treated with the restriction enzyme a protein chip (Agilent; catalogue iP/N760499) was used to Dpn1. An aliquot of 1 Jul of this PCR product was typically determine the MPB::PHI-4 polypeptide concentrations. The transformed into BL21 (DE3) cells and inoculated on Luria protein analysis was repeated at least 3 times until the final Bertani (LB) plate containing 100 g/ml amplicillin. About 10 concentration was within the predetermined deviation (less colonies in the case of single amino acid mutation or 48 or than 10%). Unless otherwise noted, the PHI-4 polypeptides more colonies for Saturation mutagenesis were selected and disclosed herein were expressed, purified and assayed for plasmid DNA was isolated for sequencing. Two step sequenc WCRW insecticidal activity as maltose binding protein ing was used, first for specific mutation site(s) with one fusions (i.e. MBP::PHI-4: SEQID NO: 6) as described above. US 2014/0274885 A1 Sep. 18, 2014 69

Example 3 insect responses were scored under a magnifying glass. The sigmoid dose-response values (Responses) were converted to Determination of WCRW Insecticidal Activity of linear probit dose-response values using SAS-JMPR), Gener MBP::PHI-4 (SEQID NO: 6) and alized Linear Model, Binomial Response, Probit). The MBP:PHI-4-SFR12-004 (SEQ ID NO:31) response for each protein in replicates was Summed, this Sum Polypeptides was compared with the probit dose-response line of the activ 0505. The activity of MBP::PHI-4 (SEQ ID NO: 6) and ity reference protein and the nominal fold improvement in MBP::PHI-4-SFR12-004 (SEQ ID NO: 31; Example 8) potency was calculated. This nominal fold improvement esti polypeptides against WCRW (western corn rootworm, mated for a given dose in a given experiment is defined as the Diabrotica virgifera virgifera) was determined in an artificial Fast Activity Evaluation Guide Number (FAEGN). For diet feeding assay essentially as described by Cong, R. et al. example, if a PHI-4 polypeptide showed a certain% response (Proceedings of the 4th Pacific Rim Conferences on Biotech value at 40 ppm and comparison to the Probit curve indicated nology of Bacillus thuringiensis and its environmental that the same response is predicted for the reference protein at impact, pp. 118-123, ed. by R. J. Akhurst, C. E. Beard and P. 100 ppm, then the FAE Guide Number is 2.5 (100/40). Hughes, published in 2002, Canberra, Australia). The assays According to this analysis, the PHI-4 polypeptide is nomi were conducted on an artificial diet containing dilutions of nally 2.5 times more potent than the reference PHI-4 polypep these polypeptides. The MBP::PHI-4-SFR12-004 polypep tide protein. The FAE assay was typically done with 2 differ tide fusion (SEQID NO:31) and MBP::PHI-4 (SEQID NO: ent doses of PHI-4 polypeptides at a time and performed in 6) fusions were prepared as above, and 10 uL of protein three independent experiments, generating 6 FAEGN for samples were mixed with 50 uL of molten (40-50° C.) artifi each mutant in a typical FAE evaluation. The mean FAEGN is cial insect diet especially prepared for Diabrotica sp. with calculated to yield the Mean FAE Index (MFI). As used herein low temperature melting agarose, whey protein and wheat “Mean FAE Index” (MFI) refers to the mean of multiple germ. The diet-PHI-4 polypeptide mixture was placed in each FAEGN (typically 6 or more); unless otherwise indicated well of a 96 well micro-titer plate. Four or more neonate MFI is understood to be an arithmetic mean of FAEGN. For WCRW larvae were placed in each well to feed for 4 days at each protein, a two sided t-test was done comparing the mul 25° C. and the response of insects towards the proteins was tiple FAEGN from the clone with FAEGN values from the scored using a 0-3 numerical scoring system based on the size reference protein (typically 48-96 FAEGN of SEQIDNO: 6). and mortality of the larvae in each well. If no response (or The two-sided t-test was done between these 48-96 FAEGN normal growth) was seen, a score of 0 was given. When the associated with the reference protein and the 6 or more growth was slightly retarded, a score of 1 was given. A score FAEGN associated with the variant of interest. The Bonfer of 2 meant that the larvae were severely retarded in growth roni correction was used to evaluate p-values (number of (close to neonate size). A score of 3 meant death to all the novel proteins/alpha) for the hypothesis that a given variant is larvae in the well. The percent response (% Response) for significantly different in insecticidal activity compared to the each treatment was calculated by dividing the total score, a reference protein, MBP::PHI-4 fusion protein (SEQID NO: sum of scores from replicated wells for each treatment, by the 6) unless otherwise noted. The Bonferroni correction was total highest possible scores and multiplying by 100 to yield used to evaluate p-values (number of novel proteins/alpha) “% Response'. For example, if one treatment (one sample, for the hypothesis that a given variant is significantly different one dose) had 6 replicated wells, the total highest possible in insecticidal activity compared to the reference protein, score would be 3x6=18. An observed set of scores of 3, 2, 2, MBP::PHI-4fusion protein (SEQID NO: 6) unless otherwise 3, 2, 2 for six wells at a given dose for a given variant would noted. result in (14/18)x100–78% Response. (0509 Effective Concentrations (EC50) evaluation: Vari 0506 Fast Activity Evaluation (FAE) Analysis: ants of particular interest were assayed at higher power in 0507. The PHI-4 polypeptides at two concentrations were more extensive dose response curves to more accurately esti assayed along with 4 doses (two-fold dilutions bracketing the mate the EC50s. The preparation of dose response, infesta EC50) of the reference MBP::PHI-4 fusion protein (SEQ ID tion, incubation and scoring were as described for the FAE NO: 6) within one 96-well assay plate. The concentrations of assay format. EC50 determinations were typically carried out the PHI-4 polypeptides were within the 4 doses of the refer with a no insecticidal protein control plus seven two-fold ence protein concentrations, preferably around the middle dilutions bracketing the expected EC50 (typically 100 ppm point of the 4 dose concentrations. Each sample plate con for MBP::PHI-4: SEQ ID NO: 6) and 24 or more replicate tained the reference MBP::PHI-4 protein (SEQID NO: 6) in wells within a given experiment. As used herein, the EC50 is a significant number of wells such as 16 wells in 4 Separate defined as the predicted point with 50% response in the scor doses. In each plate, up to 80 MBP::PHI-4 polypeptide vari ing scheme. It is a combination of growth or feeding Inhibi ants were included and assayed for activity comparison with tion and lethal responses. In order to determine EC50 values, the reference PHI-4 polypeptide protein. From a sample each treatment (one dose) was repeated 6 or more, usually 24, plate, 10 ul of samples from each well were picked by multi times. channel pipette and dispensed in one assay plate containing 0510 Data from exemplary FAE and EC50 determina 50 ul molten diet in each well and mixed on a shaker. This tions are given in FIGS. 1 and 2 respectively. MBP::PHI-4- process of producing the assay plate was repeated as many as SFR12-004 (SEQ ID NO:31; Example 8) is a variant with 6 times or more to produce a desired number of replicate improved insecticidal activity. FIG. 1 shows the primary data assay plates. After the diet was solidified and cooled to 4°C., for a typical FAE assay. Proteins were purified and quantified neOnate as described above. The % response in a typical FAE assay is 0508 WCRW larvae were placed in each well, the plate given on the y axis. The protein concentration (toxin portion was sealed with perforated Mylar film and incubated in a of the protein only) is given on the X axis. The geometric mean constant temperature incubator at 25°C. After 4 days, the FAE Index in this instance is 4.0. FIG. 2 shows the data for a US 2014/0274885 A1 Sep. 18, 2014 70 typical EC50 measurement. Proteins were purified and quan diet) is plotted on a Probit plot. The difference between tified as described above. The fractional response (multiply response expected for MBP::PHI-4 (SEQIDNO: 6) at a given by 100 to get “96 Response') is given on the y axis. The dose, based on the Probit curve, and that observed for the inferred EC50s are 245 ppm (MBP::PHI-1 of SEQID NO: 6) mean score for a PHI-4 polypeptide variant at that given dose and 48 ppm (SFR12-004; SEQID NO:31). The data indicate is calculated and this difference is defined as the Deviation that the insecticidal activity of MBP::PHI-4-SFR12-004 Score. A negative Deviation Score indicates that the response (SEQID NO:31) is increased relative to that of MBP::PHI-4 is lower than that which is expected for the relevant parental (SEQ ID NO: 6). backbone (typically MBP::PHI-4: SEQID NO: 6) at the same 0511. The MBP::PHI-4 fusion is rapidly cleaved in the concentration and indicates that the variant is nominally less presence of insect gut fluid to yield MBP and mature PHI-4 active than the parental backbone. A positive Deviation Score protein and it is believed that the insecticidal activity is due to indicates that the variant is nominally more potent than the the cleaved toxin molecules. The MBP::PHI-4 fusion protein parental backbone. As used herein, the Mean Deviation Score (SEQ ID NO:6) was digested with 1/100 (w/w) Factor Xa refers to the arithmetic mean of multiple Deviation Scores (New England Biolabs) at 25°C. overnight and the PHI-4 which are typically derived from independent experiments. polypeptide was purified by Superdex 200 column chroma The Mean Deviation Score is used to estimate rank order of tography utilizing the size difference and a weak affinity of activities associated with a set of variants within a given MBP to Superdex. The MPB::PHI-4 fusion was also cleaved experiment. The Mean Deviation Scores for the 209 variants with trypsin and the mature PHI-4 polypeptide (derived from of this example are given in Megatable 1 and is typically an SEQ ID NO: 6) was purified. The mature, purified PHI-4 average of at least three independent Deviation Score mea polypeptide derived from Factor Xa or trypsin cleavage has a SurementS. MW of -60kDa as measured on SDS-PAGEgels and is fully reactive to polyclonal antisera that also react with the MBP:: Example 5 PHI-4 fusion protein. The EC50 of this “mature” PHI-4 polypeptide fragment is within experimental error of the Single Amino Acid Substitution Mutants #2 (SFR) EC50 of the MBP::PHI-4 parental protein (SEQID NO: 6, as 0513. A BLAST search revealed that AXMI-205 is a bac calculated on the basis of ppm associated with the toxin terial perforin-like protein. Perforin proteins from Clavibac fragment and excluding ppm associated with the MBP tor michiganensis (GenBank Accession number: domain of SEQID NO: 6. YP 001223127: SEQ ID NO: 49), Laccaria bicolor (Gen Example 4 Bank Accession number:XP 001885969; SEQID NO: 48), Marinomonas sp. (GenBank Accession number: ZP 01.077945: SEQ ID NO: 38), Nematostella vectensis Single Amino Acid PHI-4 Polypeptide Variants (GenBankAccession number: XP 001617993: SEQID NO: (S1-S4) 50), Photorhabdus luminescens (GenBank Accession num 0512. A set of 209 PHI-4 polypeptide single amino acid ber: NP928713; SEQID NO: 821), and Serratia proteamacu substitution variants spread across both the N and C terminal lans (GenBank Accession number: YP 00147861; SEQ ID portions of the protein were made and characterized (Mega NO: 820) were found to be homologous to AXMI-205. The table 1: MUT ID: 1-209). The mutagenesis template was the N-terminal region of up to 311 amino acid of AXMI-205 polynucleotide of SEQ ID NO: 5 encoding PHI-4 (SEQ ID (SEQ ID NO: 35) is highly homologous to those perforin NO: 6) as an MBP fusion. The mutations were made using the proteins. Among those perforin proteins, the 3D X-ray struc QuickChange kit (Agilent; Catalogue #200524) essentially ture of Photorhabdus perforin-like protein has been pub as described in Example 1. The particular amino acid substi lished (Science 317, 1548-1551, 2007; PDB ID: 2OP2: SEQ tutions relative to PHI-4 (SEQID NO: 2) areas indicated in ID NO: 822). According to the leading theory of perforin Megatable 1. For example in Megatable 1, the PHI-4 polypep mode of action, the protein inserts 5 alpha helices of its tide identified as MUT ID: 1 has a valine substituted for the N-terminal region into the target host membrane to form a native amino acid alanine at position 202 of PHI-4 (SEQ ID large size pore (Proc. Natl. Acad. Sci. USA 102, 600-605, NO: 2) and referred to “A202V. The polypeptide variant for 2005, Immunology Today, 16, 194-201, 1995). In the Photo which activity is reported was prepared as an MBP fusion that rhabdus 2CRP2 structure, there are two loops between Alpha C is identical to SEQID NO: 6 except for this singleamino acid and D and Alpha I and J are considered to be the site for substitution. In a similar manner, MUTID: 1-872 (Megatable initiating the membrane insertion of those 5 alpha helices. 1) are all made in the context of SEQ ID NO: 6: MUT ID: These loops are called membrane insertion initiation loops. 873-910 (Megatable 1) are made in the context of SEQ ID The primary sequences of those perforin proteins listed below NO: 8: MUT ID: 911-1135 (Megatable 1) are made in the were aligned using Vector NTI Align X function in order to context of SEQID NO: 10. All polypeptides of Megatable 1 identify the membrane insertion loop sequences of AXMI were expressed and purified as MBP fusions as described in 205 (SEQID NO: 35). Amino acid sequences of V92-A103 Example 2. The PHI-4 polypeptides were expressed as MBP and G211-E220 of SEQ ID NO: 6 were aligned with the fusions and purified as described in Example 2. The assay membrane insertion initiation loops identified in the 2CRP2 protocol for WCRW insecticidal activity of the PHI-4 structure. A number of acidic, basic and other hydrophilic polypeptides was essentially as described for FAE assays in amino acids were found in the AXMI-205 (SEQID NO: 35) Example 3 using SEQID NO: 6 as the reference protein. For membrane insertion loops indicating these loops are exposed the analysis of the data a “Mean Deviation Score” was calcu to the solvent. lated rather than a mean FAE Index. This is a related metric 0514 Based on this homology model and prior mutation that is calculated as follows. Data from four two-fold dilu activity relationship data, another 664 single amino acid Sub tions of MBP::PHI-4 (typically about 25, 50, 100 and 200 stitution PHI-4 polypeptide variants were made in order to ppm final concentration of PHI-4 fragment in artificial insect assess the sequence-insecticidal activity-relationships at the US 2014/0274885 A1 Sep. 18, 2014

selected positions. This set of 664 point mutations (Megat (SEQID NO:9) as the DNA template. The PHI-4-D09 back able 1: MUT ID: 210-872) were made with MBP::PHI-1 bone contains the following substitutions relative to SEQID polypeptide of SEQID NO 5 as the DNA template. Mutations NO: 2: L401, Y98F, L145V, L163V, 1172L, V3551, and were made according to the method of Example 1. The P412A (numbers are relative to the PHI-4 polypeptide back mutagenesis oligonucleotides used to create an exemplary bone of SEQID NO: 2. Mutagenesis was done by a modifi mutant (PHI-4-R97D, SEQ ID NO: 7) are SEQID NO: 13 cation of the method of Dominy et al (Methods in Molecular and SEQID NO: 14. The other PHI-4 polypeptides (Megat Biology, Vol. 235:209-223; 2003). Briefly, “NNK' mutagen able 1: MUT ID: 210-872) were made in the same manner esis at position 396 was done as follows. A pMAL vector using mutagenesis oligonucleotides designed to create the encoding SEQID NO:9 was amplified by inverse PCR for 20 cycles using SEQ ID NO: 15 & SEQID NO: 16. The PCR selected substitutions at the desired residues of the protein. product was diluted 10-fold, subjected to one additional The resulting PHI-4 polypeptides were purified as MBP round of amplification using SEQID NO: 16 & SEQID NO: fusions and the activity measured in the FAE assay format or 17. The PCR product was purified on QuiaGuickTM column, EC50 assay format as described in Examples 2 & 3. The Mean phosphorylated with T4 polynucleotide kinase, circularized FAE Indices associated with these 664 PHI-4 polypeptides with T4DNA ligase and transformed into E. coli BL21 (DE3) are given in lines 210-872 of Megatable 1. cells. Candidate colonies were amplified by colony PCR and Example 6 the PCR product was sequenced first with a single primer to confirm the presence of the desired mutation and Subse SFR16 Point Mutants quently sequenced fully with multiple primers to identify clones with no additional mutations. All other PHI-4 polypep 0515. Another set of 38 single amino acid substitution tides of this example (Megatable 1, lines 911-1135) were PHI-4 polypeptide variants (Megatable 1: MUTID: 873-910) made by a similar manner using mutagenesis oligonucle were made with MBP::PHI-4-R97D (SEQID NO 7: Example otides designed to create the selected substitutions at the 5) as the backbone. The substitutions were selected based on desired residues of the protein. Positions with multiple the sequence-activity relationships inferred from the PHI-4 desired mutations were made with degenerate forward prim polypeptide variants of the preceding Examples. Mutations ers whereas positions with only one desired mutation were were made according to the method of Example 1. Variants made with non-degenerate primers. Clones with the desired were purified as MBP fusions and activity measured in FAE sequences were used to express protein essentially as assay format as described in Examples 2 & 3. The Mean FAE described in Example 2. Protein purification, activity mea Indices associated with these mutants are given in lines 873 surements and statistical analysis was done essentially as 910 of Megatable 1. The reference for the Mean FAE Index in described in example 3. The Mean FAE Index reflects the fold this example is SEQID NO: 8. difference relative to PHI-4 polypeptide of SEQ ID NO: 6. The Mean EC50 of PHI-4-D09 (SEQ ID NO: 10) was mea Example 7 sured at high statistical power and is 1.3-fold improved rela tive to MBP::PHI-4 (SEQID NO: 6). PHI-4 polypeptides of PSR3 Mutants this example with Mean FAE Index >1.3.x are deemed nomi 0516. The C-terminal domain of AXMI-205 shares sig nally improved relative to the parental backbone (MBP::PHI nificant homology with proteins that have f3-prism 3D struc 4-D09: SEQID NO: 10) and diversity meeting this criterion tural folding patterns. In the typical B-prism, the oligosaccha was used for production of Subsequent combinatorial ride binding site is almost always in a cavity formed between mutantS. the apex and belt-loops from the same Greek-key motif. The Example 8 primary amino-acid sequence motif has been identified as G-X-D (SEQ ID NO:39) in banana lectin, which has two Identification of Combinatorial Mutants of PHI-4 binding sites (Meagher J L et al Glycobiology 15:1033-42: Polypeptides with Improved Insecticidal Activity as 2005). The primary sequence of the PHI-4 polypeptide of Measured in an Artificial Insect Diet Feeding Assay SEQID NO: 2 contains 3 such motifs. The canonical structure 0518 Activity diversity identified in Examples 4-7 was of these motifs is indicated in FIG.3. In addition, the G-X-D used to create 192 combinatorial PHI-4 polypeptide variants motif (SEQID NO:39) can be extended toward the N-termi (SEQ ID NO: 51-242: Megatable 2). The PHI-4 polypeptide nal direction into the D-X-G-IS/T-G-X-D motif (SEQ ID variants were made by sequential point mutagenesis by the NO: 40), which are present 1, 2 or 3 times in 24 proteins that method of Example 1. In all cases, the indicated PHI-4 are orthologous to the C-terminal portion of AXMI-205 polypeptide variants were made as MBP fusions with the (GenBank accession numbers: gil 136474758; gil 136444345; same linker as is indicated in SEQID NO 6. The MBP::PHI-4 gil 136141087; gil 143658948; gil 142085802; gil 135275135: polypeptides were expressed and purified as indicated in gil 138446054; gi294814724; gil 170109524; gil 156316804: Example 2 & 3. Purified MBP::PHI-4 polypeptides were gil 156377786; gil 170109526; gi177456557; gil 1209377; assayed in FAE assays to derive a Mean FAE Index or in EC50 gi302823768; gi302532087; gi256764986; gi302787479; assays as indicated in Example 3. Thirty-seven exemplary gi302823738; gil 169762636; gi302766657; gi270056485; active PHI-4 polypeptide variants with increased Mean FAE gi302792467; gi238488445;). Each of the 3 loops in the Indices are given in Table 3, along with the sequence variation PHI-4 polypeptide of SEQID NO: 2 has potential to bind an relative to SEQID NO: 6. The substitutions relative to PHI-4 oligosaccharide, a putative binding receptor present in polypeptide of SEQ ID NO: 2 are given in the right-most WCRW mid-gut cell membrane surface. column. All proteins were expressed and purified as MBP 0517. As indicated in Megatable 1,225 PHI-4 polypeptide fusions proteins. The reference protein for the Mean FAE variants (MUT IDs: 911-1135) were made to introduce an Index is MPB::PHI-4 (SEQID NO: 6). The functional data on additional amino acid substitution into the PHI-4 polypeptide all of the PHI-4 polypeptides of this example is given in lines of PHI-4-D09 (SEQ ID NO: 10) using MBP::PHI-4-D09 51-242 of Megatable 2.

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TABLE 3-continued SEQID Ex. # NO: Alias FAE p value Mutation List (vs SEQID NO: 2) 8 80 SFR1O- 6.6 2.09E-14 RO97D, S401 H, K402H O45 8 81 SFR1O- 6.4 2.15E-06 RO97D, S401G, K402H, K52OE, 87 Q527K 8 82 SFR1O- 6.3 2.89E-08 RO97D, G462A, R464K, K465M O60 8 S1 SFRS- 6.2 1.65E-05 RO97D, R293Q, R416E, K52OE O14 8 184 SFR13- 6.1 1.84E-13 RO97D, KO99L, E220D, K289L, O18 V355I, S401G, P412A, K52OE 8 154 SFR11- 5.5 1.54E-09 RO97D, E220D O13 8 241 SFR17- 5.5 5.11 E-22 D042N, EO46N, R097D, KO99L, OO7 E220D, K289L, V355I, S401H, K402H, P412A

Example 9 164:49-53; 1995). An additional treatment was implemented as described (Saaem, I. et al. Nucleic Acids Research, Pub Identification of Combinatorial Mutants of PHI-4 lished Nov. 29, 2011, 1-8). Briefly, in a typical gene synthesis Polypeptides with Improved Insecticidal Activity as reaction a set of oligonucleotides of 120 bases each encoding Measured in an Artificial WCRW Insect Diet both top and bottom Strands of the target gene were designed. Feeding Assay Complementary oligos typically overlap by 54-65 nucle otides. Oligos to make synthetic genes are combined Such that 0519) Activity data from 315 PHI-4 polypeptide combi a final concentration of each oligo is approximately 0.05-1 natorial variants (SEQID NOS 243-558) is provided in Mega uM. Gene assembly is typically done with Herculase II (Agi table 2. Libraries were initially prepared by incorporation of lent) using the following cycling program: 98° C. 3 min diversity into SEQ ID NO: 5. The diversity was largely followed by (96° C.x30 sec, 40° C.x30 sec, 72° C.x30 sec)x derived from that described in Example 4. Oligonucleotides 24 cycles. The initial PCR is then used as template for a encoding diversity at forty positions were incorporated into second PCR in which a second pair of primers is used to SEQ ID NO: 5 in a DNA shuffling reaction essentially as amplify the fully synthetic gene product. Typical PCR con described (Ness et al., Nature Biotechnol. 20, 1251; 2002). Briefly, oligonucleotides typically of 30-45 bases in length ditions for the second synthetic gene reaction were 98°C. 3 encoding the diversity elements of interest were mixed at min followed by (96° C. 30 sec, 50° C. 30 sec, 72° C. 35 sec), 0.02-2 micromolar each in the presence of an appropriate x 24 cycles. Reactions were analyzed by 1% E-gels (Invitro concentration of fragments of SEQID NO: 5. This reaction gen). A Subsequent treatment consisting of a re-annealing was assembled, rescued and cloned essentially as described step, treatment with Cell (Transgenomics; Catalogue for synthetic genes in Example 10 and as described (Ness et #706020) and subsequent amplification (25 cycles) was done al., Nature Biotechnol. 20, 1251; 2002). Improved variants essentially as described (Saaem, I. et al. Nucleic Acids from initial libraries were subjected to family DNA shuffling Research, Published Nov. 29, 2011, 1-8). A third and final essentially as described (A. Crameri, et al Nature 391, 288: amplification of the synthetic gene was done with similar 1998). This family shuffled library was screened by methods PCR conditions in a single cycle. The product of this reaction similar to those described in Example 3 (FAE) and to those was purified and ligated by Gibson ligation method (New described in Example 4 (Mean Deviation Score). Selected, England Biolabs; Catalogue #E2611 L) to an appropriate improved PHI-4 polypeptide variants from the second round vector transformed to chemical competent BL21 (DE3) cells. of DNA shuffling were further diversified by recombining N Sequence verified clones (Lines 559-716 of Megatable 2) terminal and C terminal domains of elite clones using the comprising the PHI-4 polypeptide were expressed as MBP method of splicing by overlap extension (R. Horton, et al., fusions, purified and assayed essentially as described in Gene 77:61-68; 1989) to yield novel variants. All variants Examples 2 and 3. Table 4 shows the SEQ ID NOs and were purified by the method of Example 2 and assayed by the substitutions relative to the PHI-4 polypeptide of SEQ ID method of Mean Deviations of Example 4. PHI-4 polypep NO: 2 for twenty active variants. The mean FAE Index is tides variants identified are given in Megatable 2 (SEQ ID calculated relative to the MBP::PHI-4 backbone (SEQ ID NO: 243-558). NO: 6). Example 10 TABLE 4 SEQ Identification of Combinatorial Mutants of PHI-4 Ex. ID Mutation List Polypeptides with Improved Insecticidal Activity as # NO: Alias FAE p value (vs SEQID NO: 2) Measured in an Artificial WCRW Insect Diet 10 S59 PSR1- 25.6 3.26E-05 D042N, YO98F, L145V, L153I, Feeding Assay 1-076 I172L,Y206F, I283V, V355I, 0520. A set of 158 PHI-4 polypeptide combinatorial vari 10 S60 PSR1- 22.7 4.19E-05 D042N, YO98F, I283V, V355I ants (SEQ ID NO: 559-716) were prepared by total gene 1-074 synthesis, essentially as described by Stemmer et al (Gene US 2014/0274885 A1 Sep. 18, 2014 74

TABLE 4-continued The resulting MBP::PHI-4 polypeptide fusion proteins were expressed, purified, assayed for insecticidal activity on SEQ Ex. ID Mutation List WCRW larvae and analyzed for insecticidal relative to MBP:: # NO: Alias FAE p value (vs SEQID NO: 2) PHI-4 (SEQID NO: 6) using the Mean FAE Index metric as described in Examples 2 & 3. Table 5 shows the Mean FAE O S61 PSR1- 15.9 7.67E-06 E046N, YO98F, L145V, Y171F, Indices, SEQ ID NOs and substitutions relative to PHI-4 2-145 172L, D182O, E278N, V355I, polypeptide of SEQ ID NO: 2 for twenty active PHI-4 polypeptide variants of this example. The Mean FAE Index was calculated relative to the MBP::PHI-4 backbone (SEQID NO: 6). The insecticidal activities of the PHI-4 polypeptides in Table 5 (Mean FAE Index) reflect the arithmetic means of three independent experiments and are expressed as fold WCRW insecticidal activity improvement of the PHI-4 polypeptide variants relative to MBP::PHI-4 (SEQID NO: 6). As indicated, the mean FAE Indices range from 0.26x to >8x (fold improvement relative to MBP::PHI-4). The majority of the PHI-4 polypeptides have increased insecticidal activity relative to MBP::PHI-4 (FAE>1). The p values indicate that the measured differences relative to MBP::PHI-4 (SEQ ID NO: 6) are highly significant.

Example 11

Identification of Combinatorial Mutants of PHI-4 Polypeptides with Improved Insecticidal Activity as Measured in an Artificial Insect Diet Feeding Assay 0521. Sixty-six PHI-4 polypeptide variants (SEQID NO: 717-783), containing permutations of a number of substitu tions, were made by total gene synthesis, essentially as described in Example 10. The substitutions were done in the context of a backbone (PSR1-2-105: SEQID NO: 584 from Megatable 2) containing the following substitutions relative to PHI-4 polypeptide of SEQID NO: 2: E46N, R97N, Y98F, L145V, Y171F, I172L, V355I, I410V, V455I, and W457N.

US 2014/0274885 A1 Sep. 18, 2014 76

Example 13 the PHI-4 polypeptide protein at the C-terminal side of Lys at 520 of SEQID NO: 2. The N-terminal sequence of the 24 kDa Accordance Between FAE and EC50 Assays band was VDKVLLMD (amino acids 314 to 321 of SEQID 0523 Experimental data on Mean FAE Index and mean NO: 2). The mass-spectrometry analysis on the 24 kDa frag EC50 for twenty-five PHI-4 polypeptide variants is given in ment confirmed the C-terminal region of PHI-4 polypeptide FIG. 4. PHI-4 polypeptide variants were first tested in the of SEQID NO: 2 starting with Val at 314 relative to SEQ ID FAE assay and then selected ones were retested in a multiple NO: 2 as shown by N-terminal sequencing and ending at Lys EC50 assays. In general, the fold improvement in mean FAE at 520 relative to SEQID NO: 2. This indicated that trypsin Index is modestly larger than the fold improvement that was digested the PHI-4 polypeptide of SEQ ID NO: 2 at the subsequently measured in Mean EC50 measurements. This C-terminal side of Lys at 313 of SEQID NO: 2. overall trend is as expected from the phenomenon of regres 0526. From this experiment, it was found that there are at sion toward the mean (International Journal of Epidemiology least two protease accessible sites, Lys at 313 and Lys at 520 2005; 34:215-220). All 25 PHI-4 polypeptides elected for SEQID NO: 6. These two sites were mutated to other amino retesting in EC50 assays repeated as being significantly acid residues by Saturation mutagenesis and it was found that mutations at Lys at 313 and Lys at 520 of SEQ ID NO: 2 improved. FIG. 4 shows the EC50 measurements for repre increase the insecticidal activity significantly. For example, sentative variants from Megatable 2 (SEQID NO: 610, SEQ the activity of the PHI-4 polypeptide variant, K313Q (MUT IDNO:595, SEQIDNO:584, SEQID NO:591, SEQID NO: ID: 889), was enhanced 2.3 fold over the activity of PHI-4 576, SEQID NO: 73, SEQID NO: 74, SEQID NO: 75, SEQ polypeptide of SEQID NO: 2 as measured in an FAE assay ID NO: 79, SEQID NO: 81, SEQID NO: 150, SEQID NO: (Megatable 1). The activity of the PHI-4 polypeptide K520O 150, SEQID NO: 149, SEQ ID NO: 167, SEQID NO: 167, (MUT ID: 881) was increased 3.1 fold. Activity increases SEQID NO: 164, SEQID NO: 164, SEQID NO: 170, SEQ were also found in combinations with other mutations. For IDNO: 170, SEQIDNO:795, SEQID NO: 794, SEQID NO: example, the activity of the PHI-4 polypeptide having the 784, SEQID NO: 799, SEQID NO: 785, SEQID NO: 788, R097D and K520E substitutions (SEQID NO:52) is 3.5 fold SEQID NO: 786, SEQID NO: 796, SEQID NO: 787). higher than that of PHI-4-R097D (MUTID: 8) alone by FAE Example 14 assay. Combinatorial Substitutions Example 16 0524 Example 11 yielded numerous PHI-4 polypeptide Saturation Mutagenesis of Amino Acid Residues variants with improved insecticidal activity based on combi Selected by Site Directed Single Amino Acid natorial substitutions of the three lectin-like motifs described Mutagenesis in Example 7. A combinatorial library was prepared of 120 genes based on this diversity as follows. SEQID NOS 760 & 0527 Certain amino acid residues showed activity 761 each contains unique substitutions in loop 1. SEQ ID changes when mutated by site directed single amino acid NOs 717-726: 728-732; 734–737 & 760 contain unique sub mutagenesis. Those residues were “Selected for saturation stitutions in loop 2. SEQID NOS 761 and 758 contain unique mutagenesis. For the purposes of this example, “Selected Substitutions in loop 3. Gene synthesis was used to create a can refer to single amino acid mutations that affect the activ combinatorial library of these loop sequences essentially as ity, positively or negatively, relative to the parental backbone described in Example 10. These genes can be expressed and in which they were made. More specifically substitutions of assayed for activity on WCRW larvae by the methods Megatable 1 with Mean FAE Indices of <0.7 or of >1.3 are deemed “Selected. For example, Selected amino acid resi described above. dues were found by performing site directed mutagenesis at Example 15 certain residues such as Arg and Lys. These basic amino acid residues were mutated to either acidic (Asp, Glu) or neutral, Mutagenesis of Putative Protease Sensitive Sites of polar (e.g.: ASn and Gln) residues, and the activity of those PHI-4 Polypeptides mutants was determined by the FAE insect assay individually. Acidic amino acid residues such as Asp and Glu were 0525 Trypsin was used to identify the site(s) where pro changed to basic (e.g.: Arg, LyS) or neutral, polar (e.g.: Asn teases possibly attack (Protease Accessible Sites) the PHI-4 and Gln) residues and the mutant activity was determined. polypeptide of SEQID NO: 2. The PHI-4 polypeptide of SEQ Neutral, polar amino acid residues such as Gln and ASn were ID NO: 2 in 50 mM Tris-HCl, pH8 was mixed with 1/50 mutated to either acidic (Asp, Glu) or basic (e.g.: Arg, Lys) (weight/weight) trypsin and incubated for 1 hr at 37°C. It was amino acid residues to see if the activity of those mutants found that protein was relatively resistant to trypsin, with no were changed positively (for example mean FAE Index >1.3 immediate digestion down to the Small fragments, but pro relative to the reference protein) or negatively (for example duced a 55 kDa major band and 24 kDa minor band by mean FAE Index <0.7 relative to the reference sequence). SDS-PAGE analysis after the incubation. These two bands Another example of finding Selected amino acid residues is were excised from the geland analyzed by mass spectrometry based on the sequence-function relationship. Since AXMI and N-terminal sequencing. The N-terminal sequencing 205 is a member of the perforin family it is possible to identify revealed SAANAGQLGN (amino acids 3-12 of SEQID NO: amino acid residues of PHI-4 polypeptide of SEQID NO: 2 2) for the 55 kDa protein indicating that only two amino acid which are involved in the mode of action elements such as residues, Methionine and Alanine were missing from the membrane insertion initiation and receptor binding loops. N-terminal sequence. The mass-spectrometry, however, Amino acid residues found in those regions were considered showed a loss of C-terminal sequence from Ser at 521 to Leu Selected for Saturation mutagenesis in this Example. One can at 536 of SEQID NO: 2. This indicates that trypsin digested use the alanine Scanning to empirically define Selected resi US 2014/0274885 A1 Sep. 18, 2014 77 dues. This technique was used to find additional Selected D394, Y404, Q413, N430, Q449, D497, R500, S504. Satura amino acid residues in the putative receptor binding loops. tion or near Saturation mutagenesis at these positions can be 0528. After any amino acid residues were determined performed by the method of Example 7 or equivalent methods Selected, those residues were subjected to saturation or near and purified and screened the variant proteins for activity by saturation mutagenesis to produce a set of up to 19 mutants the methods of Example 2 & 3 or equivalent methods. for each site (20 all possible amino acids minus the amino acid found in the wild type). The insecticidal activity of all Example 17 these mutants was determined by the FAE insect assay. Satu ration mutagenesis of the Selected amino acid residues, was Transgenic Expression and Activity Evaluation useful for identifying substitutions with Mean FAE Indices of >1, in many cases >1.3. When the activity of one singleamino 0532. The PHI-4 polypeptides of SEQID NOs 22-25 were acid mutation was found to be positive by showing increased cloned under control of the maize ubiquitin promoter (Chris activity over the PHI-4 polypeptide of SEQ ID NO: 2, the tensen and Quail, (1996) Transgenic Research 5:213-218) saturation mutagenesis enabled us to find other mutation(s) into a standard vector suitable for transformation of maize by that showed further increased activity. For example, while the Agrobacterium. Transgenic maize plants were produced by FAE Index of E082O (MUTID: 370) was positive (1.37), the the method of Example 20. Selected TO plants were tested for saturation mutagenesis at this site revealed other mutations susceptibility to WCRW feeding by challenging TO plants showing much higher FAE Indices. For example, the index of with WCRW larvae. After 19-21 days of challenge, the roots E0821 (MUTID: 219) was 7.80 and that of E082L (MUTID: were visually examined and root nodal injury scores were 259) was 2.71 indicating that PHI-4 polypeptide of SEQID recorded as described (Oleson J. et al J. Economic Entomol NO: 2 hydrophobic residues are beneficial at this site as far as ogy 98:1-8; 2005). Root nodal injury scores are indicated in its insecticidal activity is concerned. FIG. 5. The data support the conclusion that the three PHI-4 0529. Other Selected amino acid substitutions resulted in polypeptide variants provide measurable in planta efficacy decreased activity. When these sites were examined further for protection of maize transgenic plants against WCRW. by saturation mutagenesis, substitutions with Mean FAE FIG. 5 shows the T0 seedlings in the V3-V4 growth stage Indices of 1 were observed. For example, the FAE Indices of were challenged as described (Oleson J. et al J. Economic K099Q (MUT ID: 677), K099E (MUT ID: 715) were 0.34 Entomology 98:1-8; 2005) and root nodal injury scores were and 0.26, respectively. This shows that Lysine at this site is recorded. functionally involved in activity and that alternative substitu tions may result in improved activity. In this example, the Example 18 saturation mutagenesis revealed Substitutions with Mean FAE Index >1. For example, the substitution K099L (MUT ID: 299) has a Mean FAE Index of 5.72 (Megatable 1). Simi In Planta Expression of Fusion Proteins lar instances were found across the entire PHI-4 polypeptide 0533 Localization of the protein can also play an impor of SEQ ID NO: 2, for example those indicated in Table 7. tant role in in planta accumulation. One can direct proteins Table 7 shows the Mean FAE Indices for nine pairs of substi Such as PHI-4 polypeptides to the chloroplast using a chlo tutions. All data is from Megatable 1. roplast targeting peptide (CTP). Additionally, one can direct expression to the apoplastic space using fusions to peptides TABLE 7 such as the barley alpha amylase-derived peptide (BAA; SEQ ID NO: 826). One may also direct transgenically expressed Substitution A Substitution B proteins for retention in the endoplasmic reticulum by fusing MUT mean FAE mean FAE to both BAA and the sequence “KDEL (SEQID NO: 828). ID Substitution Index MUTID Substitution Index Proteins can also be directed to the vacuolar space by fusion 570 KO74Q 0.7 215 KO74E 1240 with the C terminal peptide from plant defensins such as the 596 E203Q O.S9 288 E2O3T 2.18 maize defensin 20 C-terminal propeptide (SEQID NO: 824). 800 R235Q O.O6 497 R23SK 1.34 Other functionally equivalent gene elements may be com 906 K313E O.14 889 K313Q 2.29 838 D395Q O.O3 832 D39SR 1.60 bined in a similar manner. One may also direct expression 784 S398A O.11 342 S398Q 1.51 specifically to the roots with root-specific promoters. Each of 629 K402O O.47 216 K4O2F 10.2O these modifications may be made separately or in combina 842 D403Q O.O3 251 D403Y 3.03 tion and any given combination of elements to improve accu 611 D447Q O.S4 211 D447K 31.70 mulation of protein in plant tissue or in functionally improved efficacy of the expressed protein. 0530. The serine at position 98 of SEQ ID NO: 2 was Selected by alanine scanning amino acid residues found in a Example 19 region of the protein that is suspected overlap with the recep tor binding loops. Point mutants with improved potency may Transformation of Maize by Particle Bombardment then be used to prepare and screen combinatorial libraries and Regeneration of Transgenic Plants based on that diversity. 0531 Taking mean FAE Index <0.7 as a definition of 0534. Immature maize embryos from greenhouse donor Selected, the following positions are deemed Selected: P14, plants are bombarded with a DNA molecule containing the D24, Q38, E53, R55, R61, Q75, D76, E83, E118, E126, PHI-4 polypeptide of nucleotide sequence (e.g., SEQID NO: D152, R166, K188, K191, D193, K242, P243, R248, D254, 1) operably linked to an ubiquitin promoter and the selectable L266, D268, A270, D274, D298, K313, D315, K316, D321, marker gene PAT (Wohleben, et al., (1988) Gene 70:25-37), V343, S349, Q360, R361, D368, I373, D376, F378, D379, which confers resistance to the herbicide Bialaphos. Alterna US 2014/0274885 A1 Sep. 18, 2014 tively, the selectable marker gene is provided on a separate tored and scored for expression of the toxin by assays known DNA molecule. Transformation is performed as follows. in the art or as described above. Media recipes follow below. 0549 Bombardment and Culture Media 0535 Preparation of Target Tissue 0550 Bombardment medium (560Y) comprises 4.0 g/L 0536. The ears are husked and surface sterilized in 30% N6 basal salts (SIGMA C-1416), 1.0 mL/L Eriksson's Vita CLOROXTM bleach plus 0.5% Micro detergent for 20 min min Mix (1000xSIGMA-1511), 0.5 mg/L thiamine HCl, utes, and rinsed two times with sterile water. The immature 120.0 g/L sucrose, 1.0 mg/L 2,4-D and 2.88 g/L L-proline embryos are excised and placed embryo axis side down (brought to volume with deionized H0 following adjustment (scutellum side up), 25 embryos per plate, on 560Y medium to pH 5.8 with KOH); 2.0 g/L GelriteTM (added after bringing for 4 hours and then aligned within the 2.5 cm target Zone in to volume with dl H0); and 8.5 mg/L silver nitrate (added preparation for bombardment. after sterilizing the medium and cooling to room tempera 0537 Preparation of DNA ture). Selection medium (560R) comprises 4.0 g/L N6 basal 0538 A plasmid vector comprising a nucleotide sequence salts (SIGMA C-1416), 1.0 mL/L Eriksson’s Vitamin Mix (e.g., SEQ ID NO: 1) operably linked to an ubiquitin pro (1000xSIGMA-1511), 0.5 mg/L thiamine HCl, 30.0 g/L moter is made. For example, a suitable transformation vector sucrose, and 2.0 mg/L 2,4-D (brought to volume with dl H0 comprises a UBI1 promoter from Zea mays, a 5' UTR from following adjustment to pH 5.8 with KOH);3.0 g/L GelriteTM UBI1 and a UBI1 intron, in combination with a Pintermi (added after bringing to volume with dl H0); and 0.85 mg/L nator. The vector additionally contains a PAT selectable silver nitrate and 3.0 mg/L Bialaphos (both added after ster marker gene driven by a CAMV35S promoter and includes a ilizing the medium and cooling to room temperature). CAMV35S terminator. Optionally, the selectable marker can 0551 Plant regeneration medium (288J) comprises 4.3 reside on a separate plasmid. A DNA molecule comprising a g/L MS salts (GIBCO 11117-074), 5.0 mL/L MS vitamins toxin nucleotide sequence as well as a PATselectable marker stock solution (0.100 g nicotinic acid, 0.02 g/L thiamine HCl, is precipitated onto 1.1 um (average diameter) tungsten pel 0.10 g/L pyridoxine HCl, and 0.40 g/L Glycine brought to lets using a CaCl2 precipitation procedure as follows: volume with polished D-1H-0) (Murashige and Skoog. 0539 100 uL prepared tungsten particles in water (1962) Physiol. Plant. 15:473), 100 mg/L myo-inositol, 0.5 (0540 10 uL (1 lug) DNA in Tris EDTA buffer (1 lug total mg/L Zeatin, 60 g/L Sucrose, and 1.0 mL/L of 0.1 mM abscisic DNA) acid (brought to volume with polisheddl H0 after adjusting (0541 100 uL 2.5 M CaCl, to pH 5.6); 3.0 g/L GelriteTM (added after bringing to volume (0542 10 uL 0.1 M spermidine with dl H0); and 1.0 mg/L indoleacetic acid and 3.0 mg/L 0543. Each reagent is added sequentially to a tungsten Bialaphos (added after Sterilizing the medium and cooling to particle Suspension, while maintained on the multitube Vor 60 C). texer. The final mixture is sonicated briefly and allowed to 0552 Hormone-free medium (272V) comprises 4.3 g/L incubate under constant vortexing for 10 minutes. After the MS salts (GIBCO 11117-074), 5.0 mL/L MS vitamins stock precipitation period, the tubes are centrifuged briefly, liquid solution (0.100 g/L nicotinic acid, 0.02 g/L thiamine HCl, removed, washed with 500 mL 100% ethanol, and centri 0.10 g/L pyridoxine HCl, and 0.40 g/L Glycine brought to fuged for 30 seconds. Again the liquidis removed, and 105ul volume with polished d1 HO), 0.1 g/L myo-inositol, and 40.0 100% ethanol is added to the final tungsten particle pellet. For g/L sucrose (brought to volume with polished d1 H-0 after particle gun bombardment, the tungsten/DNA particles are adjusting pH to 5.6); and 6 g/L Bacto-agar (added after bring briefly sonicated and 10 uL spotted onto the center of each ing to volume with polished d1 HO), sterilized and cooled to macrocarrier and allowed to dry about 2 minutes before bom 60° C. bardment. 0544 Particle Gun Treatment Example 20 0545. The sample plates are bombarded at level #4 in particle gun iHE34-1 or iHE34-2. Agrobacterium-Mediated Transformation of Maize 0546 All samples receive a single shot at 650 PSI, with a and Regeneration of Transgenic Plants total of ten aliquots taken from each tube of prepared par 0553 For Agrobacterium-mediated transformation of ticles/DNA. maize with a toxin nucleotide sequence (e.g., SEQID NO: 1), (0547 Subsequent Treatment the method of Zhao can be used (U.S. Pat. No. 5,981,840 and 0548. Following bombardment, the embryos are kept on PCT Patent Publication Number WO 1998/32326; the con 560Y medium for 2 days, then transferred to 560R selection tents of which are hereby incorporated by reference). Briefly, medium containing 3 mg/liter Bialaphos, and Subcultured immature embryos are isolated from maize and the embryos every 2 weeks. After approximately 10 weeks of selection, contacted with a Suspension of Agrobacterium under condi selection-resistant callus clones are transferred to 288J tions whereby the bacteria are capable of transferring the medium to initiate plant regeneration. Following Somatic nucleotide sequence (e.g. SEQID NO: 1) to at least one cell embryo maturation (2-4 weeks), well-developed somatic of at least one of the immature embryos (step 1: the infection embryos are transferred to medium for germination and trans step). In this step the immature embryos can be immersed in ferred to the lighted culture room. Approximately 7-10 days an Agrobacterium Suspension for the initiation of inoculation. later, developing plantlets are transferred to 272V hormone The embryos are co-cultured for a time with the Agrobacte free medium in tubes for 7-10 days until plantlets are well rium (step 2: the co-cultivation step). The immature embryos established. Plants are then transferred to inserts in flats can be cultured on Solid medium following the infection step. (equivalent to 2.5" pot) containing potting soil and grown for Following this co-cultivation period an optional “resting 1 week in a growth chamber, Subsequently grown an addi step is contemplated. In this resting step, the embryos are tional 1-2 weeks in the greenhouse, then transferred to classic incubated in the presence of at least one antibiotic known to 600 pots (1.6 gallon) and grown to maturity. Plants are moni inhibit the growth of Agrobacterium without the addition of a US 2014/0274885 A1 Sep. 18, 2014 79 selective agent for plant transformants (step 3: resting step). (step 4: the selection step). The immature embryos are cul The immature embryos can be cultured on solid medium with tured on Solid medium with a selective agent resulting in the antibiotic, but without a selecting agent, for elimination of selective growth of transformed cells. The callus is then Agrobacterium and for a resting phase for the infected cells. regenerated into plants (step 5: the regeneration step), and Next, inoculated embryos are cultured on medium containing calli grown on selective medium can be cultured on Solid a selective agent and growing transformed callus is recovered medium to regenerate the plants.

MUT Back- Posi- EC50 Example ID NO: bone tion RefA.A. Substitution FAE P-value (ppm) Deviation i 1 PHI-4 202 A. V S4 O45 4 2 PH-4 342 A. S 29 O.32 4 3 PHI-4 417 A. S 96 -0.13 4 4 PHI-4 24 D N 96 O.23 4 S PHI-4 42 D N OO O.62 4 6 PHI-4 331 E D 22 -O.O2 4 7 PHI-4 109 F I 2O2 4 8 PHI-4 300 F Y 32 O.26 4 9 PHI-4 359 G A. 98 -0.06 4 10 PHI-4 52 I V 2O2 4 11 PHI-4 133 I L 24 -0.21 4 12 PH-4 172 I L 23 O.26 4 13 PHI-4 283 I V 87 4 14 PHI-4 410 I V OO O.OS 4 15 PHI-4 40 L I 13 4 16 PHI-4 145 L V O2 4 17 PHI-4 153 L I 79 O.22 4 18 PHI-4 163 L V O4 4 19 PHI-4 296 L I 16 O.36 4 2O PHI-4 418 L M 84 O.10 4 21 PHI-4 154 N D O8 O.O1 4 22 PH-4 346 P A. 85 4 23 PHI-4 411 P A. 91 O.O6 4 24 PHI-4 412 P A. 79 O.2O 4 2S PHI-4 34 S A. 76 4 26 PHI-4 78 S G 44 -0.23 4 27 PHI-4 335 S 60 -0.26 4 28 PHI-4 426 T S 93 -0.13 4 29 PHI-4 461 T S S4 4 3O PHI-4 343 V 89 O.39 4 31 PHI-4 355 V 78 O.39 4 32 PH-4 392 V 21 4 33 PHI-4 421 V O6 4 34 PHI-4 440 V 75 O.26 4 35 PHI-4 456 W Y 99 4 36 PHI-4 98 Y 71 4 37 PHI-4 121 Y 17 O.11 4 38 PHI-4 206 Y 82 O.S1 4 39 PHI-4 337 A. G -0.10 4 40 PHI-4 364 A. S O.09 4 41 PHI-4 371 A. G O.O2 4 42 PHI-4 371 A. O.24 4 43 PHI-4 385 A. G O.08 4 44 PHI-4 385 A. C -0.23 4 45 PHI-4 396 A. E -0.56 4 46 PEHI-4 4OS A. S O.09 4 47 PHI-4 4OS A. W -0.38 4 48 PHI-4 409 A. C O.25 4 49 PHI-4 417 A. C O.11 4 SO PHI-4 445 C O16 4 S1 PHI-4 445 C O.O6 4 52 PH-4 331 E N O.24 4 S3 PHI-4 339 E N O.04 4 S4 PHI-4 339 E Q O.34 4 SS PHI-4 344 F W O16 4 S6 PHI-4 374 F O.10 4 57 PHI-4 378 F -O.78 4 S8 PHI-4 351 G V O.10 4 S9 PHI-4 397 G R O.29 4 6O PHI-4 428 G S -0.17 4 61 PHI-4 373 I V -O-52 4 62 PHI-4 375 K R O.19 4 63 PHI-4 384 K A. O.OS 4 64 PHI-4 341 L V O.19 4 6S PHI-4 38O L G O.17 4 66 PHI-4 383 L I O.O7 4 67 PHI-4 383 L V O.17 4

US 2014/0274885 A1 Sep. 18, 2014 82

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MUT Posi EC50 Example ID NO: tion RefA.A. Substitution FAE P-value (ppm) Deviation i 214 109 14.3 215 74 12.4 216 4O2 10.2 217 336 8.0 218 527 7.8 219 82 7.8 220 109 6.8 221 97 6.7 222 220 6.6 223 16S 6.6 224 289 6.6 225 454 6.4 226 109 6.4 227 247 6.1 228 454 5.8 229 99 5.7 230 289 S.1 231 109 S.O 232 289 S.O 233 454 4.6 234 220 4.4 235 334 4.3 236 459 4.2 237 97 4.0 238 454 4.0 239 517 4.0 240 99 4.0 241 2S6 4.0 242 109 3.9 243 220 3.8 244 196 3.7 245 517 3.7 246 79 3.6 247 454 3.5 248 454 3.5 249 289 3.3 250 74 3.3 251 403 3.3 252 166 3.3 253 517 3.3 254 447 3.2 255 289 2.8 256 454 2.8 257 220 2.7 258 447 2.7 259 82 2.7 260 196 2.7 261 216 2.7 262 334 2.6 263 151 2.6 264 454 2.6 26S 16S 2.6 266 459 2.6 267 148 2.6 268 220 2.5 269 454 2.5 O.26 270 447 2.5 271 527 2.5 272 196 2.5 273 82 2.5 274 527 2.4 275 4O2 2.4 276 459 2.4 277 459 2.3 278 289 2.3 279 99 2.3 28O 289 2.3 281 220 2.3 282 459 2.3 283 462 2.3 284 99 2.2 285 289 2.2 286 109 2.2

US 2014/0274885 A1 Sep. 18, 2014 89

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MUT Posi EC50 Example ID NO: tion RefA.A. Substitution FAE P-value (ppm) Deviation i 725 113 726 235 727 403 728 2O3 729 340 730 340 731 247 732 2O3 733 148 734 466 735 332 736 458 737 235 738 340 739 466 740 109 741 398 742 99 743 466 744 340 745 332 746 403 747 113 748 403 749 109 750 332 751 340 752 2O3 753 235 754 196 755 247 756 2O3 757 466 758 2O3 O. 759 449 760 334 761 413 762 38 763 82 764 340 765 235 766 398 767 458 -0.49 768 247 769 466 770 395 771 466 772 220 773 340 774 247 775 2O3 776 334 777 247 778 458 779 403 780 403 781 403 782 403 783 2O3 784 398 785 340 786 82 787 2O3 788 447 789 109 790 247 791 2O3 792 332 793 332 794 82 795 247 796 368 797 82 US 2014/0274885 A1 Sep. 18, 2014 90

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MUT Posi EC50 Example ID NO: tion RefA.A. Substitution FAE P-value (ppm) Deviation i 798 247 799 458 800 235 8O1 398 802 332 803 247 804 332 805 395 806 334 807 53 808 97 809 458 810 398 811 109 812 403 813 497 814 458 815 458 7 816 447 O 817 395 4 18O -0.91 818 247 O 819 82 O 82O 395 O 821 398 O 822 332 O 823 403 O 824 247 O 825 398 5 826 458 6 827 398 5 828 403 O 829 82 8 830 403 O 831 247 6 832 395 5 833 398 1 834 395 7 835 395 7 836 395 7 837 97 4 838 395 O -0.82 839 332 O 840 395 7 841 332 O 842 403 O 843 458 O 844 398 6 845 458 5 846 2O3 4 847 395 6 848 82 6 849 395 5 850 97 4 851 332 O 852 395 5 853 332 O 854 398 4 855 395 5 856 458 5 857 321 9 858 82 3 859 82 3 860 332 O 861 458 O 862 398 3 863 395 9 864 395 3 865 2O3 3 866 458 4 867 403 9 868 247 9 869 376 9 870 321 9