title: Broad 4-hydroxyphenylpyruvate dioxygenase inhibitor herbicide tolerance in soybean with an optimized enzyme and expression cassette.
abstract: With an optimized expression cassette consisting of the soybean (Glycine max) native promoter modified for enhanced expression driving a chimeric gene coding for the soybean native amino-terminal 86 amino acids fused to an insensitive shuffled variant of maize (Zea mays) 4-hydroxyphenylpyruvate dioxygenase (HPPD), we achieved field tolerance in transgenic soybean plants to the HPPD-inhibiting herbicides mesotrione, isoxaflutole, and tembotrione. Directed evolution of maize HPPD was accomplished by progressively incorporating amino acids from naturally occurring diversity and novel substitutions identified by saturation mutagenesis, combined at random through shuffling. Localization of heterologously expressed HPPD mimicked that of the native enzyme, which was shown to be dually targeted to chloroplasts and the cytosol. Analysis of the native soybean HPPD gene revealed two transcription start sites, leading to transcripts encoding two HPPD polypeptides. The N-terminal region of the longer encoded peptide directs proteins to the chloroplast, while the short form remains in the cytosol. In contrast, maize HPPD was found almost exclusively in chloroplasts. Evolved HPPD enzymes showed insensitivity to five inhibitor herbicides. In 2013 field trials, transgenic soybean events made with optimized promoter and HPPD variant expression cassettes were tested with three herbicides and showed tolerance to four times the labeled rates of mesotrione and isoxaflutole and two times the labeled rates of tembotrione.
authors.affiliation: DuPont Pioneer, Hayward, California 94545 (D.L.S., Y.T., H.A., Y.D., M.H., A.M., B.L.-C., J.L., T.F., E.B., M.S., C.H., I.A.U., L.A.C.);DuPont Stein-Haskell Research Center, Newark, Delaware 19711 (J.M.G., T.H., P.P., J.C.);DuPont Experimental Station, Wilmington, Delaware 19803 (N.R., T.B., R.J.H.); andDuPont Pioneer, Johnston, Iowa 50131 (D.H.J., M.V., G.A.) dan.siehl@pioneer.com.
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Siehl
authors.affiliation: DuPont Pioneer, Hayward, California 94545 (D.L.S., Y.T., H.A., Y.D., M.H., A.M., B.L.-C., J.L., T.F., E.B., M.S., C.H., I.A.U., L.A.C.);DuPont Stein-Haskell Research Center, Newark, Delaware 19711 (J.M.G., T.H., P.P., J.C.);DuPont Experimental Station, Wilmington, Delaware 19803 (N.R., T.B., R.J.H.); andDuPont Pioneer, Johnston, Iowa 50131 (D.H.J., M.V., G.A.).
authors.forename: Yumin
authors.initials: Y
authors.lastname: Tao
authors.affiliation: DuPont Pioneer, Hayward, California 94545 (D.L.S., Y.T., H.A., Y.D., M.H., A.M., B.L.-C., J.L., T.F., E.B., M.S., C.H., I.A.U., L.A.C.);DuPont Stein-Haskell Research Center, Newark, Delaware 19711 (J.M.G., T.H., P.P., J.C.);DuPont Experimental Station, Wilmington, Delaware 19803 (N.R., T.B., R.J.H.); andDuPont Pioneer, Johnston, Iowa 50131 (D.H.J., M.V., G.A.).
authors.forename: Henrik
authors.initials: H
authors.lastname: Albert
authors.affiliation: DuPont Pioneer, Hayward, California 94545 (D.L.S., Y.T., H.A., Y.D., M.H., A.M., B.L.-C., J.L., T.F., E.B., M.S., C.H., I.A.U., L.A.C.);DuPont Stein-Haskell Research Center, Newark, Delaware 19711 (J.M.G., T.H., P.P., J.C.);DuPont Experimental Station, Wilmington, Delaware 19803 (N.R., T.B., R.J.H.); andDuPont Pioneer, Johnston, Iowa 50131 (D.H.J., M.V., G.A.).
authors.forename: Yuxia
authors.initials: Y
authors.lastname: Dong
authors.affiliation: DuPont Pioneer, Hayward, California 94545 (D.L.S., Y.T., H.A., Y.D., M.H., A.M., B.L.-C., J.L., T.F., E.B., M.S., C.H., I.A.U., L.A.C.);DuPont Stein-Haskell Research Center, Newark, Delaware 19711 (J.M.G., T.H., P.P., J.C.);DuPont Experimental Station, Wilmington, Delaware 19803 (N.R., T.B., R.J.H.); andDuPont Pioneer, Johnston, Iowa 50131 (D.H.J., M.V., G.A.).
authors.forename: Matthew
authors.initials: M
authors.lastname: Heckert
authors.affiliation: DuPont Pioneer, Hayward, California 94545 (D.L.S., Y.T., H.A., Y.D., M.H., A.M., B.L.-C., J.L., T.F., E.B., M.S., C.H., I.A.U., L.A.C.);DuPont Stein-Haskell Research Center, Newark, Delaware 19711 (J.M.G., T.H., P.P., J.C.);DuPont Experimental Station, Wilmington, Delaware 19803 (N.R., T.B., R.J.H.); andDuPont Pioneer, Johnston, Iowa 50131 (D.H.J., M.V., G.A.).
authors.forename: Alfredo
authors.initials: A
authors.lastname: Madrigal
authors.affiliation: DuPont Pioneer, Hayward, California 94545 (D.L.S., Y.T., H.A., Y.D., M.H., A.M., B.L.-C., J.L., T.F., E.B., M.S., C.H., I.A.U., L.A.C.);DuPont Stein-Haskell Research Center, Newark, Delaware 19711 (J.M.G., T.H., P.P., J.C.);DuPont Experimental Station, Wilmington, Delaware 19803 (N.R., T.B., R.J.H.); andDuPont Pioneer, Johnston, Iowa 50131 (D.H.J., M.V., G.A.).
authors.forename: Brishette
authors.initials: B
authors.lastname: Lincoln-Cabatu
authors.affiliation: DuPont Pioneer, Hayward, California 94545 (D.L.S., Y.T., H.A., Y.D., M.H., A.M., B.L.-C., J.L., T.F., E.B., M.S., C.H., I.A.U., L.A.C.);DuPont Stein-Haskell Research Center, Newark, Delaware 19711 (J.M.G., T.H., P.P., J.C.);DuPont Experimental Station, Wilmington, Delaware 19803 (N.R., T.B., R.J.H.); andDuPont Pioneer, Johnston, Iowa 50131 (D.H.J., M.V., G.A.).
authors.forename: Jian
authors.initials: J
authors.lastname: Lu
authors.affiliation: DuPont Pioneer, Hayward, California 94545 (D.L.S., Y.T., H.A., Y.D., M.H., A.M., B.L.-C., J.L., T.F., E.B., M.S., C.H., I.A.U., L.A.C.);DuPont Stein-Haskell Research Center, Newark, Delaware 19711 (J.M.G., T.H., P.P., J.C.);DuPont Experimental Station, Wilmington, Delaware 19803 (N.R., T.B., R.J.H.); andDuPont Pioneer, Johnston, Iowa 50131 (D.H.J., M.V., G.A.).
authors.forename: Tamara
authors.initials: T
authors.lastname: Fenwick
authors.affiliation: DuPont Pioneer, Hayward, California 94545 (D.L.S., Y.T., H.A., Y.D., M.H., A.M., B.L.-C., J.L., T.F., E.B., M.S., C.H., I.A.U., L.A.C.);DuPont Stein-Haskell Research Center, Newark, Delaware 19711 (J.M.G., T.H., P.P., J.C.);DuPont Experimental Station, Wilmington, Delaware 19803 (N.R., T.B., R.J.H.); andDuPont Pioneer, Johnston, Iowa 50131 (D.H.J., M.V., G.A.).
authors.forename: Ericka
authors.initials: E
authors.lastname: Bermudez
authors.affiliation: DuPont Pioneer, Hayward, California 94545 (D.L.S., Y.T., H.A., Y.D., M.H., A.M., B.L.-C., J.L., T.F., E.B., M.S., C.H., I.A.U., L.A.C.);DuPont Stein-Haskell Research Center, Newark, Delaware 19711 (J.M.G., T.H., P.P., J.C.);DuPont Experimental Station, Wilmington, Delaware 19803 (N.R., T.B., R.J.H.); andDuPont Pioneer, Johnston, Iowa 50131 (D.H.J., M.V., G.A.).
authors.forename: Marian
authors.initials: M
authors.lastname: Sandoval
authors.affiliation: DuPont Pioneer, Hayward, California 94545 (D.L.S., Y.T., H.A., Y.D., M.H., A.M., B.L.-C., J.L., T.F., E.B., M.S., C.H., I.A.U., L.A.C.);DuPont Stein-Haskell Research Center, Newark, Delaware 19711 (J.M.G., T.H., P.P., J.C.);DuPont Experimental Station, Wilmington, Delaware 19803 (N.R., T.B., R.J.H.); andDuPont Pioneer, Johnston, Iowa 50131 (D.H.J., M.V., G.A.).
authors.forename: Caroline
authors.initials: C
authors.lastname: Horn
authors.affiliation: DuPont Pioneer, Hayward, California 94545 (D.L.S., Y.T., H.A., Y.D., M.H., A.M., B.L.-C., J.L., T.F., E.B., M.S., C.H., I.A.U., L.A.C.);DuPont Stein-Haskell Research Center, Newark, Delaware 19711 (J.M.G., T.H., P.P., J.C.);DuPont Experimental Station, Wilmington, Delaware 19803 (N.R., T.B., R.J.H.); andDuPont Pioneer, Johnston, Iowa 50131 (D.H.J., M.V., G.A.).
authors.forename: Jerry M
authors.initials: JM
authors.lastname: Green
authors.affiliation: DuPont Pioneer, Hayward, California 94545 (D.L.S., Y.T., H.A., Y.D., M.H., A.M., B.L.-C., J.L., T.F., E.B., M.S., C.H., I.A.U., L.A.C.);DuPont Stein-Haskell Research Center, Newark, Delaware 19711 (J.M.G., T.H., P.P., J.C.);DuPont Experimental Station, Wilmington, Delaware 19803 (N.R., T.B., R.J.H.); andDuPont Pioneer, Johnston, Iowa 50131 (D.H.J., M.V., G.A.).
authors.forename: Theresa
authors.initials: T
authors.lastname: Hale
authors.affiliation: DuPont Pioneer, Hayward, California 94545 (D.L.S., Y.T., H.A., Y.D., M.H., A.M., B.L.-C., J.L., T.F., E.B., M.S., C.H., I.A.U., L.A.C.);DuPont Stein-Haskell Research Center, Newark, Delaware 19711 (J.M.G., T.H., P.P., J.C.);DuPont Experimental Station, Wilmington, Delaware 19803 (N.R., T.B., R.J.H.); andDuPont Pioneer, Johnston, Iowa 50131 (D.H.J., M.V., G.A.).
authors.forename: Peggy
authors.initials: P
authors.lastname: Pagano
authors.affiliation: DuPont Pioneer, Hayward, California 94545 (D.L.S., Y.T., H.A., Y.D., M.H., A.M., B.L.-C., J.L., T.F., E.B., M.S., C.H., I.A.U., L.A.C.);DuPont Stein-Haskell Research Center, Newark, Delaware 19711 (J.M.G., T.H., P.P., J.C.);DuPont Experimental Station, Wilmington, Delaware 19803 (N.R., T.B., R.J.H.); andDuPont Pioneer, Johnston, Iowa 50131 (D.H.J., M.V., G.A.).
authors.forename: Jenna
authors.initials: J
authors.lastname: Clark
authors.affiliation: DuPont Pioneer, Hayward, California 94545 (D.L.S., Y.T., H.A., Y.D., M.H., A.M., B.L.-C., J.L., T.F., E.B., M.S., C.H., I.A.U., L.A.C.);DuPont Stein-Haskell Research Center, Newark, Delaware 19711 (J.M.G., T.H., P.P., J.C.);DuPont Experimental Station, Wilmington, Delaware 19803 (N.R., T.B., R.J.H.); andDuPont Pioneer, Johnston, Iowa 50131 (D.H.J., M.V., G.A.).
authors.forename: Ingrid A
authors.initials: IA
authors.lastname: Udranszky
authors.affiliation: DuPont Pioneer, Hayward, California 94545 (D.L.S., Y.T., H.A., Y.D., M.H., A.M., B.L.-C., J.L., T.F., E.B., M.S., C.H., I.A.U., L.A.C.);DuPont Stein-Haskell Research Center, Newark, Delaware 19711 (J.M.G., T.H., P.P., J.C.);DuPont Experimental Station, Wilmington, Delaware 19803 (N.R., T.B., R.J.H.); andDuPont Pioneer, Johnston, Iowa 50131 (D.H.J., M.V., G.A.).
authors.forename: Nancy
authors.initials: N
authors.lastname: Rizzo
authors.affiliation: DuPont Pioneer, Hayward, California 94545 (D.L.S., Y.T., H.A., Y.D., M.H., A.M., B.L.-C., J.L., T.F., E.B., M.S., C.H., I.A.U., L.A.C.);DuPont Stein-Haskell Research Center, Newark, Delaware 19711 (J.M.G., T.H., P.P., J.C.);DuPont Experimental Station, Wilmington, Delaware 19803 (N.R., T.B., R.J.H.); andDuPont Pioneer, Johnston, Iowa 50131 (D.H.J., M.V., G.A.).
authors.forename: Timothy
authors.initials: T
authors.lastname: Bourett
authors.affiliation: DuPont Pioneer, Hayward, California 94545 (D.L.S., Y.T., H.A., Y.D., M.H., A.M., B.L.-C., J.L., T.F., E.B., M.S., C.H., I.A.U., L.A.C.);DuPont Stein-Haskell Research Center, Newark, Delaware 19711 (J.M.G., T.H., P.P., J.C.);DuPont Experimental Station, Wilmington, Delaware 19803 (N.R., T.B., R.J.H.); andDuPont Pioneer, Johnston, Iowa 50131 (D.H.J., M.V., G.A.).
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Howard
authors.affiliation: DuPont Pioneer, Hayward, California 94545 (D.L.S., Y.T., H.A., Y.D., M.H., A.M., B.L.-C., J.L., T.F., E.B., M.S., C.H., I.A.U., L.A.C.);DuPont Stein-Haskell Research Center, Newark, Delaware 19711 (J.M.G., T.H., P.P., J.C.);DuPont Experimental Station, Wilmington, Delaware 19803 (N.R., T.B., R.J.H.); andDuPont Pioneer, Johnston, Iowa 50131 (D.H.J., M.V., G.A.).
authors.forename: David H
authors.initials: DH
authors.lastname: Johnson
authors.affiliation: DuPont Pioneer, Hayward, California 94545 (D.L.S., Y.T., H.A., Y.D., M.H., A.M., B.L.-C., J.L., T.F., E.B., M.S., C.H., I.A.U., L.A.C.);DuPont Stein-Haskell Research Center, Newark, Delaware 19711 (J.M.G., T.H., P.P., J.C.);DuPont Experimental Station, Wilmington, Delaware 19803 (N.R., T.B., R.J.H.); andDuPont Pioneer, Johnston, Iowa 50131 (D.H.J., M.V., G.A.).
authors.forename: Mark
authors.initials: M
authors.lastname: Vogt
authors.affiliation: DuPont Pioneer, Hayward, California 94545 (D.L.S., Y.T., H.A., Y.D., M.H., A.M., B.L.-C., J.L., T.F., E.B., M.S., C.H., I.A.U., L.A.C.);DuPont Stein-Haskell Research Center, Newark, Delaware 19711 (J.M.G., T.H., P.P., J.C.);DuPont Experimental Station, Wilmington, Delaware 19803 (N.R., T.B., R.J.H.); andDuPont Pioneer, Johnston, Iowa 50131 (D.H.J., M.V., G.A.).
authors.forename: Goke
authors.initials: G
authors.lastname: Akinsola
authors.affiliation: DuPont Pioneer, Hayward, California 94545 (D.L.S., Y.T., H.A., Y.D., M.H., A.M., B.L.-C., J.L., T.F., E.B., M.S., C.H., I.A.U., L.A.C.);DuPont Stein-Haskell Research Center, Newark, Delaware 19711 (J.M.G., T.H., P.P., J.C.);DuPont Experimental Station, Wilmington, Delaware 19803 (N.R., T.B., R.J.H.); andDuPont Pioneer, Johnston, Iowa 50131 (D.H.J., M.V., G.A.).
authors.forename: Linda A
authors.initials: LA
authors.lastname: Castle
mesh_terms: D015111:4-Hydroxyphenylpyruvate Dioxygenase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D003512:Cyclohexanones / Q000737:chemistry / Q000494:pharmacology; D015870:Gene Expression; D006540:Herbicides / Q000737:chemistry / Q000494:pharmacology*; D007555:Isoxazoles; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D016415:Sequence Alignment; D013025:Glycine max / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics
chemical_list: D003512:Cyclohexanones; D006540:Herbicides; D007555:Isoxazoles; D010940:Plant Proteins; C415327:isoxaflutole; D015111:4-Hydroxyphenylpyruvate Dioxygenase