USO08097774B2

(12) United States Patent (10) Patent No.: US 8,097,774 B2 Hawkes et al. (45) Date of Patent: Jan. 17, 2012

(54) CYTOCHROME P4SO GENES CONFERRING WO WOOO.21924 4/2000 RESISTANCE WO O2/46.387 A2 6, 2002 WO WO O2/O851.18 10, 2002 WO 2007/OOOO77 A1 1, 2007 (75) Inventors: Timothy Robert Hawkes, Bracknell WO 2007 O24739 A2 3, 2007 (GB); Bernardus Theodorus Maria WO WO 2007/103567 9, 2007 Vernooij, Durham, NC (US) OTHER PUBLICATIONS (73) Assignee: Syngenta Participations AG, Basel Panet al. Aug. 2006, Plant Molecular Biology 61:933-943.* (CH) Frear, D.S. et all “N-demethylation of substituted 3-(phenyl)-1- (*) Notice: Subject to any disclaimer, the term of this methylureas: isolation and characterization of a microsomal mixed patent is extended or adjusted under 35 function oxidase from cotton'. Phytochemistry (1969) 8: 2157-2169. Grossmann, Klaus etal "On the mechanism of action and selectivity U.S.C. 154(b) by 486 days. of the corn herbicide toprameZone: a new inhibitor of 4-hydroxyphenylpyruvate dioxygenase” Pest Mgmt. Sci. (2007) (21) Appl. No.: 12/156.247 63:429-439. (22) Filed: May 30, 2008 Hawkes et al. ": Mechanism of herbicidal activity and selectivity in corn”. Proc. Brighton Pest Cont Conf. Weeds, BCPC, (65) Prior Publication Data (2001) 563-568. Shiota et al. “Herbicide-resistant tobacco plants expressing the fused US 2009/OO11936A1 Jan. 8, 2009 enzyme between rat cytochrome P450 1A1 (CYP1A1) and yeast NADPH-cytochrome P450 oxidoreductase'. Plant Physiol. (1994) Related U.S. Application Data 106:17-23. (60) Provisional application No. 60/932,560, filed on May Siminszky et al. “Expression of a soybean cytochrome P450 30, 2007. monooxygenase cDNA in yeast and tobacco enhances the metabo lism of phenylurea '. Proc. Natl. Acad. Sci USA (1999) (51) Int. Cl. 96:1750-1755. CI2N 15/82 (2006.01) Bolwell et al (1994) Phytochemistry 37: 1491-1506. (52) U.S. Cl...... 8OO/3OO Mizutani and Ohta (1998) Plant Physiol., 116, 357-367. (58) Field of Classification Search ...... None Morant et al. (2003) Current Opinion in Biotechnology, 14:151-162. See application file for complete search history. Robineau et al. (1998) Plant Physiol., 118: 1049-1056. Didierjean et al. (2002) Plant Physiol. 130:179-189. (56) References Cited Persans et al (2001) Plant physiol. 125:1126-1138. Shuler (1996) Crit. Rev. Plant. Sci, 15:235-284. U.S. PATENT DOCUMENTS Panet al. (2006) Plant Molecular Biology, 61:933-943. 4,780,127 A 10/1988 Michaely et al. 5,006,158 A 4, 1991 Carter et al. (Continued) 5,349,127 A 9, 1994 Dean 5,656,573 A 8, 1997 Roberts et al. 5,882,851 A 3, 1999 Koch Primary Examiner — David H Kruse 6,121,512 A 9/2000 Siminszky 6,300,544 B1 10/2001 Halkier (74) Attorney, Agent, or Firm — S. Matthew Edwards 6,367.429 B2 4/2002 Van Almsick et al. 6,380.465 B1 4/2002 Barrett 6,534,444 B1 3/2003 Sievernich et al. (57) ABSTRACT 6,649,814 B2 11/2003 Halkier et al. 6,844.294 B2 1/2005 Auler et al. Compositions and methods for conferring herbicide resis 7,005,283 B2 2/2006 Croteau 7,250,561 B1* 7/2007 Pallett et al...... 800/300 tance or tolerance to plants, plant cells, tissues and seeds are 7,405,057 B2 7/2008 Chappell provided. Compositions include transgenic plants, plant 7,608,564 B2 10/2009 Von Deyn et al. cells, tissues, and seeds that have been transformed with a 7,705,200 B2 * 4/2010 Dam et al...... 800,278 nucleic acid molecule encoding a cytochrome P450 or variant 2004.0034.888 A1 2/2004 Liu et al. 2004/0058427 A1 3, 2004 Andrews thereofthat confers herbicide resistance or tolerance, alone or 2005/006O767 A1 3/2005 Subramanian et al. in combination with one or more additional nucleic acid 2005/0204436 A1 9, 2005 Hammer molecules encoding polypeptides that confer desirable traits. 2005/0246800 A1 11/2005 Dunne et al. 2006/01 17406 A1 6, 2006 Schultze In particular, the cytochrome P450 or variant thereof confers 2007/OO79393 A1 4/2007 McCutchen et al. resistance or tolerance to HPPD inhibitors, benzothiadiazi 2007/0214515 A1 9, 2007 Dam nones, Sulfonylureas, and other classes of herbicides. The additional polypeptide may also confer resistance or toler FOREIGN PATENT DOCUMENTS ance to an herbicide, including HPPD inhibitors and other AU 19995.8616 5, 2000 herbicides. Methods are also provided for the production and AU T55.314 B2 12/2002 EP 1728868 12/2006 use of the herbicide resistant or tolerant plants, plant cells, WO 98/20144 A2 5, 1998 tissues and seeds of the invention. WO WO 98.31681 7, 1998 WO WO99, 65314 12/1999 WO WOOOf 15615 3, 2000 7 Claims, 4 Drawing Sheets US 8,097,774 B2 Page 2

OTHER PUBLICATIONS Gang, Panetal"Map-based cloning of a novel rice cytochrome P450 Siminszky (2006) Phytochemistry Reviews, 5:445-458. gene CYP81A6 that confers resistance to two different classes of Werck-Reichhart et al. (2000) Trends in Plant Science, 5:116-123. herbicides' Plant Molecular Biology, Aug. 1, 2006, vol. 61, No. 6, Ohkawa et al. (1999) Pesticide Science, 55:867-874. pp. 933-943. Kreuz et al. (1996) Plant Physiology, 1 11:349-353. Kawahigashi et al “Broad range of herbicide tolerance of glutinous O'Keefe et al. (1994) Plant Physiology, 105:473-482. upland rice variety Yumenohatamochi carrying human cytochrome Williams et al., Map-based cloning of the nsfl (nicosulfuron Suscep P450 genes' Plant Biotechnology, 23:227-231; p. 229 (2006). tible 1) gene of maize, P26, 48th Annual Maize Genetics Conference, Kawahigashi et al "Herbicide resistance of transgenic rice plants Mar. 9-12, 2006, Asiolmar Conference Grounds, Pacific Grove, Cali expressing human CYP1A1” Biotechnology Advances, 25 (2007) fornia. 75-84; abstract. Williams et al. (2005) HortScience, 40: 1801-1805. Letouze et al Inheritance of fenoxaprop-P-ethyl resistance in Hawkes et al., Mesotrione: Mechanism of herbicidal activity and blackgrass (Alopecurus myosuroides Huds.) population. Theor Appl selectivity in corn, The BCPC Conference Weeds 2001, Brighton Genet, 2001, 103:288-296. Metropole Hotel, Brighton, UK, Nov. 12-15, 2001. Matringe et al., “p-Hydroxyphenylpyruvate dioxygenase inhibitor Armel. Weed management in conventional, no-till, and transgenic resistant plants' Pest Management Science, 2005, 61: 269-276. corn with mesotrione combinations and other herbicides. PhD Dis Subramanian et all “Engineering selectivity in crops: a sertation (Chapter 1) Apr. 29, 2002. search for appropriate degradative enzyme(s).' Journal of Industrial Bierman et al Fungicide-herbicide interaction in Soybean (Glycine Microbiology & Biotechnology, 1997, 19:344-349; abstract. max). Crop Protection 25 (2006) 134-139. Search Report dated Jun. 10, 2009 issued in corresponding Interna Frear, etal"Metabolism offlumetSulam (DE-498) in wheat, corn, and tional Application No. PCT/US2008/006891. barley” Pesticide biochemistry and physiology (USA) Mar. 1993, 45(3):178-192: Abstract. * cited by examiner U.S. Patent Jan. 17, 2012 Sheet 1 of 4 US 8,097,774 B2

8:88-88

U.S. Patent Jan. 17, 2012 Sheet 2 of 4 US 8,097,774 B2

Figure 2

Sequence Identification Number SEQID NO:1 Maizensf CYP amino acid sequence,

deduced from the nsf1 gene sequence derived from B73 BAC clone b0159B4 SEQID NO:3 Maize CYP72A1 amino acid sequence SEQID NO:4 Optimized CYP72A1 gene sequence SEQID NO:5 Rice CYP81A6 amino acid sequence, G. Panet al. (2007) Plant Mol. Biol., 61:933

943; GenBank accession no: ABC69856

(derived from corn line SSCIC008) SEQID NO:8 Amino acid sequence from gene 136090 (derived from corn line B73, BAC b0159B4) Catharanthus CP72A1 amino acid sequence (Persans et al. (2001) Plant

Physiol., 125:1126); Genbank Accession: AAA33106 SEQID NO:10 Avena HPPD amino acid sequence SEQID NO:11 Modified Avena HPPD amino acid SeCeCe. SEQID NO:12 Modified Avena HPPD amino acid sequence SEQ ID NO:13 Modified Avena HPPD amino acid Synthetic, optimized Avena HPPD gene Sequence HPPD gene sequence

US 8,097,774 B2 1. 2 CYTOCHROME P4SO GENES CONFERRING pounds (reviewed in Bolwell et al. (1994) Phytochemistry HERBICIDE RESISTANCE 37:1491-1506; Shuler (1996) Crit. Rev. Plant. Sci. 15:235 284). Likewise, the cropfweed selectivity of certain HPPD FIELD OF THE INVENTION inhibiting herbicides such as mesotrione (Hawkes et al. (2001) Proc. Brighton Pest Cont Conf. Weeds, BCPC, 563 The present invention relates to herbicide tolerant plants 568) and toprameZone (Grossmann and Ehrhardt (2007) Pest and the use of herbicides over such plants. More particularly, Mgmt. Sci. 63:429-439) for example is largely dependent on the present invention relates to genes for the production of the activity of cytochrome P450 type enzymes. commercial plants that are resistant to certain classes of her Differential herbicide metabolizing P450 activities are bicides that inhibit hydroxyphenyl pyruvate dioxygenase 10 believed to represent one of the mechanisms that enable cer (HPPD) as well other classes of herbicides. Specifically, the tain crop species to be more tolerant of a particular herbicide present invention relates to the degradation of such herbicides than other crop or weedy species. The following patents and in plants by expression of certain cytochrome P450 genes. applications collect information useful for the background understanding of cytochrome P450 use for herbicide toler BACKGROUND OF THE INVENTION 15 ance (U.S. Pat. Nos. 6,380,465; 6,121,512; 5,349,127; and PCT Patent App. Pub. No. WO2007000077). See also U.S. Cytochrome P450 (P450) monooxygenases are ubiquitous Pat. Nos. 6,649,814 and 6,300,544 for disclosure relating to hemoproteins present in microorganisms, plants and animals. cytochrome P450 monooxygenases for obtaining transgenic Comprised of a large and diverse group of isozymes, P450s plants resistant to insects, acarids, or nematodes, or with mediate a great array of oxidative reactions using a wide improved nutritive value. range of compounds as Substrates, and including reactions Methods for providing plants which are tolerant to HPPD involved in biosynthetic processes Such as phenylpropanoid, herbicides which comprise transformation of plant material fatty acid, and terpenoid biosynthesis; metabolism of natural with polynucleotides comprising regions which encode products; and detoxification of foreign Substances (Xenobiot HPPD enzymes are known (See, e.g., U.S. Application Pub ics). See e.g., Schuler (1996) Crit. Rev. Plant Sci. 15:235-284 25 lication Number 2004/0058427; PCT application WO98/ (1996). In a typical P450 catalyzed reaction, one atom of 20144; PCT application WO 02/46387: see also U.S. Appli molecular oxygen (O) is incorporated into the Substrate, and cation Publication Number 2005/0246800 relating to the other atom is reduced to water. Electrons are supplied identification and labelling of soybean varieties as being rela through oxidation of NADPH. For most eukaryotic P450s, tively HPPD tolerant). However what has not hitherto been NADPH:cytochrome P450 reductase, a membrane-bound 30 generally recognised is that cytochrome P450 enzymes can flavoprotein, transfers the necessary two electrons from provide additional or commercial levels of tolerance to NADPH to the cytochrome P450 (Bolwell et al (1994) Phy HPPD-inhibitor and other herbicides, when expressed either tochemistry 37: 1491-1506; Mizutani and Ohta (1998) Plant in combination with HPPD enzymes, or in tolerant back Physiol., 16, 357-367). grounds, or alone in a transformed plant. While a given HPPD Plant cytochrome P450s are known to be involved in the 35 enzyme may provide a useful level of tolerance to some metabolism and detoxification of numerous pesticides as well HPPD-inhibitor herbicides it may be quite inadequate to pro as in the biosynthesis of primary and secondary metabolites. vide commercial levels of tolerance to a different, more desir Much of the evidence has been gathered via traditional chem able HPPD-inhibitor herbicide which, for example, may con istry techniques (Shuler (1996) Crit. Rev. Plant. Sci. 15:235 trola different spectrum of weeds, be cheaper to make or offer 284; Bolwell et al. (1994) Phytochemistry 37:1491-1506; 40 environmental benefits. As well as particular HPPD enzymes Frear et al. (1969) Phytochemistry 8:2157-2169) and through and the polynucleotides which encode them the current studies of mammalian or bacterial genes in plants (Shiota et invention provides a means of enhancing the effect of HPPD al. (1994) Plant Physiol. 106:17-23; O'Keefe et al. (1994) enzymes suitable for providing commercially useful levels of Plant Physiol. 105:473-482). resistance to particular HPPD-inhibitor herbicide chemistries Recently, endogenous plant P450s have been successfully 45 through the use of cytochrome P450 enzymes that degrade cloned, expressed, characterised and assayed in heterologous certain, below-specified, HPPD-inhibitor herbicide chemis systems (Siminszky et al. (1999) PNAS (USA) 96:1750 tries. In addition, expression of these cytochrome P450 1755). For example, CYP76B1 was cloned (Robineau et al. enzymes also provides a means of reducing the amount of (1998) Plant Physiol., 118: 1049-1056) from the Jerusalem parent active herbicide that persists in plant tissues and of artichoke (Helianthus tuberosus). This xenobiotic inducible 50 therefore decreasing the overall amount of parent herbicide cytochrome P450 was found to be strongly inducible and to residue entering the food and feed chain as a result of appli catalyze the rapid oxidative N-dealkylation of various pheny cation to food or feed crops. lurea herbicides to yield non-phytotoxic metabolites. Heter In addition, the genes disclosed herein also provide an ologous expression of the CYP76B1 gene in tobacco (Nicoti alternative process for providing resistance to various Xeno ana tabacum) and Arabidopsis resulted in increased rates of 55 biotics including herbicides and including some types. Such herbicide oxidation and was, by itself, sufficient to yield a 20 as sulfonylureas having a non-HPPD mode of action. fold level of tolerance to , a compound detoxified by a single dealkylation, and also a 10-fold increase intolerance to SUMMARY OF THE INVENTION isoproturon or , which need Successive catalytic steps for detoxification (Didierjean et al. (2002) Plant 60 Compositions and methods for conferring herbicide resis Physiol. 130:179-189). tance or tolerance to plants, plant cells, tissues and seeds are Frear et al. (1969) Phytochemistry 8:2157-2169 demon provided. Compositions include transgenic plants, plant strated the metabolism of monuron by a mixed-function oxi cells, tissues, and seeds that have been transformed with a dase located in a microsomal fraction of cotton seedlings. nucleic acid molecule encoding a cytochrome P450 or variant Further evidence has accumulated Supporting the involve 65 thereofthat confers herbicide resistance or tolerance, alone or ment of P450s in the metabolism and detoxification of numer in combination with one or more additional nucleic acid ous herbicides representing several distinct classes of com molecules encoding polypeptides that confer desirable traits. US 8,097,774 B2 3 4 In particular, the cytochrome P450 or variant thereof confers sequences (and expression products thereof) when used in the resistance or tolerance to HPPD inhibitors, Benzothiadiazi production of, or when produced by, the said transgenic nones, Sulfonylureas, and other classes of herbicides includ plants. Compositions of the invention include transgenic ing, for example, Imidazolinones, Triazolopyrimidines, Pyri plants, plant cells, tissues, and seeds that have been trans midinylthiobenzoates, Triazolinones, , Acetyl- 5 formed with a nucleic acid molecule encoding a cytochrome coenzyme A Carboxylase (ACCase) inhibitors, Photosystem P450 or variant thereof that confers herbicide resistance or II (PSII) inhibitors, Protoporphyrinogen Oxidase (PPO) tolerance, alone or in combination with one or more addi inhibitors, Phytoene Desaturase (PDS) inhibitors, Dini tional nucleic acid molecules encoding polypeptides that con troanalines, and Acetamides, as well as herbicides with fer desirable traits. In particular, the cytochrome P450 or unknown modes of action such as Difenzoquat and Cloma- 10 variant thereof confers resistance or tolerance to HPPD Zone. The additional polypeptide may also confer resistance inhibitors, Benzothiadiazinones, Sulfonylureas, and other or tolerance to an herbicide, including HPPD inhibitors and classes of herbicides including, for example, Imidazolinones, other herbicides. Methods are also provided for the produc Triazolopyrimidines, Pyrimidinylthiobenzoates, Triazolino tion and use of the herbicide resistant or tolerant plants, plant nes, Auxins, Acetyl-coenzyme A Carboxylase (ACCase) cells, tissues and seeds of the invention. 15 inhibitors, Photosystem II (PSII) inhibitors, Protoporphy The present invention provides gene sequences set forth in rinogen Oxidase (PPO) inhibitors, Phytoene Desaturase SEQ ID NOS:2, 4, and 6, that encode cytochrome P450 (PDS) inhibitors, Dinitroanalines, and Acetamides, as well as enzymes capable of degrading certain herbicides of the herbicides with unknown modes of action such as Difenzo HPPD classes of herbicides when expressed in plants. The quat and . The additional polypeptide may also present invention also relates to transformed plants overex- 20 confer resistance or tolerance to an herbicide, including pressing the P450s set forth in SEQ ID NOS:1, 3, and 5, HPPD inhibitors and other herbicides. Methods are also pro genetic constructs for overexpressing the genes in plants, and vided for the production and use of the herbicide resistant or to the over-the-top application of HPPD class herbicides, tolerant plants, plant cells, tissues and seeds of the invention. both alone and in combination with other herbicides, to Within the context of the present invention the terms HPPD resistant plants, especially crop plants in fields, for 25 hydroxy phenyl pyruvate (or pyruvic acid) dioxygenase weed control without damage to the crop plants. The present (HPPD), 4-hydroxyphenyl pyruvate (or pyruvic acid) dioxy invention also contemplates combining the P450 genes in the genase (4-HPPD) and p-hydroxyphenylpyruvate (or pyruvic same plant with genes conferring resistance to other herbi acid) dioxygenase (p-HPPD) are synonymous. cides, for example EPSPS genes (for instance, EPSPS genes As used herein, plants which are substantially “tolerant’ to with natural tolerance to ) conferring resistance to 30 a herbicide when they are subjected to it provide a dose/ glyphosate, and phosphinothricin acetyl transferase genes response curve which is shifted to the right when compared (PAT and BAR genes, described in U.S. Pat. Nos. 5,561,236 with that provided by similarly subjected non tolerant like and 5,276.268) conferring resistance to . Further plants. Such dose/response curves have “dose” plotted on the more, the present invention also provides plants with reduced x-axis and “percentage kill or damage”, “herbicidal effect” HPPD herbicide residue levels. 35 etc. plotted on the y-axis. Tolerant plants will typically require at least twice as much herbicide as non tolerant like BRIEF DESCRIPTION OF THE FIGURES plants in order to produce a given herbicidal effect. Plants which are substantially “resistant to the herbicide exhibit FIG. 1 shows a DNA construct comprising: 1) a cassette few, if any, necrotic, lytic, chlorotic or other lesions when having a modified Avena HPPD gene under operable expres- 40 subjected to the herbicide at concentrations and rates which sion control of an upstream promoter (e.g., a small subunit of are typically employed by the agricultural community to kill Rubisco promoter region including 5' untranslated leader weeds in the field. sequence) and a downstream nopaline synthase 3' terminator As used herein, “non-trangenic-like plants' are plants that sequence; 2) a DNA sequence encoding the maize insfl pro are similar or the same as transgenic plants but that do not tein sequence (SEQ ID NO:2) under operable control of an 45 contain a transgene conferring herbicide resistance. upstream Arabidopsis or soybean polyubiquitin promoter and As used herein, the term “confer refers to providing a 5' untranslated leader sequence and a downstream 3' termi characteristic or trait, such as herbicide tolerance or resis nator sequence from a histone gene. tance and/or other desirable traits to a plant. FIG. 2 shows a list of polynucleotide and amino acid Disclosed here is a method for rendering crop plants (corn, sequences relating to cytochrome P450 enzymes for use in the 50 rice, maize, wheat, barley, Soybean, rape/canola, cotton etc.) present invention. tolerant to herbicides that act by inhibiting the enzyme FIG.3 shows a map of binary vector 17107 harbouring the hydroxyphenylpyruvate dioxygenase (HPPD), or other her CYP72A1 coding region. bicides such as Benzothiadiazinones, Sulfonylureas, and FIG. 4 shows a map of binary vector 17108 harbouring the other classes of herbicides including, for example, Imidazol NSF coding region. 55 inones, Triazolopyrimidines, Pyrimidinylthiobenzoates, Triazolinones, Auxins, Acetyl-coenzyme A Carboxylase DETAILED DESCRIPTION (ACCase) inhibitors, Photosystem II (PSII) inhibitors, Proto porphyrinogen Oxidase (PPO) inhibitors, Phytoene Desatu Overview rase (PDS) inhibitors, Dinitroanalines, and Acetamides, as The present invention relates to recombinant DNA tech- 60 well as herbicides with unknown modes of action such as nology, and in particular to the production of: (i) transgenic DifenZoquat and ClomaZone. plants which exhibit substantial resistance or substantial tol The article “a” and “an are used herein to refer to one or erance to herbicides when compared with non-transgenic more than one (i.e., to at least one) of the grammatical object like plants; and (ii) transgenic plants having an enhanced of the article. By way of example, “an element’ means one or ability to transform certain herbicides to oxidised metabo- 65 more element. Throughout the specification the word “com lites, when likewise compared with Such non-transgenic-like prising.” or variations such as “comprises' or "comprising.” plants. The invention also relates, interalia, to the nucleotide will be understood to imply the inclusion of a stated element, US 8,097,774 B2 5 6 integer or step, or group of elements, integers or steps, but not Alternatively, candidate P450 enzymes are expressed in the exclusion of any other element, integer or step, or group of tobacco, Arabidopsis, corn or other easily transformed, her elements, integers or steps. bicide sensitive plant and the resultant transformant plants Cytochrome P450 Sequences assessed for their tolerance to HPPD inhibitor(s) (as Cytochrome P450 sequences are provided that confer her- 5 described in Didierjean et al. (2002) Plant Physiol. 130:179 bicide tolerance or resistance. Such sequences include the 189) or other herbicides of interest. Optionally the plants, or amino acid sequences set forth in SEQID NOS:1, 3, and 5, tissue samples taken from plants, are treated with herbicide and variants thereof. Also provided are polynucleotide and assayed in order to assess the rate of metabolic conversion sequences encoding Such amino acid sequences, including of parent herbicide to oxidised metabolic degradation prod SEQ ID NOS:2, 4, and 6. 10 ucts. For example, radiolabeled mesotrione is used along with According to the method of the present disclosure crop analytical methods described in Hawkes et al 2001, BCPC plants are transformed with a gene encoding a cytochrome conference Weeds, p563; Alferness and Wiebe (2002) J. P450 enzyme (CYP enzymes) capable of oxidising certain Agric. Food Chem., 50:3926-3934 and/or Gledhill et al HPPD-inhibitors and also, optionally, other types of herbi 15 (2004) Xenobiotica 31, 733-747. Suitable gene constructs for cides. The gene cassette comprises promoter and terminator expressing the cytochrome P450 encoding sequence in crop regions and, optionally, other elements (such as introns, tran plants comprise a plant Suitable promoter and 5' leader Scriptional enhancers, translational enhancers etc.) So as to sequence upstream (for example the CMV 3.5 S promoter or provide for expression of an effective amount of the P450 an actin promoter region) and a suitable terminator sequence enzyme in the crop plant. Optionally the structural gene 20 (to ensure polyadenylation) downstream (for example the nos encoding the P450 enzyme is codon optimised to remove gene 3' sequence) of the cytochrome P450 coding sequence. features inimical to expression and codon usage is optimised Optionally, constructs may comprise additional sequences for expression in the particular crop (see, for example, U.S. Such as translational and transcriptional enhancers and/or the Pat. No. 6,051,760; EP 0359472: EP 80385962; EP 0431829: DNA sequence is modified for optimal expression in the host and Perlak et al. (1991) PNAS USA 88:3324-3328; all of 25 plant. which are herein incorporated by reference). In certain As used herein, a significant rate of oxidation is defined as embodiments the P450 enzyme is selected as one capable of a catalytic rate Sufficient to provide a clear (greater than about oxidising other herbicides that include nicosulfuron and/or 2x) increase in tolerance to a given Substrate herbicide in a , and/or as capable of oxidising the HPPD-inhibitor transgenic plant, where the transgenic plant expresses a given herbicide mesotrione and/or sulcotrione. In another embodi 30 P450 enzyme at up to 0.2% of the total cellular protein rela ment, the P450 enzyme is selected as one capable of oxidising tive to a similarly treated non-transgenic control plant. HPPD-inhibitors that include, but are not limited to, Mesot In one embodiment of the present disclosure, genes encod rione, SYN449280, Isoxaflutole, Tembotrione, Topram ing suitable P450 enzymes are selected from maize or rice eZone, Pyrasulfatole, Sulcotrione, Pyrazolynate, Pyrazoxy cDNA libraries prepared by standard methods (e.g. using the fen, Isoxachlortole, Benzofenap, and Benzobicyclon. lambda-ZAPII vector kit from Stratagene) from 3-7 day old Examples of optimized genes encoding a cytochrome P450 seedlings of maize or rice plants. Optionally, Suitable candi enzyme for use in the present invention include corn insfl date P450-encoding sequences are selected from amongst (SEQ ID NO: 1), corn CYP72A1 (SEQ ID NO:3), and rice those P450s induced within approximately 24 h treatment CYP81A6 (SEQID NO:5). Other polynucleotides of interest 40 with 1-50 ppm mesotrione or other HPPD inhibitor, or with that encode a cytochrome P450 enzyme for use in the present nicosulfuron. In addition, suitable candidate P450 encoding invention include, but are not limited to, polynucleotides sequences are selected from amongst those P450s induced in encoding the amino acid sequence for corn insfl (SEQ ID maize tissues within 24 h of treatment with safening levels of NO:2); corn CYP72A1 (SEQ ID NO:4), and rice CYP81A6 cyprosulfamide, isoxadifen and/or CGA24678; or alterna (SEQ ID NO:6). 45 tively in other cereals following treatment with cloquintacet Additional polynucleotide sequences encoding a cyto and/or mefenpyr. This can be readily accomplished by mea chrome P450 enzyme for use in the present invention may be Suring (relative) transcript levels via northern blot analysis, identified using methods well known in the art based on their differential display analysis, and/or with a RNA Chip, con ability to confer resistance or tolerance to an herbicide of taining probes for all, or a majority of the P450 genes of the interest. For example, candidate P450 genes are transformed 50 plant species of interest. Alternatively, a library is screened by into and expressed in Suitable yeast strains and selected on the PCR and, for example, the incorporation of radio labelled basis of their ability to oxidise test herbicides in vitro (cf nucleotide used to detect clones comprising the characteristic Siminszky etal (1999) PNAS (USA).96:1750-1755). Suitable heme-binding region of cytochrome P450s. For example yeast strains include such as WAT11 or WAT21 which also comprise a suitable plant cytochrome P450 competent reduc- 55 degenerate 5'primers for the heme motif region (Ohbayashi et tase (e.g. as described by Denis Pompon and Phillipe Urban). all 1993) can be used along with 3' primers designed for the Following induction for a suitable period (for example, poly A tail (cf. Persans etal (2001) Plant physiol., 125: 1126). depending on the inducible promoter used in the transforma The so-produced, labelled PCR products are used to rescreen tion vector, with galactose) cells are grown up, harvested, the library to recover full-length cDNAs which are then broken, the microsome fraction prepared by the usual means 60 sequenced. Candidate P450 genes are obtained for example and assayed with NADPH for the ability to oxidise 14C by the above method or by other methods known in the art (cf labelled mesotrione. Optionally, assays are carried out using U.S. Pat. No. 6,380,465 and U.S. Pat. No. 6,121,512 and all whole cells in culture. The oxidised products with 4 and 5 references cited within; cf Persans etal (2001) Plant physiol., hydroxy modifications of the cyclohexanedione ring are eas 125:1126) and are then selected via a process of expressing ily detected and separated from unmodified parent by, for 65 them and assayed for their ability to oxidise (or provide example, RP HPLC or TLC (cf Hawkes et al. 2001, BCPC resistance) to especially mesotrione or other HPPD conference Weeds, p563). inhibitor(s) or nicosulfuron in a suitable test system. US 8,097,774 B2 7 8 An alternative method for selecting suitable P450 enzymes TABLE 1-continued

is through genetic mapping of P450 genes involved in resis tance to the herbicide of interest. If lines exist with tolerance FA3112 and lines with resistance to the herbicide, based on differen CO5041 tial P450 responses, this is possible. For an example of fine IDS4O1 structure QTL mapping and sequencing of candidate CYP IC3008 genes (readily recognisable through sequence homologies IC3008GF ID3461 and by their characteristic heme motif: see Bortiri et al. FX7.093 (2006) Current Opinion in Plant Biology 9:164-171). For 10 XDOP735 example, certain lines and hybrids of Sweetcorn (e.g. Merit) CC8962 are susceptible to mesotrione and through conventional cross OX7010 ing studies with tolerant varieties and the use of markers it is XXFOOO1 possible to map QTLs associated with this susceptibility. We CH5OO1 have previously shown that mesotrione levels in treated corn 15 plants decrease over time, due to a P450 based degradation mechanism. Merit plants appear defective in this degradation step. Thus, the QTLs associated with the differential response are expected to contain a P450 gene, or genes, that degrade mesotrione. If genomic sequences exist for the mapping inter val, candidate CYP gene can be identified. Alternatively, BACS (Bacterial Artificial Chromosome clones) encompass ing the QTL are sequenced and candidate CYP genes identi fied. By comparing the sequences of the P450 alleles in the tolerant and sensitive varieties, the candidate CYP genes be 25 identified (and particularly easily if, for example, there is an obvious gene defect and/or lack of expression in sensitive varieties). For example, relevant to the current invention, it is known 30 that a small number of corn lines are sensitive to mesotrione, CH6150 7 6 and a small number are sensitive to nicosulfuron. These 2 UM12 7 6 groups overlap partially (see HortScience (2005) 40: 1801 1805 and the list in Table 1). Table 1 is based on screening of corn lines with mesotrione and nicosulfuron (by Syngenta, 35 XICC310 7 4 Toulouse, FR). Only the lines with the highest tolerance and DH6901 7 6 highest sensitivity are shown, from screening ~400 lines. i 5 TABLE 1. 40 A general correlation exists between tolerance to mesotrione or nicosulfuron, and sensitivity to mesotrione and nicosulfuron. *Scale used for scoring: 1 to 9 scale, where 1 = most resistant lines and 9 = most susceptible lines, Unshaded boxes are genotypes most resistant to mesotrione (Callisto (R). Shaded Milagro (R) boxes are genotypes most susceptible to mesotrione (Callisto (R), Line Code treatment treatment** **Callisto (R) (Mesotrione) treatment; concentration equivalent to 60 g of active matteriha, Lice St. spray at stage V2, score 10 days after spray, 45 ***Milagro (R) (nicosulfuron) treatment: concentration equivalent to 24 g of active matter? IAFX239 1 ha, spray at stage V2, score 10 days after spray, FX7179 1 PC7505 1 Genetic analysis indicates that the genes that confer the FB7768 1 tolerance/susceptibility to mesotrione and nicosulfuron are IC3008HG 1 50 closely linked in the corn genome. M. Williams et al. (2006, FB7456 2 Corn Genetics Conference, Asiloma), used a map based clon FB7556 2 ing approach to clone the P450 gene that conditions the nico IC4612 2 Sulfuron response of corn plant, the nsfl gene, located in bin IG5226 2 5.01. One mesotrione tolerance QTL in corn lines with sus HX904 2 55 ceptibility to mesotrione and Sulcotrione (a close homolog of ICFX234 2 mesotrione) has been mapped, to corn genome position bin ICFX235 2 5.01. We sequenced this gene from B73 BAC b0159B4, and FX8236 2 deduced the amino acid sequence (SEQID NO:1). A second FX7422 i2 and third P450 gene are located on this nsf1 containing BAC FX7423 2 60 FX7425 2 (genes 160842 and 136090) SEQID NO:7 and SEQID NO:8 FX7521 2 are the deduced amino acid sequences from these 2 P450 FX7527 2 genes. ICPC407 2 Thus, all 3 P450 genes above are candidate P450 gene P7502 2i 65 capable of degrading mesotrione. In addition, all of the genes/ P8619 2 P450 enzymes listed in the Listing of Sequences (FIG. 2) are candidate genes for use in the present invention. US 8,097,774 B2 10 Suitable P450 genes are thus selected from the list com perfectly matched probe. Very stringent conditions are prising the above candidate cytochrome P450 genes obtain selected to be equal to the T for a particular probe. An able by the above described methods and including those example of stringent hybridization conditions for hybridiza from maize and from rice and also other species, including for tion of complementary nucleic acids which have more than example Catharanthus roseus (SEQ ID NO:9; see also the 100 complementary residues on a filter in a Southern or Listing of Sequences, FIG. 2). northern blot is 50% formamide with 1 mg of heparin at 42 Once one P450 gene capable of degrading these herbicides C., with the hybridization being carried out overnight. An is identified, those skilled in the art may also find further gene example of highly stringent wash conditions is 0.15M NaCl candidates based on genome synteny and sequence similarity. at 72°C. for about 15 minutes. An example of stringent wash In one embodiment, additional gene candidates can be 10 conditions is a 0.2xSSC wash at 65° C. for 15 minutes (see, obtained by hybridization or PCR using sequences based on Sambrook, infra, for a description of SSC buffer). Often, a the cytochrome P450 nucleotide sequences noted above. high Stringency wash is preceded by a low stringency wash to In a PCR approach, oligonucleotide primers can be remove background probe signal. An example medium strin designed for use in PCR reactions to amplify corresponding gency wash for a duplex of e.g., more than 100 nucleotides, DNA sequences from cDNA or genomic DNA extracted from 15 is 1xSSC at 45° C. for 15 minutes. An example low stringency any plant of interest. Methods for designing PCR primers and wash for a duplex of, e.g., more than 100 nucleotides, is PCR cloning are generally known in the art. See, for example, 4-6xSSC at 40° C. for 15 minutes. For short probes (e.g., Sambrook et al. (1989) Molecular Cloning. A Laboratory about 10 to 50 nucleotides), stringent conditions typically Manual (2d ed., Cold Spring Harbor Laboratory Press, Pla involve salt concentrations of less than about 1.0 M Naion, inview, N.Y.). See also Innis et al., eds. (1990) PCR Proto typically about 0.01 to 1.0 M Na ion concentration (or other cols. A Guide to Methods and Applications (Academic Press, salts) at pH 7.0 to 8.3, and the temperature is typically at least New York); Innis and Gelfand, eds. (1995) PCR Strategies about 30° C. Stringent conditions can also be achieved with (Academic Press, New York); and Innis and Gelfand, eds. the addition of destabilizing agents such as formamide. In (1999) PCR Methods Manual (Academic Press, New York). general, a signal to noise ratio of 2x (or higher) than that In hybridization techniques, all or part of a known poly 25 observed for an unrelated probe in the particular hybridiza nucleotide is used as a probe that selectively hybridizes to tion assay indicates detection of a specific hybridization. other corresponding polynucleotides present in a population Nucleic acids that do not hybridize to each other under strin of cloned genomic DNA fragments or cDNA fragments (i.e., gent conditions are still Substantially identical if the proteins genomic or cDNA libraries) from a chosen organism. The that they encode are substantially identical. This occurs, e.g., hybridization probes may be genomic DNA fragments, 30 when a copy of a nucleic acid is created using the maximum cDNA fragments, RNA fragments, or other oligonucleotides, codon degeneracy permitted by the genetic code. and may be labeled with a detectable group such as Porany The following are examples of sets of hybridization/wash other detectable marker. Methods for preparation of probes conditions that may be used to clone nucleotide sequences for hybridization and for construction of cDNA and genomic that are homologues of reference nucleotide sequences of the libraries are generally known in the art and are disclosed in 35 present invention: a reference nucleotide sequence preferably Sambrook et al. (1989) Molecular Cloning. A Laboratory hybridizes to the reference nucleotide sequence in 7% sodium Manual (2d ed., Cold Spring Harbor Laboratory Press, Pla dodecyl sulfate (SDS), 0.5 M NaPO 1 mM EDTA at 50° C. inview, N.Y.). with washing in 2xSSC, 0.1% SDS at 50° C., more desirably By “hybridizing to’ or “hybridizing specifically to refers in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO 1 mM to the binding, duplexing, or hybridizing of a molecule only to 40 EDTA at 50° C. with washing in 1xSSC, 0.1% SDS at 50° C., a particular nucleotide sequence under Stringent conditions more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 when that sequence is presentina complex mixture (e.g., total MNaPO 1 mM EDTA at 50° C. with washing in 0.5xSSC, cellular) DNA or RNA. “Bind(s) substantially” refers to 0.1% SDS at 50° C., preferably in 7% sodium dodecyl sulfate complementary hybridization between a probe nucleic acid (SDS), 0.5 MNaPO 1 mM EDTA at 50° C. with washing in and a target nucleic acid and embraces minor mismatches that 45 0.1xSSC, 0.1% SDS at 50° C., more preferably in 7% sodium can be accommodated by reducing the stringency of the dodecyl sulfate (SDS), 0.5 M NaPO 1 mM EDTA at 50° C. hybridization media to achieve the desired detection of the with washing in 0.1xSSC, 0.1% SDS at 65° C. target nucleic acid sequence. The present invention also relates to the use of cytochrome “Stringent hybridization conditions” and “stringent P450 or variants thereof that confer resistance or tolerance to hybridization wash conditions’ in the context of nucleic acid 50 HPPD inhibitors, benzothiadiazinones, sulfonylureas, and hybridization experiments such as Southern and Northern other classes of herbicides. 'Variants' is intended to mean hybridizations are sequence dependent, and are different Substantially similar sequences. For polynucleotides, a vari under different environmental parameters. Longer sequences ant comprises a deletion and/or addition of one or more nucle hybridize specifically at higher temperatures. An extensive otides at one or more internal sites within the native poly guide to the hybridization of nucleic acids is found in Tijssen 55 nucleotide and/or a Substitution of one or more nucleotides at (1993) Laboratory Techniques in Biochemistry and Molecu one or more sites in the native polynucleotide. As used herein, lar Biology-Hybridization with Nucleic Acid Probes part I a “native' polynucleotide or polypeptide comprises a natu chapter 2 “Overview of principles of hybridization and the rally occurring nucleotide sequence or amino acid sequence, strategy of nucleic acid probe assays' Elsevier, New York. respectively. For polynucleotides, conservative variants Generally, highly stringent hybridization and wash condi 60 include those sequences that, because of the degeneracy of tions are selected to be about 5° C. lower than the thermal the genetic code, encode cytochrome P450 enzymes melting point (T) for the specific sequence at a defined ionic described above for use in the present invention. Naturally strength and pH. Typically, under “stringent conditions” a occurring allelic variants can be identified with the use of probe will hybridize to its target subsequence, but to no other well-known molecular biology techniques, as, for example, Sequences. 65 with polymerase chain reaction (PCR) and hybridization The T is the temperature (under defined ionic strength and techniques as outlined above. Variant polynucleotides also pH) at which 50% of the target sequence hybridizes to a include synthetically derived polynucleotides, such as those US 8,097,774 B2 11 12 generated, for example, by using site-directed mutagenesis encoding polypeptides having pesticidal and/or insecticidal but which still encode a cytochrome P450 enzyme conferring activity, such as other Bacillus thuringiensis toxic proteins herbicide resistance or tolerance. Generally, variants of a (described in U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; particular polynucleotide of the invention will have at least 5,723,756; 5,593.881; and Geiser et al. (1986) Gene 48:109), about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, lectins (Van Damme et al. (1994) Plant Mol. Biol. 24:825, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, pentin (described in U.S. Pat. No. 5,981,722), and the like: 99% or more sequence identity to that particular polynucle traits desirable for disease or herbicide resistance (e.g., fumo otide. nisin detoxification genes (U.S. Pat. No. 5,792.931); aviru Variants of a particular polynucleotide encoding a cyto lence and disease resistance genes (Jones etal. (1994) Science chrome P450 that confers herbicide resistance or tolerance 10 266:789; Martinet al. (1993) Science 262:1432: Mindrinos et are encompassed by the present invention and can be evalu al. (1994) Cell 78:1089); acetolactate synthase (ALS) ated by comparison of the percent sequence identity between mutants that lead to herbicide resistance such as the S4 and/or the polypeptide encoded by a variant polynucleotide and the Hra mutations; glyphosate resistance (e.g., 5-enol-pyrovyl polypeptide encoded by the reference polynucleotide. Per shikimate-3-phosphate-synthase (EPSPS) gene, described in cent sequence identity between any two polypeptides can be 15 U.S. Pat. Nos. 4,940,935 and 5,188,642; or the glyphosate calculated using sequence alignment programs and algo N-acetyltransferase (GAT) gene, described in Castle et al. rithms described below. Where any given pair of polynucle (2004) Science, 304:1151-1154; and in U.S. Patent App. Pub. otides of the invention is evaluated by comparison of the Nos. 20070004912, 20050246798, and 20050060767)); glu percent sequence identity shared by the two polypeptides they fosinate resistance (e.g., phosphinothricin acetyl transferase encode, the percent sequence identity between the two genes PAT and BAR, described in U.S. Pat. Nos. 5,561,236 encoded polypeptides is at least about 40%, 45%, 50%, 55%, and 5,276.268); and traits desirable for processing or process 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, products such as high oil (e.g., U.S. Pat. No. 6,232,529); 94%. 95%, 96%,97%.98%, 99% or more sequence identity. modified oils (e.g., fatty acid desaturase genes (U.S. Pat. No. Methods of alignment of sequences for comparison are 5,952,544: WO 94/11516)); modified starches (e.g., ADPG well known in the art and can be accomplished using math 25 pyrophosphorylases (AGPase), starch synthases (SS), starch ematical algorithms such as the algorithm of Myers and branching enzymes (SBE), and starch debranching enzymes Miller (1988) CABIOS 4:11-17; the local alignment algo (SDBE)); and polymers or bioplastics (e.g., U.S. Pat. No. rithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the global 5,602.321; beta-ketothiolase, polyhydroxybutyrate synthase, alignment algorithm of Needleman and Wunsch (1970) J. and acetoacetyl-CoA reductase (Schubert et al. (1988).J. Bac Mol. Biol. 48:443-453; and the algorithm of Karlin and Alts 30 teriol. 170:5837-5847) facilitate expression of polyhydroxy chul (1990) Proc. Natl. Acad. Sci. USA 872264, modified as alkanoates (PHAs)); the disclosures of which are herein in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA incorporated by reference. 90:5873-5877. Computer implementations of these math Thus, in one embodiment, the polynucleotides encoding a ematical algorithms can be utilized for comparison of cytochrome P450 or variant thereof that confers herbicide sequences to determine sequence identity. Such implementa 35 resistance or tolerance are stacked with one or more poly tions include, but are not limited to: CLUSTAL in the nucleotides encoding polypeptides that confer resistance or PC/Gene program (available from Intelligenetics, Mountain tolerance to an herbicide. In one embodiment, the desirable View, Calif); the ALIGN program (Version 2.0) and GAP, trait is resistance or tolerance to an HPPD inhibitor. In another BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wis embodiment, the desirable trait is resistance or tolerance to consin Genetics Software Package, Version 10 (available 40 glyphosate. In another embodiment, the desirable trait is from Accelrys Inc., 9685 Scranton Road, San Diego, Calif., resistance or tolerance to glufosinate. USA). These stacked combinations can be created by any method Essentially similar methods of gene mutagenesis and including, but not limited to, cross-breeding plants by any selection based on improved tolerance or resistance as conventional or TopCross methodology, or genetic transfor described elsewhere herein can also be used to generate and 45 mation. If the sequences are stacked by genetically transform select improved variants of the cytochrome P450 genes of the ing the plants, the polynucleotide sequences of interest can be current invention. combined at any time and in any order. For example, a trans Gene Stacking genic plant comprising one or more desired traits can be used In certain embodiments the polynucleotides encoding a as the target to introduce further traits by Subsequent trans cytochrome P450 or variant thereof that confers herbicide 50 formation. The traits can be introduced simultaneously in a resistance or tolerance can be stacked with any combination co-transformation protocol with the polynucleotides of inter of polynucleotide sequences of interest in order to create est provided by any combination of transformation cassettes. plants with a desired trait. A trait, as used herein, refers to the For example, if two sequences will be introduced, the two phenotype derived from a particular sequence or groups of sequences can be contained in separate transformation cas sequences. For example, the polynucleotides encoding a 55 settes (trans) or contained on the same transformation cas cytochrome P450 or variant thereof that confers herbicide sette (cis). Expression of the sequences can be driven by the resistance or tolerance may be stacked with any other poly same promoter or by different promoters. In certain cases, it nucleotides encoding polypeptides that confer a desirable may be desirable to introduce a transformation cassette that trait, including but not limited to resistance to diseases, will suppress the expression of the polynucleotide of interest. insects, and herbicides, tolerance to heat and drought, 60 This may be combined with any combination of other Sup reduced time to crop maturity, improved industrial process pression cassettes or overexpression cassettes to generate the ing, such as for the conversion of starch or biomass to fer desired combination of traits in the plant. It is further recog mentable Sugars, and improved agronomic quality, Such as nized that polynucleotide sequences can be stacked at a high oil content and high protein content. desired genomic location using a site-specific recombination Exemplary polynucleotides that may be stacked with poly 65 system. See, for example, WO99/25821, WO99/25854, nucleotides encoding an herbicide resistant or tolerant cyto WO99/25840, WO99/25855, and WO99/25853, all of which chrome P450 or variant thereof include polynucleotides are herein incorporated by reference. US 8,097,774 B2 13 14 In one embodiment the crop plant is transformed not only mum inhibitory concentration) values where increases in val with a gene to express said P450 enzyme but is also trans ues correspond to increases in inherent tolerance of the formed with a gene to express an heterologous HPPD expressed HPPD. After identification of suitable resistant and enzyme. The P450 gene and the HPPD gene may be intro functional gene mutants, the encoded enzymes can be further duced into separate events, and these events or their progeny 5 analyzed by kinetic analysis, for instance for enzyme activity, crossed so as to combine the genes in a single plant. Alterna and inhibitor binding characteristics. tively, both the P450 and HPPD genes are combined within a Many combinations of host organism, indicator phenotype single DNA molecule which is transformed into the crop and control HPPD would achieve a similar scope of selection plant. In a further embodiment the HPPD gene is derived from and these are contemplated within the scope of the current a monocot plant, a cereal plant or, more preferably a wheat, 10 invention. For example, in a relatively rapid assay system Avena or corn plant and is, for example, as disclosed in PCT based upon transformation of a bacterium Such as E. coli, application WO 02/.46387. Optionally, the HPPD enzyme each HPPD encoding sequence may be expressed, for may be targeted to cellular locations, to enhance expression/ example, as a DNA sequence under expression control of a accumulation levels, or to enhance activity levels. For controllable promoter Such as the lacZ promoter and taking instance, the HPPD may be plastid-targeted through fusion 15 suitable account, for example by the use of synthetic DNA, of with a suitable transit peptide. Suitable HPPD genes, con Such issues as codon usage in order to obtain as comparable a structs, promoter regions, 5' leader sequences, terminators etc level of expression as possible of different HPPD sequences. for expressing HPPD in crop plants are, for example, also Such strains expressing polynucleotides comprising alterna described in WO 0246387, WO 0032757, and WO9904021. tive candidate HPPD sequences may be plated out on differ Optionally, the HPPD gene may be directly transformed into 20 ent concentrations of the selected herbicides in, optionally, a the plastome of the chloroplast of the target plant, for high tyrosine supplemented medium and the relative levels of level enzyme accumulation. inherent tolerance of the expressed HPPD enzymes estimated In a further embodiment the HPPD gene sequence encodes on the basis of the extent and MIC for inhibition of the the Avena HPPD enzyme sequence which is SEQID NO:10 formation of the brown, ochronotic pigment. In variations of here (SEQ ID NO:4 in WO 0246387) with the following 25 the method the cells may be permeabilized or, particularly in slight sequence modifications within the N terminal part of the case of yeast, be strains having disabled pumps in order to the protein. The sequence motif AATAAATASIPS within minimise the effects of differential uptake and export of SEQID NO: 10 is shortened to read AATAATASIPS (SEQID HPPD inhibitors into and out of the cell. In one variation, NO: 11) or altered to read AATAATTASIPS (SEQID NO:12) bacterial cells are grown almost to stationary phase in a liquid or altered to read AADAAATASIPS (SEQID NO:13) or any 30 medium, exposed to selected herbicides for a short period of combination of these 3 changes is made. The resultant one hour or less, resuspended in a similar Volume of fresh enzymes are described as "modified-Avena HPPD. In one medium and the rate of development of pigment monitored. embodiment, the crop plant is transformed with a gene encod In another variation, candidate HPPD expressing sequences ing a cytochrome P450 enzyme selected from the group con are transferred to a shuttle vector and, similar to the previous sisting of nsf1 (SEQ ID NO:1), corn CYP72A1 (SEQ ID 35 variation, are each expressed at a comparable level, but in this NO:3), and rice CYP81A6 gene (SEQID NO. 5), in combi case in a suitable Pseudomonas species such as Pseudomonas nation with a gene sequence encoding a modified-Avena fluorescens capable of being transformed and of growing on HPPD. tyrosine as sole carbon source. Preferably the endogenous In a further embodiment, the Avena gene, for example, or HPPD gene of the host Pseudomonas line is knocked out, for another plant-derived HPPD sequence is modified to enhance 40 example, by recombinational insertion of an antibiotic the tolerance to one or more HPPD inhibitors (see, e.g., U.S. marker gene. Pseudomonas lines each transformed to express Patent App. Pub. No. 2008.0076178, incorporated herein by an alternative resistant HPPD enzyme are each grown on reference in its entirety). The resultant enzymes are described different concentrations of selected HPPD inhibitors and the as modified-HPPD. Standard methodologies known in the inherent resistance of the expressed HPPD sequence in art can be used to introduce changes in the HPPD enzyme 45 respect of each HPPD inhibitor estimated upon the basis of gene, that lead to enhanced tolerance of the encoded enzyme the concentration necessary to prevent growth on a medium to HPPD enzyme inhibitors. These methodologies include containing tyrosine as Sole carbon Source. In a final selection gene shuffling, directed mutations, random mutations, and step a short list of candidate polynucleotides may be trans Gene Site Saturated Mutagenesis (GSSM). Putative HPPD formed into plant material, which material is regenerated into gene mutants can be screened in biological systems for activ 50 morphologically normal fertile plants and which plants are ity in the presence of inhibitors, present at concentrations that then measured for differential tolerance to selected HPPD inhibit wild type enzymes. inhibitor herbicides. Suitable resistant and functional HPPD gene mutants can Plant Expression Cassettes be selected by many methods well known in the art. For The compositions of the invention may additionally con example candidate HPPD encoding sequences produced by 55 tain nucleic acid sequences for transformation and expression mutagenesis are expressed in yeast, in a bacterial host strain, in a plant of interest. The nucleic acid sequences may be in an alga or in a higher plant such as tobacco or Arabidopsis present in DNA constructs or expression cassettes. "Expres and the relative levels of inherent tolerance of the HPPD sion cassette' as used herein means a nucleic acid molecule encoding sequences screened according to the observed lev capable of directing expression of a particular nucleotide els of resistance or tolerance or, alternatively, a visible indi 60 sequence in an appropriate host cell, comprising a promoter cator phenotype of the transformed Strain or plant in the operatively linked to the nucleotide sequence of interest (i.e., presence of different concentrations of the selected HPPD an herbicide resistant or tolerant cytochrome P450 polynucle inhibitors. Dose responses and relative shifts in dose otide, alone or in combination with one or more additional responses associated with these indicator phenotypes (forma nucleic acid molecules encoding polypeptides that confer tion of brown colour, growth inhibition, herbicidal effect etc) 65 desirable traits) which is operatively linked to termination are conveniently expressed in terms, for example, of GR50 signals. It also typically comprises sequences required for (concentration for 50% reduction of growth) or MIC (mini proper translation of the nucleotide sequence. The coding US 8,097,774 B2 15 16 region usually codes for a protein of interest but may also A "homologous nucleic acid (e.g. DNA) sequence is a code for a functional RNA of interest, for example antisense nucleic acid (e.g. DNA or RNA) sequence naturally associ RNA or a nontranslated RNA, in the sense orantisense direc ated with a host cell into which it is introduced. tion. The expression cassette comprising the nucleotide The choice of promoters to be included depends upon sequence of interest may be chimeric, meaning that at least several factors, including, but not limited to, efficiency, one of its components is heterologous with respect to at least selectability, inducibility, desired expression level, and cell one of its other components. The expression cassette may also or tissue-preferential expression. It is a routine matter for one be one that is naturally occurring but has been obtained in a of skill in the art to modulate the expression of a sequence by recombinant form useful for heterologous expression. Typi appropriately selecting and positioning promoters and other 10 regulatory regions relative to that sequence. The promoters cally, however, the expression cassette is heterologous with that are used for expression of the transgene(s) can be a strong respect to the host, i.e., the particular DNA sequence of the plant promoter, a viral promoter, or a chimeric promoters expression cassette does not occur naturally in the host cell composed of elements such as: TATA box from any gene (or and must have been introduced into the host cell or an ances synthetic, based on analysis of plant gene TATA boxes), tor of the host cell by a transformation event. The expression 15 optionally fused to the region 5' to the TATA box of plant of the nucleotide sequence in the expression cassette may be promoters (which direct tissue and temporally appropriate under the control of a constitutive promoter or of an inducible gene expression), optionally fused to 1 or more enhancers promoter that initiates transcription only when the host cell is (such as the 35S enhancer, FMV enhancer, CMP enhancer). exposed to some particular external stimulus. Additionally, Exemplary constitutive promoters include, for example, the promoter can also be specific to a particular tissue or organ the core promoter of the Rsyn? promoter and other constitu or stage of development. tive promoters disclosed in WO99/43838 and U.S. Pat. No. The present invention encompasses the transformation of 6,072,050; the core CaMV 35S promoter (Odellet al. (1985) plants with expression cassettes capable of expressing a poly Nature 313:810-812); rice actin (McElroy et al. (1990) Plant nucleotide of interest, i.e., a polynucleotide encoding a cyto Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant chrome P450 or variant thereof that confers herbicide resis 25 Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant tance or tolerance, alone or in combination with one or more Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor: additional nucleic acid molecules encoding polypeptides that Appl. Genet. 81:581–588); MAS (Velten et al. (1984) EMBO confer desirable traits. The expression cassette will include in J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), the 5'-3' direction of transcription, a transcriptional and trans and the like. Other constitutive promoters include, for lational initiation region (i.e., a promoter) and a polynucle 30 example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121: otide of. The expression cassette may optionally comprise a 5,569,597; 5,466,785; 5,399,680: 5,268,463; 5,608,142; and 6,177,611. transcriptional and translational termination region (i.e. ter Appropriate plant or chimeric promoters are useful for mination region) functional in plants. In some embodiments, applications such as expression of transgenes in certain tis the expression cassette comprises a selectable marker gene to 35 Sues, while minimizing expression in other tissues, such as allow for selection for stable transformants. Expression con seeds, or reproductive tissues. In some embodiments expres structs of the invention may also comprise a leader sequence sion of the cytochrome P450 gene of the current invention and/or a sequence allowing for inducible expression of the may optionally be localised to seed or fruit tissues in order to polynucleotide of interest. See, Guo et al. (2003) Plant J further oxidise away any residue of parent herbicide reaching 34:383-92 and Chen et al. (2003) Plant J 36:731-40 for 40 these tissues. Exemplary cell type- or tissue-preferential pro examples of sequences allowing for inducible expression. moters drive expression preferentially in the target tissue, but The regulatory sequences of the expression construct are may also lead to Some expression in other cell types or tissues operably linked to the polynucleotide of interest. By “oper as well. Methods for identifying and characterizing promoter ably linked' is intended a functional linkage between a pro regions in plant genomic DNA include, for example, those moter and a second sequence wherein the promoter sequence 45 described in the following references: Jordano, et al., Plant initiates and mediates transcription of the DNA sequence Cell, 1:855-866 (1989); Bustos, et al., Plant Cell, 1:839-854 corresponding to the second sequence. Generally, operably (1989); Green, et al., EMBO.J. 7,40354044 (1988); Meier, et linked means that the nucleotide sequences being linked are al., Plant Cell, 3, 309-316 (1991); and Zhang, et al., Plant contiguous. Physiology 110: 1069-1079 (1996). Any promoter capable of driving expression in the plant of 50 In other embodiments of the present invention, inducible interest may be used in the practice of the invention. The promoters may be desired. Inducible promoters drive tran promoter may be native or analogous or foreign or heterolo Scription in response to external stimuli such as chemical gous to the plant host. The terms "heterologous' and “exog agents or environmental stimuli. For example, inducible pro enous” when used herein to refer to a nucleic acid sequence moters can confer transcription in response to hormones Such (e.g. a DNA or RNA sequence) or a gene, refer to a sequence 55 as giberellic acid or ethylene, or in response to light or that originates from a source foreign to the particular host cell drought. or, if from the same source, is modified from its original form. A variety of transcriptional terminators are available for Thus, a heterologous gene in a host cell includes a gene that is use in expression cassettes. These are responsible for the endogenous to the particular host cell but has been modified termination of transcription beyond the transgene and correct through, for example, the use of DNA shuffling. The terms 60 mRNA polyadenylation. The termination region may be also include non-naturally occurring multiple copies of a native with the transcriptional initiation region, may be native naturally occurring DNA sequence. Thus, the terms refer to a with the operably linked DNA sequence of interest, may be DNA segment that is foreign or heterologous to the cell, or native with the plant host, or may be derived from another homologous to the cell but in a position within the host cell Source (i.e., foreign or heterologous to the promoter, the DNA nucleic acid in which the element is not ordinarily found. 65 sequence of interest, the plant host, or any combination Exogenous DNA segments are expressed to yield exogenous thereof). Appropriate transcriptional terminators are those polypeptides. that are known to function in plants and include the CAMV US 8,097,774 B2 17 18 35S terminator, the timl terminator, the nopaline synthase plants will be grown, harvested, and/or processed. One of terminator and the pea rbcs E9 terminator. These can be used skill will further recognize that additional factors for select in both monocotyledons and dicotyledons. In addition, a ing appropriate plant varieties for use in the present invention gene's native transcription terminator may be used. include highyield potential, good stalk strength, resistance to Generally, the expression cassette will comprise a select 5 specific diseases, drought tolerance, rapid dry down and grain able marker gene for the selection of transformed cells. quality Sufficient to allow storage and shipment to market Selectable marker genes are utilized for the selection of trans with minimum loss. formed cells or tissues. Plants according to the present invention include any plant Numerous sequences have been found to enhance gene that is cultivated for the purpose of producing plant material expression from within the transcriptional unit and these 10 that is sought after by man or animal for either oral consump sequences can be used in conjunction with the genes of this tion, or for utilization in an industrial, pharmaceutical, or invention to increase their expression in transgenic plants. commercial process. The invention may be applied to any of Various intron sequences have been shown to enhance a variety of plants, including, but not limited to maize, wheat, expression, particularly in monocotyledonous cells. For rice, barley, Soybean, cotton, Sorghum, beans in general, rape/ example, the introns of the maize Adh1 gene have been found 15 canola, alfalfa, flax, Sunflower, safflower, millet, rye, Sugar to significantly enhance the expression of the wild-type gene cane, Sugar beet, cocoa, tea, Brassica, cotton, coffee, Sweet under its cognate promoter when introduced into maize cells. potato, flax, peanut, clover, vegetables Such as lettuce, Intron 1 was found to be particularly effective and enhanced tomato, cucurbits, cassava, potato, carrot, radish, pea, lentils, expression in fusion constructs with the chloramphenicol cabbage, cauliflower, broccoli, Brussels sprouts, peppers, and acetyltransferase gene (Callis et al., Genes Develop. 1:1 183 pineapple; tree fruits such as citrus, apples, pears, peaches, 1200 (1987)). In the same experimental system, the intron apricots, walnuts, avocado, banana, and coconut; and flowers from the maize bronze 1 gene had a similar effect in enhanc Such as orchids, carnations and roses. Other plants useful in ing expression. Intron sequences have been routinely incor the practice of the invention include perennial grasses, such as porated into plant transformation vectors, typically within the Switchgrass, prairie grasses, Indiangrass, Big bluestem grass non-translated leader. 25 and the like. It is recognized that mixtures of plants may be A number of non-translated leader sequences derived from used. viruses are also known to enhance expression, and these are In addition, the term “crops” is to be understood as includ particularly effective in dicotyledonous cells. Specifically, ing crops that have been rendered tolerant to herbicides or leader sequences from Tobacco Mosaic Virus (TMV, the classes of herbicides (such as, for example, ALS inhibitors, “W-sequence”), Maize Chlorotic Mottle Virus (MCMV), and 30 for example primisulfuron, prosulfuron and trifloxysulfuron, Alfalfa Mosaic Virus (AMV) have been shown to be effective EPSPS (5-enol-pyrovyl-shikimate-3-phosphate-synthase) in enhancing expression (e.g. Gallie etal. Nucl. Acids Res. 15: inhibitors, GS(glutamine synthetase) inhibitors) as a result of 8693-8711 (1987); Skuzeski et al Plant Molec. Biol. 15: conventional methods of breeding or genetic engineering. 65-79 (1990)). Other leader sequences known in the art Examples of crops that have been rendered tolerant to herbi include but are not limited to: picornavirus leaders, for 35 cides or classes of herbicides by genetic engineering methods example, EMCV leader (Encephalomyocarditis 5' noncoding include glyphosate- and glufosinate-resistant crop varieties region) (Elroy-Stein, O., Fuerst, T. R., and Moss, B. PNAS commercially available under the trade names Roundu USA 86:6126-6130 (1989)); potyvirus leaders, for example, pReady(R) and LibertyLink R. The method according to the TEV leader (Tobacco Etch Virus) (Allison et al., 1986); present invention is especially suitable for the protection of MDMV leader (Maize Dwarf Mosaic Virus); Virology 154: 40 soybean crops which have also been rendered tolerant to 9-20); human immunoglobulin heavy-chain binding protein glyphosate and/or glufosinate and where HPPD herbicides (BiP) leader, (Maceak, D. G., and Samow, P., Nature 353: are used in a weed control programme along with other Such 90-94 (1991); untranslated leader from the coat protein herbicides (glufosinate and/or glyphosate) for weed control. mRNA of alfalfa mosaic virus (AMV RNA 4), (Jobling, S.A., It is further contemplated that the constructs of the inven and Gehrke, L., Nature 325:622-625 (1987); tobacco mosaic 45 tion may be introduced into plant varieties having improved virus leader (TMV), (Gallie, D. R. et al., Molecular Biology properties Suitable or optimal for a particular downstream of RNA, pages 237-256 (1989); and Maize Chlorotic Mottle use. For example, naturally-occurring genetic variability Virus leader (MCMV) (Lommel, S. A. et al., Virology results in plants with resistance or tolerance to HPPD inhibi 81:382-385 (1991). See also, Della-Cioppa et al., Plant Physi tors or other herbicides, and such plants are also useful in the ology 84:965-968 (1987). 50 methods of the invention. The method according to the Plants present invention can be further optimized by crossing the As used herein, the term “plant part or “plant tissue' transgenes that provide a level of tolerance, with soybean includes plant cells, plant protoplasts, plant cell tissue cul cultivars that exhibit an enhanced level of tolerance to HPPD tures from which plants can be regenerated, plant calli, plant inhibitors that is found in a small percentage of soybean lines. clumps, and plant cells that are intact in plants or parts of 55 Plant Transformation plants such as embryos, pollen, ovules, seeds, leaves, flowers, Once an herbicide resistant or tolerant cytochrome P450 branches, fruit, kernels, ears, cobs, husks, stalks, roots, root polynucleotide, alone or in combination with one or more tips, anthers, and the like. additional nucleic acid molecules encoding polypeptides that Plants useful in the present invention include plants that are confer desirable traits, has been cloned into an expression transgenic for at least a polynucleotide encoding a cyto 60 system, it is transformed into a plant cell. The receptor and chrome P450 or variant thereof that confers herbicide resis target expression cassettes of the present invention can be tance or tolerance, alone or in combination with one or more introduced into the plant cell in a number of art-recognized additional nucleic acid molecules encoding polypeptides that ways. The term “introducing in the context of a polynucle confer desirable traits. The type of plant selected depends on otide, for example, a nucleotide construct of interest, is a variety of factors, including for example, the downstream 65 intended to mean presenting to the plant the polynucleotide in use of the harvested plant material, amenability of the plant Such a manner that the polynucleotide gains access to the species to transformation, and the conditions under which the interior of a cell of the plant. Where more than one polynucle US 8,097,774 B2 19 20 otide is to be introduced, these polynucleotides can be Many vectors are available for transformation using Agro assembled as part of a single nucleotide construct, or as bacterium tumefaciens. These typically carry at least one separate nucleotide constructs, and can be located on the T-DNA border sequence and include vectors such as pBIN19 same or different transformation vectors. Accordingly, these (Bevan, Nucl. Acids Res. (1984)). For the construction of polynucleotides can be introduced into the host cell of interest vectors useful in Agrobacterium transformation, see, for in a single transformation event, in separate transformation example, US Patent Application Publication No. 2006/ events, or, for example, in plants, as part of a breeding proto 0260011, herein incorporated by reference. col. The methods of the invention do not depend on a particu Transformation without the use of Agrobacterium tumefa lar method for introducing one or more polynucleotides into ciens circumvents the requirement for T-DNA sequences in a plant, only that the polynucleotide(s) gains access to the 10 the chosen transformation vector and consequently vectors interior of at least one cell of the plant. Methods for introduc lacking these sequences can be utilized in addition to vectors ing polynucleotides into plants are known in the art including, such as the ones described above which contain T-DNA but not limited to, transient transformation methods, stable sequences. Transformation techniques that do not rely on transformation methods, and virus-mediated methods. 15 Agrobacterium include transformation via particle bombard “Transient transformation” in the context of a polynucle ment, protoplast uptake (e.g. PEG and electroporation) and otide is intended to mean that a polynucleotide is introduced microinjection. The choice of vector depends largely on the into the plant and does not integrate into the genome of the preferred selection for the species being transformed. For the plant. construction of Such vectors, see, for example, US Applica By “stably introducing or “stably introduced in the con tion No. 20060260011, herein incorporated by reference. text of a polynucleotide introduced into a plant is intended the For expression of a nucleotide sequence of the present introduced polynucleotide is stably incorporated into the invention in plant plastids, plastid transformation vector plant genome, and thus the plant is stably transformed with pPH143 (WO 97/32011, example 36) is used. The nucleotide the polynucleotide. sequence is inserted into pPH143 thereby replacing the PRO "Stable transformation” or “stably transformed' is 25 TOX coding sequence. This vector is then used for plastid intended to mean that a polynucleotide, for example, a nucle transformation and selection of transformants for spectino otide construct described herein, introduced into a plant inte mycin resistance. Alternatively, the nucleotide sequence is grates into the genome of the plant and is capable of being inserted in pPH143 so that it replaces the aadH gene. In this inherited by the progeny thereof, more particularly, by the case, transformants are selected for resistance to PROTOX progeny of multiple successive generations. 30 inhibitors. Numerous transformation vectors available for plant trans Transformation techniques for dicotyledons are well formation are known to those of ordinary skill in the plant known in the art and include Agrobacterium-based tech transformation arts, and the genes pertinent to this invention niques and techniques that do not require Agrobacterium. can be used in conjunction with any such vectors. The selec Non-Agrobacterium techniques involve the uptake of exog tion of vector will depend upon the preferred transformation 35 enous genetic material directly by protoplasts or cells. This technique and the target species for transformation. For cer can be accomplished by PEG or electroporation mediated tain target species, different antibiotic or herbicide selection uptake, particle bombardment-mediated delivery, or micro markers may be preferred. Selection markers used routinely injection. Examples of these techniques are described by in transformation include the npt11 gene, which confers resis Paszkowski et al., EMBO.J. 3: 2717-2722 (1984), Potrykus et tance to kanamycin and related antibiotics (Messing & Vierra 40 al., Mol. Gen. Genet. 199: 169-177 (1985), Reich et al., Bio Gene 19: 259-268 (1982); Bevan et al., Nature 304:184-187 technology 4: 1001-1004 (1986), and Kleinet al., Nature 327: (1983)), the pat and bar genes, which confer resistance to the 70-73 (1987). In each case the transformed cells are regener herbicide glufosinate (also called phosphinothricin; see ated to whole plants using standard techniques known in the White et al., Nucl. Acids Res 18: 1062 (1990), Spencer et al. art. Theor: Appl. Genet. 79: 625-631 (1990) and U.S. Pat. Nos. 45 Agrobacterium-mediated transformation is a preferred 5,561.236 and 5,276.268), the hph gene, which confers resis technique for transformation of dicotyledons because of its tance to the antibiotic hygromycin (Blochinger & Diggel high efficiency of transformation and its broad utility with mann, Mol. Cell. Biol. 4: 2929-2931), and the dhfr gene, many different species. Agrobacterium transformation typi which confers resistance to methatrexate (Bourouis et al., cally involves the transfer of the binary vector carrying the EMBO.J. 2(7): 1099-1104 (1983)), the EPSPS gene, which 50 foreign DNA of interest (e.g. pCIB200 or pCIB2001) to an confers resistance to glyphosate (U.S. Pat. Nos. 4,940,935 appropriate Agrobacterium strain which may depend of the and 5,188,642), the glyphosate N-acetyltransferase (GAT) complement of vir genes carried by the host Agrobacterium gene, which also confers resistance to glyphosate (Castle et strain either on a co-resident Ti plasmid or chromosomally al. (2004) Science, 304:1151-1154; U.S. Patent App. Pub. (e.g. strain CIB542 for pCIB200 and pCIB2001 (Uknes et al. Nos. 20070004912, 2005.0246798, and 20050060767); and 55 Plant Cell 5: 159-169 (1993)). The transfer of the recombi the mannose-6-phosphate isomerase gene, which provides nant binary vector to Agrobacterium is accomplished by a the ability to metabolize mannose (U.S. Pat. Nos. 5,767,378 triparentalmating procedure using E. coli carrying the recom and 5,994,629). binant binary vector, a helper E. coli strain which carries a Methods for regeneration of plants are also well known in plasmid such as pRK2013 and which is able to mobilize the the art. For example, Tiplasmid vectors have been utilized for 60 recombinant binary vector to the target Agrobacterium strain. the delivery of foreign DNA, as well as direct DNA uptake, Alternatively, the recombinant binary vector can be trans liposomes, electroporation, microinjection, and microprojec ferred to Agrobacterium by DNA transformation (Hofgen & tiles. In addition, bacteria from the genus Agrobacterium can Willimitzer, Nucl. Acids Res. 16:9877 (1988)). be utilized to transform plant cells. Below are descriptions of Transformation of the target plant species by recombinant representative techniques for transforming both dicotyledon 65 Agrobacterium usually involves co-cultivation of the Agro ous and monocotyledonous plants, as well as a representative bacterium with explants from the plant and follows protocols plastid transformation technique. well known in the art. Transformed tissue is regenerated on US 8,097,774 B2 21 22 selectable medium carrying the antibiotic or herbicide resis 102:1077-1084 (1993)) using particle bombardment of tance marker present between the binary plasmid T-DNA immature embryos and immature embryo-derived callus. A borders. preferred technique for wheat transformation, however, Another approach to transforming plant cells with a gene involves the transformation of wheat by particle bombard involves propelling inert or biologically active particles at ment of immature embryos and includes either a high Sucrose plant tissues and cells. This technique is disclosed in U.S. Pat. or a high maltose step prior to gene delivery. Prior to bom Nos. 4,945,050, 5,036,006, and 5,100,792 all to Sanford etal. bardment, any number of embryos (0.75-1 mm in length) are Generally, this procedure involves propelling inert or biologi plated onto MS medium with 3% sucrose (Murashiga & cally active particles at the cells under conditions effective to Skoog. Physiologia Plantarum 15: 473-497 (1962)) and 3 penetrate the outer surface of the cell and afford incorporation 10 mg/l 2,4-D for induction of Somatic embryos, which is within the interior thereof. When inert particles are utilized, allowed to proceed in the dark. On the chosen day of bom the vector can be introduced into the cell by coating the bardment, embryos are removed from the induction medium particles with the vector containing the desired gene. Alter and placed onto the osmoticum (i.e. induction medium with natively, the target cell can be surrounded by the vector so that Sucrose or maltose added at the desired concentration, typi the vector is carried into the cell by the wake of the particle. 15 cally 15%). The embryos are allowed to plasmolyze for 2-3 Biologically active particles (e.g., dried yeast cells, dried hours and are then bombarded. Twenty embryos per target bacterium or a bacteriophage, each containing DNA sought to plate is typical, although not critical. An appropriate gene be introduced) can also be propelled into plant cell tissue. carrying plasmid (such as pCIB3064 or pSOG35) is precipi Transformation of most monocotyledon species has now tated onto micrometer size gold particles using standard pro also become routine. Preferred techniques include direct gene cedures. Each plate of embryos is shot with the DuPont transfer into protoplasts using PEG or electroporation tech BIOLISTICSR) helium device using a burst pressure of about niques, and particle bombardment into callus tissue. Trans 1000 psi using a standard 80 mesh screen. After bombard formations can be undertaken with a single DNA species or ment, the embryos are placed back into the dark to recover for multiple DNA species (i.e. co-transformation) and both of about 24 hours (still on osmoticum). After 24hrs, the embryos these techniques are suitable for use with this invention. Co 25 are removed from the osmoticum and placed back onto induc transformation may have the advantage of avoiding complete tion medium where they stay for about a month before regen vector construction and of generating transgenic plants with eration. Approximately one month later the embryo explants unlinked loci for the gene of interest and the selectable with developing embryogenic callus are transferred to regen marker, enabling the removal of the selectable marker in eration medium (MS+1 mg/liter NAA, 5 mg/liter GA), fur Subsequent generations, should this be regarded desirable. 30 ther containing the appropriate selection agent (10 mg/l basta However, a disadvantage of the use of co-transformation is in the case of pCIB3064 and 2 mg/l methotrexate in the case the less than 100% frequency with which separate DNA spe of pSOG35). After approximately one month, developed cies are integrated into the genome (Schocher et al. Biotech shoots are transferred to larger sterile containers known as nology 4: 1093-1096 (1986)). “GA7s” which contain half-strength MS, 2% sucrose, and the Patent Applications EP 0292 435, EPO 392 225, and WO 35 same concentration of selection agent. 93/07278 describe techniques for the preparation of callus Transformation of monocotyledons using Agrobacterium and protoplasts from an elite inbred line of maize, transfor has also been described. See, WO 94/00977 and U.S. Pat. No. mation of protoplasts using PEG or electroporation, and the 5,591,616, both of which are incorporated herein by refer regeneration of maize plants from transformed protoplasts. ence. See also, Negrotto et al., Plant Cell Reports 19:798-803 Gordon-Kamm et al. (Plant Cell 2: 603-618 (1990)) and 40 (2000), incorporated herein by reference. Fromm et al. (Biotechnology 8: 833-839 (1990)) have pub For example, rice (Oryza sativa) can be used for generating lished techniques for transformation of A188-derived maize transgenic plants. Various rice cultivars can be used (Hiei et line using particle bombardment. Furthermore, WO al., 1994, Plant Journal 6:271-282: Dong et al., 1996, 93/07278 and Koziel et al. (Biotechnology 11: 194-200 Molecular Breeding 2:267-276; Hiei et al., 1997, Plant (1993)) describe techniques for the transformation of elite 45 Molecular Biology, 35:205-218). Also, the various media inbred lines of maize by particle bombardment. This tech constituents described below may be either varied in quantity nique utilizes immature maize embryos of 1.5-2.5 mm length or Substituted. Embryogenic responses are initiated and/or excised from a maize ear 14-15 days after pollination and a cultures are established from mature embryos by culturing on PDS-1000He Biolistics device for bombardment. MS-CIM medium (MS basal salts, 4.3 g/liter; B5 vitamins Transformation of rice can also be undertaken by direct 50 (200x), 5 ml/liter. Sucrose, 30 g/liter; proline, 500 mg/liter; gene transfer techniques utilizing protoplasts or particle bom glutamine, 500 mg/liter; casein hydrolysate, 300 mg/liter; bardment. Protoplast-mediated transformation has been 2,4-D (1 mg/ml), 2 ml/liter; adjust pH to 5.8 with 1 N KOH: described for Japonica-types and Indica-types (Zhang et al. Phytagel, 3 g/liter). Either mature embryos at the initial stages Plant Cell Rep 7: 379-384 (1988); Shimamoto et al. Nature of culture response or established culture lines are inoculated 338: 274-277 (1989); Datta et al. Biotechnology 8:736-740 55 and co-cultivated with the Agrobacterium tumefaciens strain (1990)). Both types are also routinely transformable using LBA4404 (Agrobacterium) containing the desired vector particle bombardment (Christou et al. Biotechnology 9.957 construction. Agrobacterium is cultured from glycerol Stocks 962 (1991)). Furthermore, WO 93/21335 describes tech on solid YPC medium (100 mg/L spectinomycin and any niques for the transformation of rice via electroporation. other appropriate antibiotic) for about 2 days at 28°C. Agro Patent Application EP 0 332 581 describes techniques for 60 bacterium is re-suspended in liquid MS-CIM medium. The the generation, transformation and regeneration of Pooideae Agrobacterium culture is diluted to an OD600 of 0.2-0.3 and protoplasts. These techniques allow the transformation of acetosyringone is added to a final concentration of 200 uM. Dactylis and wheat. Furthermore, wheat transformation has Acetosyringone is added before mixing the Solution with the been described by Vasil et al. (Biotechnology 10: 667-674 rice cultures to induce Agrobacterium for DNA transfer to the (1992)) using particle bombardment into cells of type Clong 65 plant cells. For inoculation, the plant cultures are immersed in term regenerable callus, and also by Vasil et al. (Biotechnol the bacterial Suspension. The liquid bacterial Suspension is ogy 11:1553-1558 (1993)) and Weeks et al. (Plant Physiol. removed and the inoculated cultures are placed on co-culti US 8,097,774 B2 23 24 vation medium and incubated at 22°C. for two days. The aseptically on spectinomycin-containing MS/IBA medium cultures are then transferred to MS-CIM medium with Ticar (McBride, K. E. etal (1994) PNAS 91,7301-7305) and trans cillin (400 mg/liter) to inhibit the growth of Agrobacterium. ferred to the greenhouse. For constructs utilizing the PMI selectable marker gene (Reed The genetic properties engineered into the transgenic seeds et al. In Vitro Cell. Dev. Biol.-Plant 37:127-132), cultures are and plants described above are passed on by sexual reproduc transferred to selection medium containing Mannose as a tion or vegetative growth and can thus be maintained and carbohydrate source (MS with 2% Mannose, 300 mg/liter propagated in progeny plants. Generally, maintenance and Ticarcillin) after 7 days, and cultured for 3-4 weeks in the propagation make use of known agricultural methods devel dark. Resistant colonies are then transferred to regeneration oped to fit specific purposes such as tilling, sowing or har induction medium (MS with no 2,4-D, 0.5 mg/liter IAA, 1 10 Vesting. mg/liter Zeatin, 200 mg/liter timentin 2% Mannose and 3% Use of the advantageous genetic properties of the trans Sorbitol) and grown in the dark for 14 days. Proliferating genic plants and seeds according to the invention can further colonies are then transferred to another round of regeneration be made in plant breeding. Depending on the desired proper induction media and moved to the light growth room. Regen ties, different breeding measures are taken. The relevant tech erated shoots are transferred to GA7 containers with GA7-1 15 niques are well known in the art and include but are not medium (MS with no hormones and 2% Sorbitol) for 2 weeks limited to hybridization, inbreeding, backcross breeding, and then moved to the greenhouse when they are large enough multi-line breeding, variety blend, interspecific hybridiza and have adequate roots. Plants are transplanted to Soil in the tion, aneuploid techniques, etc. Thus, the transgenic seeds greenhouse (To generation) grown to maturity, and the T and plants according to the invention can be used for the seed is harvested. breeding of improved plant lines that, for example, increase The plants obtained via transformation with a nucleic acid the effectiveness of conventional methods such as herbicide sequence of interest in the present invention can be any of a or pesticide treatment or allow one to dispense with said wide variety of plant species, including those of monocots methods due to their modified genetic properties. and dicots; however, the plants used in the method of the Many suitable methods for transformation using suitable invention are preferably selected from the list of agronomi 25 selection markers such as kanamycin, binary vectors such as cally important target crops set forth elsewhere herein. The from Agrobacterium and plant regeneration as, for example, expression of a gene of the present invention in combination from tobacco leaf discs are well known in the art. Optionally, with other characteristics important for production and qual a control population of plants are likewise transformed with a ity can be incorporated into plant lines through breeding. polynucleotide expressing the control HPPD. Alternatively, Breeding approaches and techniques are known in the art. 30 an untransformed dicot plant such as Arabidopsis or Tobacco See, for example, Welsh J. R. Fundamentals of Plant Genet can be used as a control since this, in any case, expresses its ics and Breeding, John Wiley & Sons, NY (1981); Crop own endogenous HPPD. Breeding, Wood D. R. (Ed.) American Society of Agronomy Herbicide Resistance Madison, Wis. (1983); Mayo O., The Theory of Plant Breed The present invention provides transgenic plants, plant ing, Second Edition, Clarendon Press, Oxford (1987); Singh, 35 cells, tissues, and seeds that have been transformed with a D. P. Breeding for Resistance to Diseases and Insect Pests, nucleic acid molecule encoding a cytochrome P450 or variant Springer-Verlag, NY (1986); and Wricke and Weber, Quan thereof that confers resistance or tolerance to herbicides, titative Genetics and Selection Plant Breeding, Walter de alone or in combination with one or more additional nucleic Gruyter and Co., Berlin (1986). acid molecules encoding polypeptides that confer desirable For the transformation of plastids, seeds of Nicotiana 40 traits. tabacum c.V. Xanthienc' are germinated seven per plate in a In one embodiment, the transgenic plants of the invention 1" circular array on Tagar medium and bombarded 12-14 exhibit resistance or tolerance to application of herbicide in days after sowing with 1 um tungsten particles (M10, Biorad, an amount of from about 5 to about 2,000 grams per hectare Hercules, Calif.) coated with DNA from plasmids pPH143 (g/ha), including, for example, about 5 g/ha, about 10 g/ha, and pPH145 essentially as described (Svab, Z. and Maliga, P. 45 about 15 g/ha, about 20 g/ha, about 25 g/ha, about 30 g/ha, (1993) PNAS 90,913–917). Bombarded seedlings are incu about 35 g/ha, about 40 g/ha, about 45 g/ha, about 50 g/ha, bated on T medium for two days after which leaves are about 55 g/ha, about 60 g/ha, about 65 g/ha, about 70 g/ha, excised and placed abaxial side up in bright light (350-500 about 75 g/ha, about 80 g/ha, about 85 g/ha, about 90 g/ha, umol photons/m/s) on plates of RMOP medium (Svab, Z. about 95 g/ha, about 100 g/ha, about 110 g/ha, about 120 g/ha, Hajdukiewicz, P. and Maliga, P. (1990) PNAS87,8526-8530) 50 about 130 g/ha, about 140 g/ha, about 150 g/ha, about 160 containing 500 ug/ml spectinomycin dihydrochloride g/ha, about 170 g/ha, about 180 g/ha, about 190 g/ha, about (Sigma, St. Louis, Mo.). Resistant shoots appearing under 200 g/ha, about 210 g/ha, about 220 g/ha, about 230 g/ha, neath the bleached leaves three to eight weeks after bombard about 240 g/ha, about 250 g/ha, about 260 g/ha, about 270 ment are subcloned onto the same selective medium, allowed g/ha, about 280 g/ha, about 290 g/ha, about 300 g/ha, about to form callus, and secondary shoots isolated and Subcloned. 55 310 g/ha, about 320 g/ha, about 330 g/ha, about 340 g/ha, Complete segregation of transformed plastid genome copies about 350 g/ha, about 360 g/ha, about 370 g/ha, about 380 (homoplasmicity) in independent Subclones is assessed by g/ha, about 390 g/ha, about 400 g/ha, about 410 g/ha, about standard techniques of Southern blotting (Sambrook et al., 420 g/ha, about 430 g/ha, about 440 g/ha, about 450 g/ha, (1989) Molecular Cloning: A Laboratory Manual, Cold about 460 g/ha, about 470 g/ha, about 480 g/ha, about 490 Spring Harbor Laboratory, Cold Spring Harbor). BamHI/ 60 g/ha, about 500 g/ha, about 510 g/ha, about 520 g/ha, about EcoRI-digested total cellular DNA (Mettler, I.J. (1987) Plant 530 g/ha, about 540 g/ha, about 550 g/ha, about 560 g/ha, Mol Biol Reporter 5,346349) is separated on 1% Tris-borate about 570 g/ha, about 580 g/ha, about 590 g/ha, about 600 (TBE) agarose gels, transferred to nylon membranes (Amer g/ha, about 610 g/ha, about 620 g/ha, about 630 g/ha, about sham) and probed with sup.32P-labeled random primed 640 g/ha, about 650 g/ha, about 660 g/ha, about 670 g/ha, DNA sequences corresponding to a 0.7 kb BamHI/HindIII 65 about 680 g/ha, about 690 g/ha, about 700 g/ha, about 710 DNA fragment from pC8 containing a portion of the rps 7/12 g/ha, about 720 g/ha, about 730 g/ha, about 740 g/ha, about plastid targeting sequence. Homoplasmic shoots are rooted 750 g/ha, about 760 g/ha, about 770 g/ha, about 780 g/ha, US 8,097,774 B2 25 26 about 790 g/ha, about 800 g/ha, about 810 g/ha, about 820 wherein R and R2 are hydrogen or together an ethylene g/ha, about 830g/ha, about 840 g/ha, about 850 g/ha, about bridge; 860 g/ha, about 870 g/ha, about 880 g/ha, about 890 g/ha, R is C-C alkyl, halogen, nitro, C-C alkoxy-C-C-alkyl, about 900 g/ha, about 910 g/ha, about 920 g/ha, about 930 C-Calkoxy-C-Calkoxy-C-C-alkyl or R is a group g/ha, about 940 g/ha, about 950 g/ha, about 960 g/ha, about 5 970 g/ha, about 980 g/ha, about 990 g/ha, about 1,000 g/ha, about 1,010 g/ha, about 1,020 g/ha, about 1,030 g/ha, about 1,040 g/ha, about 1,050 g/ha, about 1,060 g/ha, about 1,070 CH3 g/ha, about 1,080 g/ha, about 1,090 g/ha, about 1,100 g/ha, about 1,110 g/ha, about 1,120 g/ha, about 1,130 g/ha, about 10 N 1,140 g/ha, about 1,150 g/ha, about 1,160 g/ha, about 1,170 X g/ha, about 1,180 g/ha, about 1,190 g/ha, about 1,200 g/ha, about 1,210 g/ha, about 1,220 g/ha, about 1,230 g/ha, about 1,240 g/ha, about 1,250 g/ha, about 1,260 g/ha, about 1,270 15 g/ha, about 1,280 g/ha, about 1.290 g/ha, about 1,300 g/ha, about 1.310 g/ha, about 1,320 g/ha, about 1,330 g/ha, about 1,340 g/ha, about 1,350 g/ha, about 360 g/ha, about 1,370 R is C-C alkylsulfonyl or C-Chaloalkyl; g/ha, about 1.380 g/ha, about 1.390 g/ha, about 1,400 g/ha, Rs is hydrogen or phenylthio; X is methine, nitrogen or about 1,410 g/ha, about 1,420 g/ha, about 1,430 g/ha, about C-R, wherein R is C-Chaloalkoxy-C-C-alkyl or a 1,440 g/ha, about 1,450g/ha, about 1,460 g/ha, about 1,470 group g/ha, about 1,480 g/ha, about 1,490 g/ha, about 1,500 g/ha, about 1,510 g/ha, about 1.520 g/ha, about 1,530 g/ha, about 1,540 g/ha, about 1,550 g/ha, about 1,560 g/ha, about 1,570 g/ha, about 1,580 g/ha, about 1,590 g/ha, about 1,600 g/ha, 25 about 1,610 g/ha, about 1,620 g/ha, about 1,630 g/ha, about 1,640 g/ha, about 1,650 g/ha, about 1,660 g/ha, about 1,670 g/ha, about 1,680 g/ha, about 1,690 g/ha, about 1,700 g/ha, about 1,710 g/ha, about 1,720 g/ha, about 1,730 g/ha, about 1,740 g/ha, about 1,750 g/ha, about 1,760 g/ha, about 1,770 30 g/ha, about 1.780 g/ha, about 1,790 g/ha, about 1,800 g/ha, about 1,810 g/ha, about 1,820 g/ha, about 1,830g/ha, about 1,840 g/ha, about 1,850 g/ha, about 1,860 g/ha, about 1,870 the compounds of formula Ib g/ha, about 1,880 g/ha, about 1,890 g/ha, about 1,900 g/ha, about 1,910 g/ha, about 1.920 g/ha, about 1,930 g/ha, about 35

1,940 g/ha, about 1.950 g/ha, about 1,960 g/ha, about 1,970 (Ib) g/ha, about 1,980 g/ha, about 1,990 g/ha, or about 2,000. The average and distribution of herbicide tolerance or resistance levels of a range of primary plant transformation 40 events are evaluated in the normal manner based upon plant damage, meristematic bleaching symptoms etc. at a range of different concentrations of herbicides. These data can be expressed in terms of, for example, GR50 values derived from dose/response curves having "dose' plotted on the X-axis and 45 "percentage kill”, “herbicidal effect”, “numbers of emerging greenplants' etc. plotted on the y-axis where increased GR50 values correspond to increased levels of inherent inhibitor tolerance (increased Ki value) of the expressed HPPD. 50 The methods of the present invention are especially useful to protect soya crops from the herbicidal injury of HPPD wherein R, is methyl or chlorine; the compounds of formula inhibitor herbicides of the classes of HPPD chemistry Ic selected from the group consisting of the compounds of for mula Ia 55 (Ic)

(Ia) ORs O R3 60 21 Nx R S R4 R3 65 US 8,097,774 B2 27 28 wherein Rs is halogen or C-Chaloalkyl; the compound of the compound of formula Ih formula Id (Id) C (Ih) O CH3 5 CH3 OH H3C ls O N f \ C N C N NS N O 10 O O CH3

S-CH O-CH 21 W 3 3 15 O

CH the compound of formula II the compound of formula Ie 2O I (II) C (Ie) O CH O O sy-1 3 H3C O 25 f \ C CF N O H3C/ seso N 30 the compound of formula I

H3C 35 (I) the compound of formula If (If) C

40

n O O 45 CH3 and the compound of formula Ik

(Ik) 50 SOCH the compound of formula Ig H3C CF.

(Ig) 55 N

CH3

60 The alkyl groups appearing in the Substituent definitions may be straight-chain or branched and are, for example, methyl, ethyl, n-propyl, isopropyl. n-butyl, sec-butyl, isobutyl or tert butyl. Halogen is generally fluorine, chlorine, bromine or 65 iodine, preferably fluorine or chlorine. The same is true of halogen in conjunction with other meanings, such as and the free acid thereof, haloalkyl. US 8,097,774 B2 29 30 Haloalkyl groups preferably have a chain length of from 1 wise treated plants but lacking the P450 transgenes) the resi to 4 carbon atoms. Haloalkyl is, for example, fluoromethyl, due level of parent herbicide throughout the plant tissue and difluoromethyl, trifluoromethyl, chloromethyl, dichlorom especially in the beans. One of ordinary skill in the art will of ethyl, trichloromethyl, 2.2.2-trifluoroethyl 2-fluoroethyl, course understand that certain P450 enzymes are likely to 2-chloroethyl, pentafluoroethyl, 1,1-difluoro-2,2,2-trichloro- 5 confer resistance to certain subgroups of HPPD chemistry, ethyl, 2.2.3,3-tetrafluoroethyl or 2.2.2-trichloroethyl; prefer and one enzyme may not provide resistance to all HPPDs. ably trichloromethyl, difluorochloromethyl, difluoromethyl, In another embodiment, the present invention provides trifluoromethyl or dichlorofluoromethyl. transgenic plants that are resistant or tolerant to herbicides Alkoxyalkoxy groups preferably have a chain length of selected from the group consisting of BenzothiadiaZinones, from 1 to 8 carbon atoms. Examples of alkoxyalkoxy are: 10 Sulfonylureas, and other classes of herbicides including, for methoxymethoxy, methoxyethoxy, methoxypropoxy, example, Imidazolinones, Triazolopyrimidines, Pyrimidi ethoxymethoxy, ethoxyethoxy, propoxymethoxy and nylthiobenzoates, Triazolinones, Auxins, Acetyl-coenzyme butoxybutoxy. Alkoxyalkyl groups have a chain length of A Carboxylase (ACCase) inhibitors, Photosystem II (PSII) preferably from 1 to 6 carbon atoms. Alkoxyalkyl is, for inhibitors, Protoporphyrinogen Oxidase (PPO) inhibitors, example, methoxymethyl, methoxyethyl, ethoxymethyl, 15 Phytoene Desaturase (PDS) inhibitors, Dinitroanalines, and ethoxyethyl, n-propoxymethyl, n-propoxyethyl, isopro Acetamides, as well as herbicides with unknown modes of poxymethyl or isopropoxyethyl. action Such as DifenZoquat and ClomaZone. Alkoxy-alkoxy-alkyl groups preferably have a chain Exemplary Benzothiadiazinones include Bentazon (CAS length of from 1 to 8 carbon atoms. Examples of alkoxy Registry No. 25057-89-0; available from BASF, Research alkoxy-alkyl are: methoxymethoxymethyl, methoxy- 20 Triangle Park, N.C.). ethoxymethyl, ethoxymethoxymethyl and methoxyethoxy Exemplary Sulfonylureas include Bensulfuron (CAS Reg ethyl. istry No. 99283-01-9), Chlorsulfuron (CAS Registry No. The compounds of formula Ia to Ik are known or can be 64902-72-3), Halosulfuron (CAS Registry No. 135397-30 prepared according to known methods. Compounds of for 7), Nicosulfuron (CAS Registry No. 111991-09-4), Rimsul mula Ia and their preparation are known from WO/0015615, 25 furon (CAS Registry No. 122931-48-0), Sulfometuron (CAS WO/0015615, WO 02/085118, WO 00/021924, U.S. Pat. No. Registry No. 74223-56-6), and Triflusulfuron (CAS Registry 5,006,158 (and U.S. Pat. No. 4,780,127. Compounds of for No. 135990-29-3), as well as salts and esters thereof. Addi mula Ib are described in WO 98/31681, and mixtures of those tional Sulfonylureas include, but are not limited to, Triflox compounds with herbicides are known from WO99/65314. ysulfuron, Primisulfuron, Chlorimuron-ethyl, Amidosulfu Compounds of formula Ic are described in U.S. Pat. No. 30 ron, AZimsulfuron, Bensulfuron-methyl, CycloSulfamuron, 5,656,573. Compound of formula Id is described in The Pes Ethametsulfuron-methyl, Ethoxysulfuron, , ticide Manual 12" ed., under Entry No. 71, compound of Flupyrsulfuron-methyl, Halosulfuron-methyl, Imazosulfu formula Ie under Entry No. 663 and compound of formula If ron, Iodosulfuron, Metsulfuron-methyl, Foramsulfuron, under Entry No. 666. The compound of formula Ig (is Oxasulfuron, Prosulfuron, Pyrazosulfuron-ethyl, Sulfometu described in Chemical Abstracts under the registration num- 35 ron-methyl, Sulfosulfuron, Tritosulfuron, Thifensulfuron ber CAS 192708-91-1. The compound of formula Ih is methyl, Triasulfuron, Tribenuron-methyl, and Triflusulfu described in EP-A-0352543. The compound of formula II is ron-methyl. described in EP-A-0496 631, the compound of formula I in Exemplary Imidazolinones include, but are not limited to, WO 04/021788. The compound of formula Ik (has the ImaZamox, Imazethapyr, , ImazamethabenZ-me Chemical Abstracts registration number 365400-11-9. 40 thyl, and Imazaquin. In one aspect, the method according to the present inven Exemplary Triazolopyrimidines include, but are not lim tion is used to protect soya crops from the herbicidal injury of ited to, Flumetsulam, Diclosulam, Florasulam, Chloransu HPPD inhibitor herbicides of the classes of HPPD chemistry lam-methyl, and MetoSulam. selected from the group consisting of the compounds of for Exemplary Pyrimidinylthiobenzoates include, but are not mula Ia or Ig. In an alternative aspect the cytochrome P450 45 limited to, Bispyribac, Pyrithiobac, Pyriminobac-methyl, gene is selected from a polynucleotide sequence encoding the Pyriftalid, and . amino acid sequence of SEQID NO:1 and SEQID NO:5. In Exemplary Triazolinones include, but are not limited to, a further embodiment the HPPD inhibitor herbicide is Flucarbazone. Thiencarbazone-methyl, and Propoxycarba selected from Sulcotrione, mesotrione, tembotrione and com ZO. pounds of formula Ia where X is nitrogen and R is CF, CFH 50 Exemplary Auxins include, but are not limited to, or CFH and/or where R and R together form an ethylene Dicamba, , 2,4-D, , Aminocyclopy bridge. In a further embodiment the HPPD inhibitor herbicide rachlor, , , MCPA, MCPB, 2,4-DB, is selected from Mesotrione, SYN449280, Isoxaflutole, Tem , and . botrione, ToprameZone, Pyrasulfatole, Sulcotrione, Pyra Exemplary ACCase inhibitors include, but are not limited Zolynate, Pyrazoxyfen, Isoxachlortole, Benzofenap, and 55 to, -P-butyl, Pinoxaden, Clodinafop-propargyl, Benzobicyclon. Fenoxaprop-P-ethyl, Tralkoxydim, Diclofop-methyl, Cyha In another aspect soybean or other dicot crop plants are lofop-butyl, Haloxyfop-P-methyl, Quizalofop-P-ethyl, transformed to express both the cytochrome P450 gene Alloxydim, Butroxydim, Clethodim, and Cycloxydim. encoding the amino acid sequence of SEQID NO: 1 or SEQ Exemplary PSII inhibitors include, but are not limited to, ID NO:5 and also an HPPD gene, which is derived from a 60 Bentazon, Linuron, , , , Diu monocot plant, for example from Avena, at Sufficient levels to ron, Isoproturon, , Desmedipham, Metamitron, provide commercially useful levels of resistance to herbicides , AmicarbZone, Fluometuron, Phenmedipham, Pyri selected from Sulcotrione, mesotrione, tembotrione and com date, Ametryn, Cynazine, Dimefuron, Fluometuron, Methi pounds of formula Ia where X is nitrogen and R is CF, CFH benzuron, Metoxuron, Prometryn, , Simetryn, Ter or CFH and/or where R and R together form an ethylene 65 bacil, , Chlorotoluron, and Trietazine. bridge. The level of expression of the cytochrome P450 Exemplary PPO inhibitors include, but are not limited to, should be sufficient to reduce substantially (relative to like , , CarfentraZone, , Oxy US 8,097,774 B2 31 32 fluorfen, Flumioxazin, , , Oxadiazon, one, at least two, at least three, or at least four additional , Flufenpyr-ethyl, Flumiclorac, and Oxadiargyl. nucleic acid molecules encoding polypeptides that confer Exemplary PDS inhibitors include, but are not limited to, desirable traits. Norflurazon, Diflufenican, Fluorochloridone, Flurtamone, In one embodiment, the present invention provides trans Picolinafen, and Fluridone. 5 genic plants and methods useful for the control of unwanted Exemplary Dinitroanalines include, but are not limited to, plant species in crop fields, wherein the crop plants are made , , Orazalin, Butralin, Dinitroamine, resistant to HPPD chemistry by transformation with cyto and Ethalfluralin. chrome P450 genes expressing enzymes that degrade the Exemplary Acetamides include, but are not limited to, HPPD herbicides. Over-the-top application of HPPD herbi 10 cide in amounts capable of killing or impairing the growth of , S-, metolachlor, , unwanted plant species (weed species, or, for example, carry P-dimethenamid, Flufenacet, , , Mefen over or "rogue' or “volunteer crop plants in a field of soy acet, Pretilachlor, , and Thenylchlor. bean crop plants). The application may be pre- or post emer In another embodiment, the present invention provides gence of the crop plants or of the unwanted species, and may transgenic plants that are resistant or tolerant to herbicides 15 combine other herbicides to which the crop is naturally tol selected from the group consisting of imidazolinones, triaz erant, or to which it is resistant via expression of one or more olopyrimidines, pyrimidinyl(thio)benzoates, benzoic acids, other transgenes. See, e.g., U.S. application publication num phenoxycarboxylic acids, quinoline carboxylic acids, arylox ber 2004/0058427; PCT application WO98/20144. yphenoxypropionates, cyclohexanediones, dinitroani lines, In another embodiment, the invention also relates to a benzamides, carbamates, , amides, triazinones, phenyl method of protecting crop plants from herbicidal injury. In the pyridazines, phenylcarbamates, triazolinones, pyrimidindi cultivation of crop plants, especially on a commercial scale, ones, oxadiazoles, N-phenyl phthalimides, chloroaceta correct crop rotation is crucially important for yield stability mides, pyridaZinones, and pyridinecarboxamides. (the achievement of high yields of good quality over a long The present invention further provides a method of selec period) and for the economic Success of an agronomic busi tively controlling weeds at a locus comprising crop plants and 25 ness. For example, across large areas of the main maize weeds, wherein the plants are obtained by any of the methods growing regions of the USA (the “central corn belt), soya is of the current invention described above, wherein the method grown as the Subsequent crop to maize in over 75% of cases. comprises application to the locus of a weed controlling Selective weed control in maize crops is increasingly being amount of one or more herbicides. Any of the transgenic carried out using HPPD inhibitor herbicides. Although that plants described herein may be used within these methods of class of herbicides has excellent suitability for that purpose, it the invention. The term “locus' may include soil, seeds, and can result in agronomically unacceptable phytotoxic damage seedlings, as well as established vegetation. Herbicides can to the crop plants in subsequent crops, especially in Subse Suitably be applied pre-emergence or post-emergence. quent soya crops, because certain Soya varieties are sensitive to even very small residues of such HPPD inhibitor herbicides The term “weed controlling amount” is meant include 35 ("carry-over damage). Accordingly, the herbicide resistant functionally, an amount of herbicide which is capable of or tolerant plants of the invention are also useful for planting affecting the growth or development of a given weed. Thus, in a locus of any short term carry-over of herbicide from a the amount may be small enough to simply retard or Suppress previous application (e.g., by planting a transgenic plant of the growth or development of a given weed, or the amount the invention in the year following application of an herbicide may be large enough to irreversibly destroy a given weed. 40 to reduce the risk of damage from soil residues of the herbi Thus, the present invention provides a method of control cide). ling weeds at a locus comprising applying to the locus a The invention is made clearer through the following non weed-controlling amount of one or more herbicides, where limiting examples. The following examples are offered by the locus comprises a transgenic plant that has been trans way of illustration and not by way of limitation. formed with a nucleic acid molecule encoding a cytochrome 45 P450 or variant thereofthat confers resistance or tolerance to EXPERIMENTAL herbicides, alone or in combination with one or more addi tional nucleic acid molecules encoding polypeptides that con Example 1 fer desirable traits. In particular, the locus comprises a trans genic plant that has been transformed with a nucleic acid 50 Gene Cassettes Suitable for Engineering Tolerance molecule encoding a cytochrome P450 or variant thereofthat to HPPD Herbicides and, in Particular Mesotrione, confers resistance or tolerance to an herbicide selected from into Crop Plants the group consisting of HPPD inhibitors, Benzothiadiazino nes, Sulfonylureas, Imidazolinones, Triazolopyrimidines, For example a DNA construct is made which has 1) a Pyrimidinylthiobenzoates, Triazolinones, Auxins, ACCase 55 cassette having an Avena HPPD gene as in SEQID NO:14 or inhibitors, PSII inhibitors, PPO inhibitors, PDS inhibitors, SEQID NO:15, for example, encoding a protein of or at least Dinitroanalines, and Acetamides, as well as herbicides with 85% similar to SEQ ID NO:10, or a modified Avena HPPD unknown modes of action Such as DifenZoquat and Cloma gene (e.g., encoding the amino acid sequence set forth in SEQ Zone. In another embodiment, the desirable trait is resistance ID NO:11, 12, or 13), as described above, under operable or tolerance to an herbicide, including, for example, herbi 60 expression control of an upstream promoter, Such as a cides selected from the group consisting of an HPPD inhibi tobacco, Arabidopsis or soybean small subunit of Rubisco tor, glyphosate, and glufosinate. In another embodiment, the promoter region including 5' untranslated leader sequence locus comprises a transgenic plant that has been transformed and a downstream nopaline synthase 3' terminator sequence with any combination of nucleic acid molecules described adjacent to a second cassette 2) having a DNA sequence above, including one or more nucleic acid molecules encod 65 encoding the maize insfl or the rice CYP81.A6 protein ing a cytochrome P450 or variant thereof that confers resis sequence (SEQID NO:1 or SEQID NO: 5) under operable tance or tolerance to an herbicide in combination with at least control of an upstream Arabidopsis or soybean polyubiquitin US 8,097,774 B2 33 34 promoter and 5' untranslated leader sequence and a down nator, is excised using HindIII/EcoR1 (partial EcoR1 digest) stream 3' terminator sequence from a histone gene (See FIG. and cloned into similarly digested pBIN 19 plant transforma 1). Optionally constructs include, transcriptional enhancer tion vector and transformed into E. coli TOP 10 competent sequences (e.g. from the CMV35S promoter region) are cells. upstream of one or other of the promoters and optionally, Alternatively, double gene constructs designed as in translational enhancers such as the TMV omega sequence are example 1 are Suitably designed with restriction ends and included ahead of the translational start. Suitable sequences similarly cloned into the pBIN19 plant transformation vector and method to manipulate the DNA in order to make these and transformed into E. coli TOP 10 cells. constructs are well known in the art. Optionally they are Plant transformation vectors with the appropriate P450 partially or entirely made synthetically and pieced together 10 gene expression cassette are used to transform Agrobacte by PCR and Restriction Enzyme and ligation reactions. The rium tumefaciens LBA4404. Tobacco tissue is subjected to promoters can also be chimeric promoters, where 5' upstream Agrobacterium-mediated transformation using methods well promoter regions are fused to a TATA box, and enhancers. described in the art. Transformed shoots are regenerated from Examples of optimized genes are Avena HPPD gene (SEQID callus resistant to the selection agent (e.g., glufosinate, NO:14), “modified Avena HPPD gene” (SEQ ID NO:15), 15 butafenacil, kanamycin). Shoots are rooted on MSagar con nsf1 (SEQID NO:2), and corn CYP72A1 (SEQ ID NO:4). taining the selection agent. In further variants of the example the cytochrome P450 To determine tolerance of P450 transgenic explants (i.e. a and/or Avena genes are obtained synthetically from GeneArt, leaf plus short segment of stem containing the auxiliary bud), codon optimised for expression in Soybean and designed to these are placed into MSagar (+3% Sucrose) containing vari have 5'NdeI and 3'BamHI restriction sites for cloning (which ous concentrations of mesotrione, from 0.02 to 2 ppm. In adds no extra amino acids). Optionally, the Ndel:BamH1 tobacco, for example, untransformed explants are fully products are cloned into vectorpMCJA which is a derivative bleached at 0.02 ppm. They do not recover following pro of pMJB1 (described in WO 98/20144) modified only to longed exposure to the herbicide. In these particular experi comprise an Nde 1 rather than an Ncol site at the translation ments, only the shoot that develops from the bud is bleached, initiation codon site. pMJB1 is a PUC19 derived plasmid 25 the leaf on the explanted tissue remains green. which contains the plant operable double enhanced A number of the transgene PCR+ve transformed plants CAMV35S promoter, a TMV omega translational enhancer tolerate mesotrione with no indication of bleaching at the and the NOS transcription terminator. A schematic of the level which causes symptoms on wild-type tobacco. They plasmid is depicted in FIG. 2 of WO 98/20144. The expres root normally and are phenotypically indistinguishable from sion cassette comprising, for example, the double enhanced 30 untransformed plants. A sub-set of the transformants is toler 35S promoter, TMV omega leader, SEQ ID NO:1 cyto ant to concentrations of >0.1 ppm yielding plants looking chrome P450 coding sequence and nos 3' terminator is normal and rooting well in the presence of herbicide. Some of excised, for example as appropriate, using a HindIII/EcoR1 the transformed plants can be initially bleached when sub partial digest and cloned into similarly digested pBIN19 and jected to the herbicide at the said higher concentrations, but transformed into E. coli TOP 10 competent cells. DNA recov 35 on prolonged exposure they progressively "green up' and ered from the E. coli is then used to transform Agrobacterium “recover. Alternatively, T1 plants from events expressing a tumefaciens LBA4404 and the transformed bacteria selected P450 transgene can be sprayed with mesotrione at levels that on rifampicin and kanamycin. For example tobacco tissue is cause bleaching on new growth of non-transformed plants, then Subjected to Agrobacterium-mediated transformation but no bleaching in transgenic plants expressing a P450 trans using textbook standard methods well described in the art. 40 gene. Transformed shoots are regenerated from kanamycin resis Alternatively, HPPD sensitive corn plants can be trans tant callus. Shoots are rooted on MS agar containing kana formed with expression vectors containing the putative mycin. Surviving rooted explants are re-rooted to provide HPPD herbicide degrading P450 genes, and a selectable approximately 30-50 kanamycin resistant transformed marker gene. Events can be tested for enhanced tolerance to tobacco plants. 45 the HPPD inhibitors. Alternatively, soybean plants can be transformed with Example 2 expression vectors containing the putative HPPD herbicide degrading P450 genes, and a selectable marker gene. Events Transformation of Tobacco with Test CYP Genes can be tested for enhanced tolerance to the HPPD inhibitors. and Selection of Herbicide-Resistant Lines 50 Example 3 Candidate full length genes were synthesized, including useful restriction sites, and a translational enhancer sequence Transformation of Soybean and Selection of 5' to the start site. These gene cassettes were cloned down Herbicide-Resistant Plants stream of the FMV promoter, and these gene cassettes were 55 next cloned into transformation vectors containing either a Linear DNA suitable for use in bombardment-based plant bar or PPO selectable marker gene cassette. transformation is produced by digesting a vector comprising Alternatively, candidate full length cyp genes obtained the desired genes (e.g. as in Example 1). This desired frag from maize or rice cDNA libraries are edited by PCR to ment is then purified on an agarose gel and isolated using a include, for example, 5' Nco1 and 3' Kpn1 ends. This product 60 Biotrap. is then ligated into pMJB1.pMJB1 is a puC19 derived plas Optionally, the CYP, the HPPD gene or both together can mid which contains the plant operable double enhanced provide the means of selection and identification of trans CaMV35S promoter; a TMV omega enhancer and the NOS genic tissue. Optionally the gene for expression of CYP and, transcription terminator. A schematic representation of the for example, Avena sativa HPPD can be present in the poly resulting plasmid is shown in FIG. 2 of WO 98/20144. The 65 nucleotide alongside other sequences which provide addi expression cassette, comprising the double enhanced 35S tional means of selection/identification of transformed tissue promoter, TMV omega leader, 4-HPPD gene and nos termi including, for example, the known genes which provide resis US 8,097,774 B2 35 36 tance to kanamycin, hygromycin, phosphinothricin, Nature, 327, 70-73. Alternatively, for example, 60 mg of gold butafenacil or glyphosate. Alternatively these selectable or tungsten particles (~1.0 um) in a microcentrifuge tube are marker sequences may be present on separate polynucle washed repeatedly in HPLC-grade ethanol and then, repeat otides and a process of, for example, co-transformation and edly, insterile water. The particles are resuspended in 1 ml of co-selection is used. Alternatively, rather than a selectable sterile water and dispensed into 50 ul aliquots in microcen marker gene a scorable marker gene Such as GUS may be trifuge tubes. Gold particles are stored at 4C, tungsten par used to identify transformed tissue. Soybean plant material ticles at -20 C. 3 mg of DNA are added to each aliquot of can be suitably transformed and fertile plants regenerated by (defrosted) particles and the tubes are vortexed at top speed. many methods which are well known to the skilled man. For Whilst maintaining near continuous vortexing, 50 ul of 2.5M example, fertile morphologically normal transgenic soybean 10 CaCl and 20 ul of 0.1M spermidine is added. After 10 min plants may be obtained by 1) production of Somatic embryo utes of further Vortexing, samples are centrifuged for 5 sec genic tissue from e.g. immature cotyledon, hypocotylor other onds in an eppendorf microcentrifuge, the Supernatant is suitable tissue 2) transformation by particle bombardment or drawn off and the particles washed in successive additions of infection with Agrobacterium and 3) regeneration of plants. HPLC-grade ethanol. The particles are thoroughly resus In one example, as described in U.S. Pat. No. 5,024,944, 15 pended in 60 ul of ethanol and then dispensed in 10ul aliquots cotyledon tissue is excised from immature embryos of Soy onto the surface of each macrocarrier to be used in the bean, preferably with the embryonic axis removed, and cul PDS1000 particle gun. Components of the PDS1000 particle tured on hormone-containing medium so as to form Somatic gun are surface sterilised by immersion in 70% ethanol and embryogenic plant material. This material is transformed air-drying. Target plates prepared, as described above, with using, for example, direct DNA methods, DNA coated micro tissue arranged into an ~2.5 cm disc are placed 6 cm from the projectile bombardment or infection with Agrobacterium, stopping screen. Suitably chosen rupture discs are then used cultured on a Suitable selection medium and regenerated, for bombardment. optionally also in the continued presence of selecting agent, One week after bombardment, all tissue clumps are trans into fertile transgenic soybean plants. Selection agents may ferred onto D20 medium, buffered to pH 5.7, containing a be antibiotics such as kanamycin, hygromycin or herbicides 25 Suitable selective concentration of selecting agent (for Such as phosphonothricin or glyphosate or, alternatively, example glyphosate between 0.05 and 10 mM in the case that selection may be based upon expression of a visualisable glyphosate be used for selection and that a resistant EPSPS or marker gene Such as GUS. Alternatively target tissues for GOX encoding gene is either present on the same transform transformation comprise meristematic rather than Soma ing DNA as the gene expressing Avena sativa HPPD or, clonal embryogenic tissue or, optionally, is flower or flower 30 otherwise, is present in co-bombarded DNA). After an addi forming tissue. tional 3-4 weeks all tissue is transferred to fresh D20 medium In one example, constructs are transformed into regener containing a suitable increased concentration of selecting able embryogenic soybean tissues using either biolistic type agent. After a further 3-4 weeks, living tissue is selected and approaches (e.g. Santarem E. R. Finer, J. J. (1999) 'Transfor subcultured on every 3-4 weeks in similar D20 medium con mation of soybean (Glycine max (L.) Merrill) using prolif 35 taining selection agent. In the case that some other selectable erative embryogenic tissue maintained on a semi-solid marker than glyphosate is present then selections may be medium. In vitro Cellular and Developmental Biology-Plant made as appropriate (e.g. using increasing concentrations of 35, 451-455; U.S. Pat. No. 5,503,998, U.S. Pat. No. 5,830, hygromycin). Alternatively, all selections are made using 728) or via infection with Agrobacterium (e.g. U.S. Pat. No. HPPD inhibitor herbicides. Growing sections are thus main 5,024,944, U.S. Pat. No. 5,959,179). 40 tained and, given enough tissue, may be analysed by PCR to Proliferative embryogenic tissue can, for example, be confirm that they are transgenic for the desired DNA. maintained on a semi-solid medium. Such tissue, is, for In order to develop and mature embryos, tissue clumps are example obtained in the following way. Immature Zygotic placed onto M6 medium which comprises MS salts at pH 5.7 embryos which are 3-4 mm long are isolated from pods of for containing B5 vitamins, 6% maltose and 0.2% gelrite. 6-9 example, Glycine max (L.) Merrill, 2-3 weeks after flower 45 clumps are placed in a tall dish at 23° C. After 3-4 weeks, formation. Pods can be checked for the presence of embryos embryos elongate and can be separated and transferred to of the correct length and maturity by backlighting. Pods are another round of incubation on M6 medium. After 4-6 weeks, then sterilized. Immature embryos are removed and the axis embryos are cream-coloured and ready for desiccation. 9 removed from each. Immature embryos are then plated on such cream-coloured embryos are placed in a dry Petri dish, D40-Lite semi-solid (0.2% gelrite) MS salts medium at pH 50 sealed with parafilm and placed onto a shelf for 2-3 days. 7.0 containing B5 vitamins, 3% sucrose and 40 mg/12,4-D for Embryos should be somewhat flaccid and not “crispy 3-4 weeks. For proliferation of embryos the material is then crunchy'. transferred to D20 MS salts medium at pH 5.7 containing B5 Desiccated embryos can be germinated by plating onto vitamins, 3% sucrose, 20 mg/12,4-D and 0.2% Gelrite. Mate OMS (growth regulator-free MS medium). Following germi rial with bright green globular proliferative embryos is 55 nation which normally occurs within a week plants are trans selected and subcultured every 2-3 weeks. ferred to larger boxes and, once there is sufficient root and For bombardment, 20-25 clumps/plate of tissue are shoot formation, thence to soil. To prevent fungal contamina selected (subcultured 4-5 days prior to bombardment) and tion it is advisable to wash OMS from the roots with distilled arranged in the centre of the dish containing D20 medium. water. Plants may be kept and grown under high humidity The tissue is dried for 15 min by uncovering for 15 minutes 60 and, initially, under 24 hour lighting. Plants may be grown under a sterile hood. Gold particles coated in DNA construct until about 2 feet tall under 24 hour lighting and then encour (coated, for example, using methods described in the refer aged to flower and form pods through a shift to a 16 hour ences above) are twice bombarded into the tissue on D20 lighting regime. Seeds are collected and progeny grown on, medium using any one of a large number of commercially crossed and backcrossed into order to move the transgenes available guns. By way of further example a PDS1000 par 65 into the desired plant background using the normal methods ticle gun is used. Particles may be prepared and coated with of plant breeding. Plants are routinely analysed for the pres DNA in a similar manner to that described by Klein etal 1987, ence and expression of transgenes using the normal methods US 8,097,774 B2 37 38 of molecular biology including analysis by PCR, Southern, Regeneration and Selection of Transgenic Plants: After Western, ELISA and enzyme assay techniques. co-cultivation, elongated hypocotyls of the explants are An Agrobacterium-based method for Soybean transforma trimmed back just below the cotyledon nodes. The explants tion is as follows: are transferred on to recovery medium with antibiotics to kill Isolation of seeds from seed pods of different developing 5 Agrobacterium or inhibit Agrobacterium growth and with stages: Soybean (Glycine max cultivar Jack, Williams 82 or low or no selection agent, such as SoyR1. The plates with the S42H1) stock plants are grown in greenhouse under 16 hours explants are incubated, preferably for one week at 24° C. of day light at 24°C. Pods at developing stages R5-R7 (with under 16 hours light/8 hours dark regimen, and >80 uE/m/s. green to yellow pod color) are collected and sterilized by After the recovery period, elongated epicotyls or developing immersing in 70% ethyl alcohol for 30 second or in 20% 10 shoots are excised and transferred to regeneration media with Clorox bleach for 20 minutes. Sterilized pods are rinsed with higher concentration of selection agent, such as SoyR2, along sterile water for 4 times. Seeds are then isolated from steril with the cotyledon for two weeks. SoyR2 medium contained ized pods by hands in gloves sprayed with 70% ethyl alcohol. 6-8 mg/L glufosinate for selection. After 2 weeks in regen The isolated seeds are rinsed 3-5 times with sterile water or eration/selection media such as SoyR2, developed or devel further sterilized with 10% Clorox for ten minutes followed 15 oping multiple shoots clusters are excised and transferred to by rinsing with sterile water three times. Sterilized seeds are elongation medium, Such as SoyE1 for shoot elongation. then used directly for preparing explants for Agrobacterium SoyE1 contained 4-8 mg/L glufosinate. Subcultures to fresh mediated transformation. elongation media are performed every two weeks. Elongated Transformation vector and Agrobacterium strains: A shoots (>2 cm) are transferred to elongation media SoyE2 binary vector containing the modified HPPD gene, or a P450 without selection for two weeks. After two weeks in SoyE2 gene, with the prerequisite genetic elements needed for medium, shoots are transferred to a rooting medium, for expression, is used for transformation. These vectors are example SoyRoot. Leaves were sampled for Taqman Ranaly introduced separately into Agrobacterium tumefaciens strain sis to identify transformants that contain bar or pat gene. LB4404 or EHA101 using electroporation. Single bacterial Taqman(R) positive and rooted plantlets are rinsed with water colony containing each of these vectors is selected to confirm 25 to wash off the agar medium and transplanted to Soil and the presence of intact vector and used for further experiments. grown in green house for seeds. Agrobacterium culture is cultured on YP solid medium con The following tables provide transformation media recipes taining appropriate antibiotics and grown at 28°C. incubator. used within the methods described herein: Agrobacterium is streaked onto fresh YP medium the day before the inoculation and grown in the 28°C. incubator. For 30 plant transformation use Agrobacterium is collected from the TABLE 2 plate using a disposable plastic inoculation loop and sus SoYInf medium. pended in liquid infection medium such as SoyInfin a sterile 50 ml disposable polypropylene centrifugation tube. Shake Recipe Name SoyInf Final pH 5.4 the tube gently until Agrobacterium cells are uniformly dis 35 persed in the Suspension. The bacterial cell Suspension is then Recipe for 1 L Name of Chemical Amount Units MS Basal Salt Mixture 2.15 9. diluted to Aeo of 0.5 to 0.8 and acetosyringone is added to a B5Vitamins 200X 5 ml final concentration of 40-80 mg/L (200-400 uM) to induce Sucrose 2O 9. virulence gene expression. Glucose 10 9. Preparation of Transformation Targets: Explants are Pre 40 MES 4 9. pared from Sterilized soybean seeds isolated directly from Zeatin Riboside, Trans Isomers 1 mg/ml 2 ml pods as described in Example 1 without further germination or culture in one of the following ways: a) The hypocotyl is trimmed off just below the cotyledon TABLE 3 nodes and the seed coat is removed. One cotyledon and 45 two primary leaves are removed by breaking the adja SoYCCM medium. cent tissue with the blunt end of the scalpel. Recipe Name SoyCCM b) The hypocotyl is trimmed off just below the cotyledon Final pH 5.4 nodes and the seed coat is removed. One cotyledon and Recipe for 1 L Name of Chemical Amount Units two primary leaves are removed by breaking the adja 50 Sucrose 2O 9. cent tissue with the bluntend of the scalpel, and then the MES 4 9. plumule including the apical meristem is wounded with Purified Agar 6 9. the sharp end of blade. Evian Water 990 ml Acetosyringone 40 mg/ml 1 ml c) The seed is cut longitudinally into two halves in the BAP 1 mg/ml O.S ml middle of the embryo axis. Both primary leaves are 55 removed and then the apical region of the explant is further wounded with the sharp end of the scalpel. Both halves of the resulting explants include one cotyledon TABLE 4 and part of the immature embryo axis. Infection and Co-cultivation of soybean Seed Explants: 60 SoyCoC medium. The explants prepared as above Example 1.3 are infected with Recipe Name SoyCoC Agrobacterium by mixing the explants with bacterial Suspen Final pH 5.4 sion as prepared in Example 1.2. The mixture is incubated for Recipe for 1 L Name of Chemical Amount Units 30 minutes to 4 hours at room temperature. Following infec MS Basal Salt Mixture 2.15 9. tion, the explants are removed from the Agrobacterium Sus 65 B5Vitamins 200X 5 ml pension and placed on a co-cultivation medium such as Soy Sucrose 2O 9. CoC. The co-cultivation plates were incubated for 3-5 days. US 8,097,774 B2 40 TABLE 4-continued TABLE 7-continued

SoyCoC medium. SOVEl medium. Recipe Name SoyE1 Recipe Name SoyCoC Final pH S.6 Final pH 5.4 Recipe for 1 L Name of Chemical Amount Units Recipe for 1 L Name of Chemical Amount Units GA35 mg/ml O.1 ml Glutamine 50 mg/ml 2 ml Glucose 10 9. Asparagine 25 mg/ml 2 ml MES 4 9. 10 Zeatin Riboside, Trans Isomers 1 mg/ml 1 ml Zeatin Riboside, Trans Isomers 1 mg/ml 2 ml Purified Agar 6 9. TABLE 8

TABLE 5 15 SoyE2 medium. SoyR1 medium. Recipe Name SoyE2 Final pH 5.4 Recipe Name SoyR1 Recipe for 1 L Name of Chemical Amount Units Final pH S.6 MS Basal Salt Mixture 4.3 9. Recipe for 1 L Name of Chemical Amount Units B5Vitamins 200X 5 ml B5 Basal Salt, Gamborg's 3.1 9. MS Iron 20OX 3 ml B5Vitamins 200X 5 ml Sucrose 30 9. MS Iron 20OX 4 ml MES 590 ng Asparagine 100 ng Gelrite 3 9. Ticarcillin:Potassium Clavulanate 3 ml MES 100 mg/ml 10 ml 25 Zeatin Riboside, Trans Isomers 1 mg/ml 2 ml 15:1 100 mg/ml Sucrose 30 9. Cefotaxime 250 mg/ml 0.4 ml Purified Agar 7 9. Glufosinate ammonium ng Glutamine 50 mg/ml 2 ml Glutamine 50 mg/ml 2 ml Ticarcillin:Potassium Clavulanate 3 ml Asparagine 25 mg/ml 2 ml 15:1 100 mg/ml 30

TABLE 9 TABLE 6 Soy Root medium. SoyR2 medium. 35 Recipe Name SoyRoot-H Recipe Name SoyR2 Final pH 5.4 Final pH S.6 Recipe for 1 L Name of Chemical Amount Units Recipe for 1 L Name of Chemical Amount Units MS Basal Salt Mixture 2.2 9. B5 Basal Salt, Gamborg's 3.1 9. B5Vitamins 200X 5 ml B5Vitamins 200X 5 ml 40 MS Iron 200X 3 ml MS Iron 200X 4 ml Sucrose 2O 9. Asparagine 100 ng MES 590 ng MES 100 mg/ml 10 ml Gelrite 3 9. BAP 1 mg/ml 1 ml Glutamine 50 mg/ml 2 ml Sucrose 30 9. Asparagine 25 mg/ml 2 ml Purified Agar 7 9. 45 IBA 1 mg/ml O6 ml Glutamine 50 mg/ml 2 ml Glufosinate ammonium Ticarcillin:Potassium Clavulanate 3 ml 15:1 100 mg/ml Example 4

50 Construction of a Soybean Transformation Vector TABLE 7 Abinary vector (17107) for dicot (soybean) transformation SoyEl medium. was constructed, with the Arabidopsis UBQ3 promoter driv ing the corn CYP72A1 cytochrome P450 gene, followed by Recipe Name Soy E1 55 NOS terminator (FIG.3). The gene was codon optimized for Final pH S.6 Soybean expression based upon the predicted amino acid Recipe for 1 L Name of Chemical Amount Units sequence of the maize gene coding region. The amino acid MS Basal Salt Mixture 4.3 9. sequence of the protein encoded by the CYP72A1 gene is B5Vitamins 200X 5 ml provided in SEQID NO:3, and the nucleotide sequence of the MS Iron 20OX 3 ml 60 Sucrose 30 9. optimized CYP72A1 gene in this binary transformation vec MES 590 ng tor is provided in SEQID NO:4. The transformation vector Purified Agar 7 9. also contains two PAT gene cassettes (one with the 35S pro Ticarcillin:Potassium Clavulanate 3 ml 15:1 100 mg/ml moter and one with the CMP promoter, and both PAT genes Cefotaxime 250 mg/ml 0.4 ml are followed by the nos terminator) for glufosinate based Glufosinate ammonium ng 65 selection during the transformation process. IAA 1 mg/ml O.1 ml A similar binary vector (17108) was constructed with the corn cytochrome P450 gene “nsf instead of the CYP72A1 US 8,097,774 B2 41 42 gene (FIG. 4). The nSf gene in this vector was codon opti herbicide application. New growth that emerged from the mized for soybean expression (SEQID NO:2) based upon the plants was completely bleached on all plants. However, over predicted amino acid sequence of the maize gene coding time, leaves greened up on all plants. New growth developed region (SEQID NO:1). as partially bleached, or chlorotic, and the leaves that initially were bleached also greened up. At 1 week after spray, it was Example 5 clear that the leaves of event 17108-A turned green faster than the leaves on events with vector 17107. At 22 days after the Soybean Transformation spray, the plants were rated for symptoms on the leaves that were initially bleached. Visible chlorosis was only seen on the Transformed Soybean plants were generated with each of 10 leaves immediately above the leaves that were exposed dur the CYP vectors described in Example 4. These events were ing the spray (i.e., the leaves that had necrotic spots), and in created using the Agrobacterium method described in Some events, on leaves in sideshoots. The partial chlorosis Example 3 (see also U.S. patent application Ser. No. 1 1/716, was evident as light green patches, and often as light green 975). 15 (almost yellow) edges on the leaves. This data is shown in Example 6 Table 10 below.

TO Plant Establishment and Selection TABLE 10 Evaluation of plants following mesotrione treatment. TO plants were taken from tissue culture to the greenhouse where they are transplanted into saturated soil (Redi-EarthR) Genotype, as Number of leaves Number of leaves Plug and Seedling Mix, Sun Gro Horticulture, Bellevue, determined by with 25.0% with <50% Wash.) mixed with 1% granular Marathon(R) (Olympic Hor Event number Taqman (R) chlorosis chlorisis ticultural Products, Co., Mainland, Pa.) at 5-10 g/gal Redi 6DO32-AiiA Negative 1 1 25 6DO32-Ai14 Negative 2 O EarthR) Mix in 2" square pots. The plants were covered with 17107-A 17107 positive 2 O humidity domes and placed in a Conviron chamber (Pembina, 17107-B 17107 positive 2 2 N. Dak.) with the following environmental conditions: 24°C. 17107-C 17107 positive 1 1 day: 18°C. night; 23 hr photoperiod; 80% relative humidity. 17107-D 17107 positive 3 2 17107-E 17107 positive 2 1 After plants became established in the soil and new growth 17107-F 17107 positive 1 2 appeared (~1-2 weeks), plants were sampled and tested for 30 17107-G 17107 positive 1 2 the presence of desired transgene by Taqman Ranalysis using 17107-H 17107 positive 2 4 probes for P450 (CYP72A1), or promoters (prCMP and 17107-I 17107 positive 1 1 prUBq3). All positive plants and several negative plants were 17108-A 17108 positive O O transplanted into 4" square pots containing MetroMix R 380 soil (Sun Gro Horticulture, Bellevue, Wash.). Sierra 17-6-12 35 As shown above, only event 17108-A containing the nsf slow release fertilizer was incorporated into the soil at the P450 gene was able to fully recover from the mesotrione recommended rate. The negative plants serve as controls for injury. the spray experiment. The plants were then relocated into a standard greenhouse to acclimatize (~1 week). The environ Example 8 mental conditions were: 27° C. day: 21° C. night: 12 hr 40 photoperiod (with ambient light); ambient humidity. After Analysis of T1 Seed from 17108 Events for HPPD acclimatizing (~1 week), the plants were ready to be sprayed Herbicide Tolerance with the desired herbicides. TO transformants containing the nSf transgene (vector Example 7 45 17108) are allowed to set T1 seed, and the seed is harvested. This seed is planted, and the progeny analyzed to identify Plant Treatment and Evaluation seedlots that are derived from germline transformed plants. Transgenic progeny plants (T1 or later generations), are ana The plants from Example 6 were sprayed with mesotrione lyzed to confirm mesotrione tolerance. Plants grown from the to determine if they were tolerant to this herbicide. Callisto(R) 50 progeny seeds also display tolerance when sprayed with other (active ingredient mesotrione, Syngenta Crop Protection, HPPD herbicides (including but not limited to Tembotrione, Inc., Greensboro, N.C.) was mixed in water and X-77 surfac Isoxaflutole, ToprameZone, Pyrasulfatole, Sulcotrione, Pyra tant (0.25% V/v final concentration). Plants were placed in a Zolynate, Pyrazoxyfen, Isoxachlortole, Benzofenap, and DeVries spray chamber (DeVries Manufacturing, Holland Benzobicyclon). The plants are sprayed at 40-80 g/ha rates ale, Minn.), and the distance from the nozzle to the top of the 55 with these herbicides, or rates higher than this, as described in plants was adjusted to approximately 12 inches. The system Example 7. Plants are evaluated for bleaching, and chlorosis, was set up so that the boom moved at 2 mph, and delivered 25 as described in Example 7. gallon fluid per acre. The spray rate was calibrated to spray 52.5 g/ha mesotrione over the top of the plants. Negative T0 Example 9 transformants (i.e., negative by Taqman R for presence of 60 transgenes), TO transformants with vector 17107, and one Ability of P450 Expressing Plants to Degrade Other transformant with vector 17108 (event 17108-A), were Herbicides sprayed in this manner. After spraying, plants were placed in the greenhouse, under the same conditions as described in A number of plants have the ability to degrade herbicides Example 3 above, and evaluated frequently for symptoms. All 65 via a P450-based degradation mechanism, and it is generally plants that were sprayed developed necrotic symptoms on assumed that this degradation is partly or primarily respon small portions of the leaves that were exposed at the time of sible for the herbicide tolerance of these plants. One example US 8,097,774 B2 43 44 is the ability of the corn CYP72A1 enzyme to degrade the In the Triazolinones herbicide class, P450 enzymes may herbicides bentazon, chlortoluron, and chlorimuron. In addi degrade herbicides that include, but are not limited to, Flu tion, these plants are also able to degrade the insecticide carbazone. Thiencarbazone-methyl, and Propoxycarbazone. malathion (see, e.g., U.S. Pat. No. 6,380,465). Likewise, the In the herbicide class, P450 enzymes may degrade nSf enzyme has been shown to confer tolerance to a range of 5 herbicides that include, but are not limited to, Dicamba, Ami herbicides including HPPD herbicides, certain sulfonylureas, nopyralid, 2,4-D, Mecoprop, , Quin certain PPO herbicides, and other classes of herbicides (see, clorac, Dichlorprop, MCPA, MCPB,2,4-DB, Clopyralid, and e.g., U.S. Patent Application No. 2007/0214515A1). Another Picloram. example is the rice CYP81.A6 enzyme, that is able to degrade In the ACCase inhibitor herbicide class, P450 enzymes bentazon and certain sulfonyl herbicides (Pan et al. 10 may degrade herbicides that include, but are not limited to, (2007) Plant Molecular Biology, 61:933-943). Consequently, Fluazifop-P-butyl, Pinoxaden, Clodinafop-propargyl, when the P450s described herein are expressed at appropriate Fenoxaprop-P-ethyl, Tralkoxydim, Diclofop-methyl, Cyha levels in plants, they may provide tolerance to a variety of lofop-butyl, Haloxyfop-P-methyl, Quizalofop-P-ethyl, herbicides, including BenzothiadiaZinones, Sulfonylureas, Alloxydim, Butroxydim, Clethodim, and Cycloxydim. Imidazolinones, Triazolopyrimidines, Pyrimidinylthioben 15 In the PSII inhibitor herbicide class, P450 enzymes may Zoates, Triazolinones, Auxins, Acetyl-coenzyme A Carboxy degrade herbicides that include, but are not limited to, Ben lase (ACCase) inhibitors, Photosystem II (PSII) inhibitors, tazon, Linuron, Hexazinone, Metribuzin, Atrazine, Diuron, Protoporphyrinogen Oxidase (PPO) inhibitors, Phytoene Isoproturon, Monolinuron, Desmedipham, Metamitron, Pro Desaturase (PDS) inhibitors, Dinitroanalines, and Aceta panil, Amicarbzone, Fluometuron, Phenmedipham, Pyridate, mides, as well as herbicides with unknown modes of action Ametryn, Cynazine, Dimefuron, Fluometuron, Methibenzu Such as DifenZoquat and ClomaZone. ron, Metoxuron, Prometryn, Simazine, Simetryn, Terbacil, In the Benzothiadiazinone herbicide class, P450 enzymes Terbuthylazine, Chlorotoluron, and Trietazine. may degrade herbicides that include, but are not limited to, In the PPO inhibitor herbicide class P450 enzymes may Bentazon. degrade herbicides that include, but are not limited to, In the Sulfonylurea herbicide class, P450 enzymes may 25 Butafenacil, Fomesafen, CarfentraZone, Saflufenacil, Oxy degrade herbicides that include, but are not limited to, Nico fluorfen, Flumioxazin, SulfentraZone, Lactofen, Oxadiazon, sulfuron, Trifloxysulfuron and Rimsulfuron, and may also Acifluorfen, Flufenpyr-ethyl, Flumiclorac, and Oxadiargyl. degrade Primisulfuron, Chlorimuron-ethyl, Amidosulfuron, In the PDS inhibitor herbicide class, P450 enzymes may AZimsulfuron, Bensulfuron-methyl, Chlorsulfuron, Cyclo degrade herbicides that include, but are not limited to, Nor sulfamuron, Ethametsulfuron-methyl, Ethoxysulfuron, Fla 30 flurazon, Diflufenican, Fluorochloridone, Flurtamone, Zasulfuron, Flupyrsulfuron-methyl, Halosulfuron-methyl, Picolinafen, and Fluridone Imazosulfuron, Iodosulfuron, Metsulfuron-methyl, Foram In the Dinitroanalines herbicide class, P450 enzymes may sulfuron, Oxasulfuron, Prosulfuron, Pyrazosulfuron-ethyl, degrade herbicides that include, but are not limited to, Pen Sulfometuron-methyl, Sulfosulfuron, Tritosulfuron, Thifen dimethalin, Trifluralin, Orazalin, Butralin, Dinitroamine, and sulfuron-methyl, Triasulfuron, Tribenuron-methyl, and Tri 35 Ethalfluralin. flusulfuron-methyl. In the Acetamides herbicide class, P450 enzymes may In the Imidazolinone herbicide class, P450 enzymes may degrade herbicides that include, but are not limited to, degrade herbicides that include, but are not limited to, Acetochlor, S-metolachlor, metolachlor, Dimethenamid, ImaZamox, Imazethapyr, Imazapic, ImazamethabenZ-me P-dimethenamid, Flufenacet, Alachlor, Butachlor, Mefen thyl, and Imazaquin. 40 acet, Pretilachlor, Propachlor, and Thenylchlor. In the Triazolopyrimidines herbicide class, P450 enzymes In addition, P450 enzymes may degrade herbicides that may degrade herbicides that include, but are not limited to, include, but are not limited to, DilfenZoquat and ClomaZone, FlumetSulam, DicloSulam, Florasulam, ChloranSulam-me herbicides with unknown mode of action. thyl, and Metosulam. All publications, patents and patent applications are herein In the Pyrimidinylthiobenzoates herbicide class, P450 45 incorporated by reference to the same extent as if each indi enzymes may degrade herbicides that include, but are not vidual publication, patent or patent application was specifi limited to, Bispyribac, Pyrithiobac, Pyriminobac-methyl, cally and individually indicated to be incorporated by refer Pyriftalid, and Pyribenzoxim. CCC.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS : 15

SEO ID NO 1 LENGTH: 521 TYPE PRT ORGANISM: Zea mays <4 OOs SEQUENCE: 1 Met Asp Lys Ala Tyr Ile Ala Ala Lieu. Ser Ala Ala Ala Lieu. Phe Lieu. 1. 5 1O 15 Lieu. His Tyr Lieu. Lieu. Gly Arg Arg Ala Gly Gly Glu Gly Lys Ala Lys 2O 25 3 O

Ala Lys Gly Ser Arg Arg Arg Lieu Pro Pro Ser Pro Pro Ala Ile Pro US 8,097,774 B2 45 46 - Continued

35 4 O 45

Phe Luell Gly His Lell His Lell Wall Ala Pro Phe His Gly Ala Luell SO 55 6 O

Ala Arg Luell Ala Ala Arg His Gly Pro Wall Phe Ser Met Arg Luell Gly 65 70

Thir Arg Arg Ala Wall Wall Wall Ser Ser Pro Asp Ala Arg Glu Cys 85 90 95

Phe Thir Glu His Asp Wall Asn Phe Ala Asn Arg Pro Lell Phe Pro Ser 105 11 O

Met Arg Luell Ala Ser Phe Asp Gly Ala Met Luell Ser Wall Ser Ser Tyr 115 12 O 125

Gly Pro Trp Arg Asn Lell Arg Arg Wall Ala Ala Wall Glin Luell Luell 13 O 135 14 O

Ser Ala His Arg Wall Gly Met Ala Pro Ala Ile Glu Ala Glin Wall 145 150 155 160

Arg Ala Met Wall Arg Arg Met Asp Arg Ala Ala Ala Ala Gly Gly Gly 1.65 17O 17s

Gly Wall Ala Arg Wall Glin Lell Arg Arg Luell Phe Glu Luell Ser Luell 18O 185 19 O

Ser Wall Luell Met Glu Thir Ile Ala His Thir Thir Ser Arg Ala Glu 195 2OO

Ala Asp Ala Asp Ser Asp Met Ser Thir Glu Ala His Glu Phe Glin 21 O 215 22O

Ile Wall Asp Glu Lell Wall Pro Ile Gly Thir Ala Asn Arg Trp Asp 225 23 O 235 24 O

Luell Pro Wall Lell Arg Trp Phe Asp Wall Phe Gly Wall Arg Asn Lys 245 250 255

Ile Luell Asp Ala Wall Gly Arg Arg Asp Ala Phe Lell Gly Arg Luell Ile 26 O 265 27 O

Asp Gly Glu Arg Arg Arg Lell Asp Ala Gly Asp Glu Ser Glu Ser Lys 285

Ser Met Ile Ala Wall Lell Lell Thir Luell Glin Ser Glu Pro Glu Wall 29 O 295 3 OO

Tyr Thir Asp Thir Wall Ile Thir Ala Luell Cys Ala Asn Lell Phe Gly Ala 3. OS 310 315

Gly Thir Glu Thir Thir Ser Thir Thir Thir Glu Trp Ala Met Ser Luell Luell 3.25 330 335

Lell Asn His Arg Glu Ala Lell Lys Ala Glin Ala Glu Ile Asp Ala 34 O 345 35. O

Ala Wall Gly Thir Ser Arg Lell Wall Thir Ala Asp Asp Wall Pro His Luell 355 360 365

Thir Tyr Luell Glin Cys Ile Wall Asp Glu Thir Luell Arg Lell His Pro Ala 37 O 375

Ala Pro Luell Luell Lell Pro His Glu Ser Ala Ala Asp Thir Wall Gly 385 390 395 4 OO

Gly Asp Wall Pro Arg Gly Thir Met Luell Luell Wall Asn Wall His Ala 4 OS 415

Wall His Arg Asp Pro Ala Wall Trp Glu Asp Pro Asp Arg Phe Wall Pro 425 43 O

Glu Arg Phe Glu Gly Ala Gly Gly Ala Glu Gly Arg Luell Luell Met 435 44 O 445

Pro Phe Gly Met Gly Arg Arg Pro Gly Glu Thir Luell Ala Luell 450 45.5 460

US 8,097,774 B2 49 50 - Continued

<4 OOs, SEQUENCE: 3

Met Ala Thr Cys Asp Lell Lell Met Luell Arg Glu Ala Ser Pro Trp Ala 1. 15

Lell Ala Ala Wall Ala Ser Wall Ser Luell Luell Trp Lell Wall Ala Trp 2O 25 3O

Thir Luell Trp Ala Trp Trp Thir Pro Trp Arg Lell Asp Arg Ala Luell 4 O 45

Arg Ala Gly Lell Asn Gly Thir Arg Tyr Arg Lell Phe Thir Gly Asp SO 55 6 O

Lell Arg Thir Ala Arg Wall Asn Arg Glu Ala Arg Pro Luell 65 70

Pro Luell His Asp Ile Thir Pro Arg Wall Glin Pro Met His His 85 90 95

Ser Thir Lys Glu Tyr Gly Luell Ser Phe Thir Trp Phe Gly Pro 105 11 O

Thir Pro Wall Met Ile Pro Asp Pro Glu Luell Wall Lys Glu Wall Luell 12 O 125

Ser Asn Phe Gly His Phe Gly Pro Arg Ser Ser Arg Ile Gly 13 O 135 14 O

Arg Luell Luell Ala Asn Gly Lell Wall Asn His Asp Gly Glu Trp Ala 145 150 155 160

His Arg Arg Ile Lell Asn Pro Ala Phe His His Glu Ile 1.65 17O 17s

Gly Met Met Pro Wall Phe Ser Thir Cys Cys Ile Glu Met Ile Thir Arg 18O 185 19 O

Trp Asp Asn Ser Met Ser Ser Glu Gly Ser Ser Glu Ile Asp Wall Trp 195

Pro Glu Phe Glin Asn Lell Thir Gly Asp Wall Ile Ser Arg Thir Ala Phe 21 O 215 22O

Gly Ser Asn Tyr Glin Glu Gly Arg Arg Ile Phe Glu Lell Glin Gly Glu 225 23 O 235 24 O

Lell Ala Glu Arg Lell Ile Glin Ser Wall Glin Thir Ile Phe Ile Pro Gly 245 250 255

Trp Phe Luell Pro Thir Asn Asn Arg Arg Met Arg Ala Ile Asp 26 O 265 27 O

Wall Glu Ile Arg Ile Lell Arg Glu Ile Ile Gly Lys Arg Glu 285

Asp Thir Asn Arg Glu Thir Asn Asp Asp Lell Lell Gly Luell Luell 29 O 295 3 OO

Lell Glu Ser Asn Thir Arg Glin Ser Asn Gly ASn Ala Ser Luell Gly Luell 3. OS 310 315

Thir Thir Glu Asp Wall Ile Glu Glu Lys Luell Phe Phe Ala Gly 3.25 330 335

Met Glu Thir Thir Ser Wall Lell Luell Thir Trp Thir Lell Ile Wall Luell Ser 34 O 345 35. O

Met His Pro Glu Trp Glin Glu Arg Ala Arg Glu Glu Wall Luell Ser His 355 360 365

Phe Gly Arg Thir Thir Pro Asp Asp Ser Luell Ser Arg Luell Thir 37 O 375

Wall Thir Met Ile Lell His Glu Wall Luell Arg Luell Pro Pro Ala Thir 385 390 395 4 OO

Phe Luell Thir Arg Arg Thir Glu Met Glu Lell Gly Gly Ile 4 OS 41O 415 US 8,097,774 B2 51 52 - Continued Pro Ala Gly Val Glu Lieu. Lieu. Lieu Pro Wall Ile Phe Ile His His 425 43 O

Asp Pro Asp Ile Trp Gly Lys Asp Ala Ser Glu Phe Asn Pro Glu Arg 435 44 O 445

Phe Ala Asn Gly Ile Ser Ser Ala Thr Arg His Glin Ala Ala Phe Phe 450 45.5 460

Pro Phe Gly Gly Gly Pro Arg Ile Cys Ile Gly Glin Ser Phe Ala Lieu 465 470

Lell Glu Ala Lys Met Thr Lieu. Cys Thir Ile Lieu. Glin Arg Phe Ser Phe 485 490 495

Glu Luell Ser Pro Ser Tyr Thr His Ala Pro Tyr Thir Wall Ile Thir Lieu. SOO 505

His Pro Glin His Gly Ala Glin Ile Arg Lieu Lys Lell Ser Pro 515 525

SEQ ID NO 4 LENGTH: 1584 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Chemically Synthesized

SEQUENCE: 4 atggctacct gcgatc.ttct tatgcttaga gaggcttctic Cttgggctict tgctggtgct 6 O gttgcttctg tgtctottct ttggcttgtt gcttggactic ttgaatgggc ttggtggact 12 O

CCttggagac ttgatagggc tct tagggct Caaggactta acggalaccag gtacaggctt 18O ttcactggtg atc.ttagaga gaccgctagg gttaa.ca.gag aggctaggaa gaa.gc.cactt 24 O c cacttggat gcc acgatat tactic caagg gttcago caa tgcaccactic tac Catcaaa. 3OO gagtacggca agttgttctitt cacttggttt ggaccalactic Caagggtgat gattic cagat 360 ccagagcttg tgaaagaggit gct ct coaac aagttcggac attitcggaaa gccaaggtoc tctaggattg gaaggcttct tgctaacgga cittgttgaacc atgatggcga galagtgggct aaacacagaa ggattct caa cc.cagctitt c Caccacgaga agatcaaggg aatgatgc.ca 54 O gttittct coa cittgctgcat cgagatgatc accagatggg atalactictat gtc.ct Ctgag ggat Cttctg agattgatgt gtggc.ca.gag titcCaaalacc ttaccggcga tgttgatttct 660 agaaccocitt tcqgttccaa Ctaticaagag ggCagaagga ttitt.cgagct t calaggtgaa 72 O

Cttgctgaga ggct tatt ca gtc.cgttcag accatttitca tcc caggata ctggttccitt cctaccaaga acaacagaag gatgagggct attgatgtgg agat caggaa gatcct caga 84 O gagattatcg gcaa.gagaga gaaggatacc aagaacagag agaccaacaa ggacgat Ctt 9 OO cittggactitc ttct cq agtic talacaccagg caatctaacg gaaacgcttic t cittggacitt 96.O act accgagg atgtgatcga ggaatgcaag citct to tact tcgctggaat ggaalaccact tctgtgctitt tgacctggac cct tattgttg Ctttctatogc acccagagtg gcaagaaaga gctagggaag aggttttgtc. t catttcqga aggact accc Cagattacga ttctott to c 14 O aggcttalaga cc.gtgaccat gattctt cat gaggtgctica gactittatcc acctgct acc 2OO tt cott act a gaaggaccta caaagagatg gaact cqgag gtattaagta CCC agctggc 26 O gttgaactitc ttct cocagt gat citt catt caccacgatc cagatatttg gggaaaggac 32O gcttctgagt t caacccaga gagatticgct aacggaattit cittctgctac taggcatcag gctgctitt ct tcc catttgg aggtggacca aggatctgca ttggacagtic titt cqct citt 44 O

Ctcgaggcta agatgaccitt gtgcaccatt Ctt cagaggit t cagott cqa gctitt ct coa SOO US 8,097,774 B2 53 - Continued t cittacaccc atgctic cata caccgittatt accct tcatc. cacagcatgg togct cagatt 1560 aggcttaaga agct ct cocc ttga 1584

<210s, SEQ ID NO 5 &211s LENGTH: 513 212. TYPE: PRT <213> ORGANISM: Oryza sativa <4 OOs, SEQUENCE: 5 Met Asp Asn Ala Tyr Ile Ile Ala Ile Lieu. Ser Val Ala Ile Lieu. Phe 1. 5 1O 15 Lieu. Lieu. His Tyr Tyr Lieu. Lieu. Gly Arg Gly Asn Gly Gly Ala Ala Arg 2O 25 3O Lieu Pro Pro Gly Pro Pro Ala Val Pro Ile Leu Gly His Lieu. His Leu 35 4 O 45 Val Llys Llys Pro Met His Ala Thr Met Ser Arg Lieu Ala Glu Arg Tyr SO 55 6 O Gly Pro Val Phe Ser Lieu. Arg Lieu. Gly Ser Arg Arg Ala Val Val Val 65 70 7s 8O Ser Ser Pro Gly Cys Ala Arg Glu. Cys Phe Thr Glu. His Asp Val Thr 85 90 95 Phe Ala Asn Arg Pro Arg Phe Glu Ser Glin Lieu. Lieu Val Ser Phe Asn 1OO 105 11 O Gly Ala Ala Lieu Ala Thr Ala Ser Tyr Gly Ala His Trp Arg Asn Lieu. 115 12 O 125 Arg Arg Ile Val Ala Val Glin Lieu. Lieu. Ser Ala His Arg Val Gly Lieu 13 O 135 14 O Met Ser Gly Lieu. Ile Ala Gly Glu Val Arg Ala Met Val Arg Arg Met 145 150 155 160 Tyr Arg Ala Ala Ala Ala Ser Pro Ala Gly Ala Ala Arg Ile Glin Lieu. 1.65 17O 17s Lys Arg Arg Lieu. Phe Glu Val Ser Lieu. Ser Val Lieu Met Glu Thir Ile 18O 185 19 O Ala His Thr Lys Ala Thr Arg Pro Glu Thir Asp Pro Asp Thr Asp Met 195 2OO 2O5 Ser Val Glu Ala Glin Glu Phe Lys Glin Val Val Asp Glu Ile Ile Pro 21 O 215 22O His Ile Gly Ala Ala Asn Lieu. Trp Asp Tyr Lieu Pro Ala Lieu. Arg Trp 225 23 O 235 24 O Phe Asp Val Phe Gly Val Arg Arg Lys Ile Lieu Ala Ala Val Ser Arg 245 250 255 Arg Asp Ala Phe Lieu. Arg Arg Lieu. Ile Asp Ala Glu Arg Arg Arg Lieu 26 O 265 27 O Asp Asp Gly Asp Glu Gly Glu Lys Llys Ser Met Ile Ala Val Lieu. Lieu 27s 28O 285 Thr Lieu Gln Lys Thr Glu Pro Glu Val Tyr Thr Asp Asn Met Ile Thr 29 O 295 3 OO Ala Lieu. Thir Ala Asn Lieu. Phe Gly Ala Gly Thr Glu Thir Thr Ser Thr 3. OS 310 315 32O Thir Ser Glu Trp Ala Met Ser Lieu. Lieu. Lieu. Asn His Pro Asp Thir Lieu. 3.25 330 335 Llys Lys Ala Glin Ala Glu Ile Asp Ala Ser Val Gly Asn. Ser Arg Lieu. 34 O 345 35. O

Ile Thr Ala Asp Asp Val Thr Arg Lieu. Gly Tyr Lieu. Glin Cys Ile Val US 8,097,774 B2 55 56 - Continued

355 360 365

Arg Glu Thir Lieu. Arg Lieu. Tyr Pro Ala Ala Pro Met Lieu. Luell Pro His 37 O 375 38O

Glu Ser Ser Ala Asp Cys Llys Val Gly Gly Tyr Asn. Ile Pro Arg Gly 385 390 395 4 OO

Ser Met Luell Lieu. Ile Asn Ala Tyr Ala Ile His Arg Asp Pro Ala Wall 4 OS 41O 415

Trp Glu Glu Pro Glu Lys Phe Met Pro Glu Arg Phe Glu Asp Gly Gly 425 43 O

Asp Gly Asn Lieu Lleu Met Pro Phe Gly Met Gly Arg Arg Arg Cys 435 44 O 445

Pro Gly Glu Thir Lieu Ala Lieu. Arg Thr Val Gly Lieu Wall Lieu. Gly Thr 450 45.5 460

Lell Ile Glin Cys Phe Asp Trp Glu Arg Val Asp Gly Val Glu Val Asp 465 470

Met Thir Glu Gly Gly Gly Lieu. Thr Ile Pro Llys Wall Wall Pro Lieu. Glu 485 490 495

Ala Met Arg Pro Arg Asp Ala Met Gly Gly Val Lieu. Arg Glu Lieu. SOO 505 51O

Wall

SEQ ID NO 6 LENGTH: 1670 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Chemically Synthesized FEATURE: NAME/KEY: Cytochrome P450 ORF LOCATION: (123). . (1664)

<4 OOs, SEQUENCE: 6 ggat.cctata aataggaagt t cattt catt tggagaggaa acct coagta tttittaCaac 6 O aattacCaac aacaacaaac aacaaacaac attacaatta Ctatttacala ttacagatct 12 O c catggataa cgcttatatt attgctatt c tittctgttgc tattott titc. citt citt catt 18O atta tott ct tggaagagga aacggaggag ctgctag act tccaccagga ccaccagctg 24 O titccalatt ct tggacat citt catcttgtta agaa.gc.caat gcatgctact atgtctagac 3OO ttgctgagag atatggacca gttittctotc ttagacittgg atctagalaga gctgttgttg 360 tttgttct co aggatgcgct agaga.gtgct t cactgagca tgatgttact titcgcta aca gaccalagatt cgagt citcaa cittcttgttt Ctttcaacgg agctgct citt gct actgctt

Cttatggtgc t cattggaga aac cittagaa gaattgttgc tgttcaactt citttctgctic 54 O atagagttgg act tatgtct ggact tattg Ctggagaggt tagagctatg gttagaagaa tgtatagagc t ct coagctg gagctgctag aattcaactt. aagagaagac 660 ttitt.cgaggit ttctgtttct gttct tatgg agact attgc t catactaag gctact agac 72 O cagagactga tccagatact gatatgtctg ttgaggctica agagttcaag Caagttgttg atgagattat to Cacatatt ggagctgcta acctttggga titat cit to ca. gct cittagat 84 O ggttcgatgt titt cq9agtt agaagaaaga ttcttgctgc tgtttctaga agagatgctt 9 OO t cct tagaag act tattgat gctgagagaa gaagacittga tgatggagat gagggagaga 96.O agaagttctat gattgctgtt citt cit tactic ttcaaaagac tgagc.ca.gag gtttatactg ataa catgat tactgct citt actgctaacc tttitcggagc tggaactgag act actt Cta 108 O US 8,097,774 B2 57 - Continued c tacttctga gtgggctatg totottct to ttalaccatcC agatact citt aagaaggctic 14 O aagctgagat tgatgcttct gttggaaact ctagactitat tactgctgat gatgttact a 2OO gacttggata tottcaatgc attgttagag agact cittag actitt at CCa gctgctic caa 26 O tgcttcttico acatgagt ct tctgctgatt gCalaggttgg aggatataac att coaa.gag 32O gatctatoct tott attaac gct tatgcta tt catagaga tccagctgtt tgggaggagc cagagaagtt Catgccagag agatt.cgagg atggaggatg cgatggaaac cittct tatgc 44 O catt cqgaat gggalagalaga agatgcc.cag gagagactict tgct cittaga actgttggac SOO ttgttcttgg aactict tatt caatgctt.cg attgggaga.g agttgatgga gttgaggttg 560 atatgactga gggaggagga cit tact at to Calaaggttgt tcc acttgag gctatgtgca gaccalagaga tgctatggga ggagttctta gagagcttgt ttaagagctic 670

<210s, SEQ ID NO 7 &211s LENGTH: 513 212. TYPE : PRT <213> ORGANISM: Zea mays <4 OO > SEQUENCE: 7

Met Asp Lys Ala Tyr Val Ala Ala Lieu. Ser Wall Ala Phe Luell Phe Lieu. 1. 1O 15

Wall His Tyr Lieu Val Gly Arg Ala Ala Ala Gly Gly Gly Lys Gly Arg 25

Arg Luell Pro Pro Ser Pro Leu. Ala Ile Pro Phe Lell Gly His Lieu. 35 4 O 45

His Luell Wall Lys Thr Pro Phe His Ser Ala Lieu Gly Arg Luell Ala Glu SO 55 6 O

Arg His Gly Pro Wall Phe Ser Lieu. Arg Met Gly Arg Arg Ala Wall 65 70 7s 8O

Wall Wall Ser Ser Pro Glu. Cys Ala Arg Ala Cys Phe Thir Glu His Asp 85 90 95

Met Ser Phe Ala Asn Arg Pro Arg Phe Glu Ser Met Arg Luell Wall Ser 105 11 O

Phe Asp Gly Ala Met Lieu. Ser Wall Ser Ser Tyr Gly Pro Trp Arg 115 12 O 125

Thir Luell Arg Arg Val Ala Ala Val Gln Lieu. Lieu. Ser Ala His Arg Val 13 O 135 14 O

Ala Met Ser Pro Val Ile Cys Ala Glu Wall Arg Ala Met Val Arg 145 150 155 160

Arg Met Ala Arg Lieu Ala Ala Gly Gly Ala Ala Arg Wall Glin Lieu. Arg 1.65 17O 17s

Arg Arg Luell Phe Glu Lieu. Ser Lieu. Gly Val Lieu. Met Glu Thir Ile Ala 18O 185 19 O

Arg Thir Lys Thir Ser Arg Ser Glu Ala Cys Ala Ala Asp Thir Asp Wall 195

Ser Pro Glu Ala Ser Glu Lieu. Thr Arg Ile Ser Glu Glu Ile Met Pro 21 O 215

Tyr Luell Gly Thir Ala Asn Lieu. Trip Asp Tyr Lieu. Pro Phe Luell Arg Trp 225 23 O 235 24 O

Phe Asp Wall Phe Gly Val Arg Asn Llys Lieu Met Ala Ala Wall Arg Trp 245 250 255

Arg Asp Ala Phe Lieu. Arg Arg Lieu Ile Asp Ala Glu Arg Arg Arg Met 26 O 265 27 O

Asp Gly Asp Gly Asp Gly Glu Lys Lys Ser Met Ile Ala Wall Lieu. Luell US 8,097,774 B2 59 60 - Continued

27s 28O 285

Ser Luell Glin Ser Glu Pro Glu Luell Tyr Thir Asp Thir Met Ile Met 29 O 295 3 OO

Ala Luell Gly Asp Lell Phe Gly Ala Gly Thir Glu Thir Thir Ser Wall 3. OS 310 315

Thir Thir Glu Trp Ala Met Ser Luell Luell Luell Ser His Pro Glu Ala Luell 3.25 330 335

Ala Glin Ala Glu Ile Asp Ala Wall Wall Gly Asn Ser Arg Arg 34 O 345 35. O

Lell Ile Thir Ala Asp Asp Wall Pro Arg Luell Lell His Wall 355 360 365

Ile Asn Glu Thir Lell Arg Met Pro Ala Pro Lell Luell Luell Pro 37 O 375 38O

His Glu Ser Ala Ala Asp Wall Gly Asp Wall Pro Arg 385 390 4 OO

Gly Thir Luell Luell Ile Wall Asn Ala Ala His Arg Asp Pro Ala 4 OS 415

Wall Trp Glu Asp Pro Gly Ser Phe Luell Pro Arg Phe Glu Asp Gly 425 43 O

Ala Glu Gly Arg Lell Lell Met Pro Phe Met Gly Arg Arg 435 44 O 445

Pro Gly Glu Thir Lell Ala Luell Arg Thir Wall Gly Lell Wall Luell Ala 450 45.5 460

Thir Luell Luell Glin Phe Asp Trp Asp Thir Wall Asp Gly Ala Glu Wall 465 470 47s 48O

Asp Met Thir Glu Ser Gly Gly Luell Thir Met Pro Arg Ala Wall Pro Luell 485 490 495

Glu Ala Met Cys Lys Pro Arg Ala Ala Met Cys Asp Wall Luell Arg Glu SOO 505 51O

Lell

<210s, SEQ ID NO 8 &211s LENGTH: 542 212. TYPE : PRT &213s ORGANISM: Zea mays

<4 OOs, SEQUENCE:

Met Ala His Asp Ala Arg Ala ASn Ala Thir Arg Luell Ser 1. 15

Lell Thir Ser Met Met Asp Lell Ala Ala Ile Ala Ile Luell Ser Luell 25

Wall Phe Luell Phe Lell Lell Arg Arg Wall His Gly Lell Ala Arg Arg Asp 35 4 O 45

Gly Lys Ser Arg Ser Met Arg Arg Pro Pro Pro Ser Pro Pro Gly Ala SO 55 6 O

Wall Pro Phe Luell Gly His Lell His Luell Ile Lys Pro Phe His Ala 65 70

Thir Luell Ser Gly Lell Ala Glin Arg His Gly Pro Wall Phe Thir Luell Arg 85 90 95

Lell Gly Ser Arg Asp Ala Ala Wall Wall Thir Ser Pro Ala Cys Ala Arg 105 11 O

Glu Cys Phe Thir Glu His Asp Wall Thir Phe Ala Asn Arg Ser Luell Luell 115 12 O 125

Pro Ser Glin Arg Lell Wall Thir Phe Asp Gly Ala Ala Lell Gly Thir Ala 13 O 135 14 O US 8,097,774 B2 61 62 - Continued

Ser Gly Pro Arg Trp Arg Asp Luell Arg Arg Wall Ala Wall Wall Glin 145 150 155 160

Lell Luell Ser Ala His Arg Wall Gly Met Ser Gly Wall Ile Cys Gly 1.65 17O 17s

Glu Wall Arg Ala Met Wall Arg Arg Luell His Arg Ala Ala Ala Ala Ser 18O 185 19 O

Ala Ala Ala Gly Ala Arg Ile Glu Luell Arg Arg Lell Phe Glu Luell 195

Ser Luell Ser Wall Lell Met Glu Ala Ile Ala Glu Thir Ala 21 O 215 22O

Arg Asp Pro Asp Pro Glu Pro Asp Ala Asp Gly Thir Thir Asp Met Ser 225 23 O 235 24 O

Pro Glu Ala Glin Glu Phe Arg Wall Ile Asp Glu Wall Phe Pro Tyr 245 250 255

Wall Ser Ser Wall Lell Trp Asp Luell Pro Wall Lell Arg Trp Phe Asp 26 O 265 27 O

Wall Ala Gly Wall Arg Ser Arg Ile Luell Ala Ala Wall Ser Arg Arg Asp 28O 285

Ala Phe Luell Arg Arg Lell Ile Asp Ala Glu Arg Arg Arg Met Ala Asp 29 O 295 3 OO

Gly Wall Gly Glu Lys Ser Luell Ile Ala Wall Lell Luell Ala Luell 3. OS 310 315

Glin Luell Glu Pro Glu Wall Thir Asp Thir Wall Ile Thir Ala Phe 3.25 330 335

Ser Asn Luell Phe Gly Ala Gly Thir Glu Thir Thir Ser Thir Thir Wall 34 O 345 35. O

Glu Trp Ala Met Ser Lell Lell Luell Asn Asn Pro Gly Thir Luell Glu Lys 355 360 365

Ala Arg Ala Glu Ile Asp Ala Wall Gly His Ser Arg Luell Luell Asn 37 O 375

Ala Gly Asp Luell Pro Arg Lell Luell Arg Ile Ile Ala Glu 385 390 395 4 OO

Thir Luell Arg Luell Tyr Pro Ala Pro Luell Luell Lell Pro His Glu Ser 4 OS 415

Ser Ala Asp Cys Lys Wall Gly Tyr Asp Wall Pro Arg Gly Thir Ala 425 43 O

Lell Luell Wall Asn Wall Ala His Arg Asp Pro Ala Wall Trp Glu 435 445

Glu Pro Gly Arg Phe Wall Pro Arg Phe Glu Gly Gly Ala Glu 450 45.5 460

Gly Luell Phe Wall Ala Pro Phe Met Gly Arg Arg Pro Gly 465 470

Glu Arg Luell Ala Lell Glin Thir Wall Gly Wall Ala Lell Gly Ser Luell Ile 485 490 495

Glin Phe His Trp Asn Arg Wall Asp Gly Wall Glu Wall Asp Met Ser SOO 505 51O

Glu Gly Ser Gly Lell Thir Met Pro Ala Wall Pro Lell Glu Ala Luell 515 52O 525

Thir Thir Arg Glu Ala Met Asp Wall Luell Glin Ile 53 O 535 54 O

<210s, SEQ ID NO 9 &211s LENGTH: 524 212. TYPE : PRT US 8,097,774 B2 63 64 - Continued

&213s ORGANISM: Catharanthus roseus

<4 OOs, SEQUENCE: 9

Met Glu Met Asp Met Asp Thir Ile Arg Lys Ala Ile Ala Ala Thir Ile 1. 15

Phe Ala Luell Wall Met Ala Trp Ala Trp Arg Wall Lell Asp Trp Ala Trp 2O 25

Phe Thir Pro Ile Glu Lys Arg Luell Arg Glin Glin Gly Phe Arg 35 4 O 45

Gly Asn Pro Phe Lell Wall Gly Asp Wall Lys Glu Ser Gly SO 55 6 O

Met His Glin Glu Ala Lell Ser Pro Met Glu Phe Asn Asn Asp Ile 65 70

Wall Pro Arg Luell Met Pro His Ile Asn His Thir Ile Asn Thir Tyr Gly 85 90 95

Arg Asn Ser Phe Thir Trp Met Gly Arg Ile Pro Arg Ile His Wall Met 105 11 O

Glu Pro Glu Luell Ile Glu Wall Luell Thir His Ser Ser Glin 115 12 O 125

Asn Phe Asp Wall His Asn Pro Luell Wall Phe Lell Luell Thir Gly 13 O 135 14 O

Wall Gly Ser Phe Glu Gly Ala Trp Ser Lys His Arg Arg Ile Ile 145 150 155 160

Ser Pro Ala Phe Thir Lell Glu Luell Lys Ser Met Lell Pro Ala Phe 1.65 17O

Ala Ile Tyr His Asp Met Luell Thir Trp Glu Ile Ala Glu 18O 185 19 O

Glin Gly Ser His Glu Wall Asp Ile Phe Pro Thir Phe Asp Wall Luell 195 2O5

Thir Ser Asp Wall Ile Ser Lys Wall Ala Phe Gly Ser Thir Glu Glu 21 O 215 22O

Gly Gly Ile Phe Arg Lell Luell Glu Luell Met Asp Luell Thir Ile 225 23 O 235 24 O

Asp Met Arg Asp Wall Ile Pro Gly Trp Ser Luell Pro Thir 245 250 255

Arg Asn Lys Arg Met Glu Ile Asn Glu Ile Thir Asp Met 26 O 265 27 O

Lell Arg Phe Ile Ile Asn Arg Met Ala Lell Lys Ala Gly Glu 27s 285

Pro Gly Glu Asp Asp Lell Lell Gly Wall Luell Luell Glu Ser Asn Ile Glin 29 O 295 3 OO

Glu Ile Glin Glin Gly Asn Asp Gly Gly Met Ser Ile Asn 3. OS 310 315

Asp Wall Ile Glu Glu Luell Phe Tyr Phe Ala Gly Glin Glu Thir 3.25 330 335

Thir Gly Wall Luell Lell Thir Trp Thir Thir Ile Luell Lell Ser Lys His Pro 34 O 345 35. O

Glu Trp Glin Glu Arg Ala Arg Glu Glu Wall Luell Glin Ala Phe Gly 355 360 365

Asn Lys Pro Glu Phe Glu Arg Luell Asn His Luell Lys Wall Ser Met 37 O 375

Ile Luell Glu Wall Lell Arg Luell Pro Pro Wall Ile Asp Luell Thir 385 390 395 4 OO

Ile Wall His Lys Asp Thir Luell Gly Ser Thir Ile Pro Ala US 8,097,774 B2 65 66 - Continued

4 OS 41O 415

Gly Thir Glin Wall Met Lell Pro Thir Wall Met Luell His Arg Glu Lys Ser 425 43 O

Ile Trp Gly Glu Asp Ala Met Glu Phe Asn Pro Met Arg Phe Wall Asp 435 44 O 445

Gly Wall Ala Asn Ala Thir Lys Asn Asn Wall Thir Tyr Lell Pro Phe Ser 450 45.5 460

Trp Gly Pro Arg Wall Cys Lell Gly Glin Asn Phe Ala Lell Luell Glin Ala 465 470

Luell Gly Luell Ala Met Ile Luell Glin Arg Phe Phe Asp Wall Ala 485 490 495

Pro Ser Wall His Ala Pro Phe Thir Ile Luell Thir Wall Glin Pro Glin SOO 505 51O

Phe Gly Ser His Wall Ile Lys Luell Glu Ser 515 52O

SEQ ID NO 10 LENGTH: 44 O TYPE : PRT ORGANISM: Avena sativa

< 4 OOs SEQUENCE: 10

Met Pro Pro Thir Pro Ala Thir Ala Thir Gly Ala Ala Ala Ala Ala Wall 1. 5 15

Thir Pro Glu His Ala Ala Arg Ser Phe Pro Arg Wall Wall Arg Wall Asn 25

Pro Arg Ser Asp Arg Phe Pro Wall Luell Ser Phe His His Wall Glu Luell 35 4 O 45

Trp Cys Ala Asp Ala Ala Ser Ala Ala Gly Arg Phe Ser Phe Ala Luell SO 55 6 O

Gly Ala Pro Luell Ala Ala Arg Ser Asp Luell Ser Thir Gly Asn Ser Ala 65 70

His Ala Ser Luell Lell Lell Arg Ser Gly Luell Ala Phe Luell Phe Thir 85 95

Ala Pro Ala Pro Pro Pro Glin Glu Ala Thir Ala Ala Ala Thir 105 11 O

Ala Ser Ile Pro Ser Phe Ser Ala Asp Ala Arg Thir Phe Ala Ala 115 12 O 125

Ala His Gly Luell Ala Wall Arg Ser Wall Wall Arg Wall Ala Asp Ala 13 O 135 14 O

Ala Glu Ala Phe Wall Ser Wall Ala Gly Ala Arg Pro Ala Phe 145 150 155 160

Ala Pro Ala Asp Lell Gly His Gly Phe Luell Ala Glu Wall Glu Luell 1.65

Gly Asp Wall Wall Lell Arg Phe Wall Pro Asp Glu Thir Asp 18O 185 19 O

Lell Pro Phe Luell Pro Gly Phe Glu Arg Wall Ser Ser Pro Gly Ala Wall 195

Asp Tyr Gly Luell Thir Arg Phe Asp His Wall Wall Gly Asn Wall Pro Glu 21 O 215 22O

Met Ala Pro Wall Ile Asp Met Gly Phe Lell Gly Phe His Glu 225 23 O 235 24 O

Phe Ala Glu Phe Thir Ala Glu Asp Wall Gly Thir Thir Glu Ser Gly Luell 245 250 255

Asn Ser Wall Wall Lell Ala Asn Asn Ser Glu Ala Wall Lell Luell Pro Luell US 8,097,774 B2 67 68 - Continued

26 O 265 27 O

Asn Glu Pro Wall His Gly Thir Lys Arg Arg Ser Glin Ile Glin Thir Tyr 27s 285

Lell Glu Tyr His Gly Gly Pro Gly Wall Glin His Ile Ala Luell Ala Ser 29 O 295 3 OO

Asn Asp Wall Luell Arg Thir Lell Arg Glu Met Arg Ala Arg Thir Pro Met 3. OS 310 315

Gly Gly Phe Glu Phe Met Ala Pro Pro Glin Ala Glu Gly 3.25 330 335

Wall Arg Arg Ile Ala Gly Asp Wall Luell Ser Glu Glu Glin Ile Glu 34 O 345 35. O

Glin Glu Luell Gly Wall Lell Wall Asp Arg Asp Asp Glin Gly Wall Luell 355 360 365

Lell Glin Ile Phe Thir Pro Wall Gly Asp Arg Pro Thir Phe Phe Luell 37 O 375

Glu Met Ile Glin Arg Ile Gly Met Glu Lys Asp Glu Wall Gly Glin 385 390 395 4 OO

Glu Glin Gly Gly Gly Gly Phe Gly Gly Asn Phe Ser 4 OS 415

Glu Luell Phe Lys Ser Ile Glu Asp Tyr Glu Lys Ser Lell Glu Wall Lys 42O 425 43 O

Glin Ser Wall Wall Ala Glin Ser 435 44 O

<210s, SEQ ID NO 11 &211s LENGTH: 439 212. TYPE : PRT <213> ORGANISM: Avena sativa

<4 OOs, SEQUENCE: 11

Met Pro Pro Thir Pro Ala Thir Ala Thir Gly Ala Ala Ala Ala Ala Wall 1. 5 15

Thir Pro Glu His Ala Ala Arg Ser Phe Pro Arg Wall Wall Arg Wall Asn 25

Pro Arg Ser Asp Arg Phe Pro Wall Luell Ser Phe His His Wall Glu Luell 35 4 O 45

Trp Cys Ala Asp Ala Ala Ser Ala Ala Gly Arg Phe Ser Phe Ala Luell SO 55 6 O

Gly Ala Pro Luell Ala Ala Arg Ser Asp Luell Ser Thir Gly Asn Ser Ala 65 70

His Ala Ser Luell Lell Lell Arg Ser Ala Luell Ala Phe Luell Phe Thir 85 90 95

Ala Pro Ala Pro Pro Pro Glin Ala Ala Thir Ala Ala Thir Ala 1OO 11 O

Ser Ile Pro Ser Phe Ser Ala Asp Ala Arg Thir Phe Ala Ala Ala 115 12 O 125

His Gly Luell Ala Wall Arg Ser Wall Wall Arg Wall Ala Asp Ala Ala 13 O 135 14 O

Glu Ala Phe Arg Wall Ser Wall Ala Gly Ala Arg Pro Ala Phe Ala 145 150 155 160

Pro Ala Asp Luell Gly His Gly Phe Luell Ala Glu Wall Glu Luell Tyr 1.65 17O 17s

Gly Asp Wall Wall Lell Arg Phe Wall Ser Pro Asp Glu Thir Asp Luell 18O 185 19 O

Pro Phe Luell Pro Gly Phe Glu Arg Wall Ser Ser Pro Gly Ala Wall Asp US 8,097,774 B2 69 70 - Continued

195

Gly Luell Thir Phe Asp His Wall Wall Gly Asn Wall Pro Glu Met 21 O 215 22O

Ala Pro Wall Ile Asp Tyr Met Gly Phe Luell Gly Phe His Glu Phe 225 23 O 235 24 O

Ala Glu Phe Thir Ala Glu Asp Wall Gly Thir Thir Glu Ser Gly Luell Asn 245 250 255

Ser Wall Wall Luell Ala Asn Asn Ser Glu Ala Wall Lell Lell Pro Luell Asn 26 O 265 27 O

Glu Pro Wall His Gly Thir Arg Arg Ser Glin Ile Glin Thir Luell 285

Glu Tyr His Gly Gly Pro Gly Wall Glin His Ile Ala Lell Ala Ser Asn 29 O 295 3 OO

Asp Wall Luell Arg Thir Lell Arg Glu Met Arg Ala Arg Thir Pro Met Gly 3. OS 310 315

Gly Phe Glu Phe Met Ala Pro Pro Glin Ala Glu Gly Wall 3.25 330 335

Arg Arg Ile Ala Gly Asp Wall Luell Ser Glu Glu Glin Ile Lys Glu Cys 34 O 345 35. O

Glin Glu Luell Gly Wall Lell Wall Asp Arg Asp Asp Glin Gly Wall Luell Luell 355 360 365

Glin Ile Phe Thir Lys Pro Wall Gly Asp Arg Pro Thir Phe Phe Luell Glu 37 O 375

Met Ile Glin Arg Ile Gly Met Glu Asp Glu Wall Gly Glin Glu 385 390 395 4 OO

Glin Gly Gly Gly Gly Phe Gly Gly Asn Phe Ser Glu 4 OS 41O 415

Lell Phe Ser Ile Glu Asp Glu Ser Lell Glu Wall Glin 425 43 O

Ser Wall Wall Ala Glin Ser 435

<210s, SEQ ID NO 12 &211s LENGTH: 44 O 212. TYPE : PRT <213> ORGANISM: Avena sativa

<4 OOs, SEQUENCE: 12

Met Pro Pro Thir Pro Ala Thir Ala Thir Gly Ala Ala Ala Ala Ala Wall 1. 5 15

Thir Pro Glu His Ala Ala Arg Ser Phe Pro Arg Wall Wall Arg Wall Asn 25

Pro Arg Ser Asp Arg Phe Pro Wall Luell Ser Phe His His Wall Glu Luell 35 4 O 45

Trp Cys Ala Asp Ala Ala Ser Ala Ala Gly Arg Phe Ser Phe Ala Luell SO 55 6 O

Gly Ala Pro Luell Ala Ala Arg Ser Asp Luell Ser Thir Gly Asn Ser Ala 65 70

His Ala Ser Luell Lell Lell Arg Ser Gly Ala Luell Ala Phe Luell Phe Thir 85 90 95

Ala Pro Ala Pro Pro Pro Glin Glu Ala Ala Thir Ala Ala Thir Thir 105 11 O

Ala Ser Ile Pro Ser Phe Ser Ala Asp Ala Ala Arg Thir Phe Ala Ala 115 12 O 125

Ala His Gly Luell Ala Wall Arg Ser Wall Gly Wall Arg Wall Ala Asp Ala US 8,097,774 B2 71 72 - Continued

13 O 135 14 O

Ala Glu Ala Phe Wall Ser Wall Ala Gly Gly Ala Arg Pro Ala Phe 145 150 155 160

Ala Pro Ala Asp Lell Gly His Gly Phe Gly Luell Ala Glu Wall Glu Luell 1.65 17O

Gly Asp Wall Wall Lell Arg Phe Wall Ser Pro Asp Glu Thir Asp 18O 185 19 O

Lell Pro Phe Luell Pro Gly Phe Glu Arg Wall Ser Ser Pro Gly Ala Wall 195

Asp Tyr Gly Luell Thir Arg Phe Asp His Wall Wall Gly Asn Wall Pro Glu 21 O 215 22O

Met Ala Pro Wall Ile Asp Met Gly Phe Lell Gly Phe His Glu 225 23 O 235 24 O

Phe Ala Glu Phe Thir Ala Glu Asp Wall Gly Thir Thir Glu Ser Gly Luell 245 250 255

Asn Ser Wall Wall Lell Ala Asn Asn Ser Glu Ala Wall Lell Luell Pro Luell 26 O 265 27 O

Asn Glu Pro Wall His Gly Thir Lys Arg Arg Ser Glin Ile Glin Thir Tyr 28O 285

Lell Glu His Gly Gly Pro Gly Wall Glin His Ile Ala Luell Ala Ser 29 O 295 3 OO

Asn Asp Wall Luell Arg Thir Lell Arg Glu Met Arg Ala Arg Thir Pro Met 3. OS 310 315

Gly Gly Phe Glu Phe Met Ala Pro Pro Glin Ala Glu Gly 3.25 330 335

Wall Arg Arg Ile Ala Gly Asp Wall Luell Ser Glu Glu Glin Ile Glu 34 O 345 35. O

Glin Glu Luell Gly Wall Lell Wall Asp Arg Asp Asp Glin Gly Wall Luell 355 360 365

Lell Glin Ile Phe Thir Pro Wall Gly Asp Arg Pro Thir Phe Phe Luell 37 O 375

Glu Met Ile Glin Arg Ile Gly Met Glu Lys Asp Glu Wall Gly Glin 385 390 395 4 OO

Glu Glin Gly Gly Gly Gly Phe Gly Gly Asn Phe Ser 4 OS 415

Glu Luell Phe Lys Ser Ile Glu Asp Tyr Glu Ser Lell Glu Wall Lys 42O 425 43 O

Glin Ser Wall Wall Ala Glin Ser 435 44 O

<210s, SEQ ID NO 13 &211s LENGTH: 44 O 212. TYPE : PRT <213> ORGANISM: Avena sativa

<4 OOs, SEQUENCE: 13

Met Pro Pro Thir Pro Ala Thir Ala Thir Gly Ala Ala Ala Ala Ala Wall 1. 5 15

Thir Pro Glu His Ala Ala Arg Ser Phe Pro Arg Wall Wall Arg Wall Asn 2O 25 3O

Pro Arg Ser Asp Arg Phe Pro Wall Luell Ser Phe His His Wall Glu Luell 35 4 O 45

Trp Cys Ala Asp Ala Ala Ser Ala Ala Gly Arg Phe Ser Phe Ala Luell SO 55 6 O

Gly Ala Pro Luell Ala Ala Arg Ser Asp Luell Ser Thir Gly Asn Ser Ala US 8,097,774 B2 73 - Continued

His Ala Ser Lieu Lleu Lieu. Arg Ser Gly Ala Lieu Ala Phe Lieu. Phe Thr 85 90 95 Ala Pro Tyr Ala Pro Pro Pro Glin Glu Ala Ala Asp Ala Ala Ala Thr 1OO 105 11 O Ala Ser Ile Pro Ser Phe Ser Ala Asp Ala Ala Arg Thr Phe Ala Ala 115 12 O 125 Ala His Gly Lieu Ala Val Arg Ser Val Gly Val Arg Val Ala Asp Ala 13 O 135 14 O Ala Glu Ala Phe Arg Val Ser Val Ala Gly Gly Ala Arg Pro Ala Phe 145 150 155 160 Ala Pro Ala Asp Lieu. Gly His Gly Phe Gly Lieu Ala Glu Val Glu Lieu 1.65 17O 17s Tyr Gly Asp Val Val Lieu. Arg Phe Val Ser Tyr Pro Asp Glu Thir Asp 18O 185 19 O Lieu Pro Phe Leu Pro Gly Phe Glu Arg Val Ser Ser Pro Gly Ala Val 195 2OO 2O5 Asp Tyr Gly Lieu. Thir Arg Phe Asp His Val Val Gly Asn Val Pro Glu 21 O 215 22O Met Ala Pro Val Ile Asp Tyr Met Lys Gly Phe Leu Gly Phe His Glu 225 23 O 235 24 O Phe Ala Glu Phe Thr Ala Glu Asp Val Gly Thr Thr Glu Ser Gly Lieu. 245 250 255

Asn Ser Wal Wall Lieu Ala Asn. Asn. Ser Glu Ala Wall Lieu. Lieu Pro Lieu. 26 O 265 27 O Asn Glu Pro Val His Gly Thr Lys Arg Arg Ser Glin Ile Glin Thr Tyr 27s 28O 285 Lieu. Glu Tyr His Gly Gly Pro Gly Val Glin His Ile Ala Lieu Ala Ser 29 O 295 3 OO Asn Asp Val Lieu. Arg Thr Lieu. Arg Glu Met Arg Ala Arg Thr Pro Met 3. OS 310 315 32O Gly Gly Phe Glu Phe Met Ala Pro Pro Glin Ala Lys Tyr Tyr Glu Gly 3.25 330 335 Val Arg Arg Ile Ala Gly Asp Val Lieu. Ser Glu Glu Glin Ile Lys Glu 34 O 345 35. O Cys Glin Glu Lieu. Gly Val Lieu Val Asp Arg Asp Asp Glin Gly Val Lieu. 355 360 365 Lieu. Glin Ile Phe Thr Lys Pro Val Gly Asp Arg Pro Thr Phe Phe Leu 37 O 375 38O Glu Met Ile Glin Arg Ile Gly Cys Met Glu Lys Asp Glu Val Gly Glin 385 390 395 4 OO Glu Tyr Gln Lys Gly Gly Cys Gly Gly Phe Gly Lys Gly Asn Phe Ser 4 OS 41O 415 Glu Lieu. Phe Llys Ser Ile Glu Asp Tyr Glu Lys Ser Lieu. Glu Val Lys 42O 425 43 O Glin Ser Val Val Ala Glin Llys Ser 435 44 O

<210s, SEQ ID NO 14 &211s LENGTH: 1323 &212s. TYPE: DNA <213> ORGANISM: Avena sativa

<4 OOs, SEQUENCE: 14 atgcct coaa caccagotac togctactgga gctgctgctg ctg.ccgttac accagaacat 6 O

US 8,097,774 B2 77 78 - Continued cgat ct caga t coaaaccta cct cqaatac catggtggac caggagttca acacatcgca 9 OO ttggct tcta acgatgtgct tcgaactict c agggaaatga gagcc agaac tocaatggga 96.O gggttcgaat titatggct Co. tccacaagcc aagtact atg aaggagt ccg tagaatcgct 1 O2O ggagatgtct tt cagagga acagatcaag gagtgtcaag alactgggtgt t ct cqttgat 108 O cgagacgatc aaggtgtgct act coagatc titcaccaaac cagttggtga t cqtcc.cact 114 O tttitt cct cq aaatgattica gcgaatagga tigcatggaga aggatgaagt tdggcaa.gag 12 OO taccagaaag gtggatgtgg togggtttgga aaggggaact titt Cogagtt gttcaagttcC 126 O atagaggact acgagaagtic actggaagtic aag cagtctg. tcgttgctica galaga.gctaa 132O

That which is claimed: c) Sulcotrione; 1. A method of controlling weeds at a locus, said method d) mesotrione; comprising applying to said locus a weed-controlling amount e) tembotrione: of one or more herbicides, wherein said locus comprises a f) any PPO inhibitor; plant comprising a heterologous polynucleotide sequence g) Saflufenacil; encoding a cytochrome P450, wherein said polynucleotide h) butafenacil; sequence is selected from the group consisting of i) flumioxazin; and a) the nucleotide sequence set forth as nucleotides 123 25 j) SulfentraZone. 1664 in SEQID NO: 6; 2. The method of claim 1, wherein said nucleic acid con b) a nucleotide sequence encoding a polypeptide compris struct further comprises a second polynucleotide sequence ing the amino acid sequence set forth in SEQID NO: 5 wherein the one or more herbicides are selected from the encoding a polypeptide that confers a desirable trait. group consisting of: 3. The method of claim 2, wherein said desirable trait is 30 resistance or tolerance to an herbicide selected from the group a) any HPPD inhibitor consisting of an HPPD inhibitor, glyphosate, and glufosinate. b) the compounds of formula Ia 4. The method according to claim 1, wherein the one or more herbicides is mesotrione. 5. The method of claim 1, wherein said plant is a rice, (Ia) OR5 O R3 35 barley, potato, Sweet potato, canola, Sunflower, rye, oats, wheat, corn, soybean, Sugar beet, tobacco, Miscanthus grass, Switch grass, safflower, trees, cotton, cassava, tomato, Sor a Nx ghum, alfalfa, Sugar beet, or Sugarcane plant. R 6. The method of claim 5 wherein said plant is a soybean S R4, 40 plant. R 7. The method claim 1, wherein the one or more herbicides is saflufenacil. wherein X is nitrogen and R is CF, CFH or CFH and/or R and R together form an ethylene bridge;