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(12) Patent Application Publication (10) Pub. No.: US 2014/0274885 A1 Cong Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2014/0274885 A1 Cong Et Al 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
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