|HAO WANATA MARIAUS009783820B2 DEL DIA DE LA UNIT (12 ) United States Patent ( 10 ) Patent No. : US 9 , 783, 820 B2 Cong et al. (45 ) Date of Patent: Oct . 10, 2017 ( 54 ) METHODS AND COMPOSITIONS TO (58 ) Field of Classification Search ENHANCE ACTIVITY OF CRY None ENDOTOXINS See application file for complete search history . (71 ) Applicant: PIONEER HI BRED (56 ) References Cited INTERNATIONAL INC , Johnston , IA U . S . PATENT DOCUMENTS (US ) 9 , 109 , 231 B2 * 8 / 2015 Bermudez . .. .. .. .. CO7K 14 / 325 (72 ) Inventors : Ruth Cong, Palo Alto , CA (US ) ; 2011 / 0223686 AL 9 /2011 Sakai et al . Takashi Yamamoto , Dublin , CA (US ) ; SÊ@ Yi Zheng , Palo Alto , CA (US ) FOREIGN PATENT DOCUMENTS WO 2008121633 AL 9 /2008 (73 ) Assignee : PIONEER HI- BRED WO 2012135436 AL 4 /2012 INTERNATIONAL , INC ., Johnston , wo 2012109515 A1 8 / 2012 IA (US ) ( * ) Notice : Subject to any disclaimer , the term of this OTHER PUBLICATIONS patent is extended or adjusted under 35 Pardo -Lopez et al . Peptides (2009 ) 30 , pp . 589 -595 . * U . S . C . 154 (b ) by 0 days . Griko et al . Biochemistry (2007 ) , vol. 46 , pp . 10001 - 10007. * Hire , R . S . et al ; “ Expression , purification and characterization of the (21 ) Appl. No. : 14 /435 , 381 Cry2Aa14 toxin from Bacillus thuringiensis subsp . kenyae” , Toxicon , vol. 54 , No. 4 ; pp . 519 - 524 (2009 ) . ( 22 ) PCT Filed : Oct. 14 , 2013 Vazquez - Padron , R . I. et al; “ Cryptic endotoxic nature of Bacillus thuringiensis Cryl Ab insecticidal crystal protein ” , FEBS Letters , ( 86 ) PCT No .: PCT/ US2013 / 064784 vol . 570 , No. 1 - 3 , pp . 30 - 36 ( 2004 ) . Sachdev, D . et al; “ Solubility of Proteins Isolated from Inclusion $ 371 (c )( 1 ), Bodies is Enhanced by Fusion to Maltose - Binding Protein or ( 2 ) Date : Apr. 13 , 2015 Thioredoxin ” , Protein Expression and Purification Academic Press ; vol. 12 , No. 1 , pp . 122 - 132 ( 1998 ) . (87 ) PCT Pub . No .: WO2014 / 062544 International Search Report and Written Opinion for International PCT Pub . Date : Apr. 24 , 2014 Application No. PCT/ US2013 / 064784 completed Aug . 4 , 2014 . (65 ) Prior Publication Data * cited by examiner Primary Examiner — Medina A Ibrahim US 2015 /0232877 A1 Aug . 20 , 2015 (74 ) Attorney , Agent, or Firm — Pioneer Hi- Bred Int' l ( 57 ) ABSTRACT Related U . S . Application Data Methods and compositions for enhancing the resistance of plants to plant pests are provided . Chimeric pesticidal poly (60 ) Provisional application No . 61/ 713 , 844 , filed on Oct. peptides and nucleic acid molecules encoding the chimeric 15 , 2012 pesticidal polypeptides are provided . The chimeric pesti (51 ) Int. CI. cidal polypeptides comprising a solubility - enhancing poly CO7K 14 /325 ( 2006 .01 ) peptide operably linked to a polypeptide comprising pesti C12N 15 / 82 ( 2006 .01 ) cidal activity . The nucleic acid molecules can be used in CO7K 14 /245 ( 2006 .01 ) expression cassettes for making transformed plants with ( 52 ) U . S . CI. enhanced resistance to plant pests . Further provided are CPC . .. C12N 15 / 8286 (2013 . 01 ) ; CO7K 14 /245 transformed plants , plant tissues , plant cells , other host cells , ( 2013. 01 ) ; C07K 14 / 325 (2013 . 01 ) ; COOK and seeds as well as pesticidal compositions . 2319 / 35 (2013 . 01 ) ; CO7K 2319/ 55 (2013 .01 ) 3 Claims, 1 Drawing Sheet U . S . Patent Oct. 10 , 2017 US 9 ,783 , 820 B2 N - terminus C -terminus MBP Bt Cry Protein E . coli MBP - BT Cry protein fusion US 9 , 783 ,820 B2 METHODS AND COMPOSITIONS TO ins is their expression during the stationary phase of growth , ENHANCE ACTIVITY OF CRY as they generally accumulate in a mother cell compartment ENDOTOXINS to form a crystal inclusion that can account for 23- 30 % of the dry weight of sporulated cells . The Cry endotoxins CROSS REFERENCE TO RELATED 5 initially are produced in an inactive protoxin form , which are APPLICATIONS proteolytically converted into an active endotoxin through the action of proteases in an insect’ s gut. Once active , the This application claims the benefit U . S . Provisional endotoxins bind to the gut epithelium and form cation Application No . 61 /713 , 844 , filed Oct . 15 . 2012 , which is selective channels that cause cell lysis and subsequent death . incorporated he rein by reference in their entirety . 10 See , Carroll et al. ( 1997 ) J. Invertebr. Pathol. 70 : 41 -49 ; Oppert ( 1999 ) Arch . Insect Biochem . Phys. 42 : 1 - 12 ; and REFERENCE TO SEQUENCE LISTING Rukmini et al . ( 2000 ) Biochimie 82 : 109 - 116 . SUBMITTED ELECTRONICALLY Although Cry endotoxins often are highly effective against insect pests , some insect pests are not affected by An official copy of the Sequence Listing submitted elec - 15 them or show low susceptibility . Likewise , some insect pests tronically as an ASCII formatted Sequence Listing with a have developed resistance to the Cry endotoxins, which file named “ 3113PCT_ SequenceListing .txt , ” created on Sep . threatens their effectiveness . Methods to address these prob 17 , 2013 , having a size of 158 kb and filed concurrently with lems include enhancing or expanding Cry endotoxin activity the Specification is part of the Specification and is incorpo by site - directed mutagenesis , by introducing cleavage sites rated herein by reference as if set forth in its entirety . 20 in specific regions of the endotoxin or by deleting small fragments from the amino - terminus of the endotoxin . See , FIELD OF THE INVENTION e . g ., Abdullah & Dean ( 2004 ) Appl. Environ . Microbiol. 70 : 3769 -3771 ; Pardo - López et al . ( 2009 ) Peptides 30 : 589 The invention relates generally to plant molecular biology 595 ; Rajamohan et al . (1996 ) Proc. Natl . Acad. Sci. USA and plant pest control, and more particularly to compositions 25 93 : 14338 - 14343 ; and Wu et al. ( 2000 ) FEBS Lett . 473 and methods for enhancing the activity of pesticidal poly - 227 - 232 . These methods , however , can be time consuming peptides from Bacillus spp . and for protecting a plant from and not certain to produce a desired result . a plant pest , particularly an insect pest . For the foregoing reasons , there is a need for composi tions and methods to enhance the pesticidal activity of Cry BACKGROUND OF THE INVENTION 30 endotoxins. Pests, such as insect pests , are a major factor in the loss BRIEF SUMMARY OF THE INVENTION of the world ' s agricultural crops. For example, corn root worm and boll weevil damage can be economically devas Compositions and methods are provided for enhancing tating to agricultural producers . Insect pest -related agricul - 35 pesticidal activity of Cry endotoxins and for protecting a tural crop loss from corn rootworm alone has reached one plant from plant pest such as insect pests . The compositions billion dollars a year. comprise chimeric pesticidal polypeptide- encoding nucleic Traditionally , the primary methods for controlling insect acid molecules, variants and fragments thereof, as well as pests , such as corn rootworm , are crop rotation and appli chimeric pesticidal polypeptides, active variants and frag cation of broad - spectrum , synthetic , chemical pesticides . 40 ments thereof. The chimeric pesticidal polypeptides of the However , consumers and government regulators alike are invention comprise a solubility - enhancing polypeptide fused becoming increasingly concerned with environmental haz - to a Cry endotoxin or biologically active fragment thereof. ards associated with producing and using chemical pesti- Also provided are expression cassettes or polynucleotide cides. Because of such concerns, regulators have banned or constructs comprising a nucleotide sequence encoding a limited the use of some of the more hazardous chemical 45 chimeric pesticidal polypeptide of the invention , as well as pesticides. Thus , there is substantial interest in developing bacteria , plants , plant organs , plants tissues , plant parts , alternatives to chemical pesticides that present a lower risk plant cells and seeds comprising the expression cassette or of pollution and environmental hazards and that provide a polynucleotide nucleotide construct encoding the chimeric greater target specificity than is characteristic of chemical pesticidal polypeptide . Further provided are pesticidal com pesticides . 50 positions comprising at least one chimeric pesticidal poly Certain species in the genus Bacillus have polypeptides peptide of the invention . that possess pesticidal activity against a broad range of Methods are provided for enhancing the pesticidal activity insect pests including those in the orders Lepidoptera , Dip - of Cry endotoxins . The methods involve making a chimeric tera , Coleoptera , Hemiptera and others . For example , Bacil pesticidal polypeptide comprising the amino acid sequence lus thuringiensis and Bacillus popilliae are among the most 55 of a solubility - enhancing polypeptide operably linked to an successful species discovered to date having pesticidal activ amino acid sequence of a Cry endotoxin or biologically ity . Such pesticidal activity also has been attributed to strains active fragment thereof. Such chimeric pesticidal polypep of Bacillus larvae , Bacillus lentimorbus, Bacillus sphaeri tide can be produced by fusing amino acid sequence of the cus and Bacillus cereus . See , Biotechnology Handbook 2 : solubility - enhancing polypeptide to the amino acid sequence Bacillus ( Harwood ed . , Plenum Press 1989 ) ; and Int' l Patent 60 of a Cry endotoxin or biologically active fragment thereof. Application Publication No . WO