BPMV-based viral constructs useful for VIGS and expression of heterologous proteins in legumes

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Iowa State University Patents Iowa State University Research Foundation, Inc. 10-29-2013 BPMV-based viral constructs useful for VIGS and expression of heterologous proteins in legumes John H.

1 Iowa State University Patents Iowa State University Research Foundation, Inc BPMV-based viral constructs useful for VIGS and expression of heterologous proteins in legumes John H. Hill Iowa State University, Chunquan Zhang Iowa State University Steven A. Whitham Iowa State University, Follow this and additional works at: Part of the Agriculture Commons, Agronomy and Crop Sciences Commons, Plant Breeding and Genetics Commons, and the Plant Pathology Commons Recommended Citation Hill, John H.; Zhang, Chunquan; and Whitham, Steven A., "BPMV-based viral constructs useful for VIGS and expression of heterologous proteins in legumes" (2013). Iowa State University Patents This Patent is brought to you for free and open access by the Iowa State University Research Foundation, Inc. at Iowa State University Digital Repository. It has been accepted for inclusion in Iowa State University Patents by an authorized administrator of Iowa State University Digital Repository. For more information, please contact

2 BPMV-based viral constructs useful for VIGS and expression of heterologous proteins in legumes Abstract The invention provides Bean pod mottle virus (BPMV) vectors useful for expression of heterologous proteins in plants such as soybean. The BPMV vectors are also useful for virus-induced gene silencing. The vectors of the invention include modifications of BPMV RNA1 sequences so that infection with the vectors produces only moderate symptoms. The vectors also comprise novel RNA2 vectors which specifically provide for nontranslated VIGS constructs and further which do not require in frame insertion of heterologous sequences to be expressed. Disciplines Agriculture Agronomy and Crop Sciences Plant Breeding and Genetics Plant Pathology This patent is available at Iowa State University Digital Repository:

US008569579B2 (12) United States Patent Hill et a1. (10) Patent N0.: (45) Date of Patent: US 8,569,579 B2 Oct.

3 US B2 (12) United States Patent Hill et a1. (10) Patent N0.: (45) Date of Patent: US 8,569,579 B2 Oct. 29, 2013 (54) (75) (73) (*) (21) (22) (65) BPMV-BASED VIRAL CONSTRUCTS USEFUL FOR VIGS AND EXPRESSION OF HETEROLOGOUS PROTEINS IN LEGUMES Inventors: John H. Hill, Ames, IA (US); Chunquan Zhang, Ames, IA (US); Steve Whitham, Ames, IA (US) Assignee: Iowa State University Research Foundation, Inc., Ames, IA (US) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days. Appl. N0.: 12/927,579 Filed: Nov. 18, 2010 Prior Publication Data US 2011/ A1 Jul. 14,2011 FOREIGN PATENT DOCUMENTS CA /2000 WO WO00/ /2000 OTHER PUBLICATIONS Gu et al. (2005) Virology 333: * Zhang, Development and Use of an Ef?cient DNA-BasedViral Gene Silencing Vector for Soybean, The American Phytopathological Society, vol. 22, No. 2, 2009, pp Zhang, Development of Bean Pod Mottle Virus-Based Vectors for Stable Protein Expression and Sequence-Speci?c Virus-Induced Gene Silencing in Soybean, Virology 344 (2006), pp Zhang, The Development of an Ef?cient Multipurpose Bean Pod Mottle Virus Viral Vector Set for Foreign Gene Expression and RNA Silencing, Plant Physiology, May 2010, vol. 153, pp Zhang, Development of a high throughput Bean pod mottle virus (BPMV) based gene expression and VIGS vector for soybean host pathogen interaction study. Poster. American Phytopathological Society (APS) annual meeting, 2007, San Diego, CA, Jul. 28-Aug. l, Zhang, Development of a high throughput Bean pod mottle virus (BPMV) based gene expression and VIGS vector for soybean host pathogen interaction study, Phytopathology, vol. 97 No. 7 (Supple ment), S129, 2007 Abstract. (60) (51) (52) (58) (56) 7,005,559 B1 7,618,815 B2 2007/ A1 Related US. Application Data Provisional application No. 61/281,469,?led on Nov. 18, Int. Cl. C12N15/83 ( ) C12N15/33 ( ) C12N 5/10 ( ) US. Cl. USPC /280; 435/320.1; 800/312; 536/2372 Field of Classi?cation Search None See application?le for complete search history. References Cited U.S. PATENT DOCUMENTS 2/2006 Sela 11/2009 Ghabrial 9/2007 Ghabrial * cited by examiner Primary Examiner * David T Fox Assistant Examiner * Steven Bernacki (74) Attorney, Agent, or Firm * McKee, Voorhees & Sease, P.L.C. (57) ABSTRACT The invention provides Bean pod mottle virus (BPMV) vec tors useful for expression of heterologous proteins in plants such as soybean. The BPMV vectors are also useful for virus induced gene silencing. The vectors of the invention include modi?cations of BPMV RNAl sequences so that infection With the vectors produces only moderate symptoms. The vectors also comprise novel RNA2 vectors Which speci?cally provide for non-translatedvigs constructs and further Which do not require in frame insertion of heterolo gous sequences to be expressed. 4 Claims, 44 Drawing Sheets (38 of 44 Drawing Sheet(s) Filed in Color)

4 US. Patent 0a. 29, 2013 Sheet 1 0f 44 US 8,569,579 B2 emf gun a,

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7 US. Patent 0a. 29, 2013 Sheet 4 0f 44 US 8,569,579 B2 / mu mwm a, mm Em,

8 US. Patent 0a. 29, 2013 Sheet 5 0f 44 US 8,569,579 B2 3mm.

9 US. Patent 0a. 29, 2013 Sheet 6 0f 44 US 8,569,579 B2

10 US. Patent 0a. 29, 2013 Sheet 7 0f 44 US 8,569,579 B2

14 KG? K-G7 (1) - ~ ~ ~ ~ - ~ ~ ~ ~ ~ ~ ~ ~ - ~ ~ ~ ~ ~ ~» ~ ~ ~ ~ - ~ ~ ~ ~ - ~ ~ ~ ~ _ - _.. US. Patent 0a. 29, 2013 Sheet US 8,569,579 B2 Section 1 (1) 1 1O 2O 3O Q 80 ((-67 (1) - pbpmv.r1a (1) TATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGAC IGGGAAAACCCTGGCGTTACCCAACTTAA Consensus (1) Section 2 (81) K-G? (1) pbpm\/.r1a (81) TCGCCT'1 GCAGCACATCCCCC'1 TTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGA'I'CGCCCTTCCCAACAGTTGC Consensus (81) < Section 3 ( K-G7 (1) - pgpm\/_r1a (151) SCAGCCTGAATGGCGAATGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGG'IGTGGTGGTTACGCGCAGCGTGACCG Consensus (161) Section 4 (241 ' ) G pgpmvf imgz (24(4;CTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCC'1'TTC'ICGCCACGTTCGCCGGCTTTCCCCGT Consensus (241) Section 5 (321) , KC? ( 1 ) pbpm\/.r1a CAAGC'1 CTAAATCGGGGGCTCCCTT'I'AGGG'ITCCGATTTAGTGC'1'TTACGGCACCTCGACCCCAAAAAACTTGATTAGGG Consensus (321 1 Section G (401 ) G A40 A A K-GT ( ) ~ 1 pbpmv.r1a (401) TGATGGTTCACGTAG'I GGGCCATCGCCCTGA'X AGACGGTTT'X TCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTG Consensus (401 ) Section 7 (481) G , K-GT (1) ~ GACTC'1 TGTTCCAAAC'RGGAACAACAC'I CAACCCTATCTCGG I'CTAT'I CTTTTGATTTATAAGGGATTTTGCCGATT'PCG Consensus (481) Section 8 (561) 561, ,600,610,620, pbpm\/_r1a GCCTATTGGTTAAAAAATGAGCTGA'I'TTAACAAAAATTTAACGCGAATTTTAACAAAATA'I'TAACGCTTACAATTTCCTG Consensus (561) Section 9 (641 ) 641, ' K~G7 (1) ~ Section 10 (721') 721,7130 1'40 1' TFO, K~G7 (1) ~ PBpM\/_R1A (721) CGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTC'I'GCTCCCGGCATCCGCTT Consensus (721) Section 11 (301 J 801, B40 B50,860, (1) ~ ~ ~ ~ - ~ ~ (801') ACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCA I'CACCGAAACGCGCGAGACGAAAG Consensus (801) FIG.

15 K-G7 (1) - - ~ ~ - - ~ - - ~ _ - - _ US. Patent 0a. 29, 2013 Sheet 12 0f 44 US 8,569,579 B2 Section 12 (881) K-G7 r11 - ~ GGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACG'I'CAGGTGGCACT'I TTCGGGG Consensus (881) Section 13 ( ,7 970 ~ ~ 7, K_G7 1.._ ,,._,_... _._, pbpmv-r1a Consensus (961) Section 14 (1941) KG? (1) AAA'I'GC'X TCAATAA'I ATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCG'I'GTCGCCCTTATTCCCTTTTTTGCGGCA Consensus (1041) Section 15 mg m K-G7 (1121) 'I TT'I'GCC'l'TCCTGTT'l'TTGCTCACCCAGILAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGG Consensus (1121) Section 16 (1201) pbpm\/_r1a (1201) 1"I'ACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTT Consensus (1201) Section 17 (1231) ( K-G? m ~ pbpmv-r1a (1281) TTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCT Consensus (1281) Section 18 (1361) K-G7 1 1 ) _ pbpm\/_r1a (1361) CAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCA'ICTTACGGA'I'GGCATGACAGTAAGAGAATTATGCAGTGC Consensus (1361) Section 19 (1441) v , K-G7 ( 1 ~ 1 Consensus (1441) TGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTT 7 Section 20 (1521) , K~G7 ) ~ 1 DBPMVRMA (1521) 'I GCACAACATGGGGGATCATGTAAC'I CGCC'I TGATCG'I TGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGT Consensus (1521) Section 21 (1601) , Consensus (1601) Section 22 (1531) , (1) - KG? PBPMV.R1A (1531) ACAATTAATAGAC'I GGATGGAGGCGGATAAAGTTGCAGGACCAC'I'TCTGCGCTCGGCCCTTCCGGC TGGCTGG'I'TTATTG Consensus (1681)

16 US. Patent 0a. 29, 2013 Sheet 13 0f 44 US 8,569,579 B2 ' Sec1ion23 (1761) (1) " K~G7 PBPMVRM (1761) CTGA'X'AAATCTGGAGCCGG'I GAGCGTGGG'I CTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCG'X'ATC Consensus (1761) Section 24 ((841) (1) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~~ K~G7 p5pmv.r1a (1341) GTAGTTATCTACACGACGGGGAGTCAGGCAAC YATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGAT Consensus (1841) V Seckion 25 (1921) , K~G7 (1) ~ pbpm\/_r1a (1921) TAAGCATTGGTAAC'I'GTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGA Consensus (1921) Section 26 (2001) K-G7 > ( 1 ) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ p5pm\/_r1a (2661)'I'CTAGGTGAAGATCCTTTTTGATAATCTCA'I'GACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCG'I'CAGACCCC Consensus (2001) Section 27 (2081) K-G7 m ~ GTAGAAAAGA'I'CAAAGGATCTTCTTGAGATCCTTT'I'TTTCTGCGCGTAATC I GCTGCTTGCAAACAAAAAAACCACCGCT Consensus (2081) Section pbpmvrm (216(1)ACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTT'I'I'CCGAAGGTAACTGGC'I TCAGCAGAGCGCAGATAC Consensus (2161) Section 29 (2241) KC? pbpmv.r1a (22151: CAAA'I ACTGT'I'CTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGC'I'CTG Consensus (2241) Section 30 (2321 ) K-G7 Section 31 (240;) KC? ( ) ' 1 pbpmv-r IA TAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGAT Consensus (2401) Section 32 (2481 ) K-G? m " ACCTACAGCG'I GAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTA'I'CCGG'I'AAGCGGCAGGGTC Consensus (2481) Section 33 (256 1 ) (1) e - KC? (2561) GGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTG'ICGGGTTTCGCCACCTCTG Consensus (2561) HQ. 95

17 US. Patent 0a. 29, 2013 Sheet 14 0f 44 US 8,569,579 B2 Section 34 (2641 ) K-G7 ~ ~ ~ ~ ~ ~ ~ - ~ ~ -» ~ ~ > _ Consensus (2643) Section 35 ( ( 3 ~ ~ ~ - 1 ~ ~ ~ > ~ ~ ~ ~ ~ ~ - - ~ 'I CCTGGCC'I TTTGCTGGCCTTT'I GCTCACATGTTCTTTCCTGCGT'I ATCCCCTGATTCTGTGGATAACCGTATTACCGCC Consensus (2723') Section 36 ( K-G7 ( 1 ) o 'l'ttgagtgagctga'i'accgctcgccgcagccgaacgaccgagcgcagcgag'i'cagtgagcgaggaagcggaagagcgccc Consensus (2801) Section 37 (2883 -, K-G7 (1) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ pbpm\/_r1a (2881) AATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCAT I'AATGCAGCTGGCACGACAGGT PTCCCGAC I'GGAAAGCGG Consensus (2881) Section 38 (2961) K-G7 (M ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ H p5pmv.r1a (2951') GCAGTGAGCGCAACGCAATTAATGTGAGT'JJAGCTCACTCATTAGGCACCCCAGGCTT'I ACAC'I'TTATSCTTCCGGCTCGT Consensus (2961) Section 39 (3041) pbpmv~r1a (3041) ATG'I TG'IGTGGAATTG'I'GAGCGGATAACAA'I'TTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTATTTAGGTG Consensus (3043) Section40 (3121) $ , DBPN1VE<R$A7 (312(1)) ACACTATAGAATACTCAAGCGGCCGCCTACTCCAAGAATATCAAAGATACAGTC'I CAGAAGACCAAAGGGCTATTGAGAC Consensus (3121) Section 41 (320;) pgpmv?z? (326;; 'l'tttcaacaaaggg'i'aatatcgggaaacctcctcgga'i'tccattgcccagc'i'atctgtcacttca'i'caaaaggacagtag Consensus (3201) 7 Section 42 ( Consensus (3281) Section 43 (3361) K-GT (M ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ H p5pmv.r1a (3351') CCCAAAGATGGACCCCCACCCACGAGGAGCATCG'I GGAAAAAGAAGACGTTCCAACCACGTC'I'TCAAAGCAAGTGGATTG Consensus (3361) Section Consensus (3443) FIG. 9

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(12) United States Patent Davido et al.

USOO8802109B2 (12) United States Patent Davido et al. (10) Patent N0.: (45) Date of Patent: US 8,802,109 B2 Aug. 12, 2014 (54) HERPES SIMPLEX VIRUS MUTANT ICPO (75) Inventors: David Davido, Lawrence, KS