Alteration of Tobacco Alkaloid Content Through Modification of Specific Cytochrome P450 Genes

81 Pages·2016·1.31 MB·English

UUnniivveerrssiittyy ooff KKeennttuucckkyy
UUKKnnoowwlleeddggee
Plant and Soil Sciences Faculty Patents Plant and Soil Sciences
1-5-2016
AAlltteerraattiioonn ooff TToobbaaccccoo AAllkkaallooiidd CCoonntteenntt TThhrroouugghh MMooddiifificcaattiioonn ooff
SSppeecciifificc CCyyttoocchhrroommee PP445500 GGeenneess
Ralph E. Dewey
Balazs Siminszky
University of Kentucky
Steven W. Bowen
Lily Gavilano
University of Kentucky
Follow this and additional works at: https://uknowledge.uky.edu/pss_patents
Part of the Plant Sciences Commons
RRiigghhtt cclliicckk ttoo ooppeenn aa ffeeeeddbbaacckk ffoorrmm iinn aa nneeww ttaabb ttoo lleett uuss kknnooww hhooww tthhiiss ddooccuummeenntt bbeenneefifittss yyoouu..
RReeccoommmmeennddeedd CCiittaattiioonn
Dewey, Ralph E.; Siminszky, Balazs; Bowen, Steven W.; and Gavilano, Lily, "Alteration of Tobacco Alkaloid
Content Through Modification of Specific Cytochrome P450 Genes" (2016). Plant and Soil Sciences
Faculty Patents. 42.
https://uknowledge.uky.edu/pss_patents/42
This Patent is brought to you for free and open access by the Plant and Soil Sciences at UKnowledge. It has been
accepted for inclusion in Plant and Soil Sciences Faculty Patents by an authorized administrator of UKnowledge.
For more information, please contact [email protected].
111111 1111111111111111111111111111111111111111111111111111111111111
US009228195B2
United States Patent
c12) (10) Patent No.: US 9,228,195 B2
Dewey et al. (45) Date of Patent: *Jan. 5, 2016
(54) ALTERATION OF TOBACCO ALKALOID 5,149,645 A 9/1992 Hoekema et al.
CONTENT THROUGH MODIFICATION OF 5,159,135 A 10/1992 Umbeck
SPECIFIC CYTOCHROME P450 GENES 5,177,010 A 111993 Goldman eta!.
5,231,019 A 7/1993 Paszkowski et a!.
5,302,523 A 4/1994 Coffee eta!.
(75) Inventors: Ralph E. Dewey, Apex, NC (US);
5,352,605 A 10/1994 Fraley eta!.
Balazs Siminszky, Neuchatel (CH);
5,378,619 A 111995 Rogers
Steven W. Bowen, Raleigh, NC (US); 5,384,253 A 111995 Krzyzek et a!.
Lily Gavilano, Raleigh, NC (US) 5,463,174 A 10/1995 Moloney et a!.
5,464,763 A 1111995 Schilperoort et a!.
(73) Assignees: North Carolina State University, 5,464,765 A 1111995 Coffee eta!.
Raleigh, NC (US); University of 5,469,976 A 1111995 Burchell
Kentucky Research Foundation, 5,472,869 A 12/1995 Krzyzek et a!.
Lexington, KY (US) 5,583,021 A 12/1996 Dougherty et a!.
5,595,733 A 111997 Carswell et a!.
( *) Notice: Subject to any disclaimer, the term of this 5,614,399 A 3/1997 Quail eta!.
patent is extended or adjusted under 35 5,641,664 A 6/1997 D'Halluin eta!.
U.S.C. 154(b) by 640 days. 5,668,295 A 9/1997 Wahab eta!.
5,679,558 A 10/1997 Gobel eta!.
This patent is subject to a terminal dis 5,684,241 A 1111997 Nakatani et al.
claimer. 5,712,135 A 111998 D'Halluin eta!.
5,713,376 A 2/1998 Berger
(21) Appl. No.: 13/361,235 5,766,900 A 6/1998 Shillito et al.
(Continued)
(22) Filed: Jan.30,2012
FOREIGN PATENT DOCUMENTS
(65) Prior Publication Data
US 2012/0118308 Al May 17,2012 EP 0 120 516 A3 10/1984
EP 0 267 159 A3 5/1988
(Continued)
Related U.S. Application Data
(62) Division of application No. 12/269,531, filed on Nov. OTHER PUBLICATIONS
12, 2008, now Pat. No. 8,124,851.
Demole eta! (Helvetica ChimicaActa-vol. 55, Fasc. 6 (1972) Nbr.
(60) Provisional application No. 60/987,243, filed on Nov.
176).*
12, 2007.
(Continued)
(51) Int. Cl.
A01H 1106 (2006.01)
A01H5112 (2006.01) Primary Examiner-Lee A Visone
C12N 15105 (2006.01) (74) Attorney, Agent, or Firm-Alston & Bird LLP
C12N9/06 (2006.01)
C12N 15182 (2006.01)
C12N9102 (2006.01) (57) ABSTRACT
(52) U.S. Cl.
CPC .............. C12N 1518243 (2013.01); A01H 1106 Compositions and methods for reducing the level of nomico
(2013.01); A01H 5112 (2013.01); C12N9/0073 tine and N'-nitrosonornicotine (NNN) inNicotiana plants and
(2013.01) plant parts thereof are provided. The compositions comprise
(58) Field of Classification Search isolated polynucleotides and polypeptides for cytochrome
None P450s that are involved in the metabolic conversion of nico
See application file for complete search history. tine to nornicotine in these plants. Expression cassettes, vec
tors, plants, and plant parts thereof comprising inhibitory
(56) References Cited sequences that target expression or function of the disclosed
cytochrome P450 polypeptides are also provided. Methods
U.S. PATENT DOCUMENTS
for the use of these novel sequences to inhibit expression or
4,693,976 A 9/1987 Schilperoort et a!. function of cytochrome P450 polypeptides involved in this
4,732,856 A 3/1988 Federoff metabolic conversion are also provided. The methods find use
4,762,785 A 8/1988 Comai in the production of tobacco products that have reduced levels
4,801,540 A 111989 Hiatt eta!.
of nornicotine and its carcinogenic metabolite, NNN, and
4,940,838 A 7/1990 Schilperoort et a!.
4,945,050 A 7/1990 Sanford et a!. thus reduced carcinogenic potential for individuals consum
5,004,863 A 4/1991 Umbeck ing these tobacco products or exposed to secondary smoke
5,013,658 A 5/1991 Dooner eta!. derived from these products.
5,034,323 A 7/1991 Jorgensen eta!.
5,104,310 A 4/1992 Sal tin
5,107,065 A 4/1992 Shewmaker et al.
5,141,131 A 8/1992 Miller eta!. 12 Claims, 4 Drawing Sheets
US 9,228,195 B2
Page 2
(56) References Cited wo WO 2005/116199 A2 12/2005
wo WO 2006/022784 A1 3/2006
U.S. PATENT DOCUMENTS wo WO 2006/091194 A1 8/2006
wo WO 2006/120570 A2 1112006
5,929,304 A 7/1999 Radin eta!. wo WO 2008/070274 A2 6/2008
6,002,070 A 12/1999 D'Halluin et al. wo WO 2008/076802 A2 6/2008
6,074,877 A 6/2000 D'Halluin et al. wo WO 2009/064771 A2 5/2009
6,907,887 B2 6/2005 Conkling wo WO 20111088180 A1 7/2011
6,953,040 B2 10/2005 Atchley et al. wo WO 2012/118779 A1 9/2012
7,032,601 B2 4/2006 Atchley et al.
OTHER PUBLICATIONS
7,700,834 B2 4/2010 Xu eta!.
7,700,851 B2 4/2010 Xu
7,812,227 B2 10/2010 Xu Xu eta! (Physiologia Plantarum 129: 307-319. 2007, first published
7,855,318 B2 12/2010 Xu online: Dec. 20, 2006).*
7,884,263 B2 212011 Dewey et al. Chakrabarti et al (New Phytologist (2007) 175: 565-574, first pub
8,058,504 B2 1112011 Xu lished online: May 30, 2007). *
8,124,851 B2 * 212012 Dewey et al. 800/317.3
Uniprot Q38Q86 Cytochrome P450 monooxygenase CYP82E4v1-
8,319,011 B2 1112012 Xu eta!.
first available online: Nov. 22, 2005. *
2002/0042934 A1 4/2002 Staub et al.
2004/0103449 A1 5/2004 Xu Siminszky et al (PNAS (2005) 102(41) p. 14919-14924).*
2004/01117 59 A1 6/2004 Xu Chakrabarti, M., et al., "Inactivation of the Cytochrome P450 Gene
2004/0117869 A1 6/2004 Xu CYP82E2 by Degenerative Mutations Was a Key Event in the Evo
2004/0162420 A1 8/2004 Xu lution of the Alkaloid Profile of Modern Tobacco," New Phytol., May
2005/0132444 A1 6/2005 Xu
30, 2007, pp. 565-574, vol. 175, No.3.
2005/0160493 A9 7/2005 Ratcliffe et a!.
Chintapakorn, Y., et al., "Antisense-Mediated Down-Regulation of
2005/0178398 A1 8/2005 Breslin et a!.
2005/0223442 A1 10/2005 Xu Putrescine N-Methyltransferase Activity in Transgenic Nicotiana
2005/0244521 A1 1112005 Strickland et a!. Tabacum L. Can Lead to Elevated Levels ofA natabine at the Expense
2006/0037096 A1 2/2006 Xu of Nicotine," Plant Molecular Biology, 2003, pp. 87-105, vol. 53.
2006/0037623 A1 2/2006 Lawrence Gavilano, L.B., "Isolation, Cloning and Characterization of Novel
2006/0041949 A1 2/2006 Xu Tobacco Cytochrome P450 Genes Involved in Secondary Metabo
2006/0157072 A1 7/2006 Albino eta!.
lism," Plant Biology Meeting, Jul. 24-28, 2004, Lake Buena Vista,
2006/0185686 A1 8/2006 Lawrence
Florida, Abstract No. 992, Retrieved from the Internet: URL: http://
2006/0191548 A1 8/2006 Strickland et a!.
2007/0149408 A1 6/2007 Thomas eta!. abstracts.aspb.org/pb2004/public/P7 5/8027 .html.
2007/0199097 A1 * 8/2007 Xu eta!. C12N 9/0073 Gavilano, L.B., et a!., "Genetic Engineering of Nicotiana tabacurn
800/278 for Reduced Nornicotine Content," J Agric. Food Chern., Jul. 11,
2007/0292871 A1 12/2007 Xu 2006, pp. 9071-9078, vol. 54, No. 24.
2008/0076126 A1 3/2008 Xu Gavilano, L.B., and B. Siminszky, "Isolation and Characterization of
2008/0202541 A1 8/2008 Dewey et al. the Cytochrome P450 Gene CYP82E5v2 that Mediates Nicotine to
2008/0245377 A1 10/2008 Marshall et a!. Nornicotine Conversion in the Green Leaves ofTobacco," Plant Cell
2009/0119788 A1 5/2009 Mallmann et a!. Physiol., Oct. 8, 2007, pp. 1567-1574, vol. 48, No. 11.
2009/0205072 A1 8/2009 Dewey et al. Gavilano, L.B., eta!., "Functional Analysis ofNicotine Demethylase
2010/0218270 A1 8/2010 Xu eta!. Genes Reveals Insights into the Evolution of Modern Tobacco," J
2010/0235938 A1 9/2010 Xu eta!. Bioi. Chern, Jan. 5, 2007, pp. 249-256, vol. 282, No. 1.
2010/0235945 A1 9/2010 Xu eta!. Siminszky, B., et a!., "Conversion of Nicotine to Nornicotine in
2010/0235952 A1 9/2010 Xu eta!. Nicotiana tabacurn is Mediated by CYP82E4, a Cytochrome P450
201110048437 A1 3/2011 Xu Monooxygenase," PNAS, Oct. 11, 2005, pp. 14919-14924, vol. 102,
201110078817 A1 3/2011 Xu No. 41.
201110174322 A1 7/2011 Dewey et al. Tang & Galili, "Using RNAi to Improve Plant Nutritional Value:
201110263328 A1 10/2011 Ya rnashita et al. From Mechanism to Application," Trends in Biotechnology, 2004,
2012/0117933 A1 * 5/2012 Dewey et al. 56/27.5 pp. 463-469, vol. 22, No.9.
2012/0118308 A1 5/2012 Dewey et al. EMBL Database Report for Accession No. DQ350312, Dec. 31,
2006 (XP002511577).
FOREIGN PATENT DOCUMENTS EMBL Database Report for Accession No. EU182719, Dec. 2, 2007
(XP002511576).
EP 0 292 435 B1 1111988 Gavilano, et al., "Isolation and Characterization of the Cytochrome
EP 0 320 500 B1 6/1989 P450 Gene CYP82E5v2 that Mediated Nicotine to Nornicotine Con
EP 0116718 5/1990
version in the Green Leaves of Tobacco," Plant Cell Physiol., 2007,
EP 0 159 418 B1 5/1990
vol. 48(11), pp. 1567-1574.
EP 0 176 112 B1 5/1990
Extended European Search Report for European Application No.
EP 0 131 624 B1 9/1992
EP 0 627 752 B1 7/1997 12199188.9; date of mailing Feb. 22,2013.
EP 1 033 405 A3 9/2000 U.S. Appl. No. 60/337,684, filed Nov. 13, 2001, Xu.
EP 0 290 799 B9 1112003 U.S. Appl. No. 60/347,444, filed Jan. 11, 2012, Xu.
wo WO 87/06614 A1 1111987 U.S. Appl. No. 60/363,684, filed Mar. 12, 2002, Xu.
wo WO 92/09696 A1 6/1992 U.S. Appl. No. 60/418,933, filed Oct. 16, 2002, Xu.
wo WO 93/21335 A2 10/1993 U.S. Appl. No. 60/485,368, filed Jul. 8, 2003, Xu.
wo WO 94/01930 A1 111994 U.S. Appl. No. 60/503,989, filed Sep. 18, 2003, Xu.
wo WO 00/67558 A1 1112000
wo WO 02/072758 A2 9/2002 U.S. Appl. No. 60/566,235, filed Apr. 29,2004, Xu.
wo WO 02/100199 A2 12/2002 U.S. Appl. No. 60/607,357, filed Sep. 3, 2004, Xu.
wo WO 03/078577 A2 9/2003 U.S. Appl. No. 60/646,764, filed Apr. 24,2005, Xu.
wo WO 2004/035745 A2 4/2004 U.S. Appl. No. 60/665,097, filed Mar. 24, 2005, Xu.
wo WO 2005/038018 A2 4/2005 U.S. Appl. No. 60/665,451, filed Mar. 24, 2005, Xu.
wo WO 2005/038033 A2 4/2005 Adams eta!., "Genes duplicated by polyploidy show unequal contri
wo WO 2005/046363 A2 5/2005 butions to the transcriptome and organ-specific reciprocal silencing,"
wo W02005/111217 A2 1112005 PNAS, 100(8):4649-4654 (2003).
US 9,228,195 B2
Page 3
(56) References Cited dihydroflavonol reductase down-regulates tannin accumulation in
"hairy root" cultures," Theoretical and Applied Genetics, 87(8):
OTHER PUBLICATIONS 1006.
Caruthers, "Chapter 1: New Methods for Chemically Synthesizing
Allen et a!., "RNAi-mediated replacement of morphine with the Deoxyoligonucleotides," Methods of DNA and RNA Sequencing,
nonnarcotic alkaloid reticulin in opium poppy," Nature Biotechnol Weissman (ed.), Praeger Publishers, New York, pp. 1-22 (1983).
ogy, 22(12):1559-1566 (2004). Chai eta!., "Reducing the maize amylopectin content through RNA
Alonso eta!., "A Hox gene mutation that triggers nonsense-mediated interference manipulation," Zhi Wu Sheng Li Yu Fen Zi Sheng WuXue
RNA decay and affects alternative splicing during Drosophila devel
Xue Buo, 31:625-630 (2005) (English Abstract only).
opment," Nucleic Acids Research, 31(14):3873-3880 (2003).
Chakrabarti eta!., "CYP82E4-mediated nicotine to nornicotine con
Arciga-Reyes et a!., "UPF1 is required for nonsense-mediated
version in tobacco is regulated by a senescence-specific signaling
mRNA decay (NMD) and RNAi inArabidopsis" The Plant Journal,
pathway," Plant Mol. Bioi., 66: 415-427 (2008).
47:480-489 (2006).
Chang et a!., "Predicting and Testing Physical Locations of Geneti
Arndt eta!., "Colocalization of antisense RNAs and ribozymes with
cally Mapped Loci on Tomato Pachytene Chromosome," Genetics,
their target mRNAs," Genome, 40:785-797 (1997).
ARS-GRlN: PI 551280, "Nicotiana tabacum," http://www.ars-grin. 176:2131-2138 (2007).
gov/cgi-bin/npgs/acc/display.pl? 1446216, accessed Feb. 2009). Chao et a!., "A silent mutation induces exon skipping in the
Bak et a!., Transgenic Tobacco and Arabidopsis Plants Expressing phenylalanine hydroxylase gene in phenylketonuria," Hum. Genet.,
the Two Multifunctional Sorghum Cytochrome P450 Enzymes, 108:14-19 (2001).
CYP79 A1 and CYP71 E 1, Are Cyanogenic and Accumulate Metabo Chappell, "Biochemistry and Molecular Biology of the Isoprenoid
lites Derived from Intermediates in Dhurrin. Biosynthetic Pathway in Plants," Annu. Rev. Plant Physiol. Plant
Bartoszewski et al., "Cloning of a Wound Inducible Lycopersicon Mol. Bioi., 46:521-547 (1995).
esculentum Cytochrome P450 Gene and Lack of Regeneration of Chapple, "Molecular-Genetic Analysis of Plant Cytochrome P450-
Transgenic Plants with Sense or Antisense Constructs," J Am. Soc. Dependent Monooxygenases," Annu. Rev. Plant Physiol. Plant Mol.
Hart. Sci., 127(4):535-539 (2002). Bioi., 49:311-343 (1998).
Baseggio eta!., "Size and genomic location of the pMGA multigene Chelvarajan et a!., "Study of Nicotine Demethylation in Nicotiana
family of Mycoplasma gallisepticum," Microbiology, 142:1429- otophora," J Agric. Food Chern., 41:858-862 (1993).
1435 (1996). Chen et a!., "Toxicological analysis of low-nicotine and nocotine
Batard et al., "Increasing Expression of P450 and P450-Reductase free cigarettes," Toxicology, 249: (2008).
Proteins from Monocots in Heterologous Systems," Arch. Biochem. Cheung et a!., "A Floral Transmitting Tissue-Specific Glycoprotein
Biophys., 379:161-169 (2000). Attracts Pollen Tubes and Stimulates Their Growth," Cell, 82:383-
Baulcombe, "Fast Forward Genetics Based on Virus-Induced Gene 393 (1995).
Silencing," Current Opinion in Plant Biology, 2:109-113 (1999). Cho eta!., "Transriptome Analysis and Physical Mapping of Barley
Bolitho eta!., "Antisense apple ACC-oxidase RNA reduces ethylene Genes in Wheat-Barley Chromosome Addition Lines," Genetics,
production in transgenic tomato fruit," Plant Science, 122:91-99 172:1277-1285 (2006).
(1997). Chou et al., "Chromosome Rearrangements inArabidopsis thaliana
Bosher eta!., "RNA interference: genetic wand and genetic watch Generated Through Cre-lox Site Specific Recombination," Plant and
dog," Nat. Cell Bioi., 2:E31-E36 (2000). Animal Genome VII Conference, Abstract No. Pl33.
Bosl et al., "The role of noise and positive feedback in the onset of Chuang eta!., "Specific and heritable genetic interference by double
autosomal dominant diseases," BMC Systems Biology, 4:1-15 (20 10). stranded RNA in Arabidopsis thaliana," PNAS, 97(9):4985-4990
Boyette et a!., "Results of year 2000 TSNA sampling program in (2000).
flue-cured tobacco," Recent Advances in Tobacco Science, 27: 17-22 Cogoni eta!., "Post-transcriptional gene silencing across kingdoms,"
(200 1). Curr. Opin. Genet. Dev., 10:638-643 (2000).
Branch, "A good antisense molecule is hard to find," TIES, 23:45-50 Colbert eta!., "High-throughput screening for induced point muta
(1998). tions," Plant Physiology, 126:480-484 (2001).
Brignetti eta!., "Viral pathogenicity determinants are suppressors of Collier et a!., "A Method for Specific Amplification and PCR
transgene silencing in Nicotiana benthamiana," EMBO J., Sequencing oflndividual Members ofMultigene Families. Applica
17(22):6739-6746 (1998). tion to the Study of Steroid 21-Hydroxylase Deficiency," PCR Meth
Burns eta!., "Large-scale analysis of gene expression, protein local ods and Applications, 1:181-186 (1992).
ization, and gene disruption in Saccharomyces cerevisiae," Genes Colliver et a!., "Differential modification of flavonoid and
Dev., 8:1087-1105 (1994). isoflavonoid biosynthesis with an antisense chalcone synthase con
Burton et al., Changes in Chemical Composition of Burley Tobacco struct in transgenic Lotus corniculatus," Plant Mol. Bioi., 35(4):509-
During Senescence and Curing. 2. Acylated Pyridine Alkaloids, 522 (1997).
American Chemical Society, pp. 579-583 (1988). Crookshanks eta!., "The potato tuber transcriptome: analysis of 6077
Burton eta!., "Distribution of Tobacco Constituents in Tobacco Leaf expressed sequence tags," FEES Lett., 506: 123-126 (200 1) .
Tissue. 1. Tobacco-Specific Nitrosamines, Nitrate, Nitrite, and Alka Davuluri eta!., "Fruit-specific RNAi-mediated suppression ofDET 1
loids," J Agric. Food Chern., 40:1050-1055. enhances carotenoid and favonoid content in tomatoes," Nat.
Burton eta!., "Changes in Chemical Composition ofBurley Tobacco Biotechnol., 23:890-895 (2005).
during Senescence and Curing. 2. Acylated Pyridine Alkaloids," J Dekeyser eta!., "Transient Gene Expression in Intact and Organized
.A gric. Food Chern., 38(3):579-584 (1998). Rice Tissues," Plant Cell, 2:591-602 (1990) .
Bush eta!., "Formation of tobacco-specific nitrosamines in air-cured Dewey eta!., Meeting Abstract dated Sep. 27, 2005, 1 page.
tobacco," Rec. Adv. Tab. Sci, 27:23-46 (2001). Dewey et a!., Power point presentation titled "Functional character
Byers et a!., "Killing the messenger: new insights into nonsense ization of the nicotine N-Demethylase gene of tobacco," Philip Mor
mediated mRNA decay" The Journal of Clinical Investigation, ris USA, 21 pages, 2006.
10 9( 1) :3-6 (2002). Donato et al., "Fluorescence-Based Assays in Intact Cells Expressing
Byzova et a!., "Transforming petals into sepaloid organs in Indicidual Activities for Screening Nine Cytochrome P450 (P450)
Arabidopsis and oilseed rape: implementation of the hairpin RNA Huanm P450 Enzymes," Drug Metab. Dispos., 32(7):699-706
mediated gene silencing technology in an organ-specific manner," (2004).
Planta, 218:379-387 (2004). D'Souza et al., "Missense and silent tau gene mutations cause
Callis et a!., "Introns increase gene expression in cultured maize frontotemporal dementia with parkinsonism-chromosome 17 type,
cells," Genes and Dev., 1:1183-1200 (1987). by affecting multiple alternative RNA splicing regulatory elements"
Carron et al., "Genetic modification of condensed tannin PNAS, 96:5598-5603 (1999).
biosynthesis in Lotus corniculatus. 1. Heterologous antisense EBI Accession AV557806, dated Jun. 16, 2000, 2 pages.
US 9,228,195 B2
Page 4
(56) References Cited GenBankAccession No. DQ350312, dated Dec. 31,2006,2 pages.
GenBankAccession No. DQ350313, dated Dec. 31,2006,2 pages.
OTHER PUBLICATIONS GenBankAccession No. DQ350314, dated Dec. 31,2006,2 pages.
GenBankAccession No. DQ350315, dated Dec. 31,2006,2 pages.
Einset, "Differential expression of antisense in regenerated tobacco GenBankAccession No. DQ350316, dated Dec. 31,2006,2 pages.
plants transformed with an antisense version of a tomato ACC GenBankAccession No. DQ350317, dated Dec. 31, 2006, 2 pages.
oxidase gene," Plant Cell Tissue and Organ Culture, 46(2): 137-141 GenBankAccession No. DQ350318, dated Dec. 31,2006,2 pages.
(1996). GenBankAccession No. DQ350319, dated Dec. 31,2006,2 pages.
Elkind eta!., "Abnormal plant development and down-regulation of GenBankAccession No. DQ350320, dated Dec. 31,2006,2 pages.
phenylpropanoid biosynthesis in transgenic tobacco containing a GenBankAccession No. DQ350321, dated Dec. 31,2006,2 pages.
heterologous phenylalanine anunonia-lyase gene," PNAS, GenBankAccession No. DQ350322, dated Dec. 31,2006,2 pages.
87(22):9057-61 (1990). GenBankAccession No. DQ350323, dated Dec. 31,2006,2 pages.
Escobar et a!., "RNAi-mediated oncogene silencing confers resis GenBankAccession No. DQ350324, dated Dec. 31,2006,2 pages.
tance to crown gall tumorigenesis," PNAS, 98: 13437-13442 (200 1). GenBankAccession No. DQ350325, dated Dec. 31,2006,2 pages.
European Search Report completed on Feb. 10, 2010, in European GenBankAccession No. DQ350326, dated Dec. 31,2006,2 pages.
Application No. EP 07 86 5628, 4 pages. GenBankAccession No. DQ350327, dated Dec. 31,2006,2 pages.
European Search Report completed on Mar. 31, 20 11, in European GenBankAccession No. DQ350328, dated Dec. 31,2006,2 pages.
Application No. EP 10 01 5540, 8 pages. GenBankAccession No. DQ350329, dated Dec. 31,2006,2 pages.
Falcon-Perez eta!., "Functional Domain Analysis of the Yeast ABC GenBankAccession No. DQ350330, dated Dec. 31,2006,2 pages.
Transporter Ycflp by Site-directed Mutagenesis," J Bioi. Chern., GenBankAccession No. DQ350331, dated Dec. 31,2006,2 pages.
274(33):23584-23590 (1999). GenBankAccession No. DQ350332, dated Dec. 31,2006,2 pages.
Fang eta!., "Multiple cis regulatory elements for maximal expression GenBankAccession No. DQ350333, dated Dec. 31,2006,2 pages.
of the cauliflower mosaic virus 35S promoter in transgenic plants," GenBankAccession No. DQ350334, dated Dec. 31,2006,2 pages.
Plant Cell, 1:141-150 ( 1989). GenBankAccession No. DQ350335, dated Dec. 31,2006,2 pages.
Fannin et al., "Nicotine demethylation inNicotiana," Med. Sci. Res., GenBankAccession No. DQ350336, dated Dec. 31,2006,2 pages.
20:807-808 ( 1992). GenBankAccession No. DQ350337, dated Dec. 31,2006,2 pages.
Faske eta!., "Transgenic Tobacco Plants Expressing Pea Chloroplast GenBankAccession No. DQ350338, dated Dec. 31,2006,2 pages.
Nmdh eDNA in Sense and Antisense Orientation," Plant Physiol, GenBankAccession No. DQ350339, dated Dec. 31,2006,2 pages.
115(2): 705-715 ( 1997). GenBankAccession No. DQ350340, dated Dec. 31,2006,2 pages.
Fedoroff eta!., "Cloning of the bronze locus maize by a simple and GenBankAccession No. DQ350341, dated Dec. 31,2006,2 pages.
generalizable procedure using the transposable controlling element GenBankAccession No. DQ350342, dated Dec. 31,2006,2 pages.
Activator (Ac)," PNAS, 81:3825-3829 (1984). GenBankAccession No. DQ350343, dated Dec. 31,2006,2 pages.
Fire et a!., "Potent and specific genetic interference by double GenBankAccession No. DQ350344, dated Dec. 31,2006,2 pages.
stranded RNA in Caenorhabditis elegans," Nature, 391:806-811 GenBankAccession No. DQ350345, dated Dec. 31,2006,2 pages.
(1998). GenBankAccession No. DQ350346, dated Dec. 31,2006,2 pages.
Force eta!., "Preservation of Duplicate Genes by Complementary, GenBankAccession No. DQ350347, dated Dec. 31,2006,2 pages.
Degenerative Mutations," Genetics, 151:1531-1545 (1999). GenBankAccession No. DQ350348, dated Dec. 31,2006,2 pages.
Forsthoefel et a!., "T-DNA Insertion Mutagenesis in Arabidopsis: GenBankAccession No. DQ350349, dated Dec. 31,2006,2 pages.
Prospects and Perspectives," Aust. J Plant Physiol., 19:353-366 GenBankAccession No. DQ350350, dated Dec. 31,2006,2 pages.
(1992). GenBankAccession No. DQ350351, dated Dec. 31,2006,2 pages.
Frank et al., "Cloning of Wound-Induced Cytochrome P450 GenBankAccession No. DQ350352, dated Dec. 31,2006,2 pages.
Monooxygenases Expressed in Pea," P !ant Physiol., 110: 103 5-1046 GenBankAccession No. DQ350353, dated Dec. 31,2006,2 pages.
(1996). GenBankAccession No. DQ350354, dated Dec. 31,2006,2 pages.
Freeman et a!., "Quantitative RT-PCR: Pitfalls and Potential," GenBankAccession No. DQ350355, dated Dec. 31,2006,2 pages.
BioTechniques, 26:112-125 (1999). GenBankAccession No. DQ350356, dated Dec. 31,2006,2 pages.
Fromm eta!., "An octopine synthase enhancer element directs tissue GenBankAccession No. DQ350357, dated Dec. 31,2006,2 pages.
specific expression and binds ASF -1, a factor from tobacco nuclear GenBankAccession No. DQ350358, dated Dec. 31,2006,2 pages.
extracts," Plant Cell, 1:977-984 ( 1989). GenBankAccession No. DQ350359, dated Dec. 31,2006,2 pages.
GenBankAccession No. CAA64635, dated Sep. 12, 1996,2 pages. GenBankAccession No. DQ350360, dated Dec. 31,2006,2 pages.
GenBankAccession No. BAA35080, dated Sep. 26, 2000,2 pages. GenBankAccession No. DQ350361, dated Dec. 31,2006,2 pages.
GenBankAccession No. AAK62347, dated Jun. 14, 2001, 2 pages. GenBankAccession No. DQ350362, dated Dec. 31,2006,2 pages.
GenBank Accession No. AAK62343, dated Feb. 11, 2002, 2 pages. GenBankAccession No. DQ350363, dated Dec. 31,2006,2 pages.
GenBank Accession No. AAK62346, dated Feb. 11, 2002, 2 pages. GenBankAccession No. DQ205656, dated Jan. 18, 2007,2 pages.
GenBank Accession No. AEK08729 dated Feb. 23, 2005, 2 pages. GenBankAccession No. ABA07806, dated Mar. 7, 2007,2 pages.
GenBankAccession No. AAK62342, Sep. 20, 2005, 2 pages. GenBankAccession No. DQ131887, dated Mar. 7, 2007, 2 pages.
GenBank Accession No. ABA07804, dated Oct. 13, 2005, 2 pages. Ghosh, "Polyamines and plant alkaloids," Indian J Exp. Bioi.,
GenBank Accession No. ABA07805, dated Oct. 13, 2005, 2 pages. 38:1086-1091 (2000).
GenBankAccession No. ABA07807, dated Oct. 13,2005, 2 pages. Goldrick et a!., "Molecular Genetic Analysis of the User Group
GenBankAccession No. DQ131885, dated Oct. 13, 2005, 2 pages. Associated with Two Mouse Light Chain Genetic Markers," J Exp.
GenBankAccession No. DQ131886, dated Oct. 13, 2005, 2 pages. Med., 162:713-728 (1985).
GenBankAccession No. DQ131888, dated Oct. 13, 2005, 2 pages. Graham-Lorence eta!., "P450s: Structural similarities and functional
GenBankAccession No. DQ219341, dated Oct. 1, 2006,2 pages. differences," FASEB J., 10:206-214 (1996).
GenBankAccession No. DQ219342, dated Oct. 1, 2006,2 pages. Guo et a!., "Protein Tolerance to Random Amino Acid Change,"
GenBankAccession No. DQ219343, dated Oct. 1, 2006,2 pages. PNAS, 101(25):9205-9210 (2004).
GenBankAccession No. DQ219344, dated Oct. 1, 2006,2 pages. Hao eta!., "Mechanism of Nicotine N-Demethylation in Tobacco
GenBankAccession No. DQ219345, dated Oct. 1, 2006,2 pages. Cell Suspension Cultures," Phytochemistry, 41(2):477-482 (1995).
GenBankAccession No. DQ219346, dated Oct. 1, 2006,2 pages. Hao eta!., "Nicotine N-Demethylase in Cell-Free Preparations from
GenBankAccession No. DQ219347, dated Oct. 1, 2006,2 pages. Tobacco Cell Cultures," Phvtochemistry, 42(2):325-329 (1996).
GenBankAccession No. DQ219348, dated Oct. 1, 2006,2 pages. Hao eta!., "Evidence in Favour of an Oxidative N -Demethylation of
GenBankAccession No. DQ219349, dated Oct. 1, 2006,2 pages. Nicotine to Nornicotine in Tobacco Cell Cultures," Journal Plant
GenBankAccession No. DQ219350, dated Oct. 1, 2006,2 pages. Physiology, 152:420-426 (1998).
GenBankAccession No. DQ219351, dated Oct. 1, 2006,2 pages. Haseloff eta!., "Simple RNA enzymes with new and highly specific
GenBankAccession No. DQ219352, dated Oct. 1, 2006,2 pages. endoribonuclease activities," Nature, 334:585-591 (1998).
US 9,228,195 B2
Page 5
(56) References Cited Julio et a!. "Reducing the content of nornicotine in tobacco via
targeted mutation breeding," Mol. Breeding, 21:369-381 (2008).
OTHER PUBLICATIONS Julio et a!., "Targeted Mutation Breeding as a tool for tobacco crop
improvement," presentation made in Oct. 2008.
Hayes eta!., "Blotting techniques for the study of DNA, RNA, and Kafri et a!., "The regulatory utilization of genetic redundancy
proteins," BMJ, 299(14):965-968 (1989). through responsive backup circuits," PNAS, 10 3(31 ): 11653-11658
Hecht et a!., "The relevance of tobacco-specific nitrosamines to
(2006).
human cancer," Cancer Surveys, 8(2):273-294 (1989). Kempin et a!., "Targeted disruption in Arabidopsis," Nature,
Hecht, "Biochemistry, Biology, and Carcinogenicity of Tobacco
389:802-803 (1997).
Specific N-Nitrosamines," Chemical Research in Toxicology, Keskin eta!., "A new, structurally nonredundant, diverse data set of
11(6):559-603 (1998). protein-protein interfaces and its implications," Protein Science,
Helene eta!., "Control of Gene Expression by Triple Helix-Forming 13:1043-1055 (2004).
Oligonucleotides," Ann. NY Acad. Sci., 660:27-36 (1992). Kim et al., "Arabidopsis CYP85A2, a Cytochrome P450, Mediates
Helene, "The anti -gene strategy: control of gene expression by tri the Baeyer-Villiger Oxidation of Castasterone to Brassinolide in
plex-forming-oligonucleotides," Anti-Cancer Drug Des., 6:569-584 Brassinosteroid Biosynthesis," Plant Cell, 17:2397-2412 (2005).
(1991). Klahre et al., "High molecular weight RNAs and small interfering
Helliwell eta!., "High-throughputvectors for efficient gene silencing
RNAs induce systemic posttranscriptional gene silencing in plants,"
in plants," Funct. Plant Bioi., 29:1217-1225 (2002). PNAS, 99:11981-11986 (2002).
Henikoff et a!., "Single-Nucleotide Mutations for Plant Functional Klink et a!., "The Efficacy of RNAi in the Study of the Plant
Genomics,"Annu. Rev. Plant Bioi., 54:375-401 (2003). Cytoskeleton," J Plant Growth Regul., 19:371-384 (2000).
Herbik et a!., "Isolation, characterization and eDNA cloning of Koornneef, "Chapter 1: Classical mutagenesis in higher plants,"
nicotianamine synthase from barley," Eur J Biochem, 265( 1) : 231-9 Molecular Plant Biology, Gilmartin and Bowler, ed., Oxford Univer
(1999). sity Press, pp. 1-11 (2002).
Hibino et al., "Increase of Cinnamaldehyde Groups in Lignin of Koshinsky et a!., "Cre-lox site-specific recombination between
Transgenic Tobacco Plants Carrying an Antisense Gene for Cin Arabidopsis and tobacco chromosomes," Plant J., 23(6):715-722
namyl Alcohol Dehydrogenase," Biosci. Biotec. Biochem, 59:929- (2000).
931 (1995). Kusaba et a!., "Low glutelin content!: A Dominant Mutation That
Hildering et al., "Chimeric Structure of the Tomato Plant After Seed
Suppresses the Glutelin Multigene Family via RNA Silencing in
Treatment with EMS and X-Rays," The Use oflnduced Mutations in
Rice," Plant Cell, 15:1455-1467 (2003).
Plant Breeding, Pergamon Press, pp. 317-320 (1965).
Kynast et al., "Dissecting the maize genome by using chromosome
Hill et a!., "Functional analysis of conserved histidines in ADP
addition and radiation hybrid lines," PNAS, 101(26):9921-9926
glucose pyrophosphorylase from Escerichia coli," Biochem.
(2004).
Biophys. Res. Commun., 244:573-577 (1998) (Abstract only).
Hoekema eta!., "A binary plant vector strategy based on separation of Lazaret al., "Transforming Growth Factor a Mutation of Aspartic
the vir-and T-region oftheAgrobacterium tumefaciens Ti-plasmid," Acid 47 and Leucine 48 Results in Different Biological Activities,"
Nature, 303:179-180 (1983). Mol. Cell. Bioi., 8(3):1247-1252 (1988).
Hoffmann et a!., "Tobacco-specific N-nitrosamines and Areca-de Levin eta!., "Methods of double-stranded RNA-mediated gene inac
rived N-nitrosamines: .chemistry, biochemistry, carcinogenicity, and tivation inArabidopsis and their use to define an essential gene in the
relevance to humans," Journal of Toxicology and Environmental methionine biosynthesis," Plant Mol. Bioi., 44:759-775.
Health, 41:1-52 (1994). Lewis eta!., "Three nicotine demethylase genes mediate nornicotine
Huang et a!., "Insights into Regulation and Function of the Major biosynthesis inNicotiana tabacum L.: Functional characterization of
Stress-Induced hsp70 Molecular Chaperone In Vivo: Analysis of the CYP82E10 gene, "Phytochemistry, 71:1988-1998 (2010).
Mice with Targeted Gene Disruption of the hsp70. 1 or hsp70.3 Lewis, eta!. "RNA interference (RNAi)-induced suppression ofn ico
Gene," Mol Cell Bioi, 21(24):8575-8591 (2001). tine demethylase activity reduces levels of a key carcinogen in cured
Ingelbrecht et a!., "Posttranscriptional silencing of reporter tobacco leaves." Plant Biotechnology Journal, 6:1-9 (2008).
transgenes in tobacco correlates with DNA methylation," PNAS, Liu et al., "High-Stearic and High-Oleic Cottonseed Oils Produced
91:10502-10506 (1994). by Hairpin RNA-Mediated Post-Transcriptional Gene Silencing,"
International Preliminary Report on Patentability in PCT/US07/ Plant Physiol., 129:1732-1743 (2002).
087386 mailed Jun. 25, 2009, 6 pages. Liu et al., "Identification and characterization ofHTD2: a novel gene
International Search Report and the Written Opinion of the Interna negatively regulating tiller bud outgrowth in rice," Planta,
tional Searching Authority, or the Declaration mailed on May 4, 230(4):649-658 (2009).
2012, in International Application No. PCT/US2012/026864 (13 Liu et al "Genetic and transformation studies reveal negative regula
pages). tion ofERS 1 ethylene receptor signaling inArabidopsis, "BMC Plant
International Search Report and the Written Opinion of the Interna Bioi, 10:60-73 (2010).
tional Searching Authority, or the Declaration mailed on Jul. 4, 2012, Maher, "DNA Triple-Helix Formation: An Approach to Artificial
in International Application No. PCT/US2012/026795 (15 pages). Gene Repressors?" BioEssays, 14(12):807-815 (1992).
International Search Report mailed on Apr. 23, 20 14, in International Maniatis et a!., "Regulation of inducible and tissue-specific gene
Patent Application No. PCT/US2014/011035. expression," Science, 236:1237-1245 (1987).
Invitation to Pay Additional Fees issued on Jun. 23, 2014, in Inter Mansoor et a!. "Engineering novel traits in plants through RNA
national Patent Application No. PCT/US20 14/019381, 8 pages. interference," Trends in Plant Science, 11( 11 ): 1-7 (2006).
Isshiki eta!., "Nonsense-mediated decay of mutant waxy mRNA in Maquat, "Nonsense-mediated mRNA decay," Curr. Bioi.,
rice," Plant Physiology, 125:1388-1395 (200 1). 12(6):R196-R197 (2002).
Jacket a!., "Relative stability of nicotine to nornicotine conversion in Matthew, "RNAi for plant functional genomics," Comparative and
three burley cultivars," Coresta Congress, Kyoto, Agro-Phyto groups, Functional Genomics, 5:240-244 (2004).
Abstract AP2 (2004). McDougall et a!., "Detection of Viral DNA and RNA by In Situ
Johnston eta!., "Dosage-sensitive function of retinoblastoma related Hybridization," J Histochem. Cytochem., 34:33-38 (1986).
and convergent epigenetic control are required during the McKinney eta!., "Sequence-based identification ofT-DNA insertion
Arabidopsis life cycle," PLoS Genet, 6(6):e1000988 (2010). mutations inArabidopsis: actin mutants act2-1 and act4-1 ,"Plant
Jorgensen et a!., "Chalcone synthase cosuppression phenotypes in J., 8(4):613-622 (1995).
petunia flowers: comparison of sense vs. antisense constructs and Mesnard eta!., "Evidence for the involvement oftetrahydrofolate in
single-copy vs. complex T-DNA sequences," Plant Mol. Bioi., the demethylation of nicotine by Nicotiana plumbaginifolia cell
31:957-973 (1996). suspension cultures," Planta, 214:911-919.
US 9,228,195 B2
Page 6
(56) References Cited Reid et a!., "Studies on the Fermentation of Tobacco 1. The
Microflora of Cured and Fermenting Cigar-leaf Tobacco," Bulletin
OTHER PUBLICATIONS 356, Pennsylvania Agricultural Experiment Station, State College,
PA, 18 pages (1938).
Mette et a!., "Transcriptional silencing and promoter methylation Rodermel et al., "Nuclear-Organelle Interactions: Nuclear Antisense
triggered by double-stranded RNA," EMBO J., 19(19):5194-5201 Gene Inhibits Ribulose Biphosphate Carboxylase Enzyme Levels in
(2000). Transformed Tobacco Plants," Cell, 55:673-681(1988).
Mol eta!., "Regulation of plant gene expression by antisense RNA," Rohr et al., "Tandem inverted repeat system for selection of effective
FEES Lett., 268(2):427 -430 ( 1990). transgenic RNAi strains of Chlamydomonas," Plant J., 40:611-621
Napoli eta!., "Introduction of a Chimeric Chalcone Synthase Gene (2004).
into Petunia Results in Reversible Co-Suppression of Homologous Ruiz eta!., "Nicotine-free and salt-tolerant tobacco plants obtained
Genes in trans," Plant Cell, 2:279-289. by grafting to salinity-resistant rootstocks of tomato," Physiologia
Nawrath et a!., "Salicylic Acid Induction-Deficient Mutants of Plantarum, 24:(4):465-475 (2005).
Salehuzzaman et a!., "Isolation and characterization of a eDNA
Arabidopsis Express PR-2 andPR-5 and Accumulate High Levels of
encoding granule-bound starch synthase in cassava (Ma ni hot
Camalexin after Pathogen Inoculation," Plant Cell, 11:1393-1404
esculenta Crantz) and its antisense expression in potato," Plant Mol
(1999).
Bioi, 23(5):947-62 (1993).
Needleman eta!., "A General Method Applicable to the Search for
Schenk et a!., "Coordinated plant defense responses in Arabidopsis
Similarities in the Amino Acid Sequence of Two Proteins," J Mol.
revealed by microarray analysis," PNAS, 97(21): 11655-11660
Bioi., 48:443-453 (1970).
(2000).
Nelson et a!., "Comparative Genomics of Rice and Arabidopsis. Schnable eta!., "Genetic recombination in plants," Curr. Opin. Plant
Analysis of 727 Cytochrome P450 Genes and Pseudogenes from a Bioi., 1:123-129 (1998).
Monocot and a Dicot," Plant Physiol., 135:756-772. Schopfer et al., "Identification of elicitor-induced cytochrome p450s
Nelson eta!., "Comparison of cytochrome P450 (CYP) genes from of soybean (Glycine max L.) using differential display of mRNA,"
the mouse and human genomes, including nomenclature recommen Mol. Gen. Genet., 258:315-322 (1998).
dations for genes, pseudogenes and alternative-splice variants," Seal eta!., "Isolation of a Pseudomonas solanacearum-Specific DNA
Pharmacogenetics, 14:1-18 (2004). Probe by Subtraction Hybridization and Construction of Species
Ng et al., "Specific Detection and Confirmation of Campylobacter Specific Oligonucleotide Primers for Sensitive Detection by the
jejuni by DNA Hybridization and PCR," Appl. Environ. Microbial., Polymerase Chain Reaction," Appl. Environ. Microbial.
63(11):4558-4563 (1997). Sequence 6912fl obtained from the Internet at http://rmg.pscsiken.
Nishihara eta!., "Flavanoid components and flower color change in go.ip/nicotiana/menu/069.htrnl on Dec. 6, 2007, 1 page.
transgenic tobacco plants by suppression of chalcone isomerase Shah et a!., "Expression of Silent Mutations in Disease Phenotype,"
gene," FEES Lett., 579:6074-6078 (2005). Abstract for presentation at 111h International Congress of Human
Odell et al., "Identification ofDNA sequences required for activity of Genetics, 1 page, (2006).
the cauliflower mosaic virus 35S promoter," Nature, 313:810-812 Shen et a!., "Resistance Gene Candidates Identified by PCR with
(1985). Degenerate Oligonucleotide Primers Map to Clusters of Resistance
OfficeActionmailedon Oct. 18,2006, in U.S. Appl. No. 10/293,252. Genes in Lettuce," Molecular Plant-Microbe Interactions,
OfficeActionmailedon Oct. 30,2006, in U.S. Appl. No. 10/686,947. 11(8):815-823 (1998).
OfficeActionmailedonNov.14, 2006, in U.S.Appl. No.10/387,346. Shew eta!. (Eds. ), "Compendium ofTobacco Diseases," published by
OfficeActionmailedonNov.14, 2006, in U.S.Appl. No.10/340,861. American Phytopathology Society, 99 pages (1991).
Office Action mailed on May4, 2007, in U.S. Appl. No. 10/943,507. Sinvany-Villalobo eta!., "Expression in Multi gene Families Analysis
OfficeActionmailedon Jun. 12,2007, in U.S.Appl. No. 10/934,944. of Chloroplast and Mitochondrial Proteases," Plant Physiol,
Ogita et al., "Application of RNAi to confirm theobromine as the 135:1336-1345 (2004).
major intermediate for caffeine biosynthesis in coffee plants with Skarnes, "Entrapment Vectors: A New Tool for Mammalian Genet
potential for construction of decaffeinated varieties," Plant Mol. ics," Bio/Technology, 8:827-831 (1990).
Bioi., 54:931-941 (2004). Smith et a!., "Comparison of Biosequences," Adv. Appl. Math.,
Ohshirna et al., "Nucleotide sequence of the P R -1 gene of Nicotiana 2:482-489 (1981).
tabacum," FEES Letters, 225:243-246 (1987). Smith et al., "Total silencing by intron-spliced hairpin RNAs,"
Oliver et a! , "Inhibition of tobacco NADH-hydroxypyruvate Nature, 407:319-320 (2000).
reductase by expression of a heterologous antisense RNA derived Spradling eta!., "Gene disruptions using P transposable elements: An
from a cucumber eDNA: Implications for the mechanism of action of integral component of the Drosophila genome project," PNAS,
antisense RNAs," Mol Gen Genet, 239(3):425-34 (1993). 92:10824-10830 (1995).
Pearson eta!., "Improved tools for biological sequence comparison," Stalberg eta!., "Deletion analysis of a 2S seed storage protein pro
PNAS, 85:2444-2448 (1988). moter of Brassica napus in transgenic tobacco," Plant Mol. Bioi.,
Peele et a!., "Formation of Tobacco-specific Nitrosamines in Flue 23:671-683 (1993).
cured Tobacco," CORESTA Meeting, Agro-Phyto Groups, Suzhou, Sundaresan et a!., "Patterns of gene action in plant development
China (1999). revealed by enhancer trap and gene trap transposable elements,"
Pickett et a!., "Seeing Double: Appreciating Genetic Redundancy," Genes Dev., 9:1797-1810 (1995).
Plant Cell, 7:1347-1356 (1995). Sureka et al., "Positive Feedback and Noise Activate the Stringent
Plant Variety Protection Office (USDA-AMS, Beltsville, MD, http:/I Response Regulator Rei in Mycobacteria," PLoS One, 3(3):e1771
www.arsgrin.gov/cgi-bin/npgs/htrnllpvp.pl?Tobbaco, accessed Feb. (2008).
2009). Takeda et al., "Differentiation between Wild and Vaccine-Derived
Puchta et a!., "Two different but related mechanisms are used in Strains of Poliovirus by Stringent Microplate Hybridization ofPCR
plants for the repair of genomic double-strand breaks by homologous Products," J Clin. Microbial., 32:202-204 (1994).
recombination," PNAS, 93:5055-5060 (1996). Takemoto eta!., "Molecular Cloning of a Defense-Response-Related
Qin et a!., "Cre recombinase-mediated site-specific recombination Cytochrome P450 Gene from Tobacco," Plant Cell Physiol.,
between plant chromosomes," PNAS, 91: 1706-1710 ( 1994). 40(12):1232-1242 (1999).
Qiu eta!. "A computational study of off-target effects of RNA inter Takken et al. "A functional cloning strategy, based on a binary PYX
ference." Nucleic Acids Research, 33(6)1834-1847 (2005). expression vector, to isolate HR-inducing cDNAs of plant patho
Ralston et a!., "Cloning, Heterologous Aristolochene-1,3- gens." The Plant Journal, 24(2): 275-283 (2000).
Dihydroxylase from Expression, and Functional Characterization of Tavernarakis eta!., "Heritable and inducible genetic interference by
5-epi-Tobacco (Nicotiana tabacum)," Arch. Biochem. Biophys., double-stranded RNA encoded by transgenes," Nat. Genet., 24:180-
393(2):222-235 (200 1) . 183 (2000).
US 9,228,195 B2
Page 7
(56) References Cited Wang et a!., "Isolation and characterization of the CYP71Dl6
trichome-specific promoter from Nicotania tabacum L," J Exp.
OTHER PUBLICATIONS Botany, 53(376):1891-1897 (2002).
Wang et a!., "Elucidation of the functions of genes central to
Temple eta!., "Modulation of glutamine synthetase gene expression diterpene metabolism in tobacco trichomes using posttranscriptional
in tobacco by the introduction of an alfalfa glutamine synthetase gene
gene silencing," Planta, 216:686-691 (2003).
in sense and antisense orientation: molecular and biochemical analy
Waterhouse et al., "Virus resistance and gene silencing in plants can
sis," Mol Gen Genet, 236(2-3):315-25 (1993).
be induced by simultaneous expression of sense and antisense RNA,"
Thomas et a!., "Size constraints for targeting post-transcriptional
PNAS, 95:13959-13964 (1998).
gene silencing and for RNAdirected methylation in Nicotiana
Weigel eta!., "A developmental switch sufficient for flower initiation
benthamiana using a potato virus X vector," Plant J., 25(4):417-425
in diverse plants," Nature, 377:495-500 (1995).
(200 1).
Thornton et al., "From structure to function: Approaches and limita Wei sing et al., "Foreign Genes in Plants: Transfer, Structure, Expres
tions," Nature Structural Biology, Structural Genomics Supplement, sion, and Applications," Ann. Rev. Genetics, 22:421-477 (1988).
pp. 991-994 (2000). Werck-Reichhart et al., "Cytochromes P450: a success story,"
Till eta!., "Discovery of induced point mutations in maize genes by Genome Biology, 1(6):reviews3003.1-3003.9 (2000).
Tilling," BMC Plant Biology, 4:12 (2004). Werck-Reichhart et a!., "Cytochromes P450," The Arabidopsis
Toscano eta!., "A silent mutation (2939G>A, exon 6; CYP2D6*59) Book, American Society of Plant Biologists, 28 pages (2002).
leading to impaired expression and function of CYP2D6," Wernsman eta!., "Time and site of nicotine conversion in tobacco,"
Pharmacogenet. Genomics, 16(10):767-770 (2006). Tobacco Science, 167(22):226-228 (1968).
Travella, eta!. "RNA Interference-Based Gene Silencing as an Effi Wernsman eta!., "Relative Stability of Alleles at the Nicotine Con
cient Tool for Functional Genomics in Hexaploid Bread Wheat." version Locus of Tobacco," Tobacco Science, 14:34-36 (1970).
Plant Physiology, 142:6-20 (2006). Wernsman eta!., "Chapter Seventeen: Tobacco." Cultivar Develop
Trevanion et a!., "NADP-Malate Dehydrogenase in the C4 Plant ment. Crop Species., W. H. Fehr (ed.), MacMillan Publishing Go.,
Flaveria bidentis," Plant Physiol, 113(4):1153-1165 (1997).
Inc., New York, N.Y., pp. 669-698 (1987).
Turner eta!., "Post-transcriptional gene-silencing and RNA interfer
Wesley et a!., "Construct design for efficient, effective and high
ence: genetic immunity, mechanisms and applications," J Chern.
throughput gene silencing in plants," The Plant Journal, 27(6): 581-
Techno!. Biotechnol., 75:869-882 (2000).
590 (2001).
United States, "Tobacco in the United States," Miscellaneous Publi
Wetmur, "DNA Probes: Applications of the Principles of Nucleic
cation No. 867, U.S. Dept. of Agriculture, Agricultural Marketing
Acid Hybridization" Critical Reviews in Bio. and Mol. Bioi., 26:227-
Service, 27 pages (1979).
Vaistij eta!., "Spreading of RNA Targeting and DNA Methylation in 259, (1991).
RNA Silencing Requires Transcription of the Target Gene and a Whitbred et al., "Molecular Characterization of CYP73A9 and
Putative RNA-Dependent RNA Polymerase," Plant Cell, 14:857-867 CYP82Al P450 Genes Involved in plant Defense in Pea," Plant
(2002). Physiol., 124:47-58 (2000).
Van der Krol et al., "An anti-sense chalcone synthase gene in Written Opinion of the International Searching Authority mailed on
transgenic plants inhibits flower pigmentation," Nature, 333:866-869 Apr. 23, 20 14, in International Patent Application No. PCT /US20 14/
(1988). 011035.
Vander Krol et al., "Antisense genes in plants: an overview," Gene, Wu et al. "Herbivory Rapidly Activates MAPK Signaling in Attacked
72:45-50 (1988). and Unattacked Leaf Regions but Not between Leaves of Nicotiana
Vaucheret et al., "Post-transcriptional gene silencing in plants," J attenuata." The Plant Cell, 19:1096-1122 (2007).
Cell Sci., 114:3083-3091 (2001). Xiong et a!., "Different effects on ACC oxidase gene silencing trig
Veena eta!., "Glyoxalase I fromBrassicajuncea: molecular cloning, gered by RNA interference in transgenic tomato," Plant Cell, 23:639-
regulation and its overexpression confer tolerance in transgenic
646 (2004).
tobacco under stress," Plant Journal, 17(4):385-395 (1999).
Xu et a!. "Computational Estimation and Experimental Verification
Verdaguer eta!., "Functional organization of the cassava vein mosaic
of Off-Target Silencing during Posttranscriptional Gene Silencing in
virus (CsVMV) promoter," Plant Mol. Bioi., 37(6):1055-1067
Plants," Plant Physiology, 142:429-440 (2006).
(1998).
Xu et a!., "Biochemical and molecular characterizations of nicotine
Verkerk, "Chimerism of the tomato plant after seed irradiation with
demethylase in tobacco," Physiologia Plantarum, 129(2):307-319
fast neutrons," Neth. J Agric. Sci., 19:197-203 (1971).
Voss et a!., "The role of enhancers in the regulation of cell-type (2007).
specific transcriptional control," Trends Biochem. Sci., 11(7):287- Zwart et a!., "Rapid Screening for Freshwater Bacterial Groups by
289 (1986). Using Reverse Line Blot Hybridization," Appl. Environ. Microbial.,
Wang et a!., "Suppression of a P450 hydroxylase gene in plant 69(10):5875-5883 (2003).
trichome glands enhances natural product-based aphid resistance,"
Nat. Biotechnol., 19:371-374 (2001). * cited by examiner
U.S. Patent Jan.5,2016 Sheet 1 of 4 US 9,228,195 B2
FIG. lA
1 50
-~~~-XI:~Q~~?:~~:~~-~~~~? HLSPIEAIVG LVTFTFLFFF LWTKKSQKPS KPLPPKIPGG WPVIGHLFHF
CYP82E4v6 HLSPIEAIVG LVTFTFLFFF LWTKKSQKPS KPLPPKIPGG WPVIGHLFHF
CYP82E4vl2 HLSPIEAIVG LVTFTFLFfF LWTKKSQKPS KPLPPKIPGG WPVIGHLFHF
58-166 H)/FPIEAIVG LVTFTFLFYF LWTKKSQKPS KPLPPKIPGG WPVIGHLFHF
CYP82E3 r-rif:lhVEAIVG LVTFTFLFYF LWTKKSQKPS KPLPPKIPGG WPVIGHLFXF
CYP82E2v1 r-rlhh IEAFVG LVTFTFLLYF LWTKKSQKLE KPLPPKIPGG WPVIGHLFHF
CYP82E2v2 HLSPIEAFVG LVTFTFLLYF LWTKKSQKLE KPLPPKIPGG WPVIGHLFHF
-~~!~~Efl.~~?~??~;~t~t~~~? HVSPVEAIVG LVTt;Tt;LFYF L wJ[lKKfQlPS KPLPPKIPGG WPVIGHLF$.F
51 100
----~~~~~'f:_ft~~~~~=~~E~~f NDDGDDRPLA RKLGDLADKY GPVFTFRLGL PLVLVVSSYE AVKDCFSTND
CYP82E4v6 NDDGDDRPLA RKLGDLADKY GPVFTFRLGL PLVLVVSSYE AVKDCFSTND
CYP82E4v12 NDDGDDRPLA RKLGDLADKY GPVFTFRLGL PLVLVVSSYE AVKDCFSTND
58-166 NDDGNDRPLA RKLGDLADKY GPVFTFRLGL PLVLVVSSYE AlKDCFSTND
CYP82E3 P])DGDDRPLA RKLGDLADKY GPVFTFRLGL PLVLVVSSYE A~KDCFSTND
CYP82E2vl N'NDGDDRPLA RKLGDLADKY GPVFTFRLGL PLVLVVSSYE AJ'KDCFSTND
CYP82E2v2 NNDGDDRPLA RKLGDLADKY GPVFTFRLGL PLVLVVSSYE A$.KDCFSTND
-~~~(~~_?_;~~!~~;~:l~~? PDDGDDRPLA RKLGDLADKY GPVFTFRLGL PLVLVVSSYE AVKDCFSTND
101 150
~~~J~.$~~~!~~-~~~.:~~?. AIFSNRPAFL YGDYLGYNNA JVILFU'I.NYGPY WRKNRKLVIQ EVLSl\.SRLEK
CYP82E4v6 AIFSNRPAFL YGjijyLGYNNA MLFL}'I.NYGPY WRKNRKLVIQ EVLSl\.SRLEK
CYP82E4v12 AIFSNRPAFL YGPYLGYNNA JVILFU'I.NYGPY WRKNRKLVIQ EVLSl\.SRLEK
58-166 AIFSNRPAFL YGEYLGYNNA JVILFLANYGPY WRKNRKLVIQ EVLSl\.SRL~K
CYP82E3 AIFSNRPAFL YGEYLGY)1N_I'I: JVILFL.ZI,.NYG$Y WRKNRKLW.IQ EVLSl\.SRLEK
CYP82E2vl AIFSNRPAFL YGEYLGYNN:'r JVILFL.ZI,.NYGPY WRKNRKLVIQ EVLSl\.SRLEK
CYP82E2v2 AIFSNRPAtiL YGEYLGYNNT JVILFL.ZI,.NYGPY WRKNRKLVIQ EVLSl\.SRLEK
_s;;_?;J~~~-~f~~~~!-::~~? AIFSNRPAFL YGEYLGY$.NA JVILFLW¥.'YGPY WRKNRKLVIQ EVLSl\.SRLEK
151 200
_(~1~E5$.:~~:~~~~-,~~~~~ FKHVRFARIQ @SIKNLYTRI DGNSSTINLT DWLEELNFGL IVKHIAGKNY
CYP82E4v6 FKHVRFARIQ ASIKNLYTRI DGNSSTINLT DWLEELNFGL IVKHIAGKNY
CYP82E4v12 FKHVRFARIQ $SIKNLYTRI DGNSSTINLT DWLEELNFGL IVKHIAGKNY
58-166 FKHVRFARIQ TSIKNLYTRI D$NSSTINLT DWLEELNFGL IVKHIAGKNY
CYP82E3 FKHVRFARIQ TSIKNLYTRI DGNSSTINLT DWLEELNFGL IVKHIAGKNY
CYP82E2vl FKQVRF(ilRIQ TSIKNLYTRI l4GNSSTINLT DWLEELNFGL IVKHIAGKNY
CYP82E2v2 FKQVRF~RIQ TSIKNLYTRI ~\fGNSSTINLT DWLEELNFGL IVKHIAGKNY
-~~~J?fl_~~;~~;;~_'§:~:~~~ PKHVRFG}.j;IQ TSIK$.LYTRI DGNSSTINLT DWLEELNFGL IVKHIAGKNY
U.S. Patent Jan.5,2016 Sheet 2 of 4 US 9,228,195 B2
FIG. lB
201 250
ESGKGDEQVE RFKKAFKDFM ILSI'1EFVLWD AFPIPLFKWV DFQGHVKAJVIK
-~~g~~~~~~~~~~~~~-~~:~?
CYP82E4v6 ESGKGDEQVE RFKKAFKDFM ILSI'1EFVLWD AFPIPLFKWV DFQGHVKAJVIK
CYP82E4v12 ESGKGDEQVE RFKKAFKDFM ILSI'1EFVLWD AFPIPLFKWV DFQGHVKAJVIK
58-166 ESGKGDEQVE RFKKAFKDFM If.Sl'1EFVLWD AFPIPLFKWV DFQGHVKAJVIK
CYP82E3 ESGKGDEQVE RFKKAFKDFM ILSI'1EFVLWD AFPIPLFKWV DFQGHVKAJVIK
CYP82E2v1 ESGKGDEQVE RFK~AFKDFM VLSI'1EFVLWD AFPIPLFKWV DFQGHIKAJVIK
CYP82E2v2 ESGKGDEQVE RFK!:\tAFKDFM VLSI'1EFVLWD AFPIPLFKWV DFQGHlKAJVIK
ESGKGDEQVE RFRKAFKDFI ILSI'1EFVLWD AFPIPLFKWV DFQGHVKAJVIK
~~~~~~~-~~~~:~1~;~~·:~~~~
251 300
RTFKDIDSVF REK.MEVNAE GNEQDFIDVV LSKJVISNEYLG
s~~:X}~:3:~~~~--~~~~~~? QN~"i'LEEHINK
CYP82E4v6 RTFKDIDSVF REK.MEVNAE GNEQDFIDVV LSKJVISNEYLG
QN~"i'LEEHINK
CYP82E4vl2 RTFKDIDSVF REK.MEVNAE GNEQDFIDVV LSKJVISNEYLG
QN~"i'LEEHINK
58-166 RTFKDIDSVF REK.MEVNAE GNEQDFIDVV LSKJVISNEYLG
QN~"i'LEEHINK
CYP82E3 RTFKDIDSVF QN~"i'LEEHikK REKiMEVGTE GNEQDFIDVV LSKJVISNEYLG
CYP82E2vl RTFKDIDSVF REK:MEVGAE GNEQDFIDVV LSKLSKEYLD
QN~"i'LEEHINK
CYP82E2v2 RTFKDIDSVF REK.iEVGAE GNEQDFIDVV LSKLSKEYLD
QN~"i'LEEHINK
J~?~e§:=~~~~;I~~~~~ RTFKDIDSVF QN~"i'LEEH)(ij:gK REK. MEVi~AQ GNEQDFIDVV LSK~iSNEYLQ
301 350
EGYSRDTVIK ATVFSLVLDA GJVIALL INNQ'l{ l\.LjlKAQEEID
-~;_~~~f_~~~~~~l~:~~~-y:~? ADTVALHIN~iJ
CYP82E4v6 EGYSRDTVIK ATVFSLVLDA GJVIALLINNQK l\.LWKAQEEID
ADTVALHIN~iJ
CYP82E4v12 EGYSRDTVIjlj ATVFSLVLDA GJVIALLINNQ:f{ l\.LWKAQEEID
ADTVALHIN~iJ
58-166 EGYSRDTVIK ATVFSLVLDA GJVIALLINNQN l\.LKKAQEEID
ADTVALHIN~iJ
CYP82E3 EGYSRDTVIK ATVFSLVLDA ADTVALHINQ GJVIALLINNQN l\.LKKAQEEID
CYP82E2v1 EGYSRDTVIK ATVFSLVLDA GJVI!JLLINNQN l\.LijKAQEEID
ADTVALHIN~iJ
CYP82E2v2 EGYSRDTVIK ATVFSLVLDA GJVJfr:LLINNQN l\.Li0KAQEEID
ADTVALHIN~iJ
~~~~~~~-~~~~:~1~;~~·:~~~~ EGYSRDTVIK ATVFSLVLDA ADTVALHi)itN~iJ GJVIALLINNQff l\.LKKAQEEID
351 400
TKVGKDRWVE ESDIKDLVYL QAIVKEVLRL YPPGPLLVPH ENVEDCVVSG
~~~~~~_?_;~~g;_-~~·:~~?
CYP82E4v6 TKVGKDRWVE ESDIKDLVYL QAIVKEVLRL YPPGPLLVPH ENVEDCVVSG
CYP82E4vl2 TKVCKDRWVE ESDIKDLVYL QAIVKEVLRL YPPGPLLVPH ENVEDCVVSG
58-166 TlVGKDRWVE ESDIKDLVYL QAIVKEVLRL YPPGPLLVPH EN¢EDCVVSG
CYP82E3 TKVGKDRWVE ESDIKDLVYL QAIVKEVLRL YPPGPLLVPH ENVEDCVVSG
CYP82E2vl TKVGKDRWVE ESDIKDLVYL QAIVKKVLRL YPPGPLLVPH ENV;DcvvsG
CYP82E2v2 TKVGKDRWVE ESDIKDLVYL QAIVKkVLRL YPPGPLLVPH ENVI)'DCVVSG
J~?~e§:=~~~~;I~~~~~ ~KVGK)l.:lRWVE ESDIKDLVYL QAIVKEVLRL YPPGPLLVPH ENVEDCVVSG
401 450
_r;_I;f~~~=~;~~:~_Y::;~ YHIPKGTRLF ANVJVIKLQRDP KLli'/SDPDTFD PERFIATDID FRGQYYKYIP
CYP82E4v6 YHIPKGTRLF ANVJVIKLpRDP KL li'/FDP DTFD PERFIATDID FRGQj(yfi:YIP
CYP82E4v12 YHIPKGTRLF ANVJVIKLQRDP KLli'/SDPDifFD PERFIATDID FRGQihKYIP
58-166 YXISKGTRLF ANVJVIKLQRDP KLli'/PNPDNFD FRGQHYEYIP
~~~~~~·~~~
CYP82E3 YHIPKGTRLF ANVJVIKLQRDP KLli'/SNPDKFN FBGQHYEYIP