WO1998040504A1 - Procedes de modification des concentrations en benzoxazinone de plantes - Google Patents

Procedes de modification des concentrations en benzoxazinone de plantes Download PDF

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WO1998040504A1
WO1998040504A1 PCT/US1998/004165 US9804165W WO9840504A1 WO 1998040504 A1 WO1998040504 A1 WO 1998040504A1 US 9804165 W US9804165 W US 9804165W WO 9840504 A1 WO9840504 A1 WO 9840504A1
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plant
gene
dimboa
biosynthesis
genes
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PCT/US1998/004165
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Alfons Gierl
Monika Frey
Robert Meeley
Steven P. Briggs
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Pioneer Hi-Bred International, Inc.
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Priority to AU67579/98A priority Critical patent/AU6757998A/en
Publication of WO1998040504A1 publication Critical patent/WO1998040504A1/fr

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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
    • C12N15/8254—Tryptophan or lysine
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the invention relates to the genetic manipulation of plants, particularly to increasing insect and disease resistance in plants.
  • Hydroxamic acids are known to function in the chemical defense of many cereal crops against a diverse group of pests including insects, fungi and bacteria. Efforts to exploit hydroxamic acids as natural pesticides have met with limited success despite their widespread occurrence in the gramineae. Besides being associated with resistance to pests, hydroxamic acids have been associated with triggering the reproduction of grass-feeding mammals and with allelopathic effects of cereals. The presence of hydroxamic acids has also been related to the detoxification of herbicides and pesticides, and to the mineral nutrition of the plant.
  • Hydroxamic acid concentrations are high in certain maize seedlings and provide substantial resistance against the first brood European corn borer, Ostrinia nubilalis, but may not be effective against the second brood European cor borer, which feeds after hydroxamic acid levels have dropped to lower levels. Attempts at selecting maize lines which maintain high hydroxamic acid concentrations throughout development have been unsuccessful.
  • Plant pests result in losses to farmers which run into multi- millions of dollars per year.
  • a mechanism is needed to protect plants against pest attack.
  • plants, plant cells, and seeds having altered hydroxamic acid levels are provided. Additionally, plants, plant cells, and seeds of the invention may have altered levels of tryptophan.
  • the plants, plant cells, and seeds are transformed with at least one hydroxamic acid biosynthesis gene.
  • the compositions and methods of the invention find use in altering the levels of particular benzoxazinones in plants as a plant defense mechanism for insect and disease resistance and for increasing herbicide tolerance in the plant. At the same time, the transformed plants can provide a source for tryptophan.
  • Fig. 1 sets forth the structure and chromosomal location of the Bx genes.
  • A Schematic representation of the Bx gene cluster on chromosome 4. Genetic distance is indicated in centi Morgan.
  • B Exon/intron structure of Bx2 to Bx5. Exons are represented by boxes. Translation start and stop codons and poly(A) addition sites are shown. The insertion of a Mu element in the bx3: :Mu allele is designated by an arrow. The complete sequences of the genes have been deposited in the EMBL data bank (Accession numbers Bx2: Y11368, Bx3: Y11404; Bx4: X81828; Bx5: Y11403).
  • Fig. 2 sets forth the detection of metabolites of the DIMBOA pathway by HPLC. Metabolites are indicated at the position of chromatographic peaks. S represents the solvent peak.
  • Fig. 3 sets forth the pathway for DIMBOA biosynthesis and tryptophan biosynthesis and their relationship.
  • the method involves transforming a plant with at least one hydroxamic acid biosynthesis gene. This has the effect of altering hydroxamic acid biosynthesis, particularly increasing the production of downstream products. Additional genes can then be utilized to shunt the metabolic activity to the production of particular benzoxazinones. Of particular interest are the benzoxazinones which occur in hydroxamic acid and lactam forms. Such compounds are a part of the biosynthesis pathway of DIMBOA. See Fig. 3 for the pathway of DIMBOA biosynthesis and its relationship with the tryptophan synthesis pathway.
  • the hydroxamic acid forms of benzoxazinones possess an N- hydroxyl group and are generally found in high concentrations. Lactam members lack the N-hydroxyl group and occur in lower concentrations.
  • the hydroxamic acid 2,4-dihyroxy-l,4-benzoxazin-3-one (DIBOA) has as its lactam counterpart 2-hydroxy-l,4-benzoxazin-3-one (HBOA).
  • the hydroxamic acids of the invention include the defense chemicals, the benzoxazinones, and more particularly the intermediates in the DIMBOA biosynthesis pathway, as well as DIMBOA.
  • Such intermediate compounds include HMBOA, DIBOA, HBOA, 3-hydroxy-indolin-2-one, indolin-2-one, indole, and the like.
  • biosynthesis genes are the genes which encode the DIMBOA biosynthesis enzymes, referred to herein as biosynthesis genes. Such genes can be used to produce transformed plants with increased or altered benzoxazinones.
  • biosynthesis genes of the invention include the CYP71C (Bx) genes included in DIMBOA biosynthesis. These genes are described in Frey et al. (1995) Mol. Gen. Genet. 246: 100-109, and are designed as the CYPzm genes, CYPzm 1-4.
  • the biosynthesis genes of the invention also encompass other genes encoding cytochrome P450 enzymes and P450 reductases.
  • Cytochrome P450 enzymes are membrane-bound, heme-containing enzymes implicated in a variety of biosynthetic reactions. See, for example, Donaldson and Luster (1991) Plant Physiol 96:669-674; West, CA (1980) m Davis DD (ed), The Biochemistry of Plants. Vol. 2, Academic Press, New York, pp. 317-364; and Butt and Lamb (1981) In: Conn E.E. (ed), The Biochemistry of Plants. Vol. 7, Academic Press, New York, pp. 627-665. Gene sequences for several P450 enzymes are available.
  • biosynthesis genes are other alkalaid biosynthetic genes. See, Kutchan, T.M. 196) Gene 179:73-81, and the references cited therein.
  • sources are available for the biosynthesis genes of the invention and, for the most part, a gene from any source can be utilized. In fact, because of the similarity of the P-450 systems in bacteria, yeast, plants, and mammals, genes from any of these sources can be utilized. It is recognized that because of co-suppression, the native plant gene or one having high homology due to the plant gene may not be preferred when increased expression of a compound is intended.
  • Transformation of a plant with an early DIMBOA biosynthesis gene leads to significant increases in the production of DIMBOA in the transformed plant.
  • the transformed plant has a heightened defense mechanism for insect and disease resistance.
  • compositions and methods of the invention also find use in altering allelopathic effects of plants. DIBOA and BOA have been shown to be involved in the allelopathic effects of rye. See, Fuerst and Putman (1983) J. Chem. Ecol. 9:937; and Barnes and Putman (1987) J. Chem. Ecol. 13:889. DIBOA has been shown to be the most active compound against monocots while BOA was most active against dicots. Barnes and Putnam (1987) J. Chem. Ecol. 13:889.
  • compositions and methods also find use in the detoxification of herbicides and pesticides.
  • Detoxification of the 2-chloro-s-triazine derived herbicides occur by hydroxylation, dealkylation, or glutathione conjugation. Altering the hydroxamic acid levels in the plant can increase tolerance to these and other herbicides.
  • Plants can be transformed with biosynthesis genes and tested for tolerance to the herbicide of interest. Hurter, J. (1966) Experimentia 22:741; Shimabukuro, R.H. (1967) Plant Physiol. 42: 1269; Shimabukuro et al. (1970) Plant Physiol. 46: 103; Malan et al. (1986) S. Afr. J.
  • Plant Soil 3 115; Malan et al. (1984) S. Afr. J. Plant Soil 1 : 103; Hamilton and Moreland (1962) Science 135:373; Nakano et al. (1973) J. Org. Chem. 38:4396; Anderson, R.N. (1964) Weeds 12:60; and Palmer and Grogan (1965) Weeds 13:219.
  • transformation of the plant with at least one biosynthesis gene which encodes an enzyme which catalyzes an early step in the pathway is sufficient.
  • Such enzymes include, but are not limited to, Bx2, Bx3, Bx4, Bx5, the CYPzm genes, etc.
  • the DIMBOA biosynthesis pathway can also be directed for the production of a particular compound or compounds.
  • a plant is transformed with an early DIMBOA biosynthesis gene, such as Bx2 or Bx3. Transformation with such an early gene (referred to also as the primary gene) increases the metabolic activity for the production of downstream compounds.
  • the pathway can be diverted for the production of specific compounds.
  • the diversion involves the action of at least one second gene of interest (the secondary gene).
  • the secondary gene can encode an enzyme to force the production of a particular compound or alternatively can encode an antisense RNA to stop the pathway for the accumulation of a particular compound. For example, for the production of HBOA, antisense Bx5 is utilized as the secondary gene.
  • the pathway can be modified for high production of particular compounds of interest.
  • Such plants can be tested for increased resistance to particular pathogens.
  • the DIMBOA biosynthesis genes can also be used in combination with any of the genes from the tryptophan biosynthetic pathway. Because DIMBOA synthesis has some intermediates in common with the tryptophan biosynthesis pathway, early genes in the tryptophan pathway can be utilized to prime the pathway for the production of DIMBOA. For example, the anthranilate synthase trp4 can be utilized to increase the biosynthesis of tryptophan. However, before indole is converted into tryptophan, the indole can be diverted to DIMBOA biosynthesis by transforming the plant with Bx2.
  • tryptophan pathway can be utilized including phosphoribosylanthranilate synthase (trp 1), phosphoribosylanthranilate isomerase; indole-3-glycerolphosphate synthase; tryptophan synthase (trp 3); and the like.
  • trp 1 phosphoribosylanthranilate synthase
  • phosphoribosylanthranilate isomerase phosphoribosylanthranilate isomerase
  • indole-3-glycerolphosphate synthase tryptophan synthase
  • tryptophan synthase tryptophan synthase
  • the plant can be transformed with a gene encoding a P-450 reductase.
  • genes have been cloned from yeast and maize and are available in the art. See, for example, U.S. Patent No. 5,114,852; Murakami et al. (1990) J. Biochem 108:859-865, Tokyo: Japanese Biochemical Society. Any means for producing a plant comprising both the primary and secondary genes, or the biosynthesis gene and any of the other genes described herein, are encompassed by the present invention.
  • the secondary gene of interest can be used to transform a plant at the same time as the primary gene (cotransformation).
  • the secondary gene can be introduced into a plant which has already been transformed by the primary gene.
  • transformed plants one expressing the primary gene and one expressing the secondary gene can be crossed to bring the genes together in the same plant.
  • the hydroxamic acid levels can be altered in particular tissues of the plant. While any promoter or promoter element capable of driving expression of a coding sequence can be utilized, of particular interest are root promoters (Bevan et al. (1993) in Gene Conservation and Exploitation. Proceedings of The 20th Stadler Genetics Symposium. Gustafson et al. (eds.), Plenum Press, New York pp. 109-129; Brears et al. (1991) Plant J. 1 :235-244; Lorenz et al. (1993) Plant J. 4:545-554; U.S. Patent Nos.
  • the primary, secondary, or other genes encoding the enzymes of interest can be used in expression cassettes for expression in the transformed plant tissues.
  • the plant is transformed with at least one expression cassette comprising a transcriptional initiation region linked to the gene of interest.
  • Such an expression cassette is provided with a plurality of restriction sites for insertion of the gene of interest to be under the transcriptional regulation of the regulatory regions.
  • the transcriptional initiation region may be native or analogous or foreign or heterologous to the host. By foreign is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced.
  • a chimeric gene comprises a coding sequence operably linked to transcription initiation region which is heterologous to the coding sequence.
  • the transcriptional cassette will include the in 5 '-3' direction of transcription, a transcriptional and translational initiation region, a DNA sequence of interest, and a transcriptional and translational termination region functional in plants.
  • the termination region may be native with the transcriptional initiation region, may be native with the DNA sequence of interest, or may be derived from another source.
  • Convenient termination regions are available from the Ti-plasmid of A. tunefaciens, such as the octopine synthase and nopaline synthase termination regions. See also,
  • the nucleotide sequences encoding the proteins or polypeptides of the invention are useful in the genetic manipulation of plants.
  • the genes of the invention are provided in expression cassettes for expression in the plant of interest.
  • the cassette will include 5' and 3' regulatory sequences operably linked to the gene of interest.
  • the cassette may additionally contain at least one additional gene to be cotransformed into the organism.
  • the gene(s) of interest can be provided on another expression cassette.
  • the gene(s) may be optimized for increased expression in the transformed plant.
  • mammalian, yeast, or bacterial P450 enzymes are used in the invention, they can be synthesized using plant preferred codons for improved expression. Methods are available in the art for synthesizing plant preferred genes. See, for example, U.S. Patent Nos. 5,380,831, 5,436, 391, and Murray et al. (1989) Nucleic Acids Res. 17:477- 498, herein incorporated by reference.
  • the expression cassettes may additionally contain 5' leader sequences in the expression cassette construct.
  • leader sequences can act to enhance translation.
  • Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein, O. , Fuerst, T.R. , and Moss, B. (1989) PN S USA 56:6126-6130); poty virus leaders, for example, TEV leader (Tobacco Etch Virus) (Allison et al. (1986); MDMV leader (Maize Dwarf Mosaic Virus); Virology 154:9-20), and human immunoglobulin heavy-chain binding protein (BiP), (Macejak, D.G.
  • EMCV leader Engelphalomyocarditis 5' noncoding region
  • poty virus leaders for example, TEV leader (Tobacco Etch Virus) (Allison et al. (1986); MDMV leader (Maize Dwarf
  • organelles such as chloroplasts (Lancelin et al. (1994) EE ⁇ S Letters 343:261-266; Tsutsumi et al. (1994) Gene 141 :215-220; Kubo et al. (1993) Platn and Cell Physiol 34: 1259-1266; Tang et al. (1994) Plant Physiol 104: 1081-1082; U.S. Patent Nos. 5,45,818; 5,545,817; 5,608, 149); and mitochondria (Arai et al. (1996) Biochem. Biophys. Res. Com.
  • the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame.
  • adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like.
  • in vitro mutagenesis, primer repair, restriction, annealing, resection, ligation, PCR, or the like may be employed, where insertions, deletions or substitutions, e.g. transitions and trans versions, may be involved.
  • compositions and methods of the invention can be used to transform any plant. It is recognized that DIMBOA biosynthesis genes are present in cereals. Thus, the DIMBOA biosynthesis of such plants can be modified by transformation of the plant with a single or several genes. For those plants, including dicots, which do not natively synthesize DIMBOA, a pathway for the synthesis of a particular compound or compounds can be constructed. Such pathways provide new approaches to insect and disease resistance in these plants. As discussed, the compositions and methods of the present invention can be used to transform any plant. In this manner, genetically modified plants, plant cells, plant tissue, seed, and the like can be obtained. Transformation protocols may vary depending on the type of plant or plant cell, i.e. monocot or dicot, targeted for transformation.
  • Suitable methods of transforming plant cells include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium mediated transformation (Hinchee et al. (1988) Biotechnology 6:915-921), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for example, Sanford et al. , U.S. Patent 4,945,050; and McCabe et al. (1988) Biotechnology 6:923-926).
  • nucleotide sequences of the invention can be utilized to protect plants from insect and disease pests.
  • pests include but are not limited to insects, pathogens including fungi, bacteria, nematodes, viruses or viroids, and the like.
  • Insect pests include insects selected from the orders Coleoptera, Diptera,
  • Hymenoptera Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc. , particularly Coleoptera and Lepidoptera.
  • Insect pests of the invention for the major crops include: Maize: Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm; Helicoverpa zea, corn earworm; Spodoptera frugiperda, fall armyworm; Diatraea grandiosella, southwestern corn borer; Elasmopalpus lignosellus, lesser cornstalk borer; Diatraea saccharalis, surgarcane borer; Diabrotica virgifera, western corn rootworm; Diabrotica longicornis barberi, northern corn rootworm; Diabrotica undecimpunctata howardi, southern corn rootworm; Melanotus spp.
  • Viruses include tobacco or cucumber mosaic virus, ringspot virus, necrosis virus, maize dwarf mosaic virus, etc.
  • Specific pathogens for the major crops include: Soybeans: Phytophthora megasperma fsp. glycinea, Macrophomina phaseolina, Rhizoctonia solani, Sclerotinia sclerotiorum, Fusarium oxysporum, Diaporthe phaseolorum var. sojae (Phomopsis sojae), Diaporthe phaseolorum var.
  • phaseoli Microsphaera diffusa, Fusarium semitectum, Phialophora gregata, Soybean mosaic virus, Glomerella glycines, Tobacco Ring spot virus, Tobacco Streak virus, Phakopsora pachyrhizi, Pythium aphanidermatum, Pythium ultimum, Pythium debaryanum, Tomato spotted wilt virus, Heterodera glycines Fusarium solani; Canola: Albugo Candida, Alternaria brassicae, Leptosphaeria maculans, Rhizoctonia solani, Sclerotinia sclerotiorum, Mycosphaerella brassiccola, Pythium ultimum, Peronospora parasitica, Fusarium roseum, Alternaria alternata; Alfalfa: Clavibater michiganese subsp.
  • medicaginis Cercospora medicaginis, Pseudopeziza medicaginis, Leptotrochila medicaginis, Fusarium oxysporum, Rhizoctonia solani, Uromyces striatus, Colletotrichum trifolii race 1 and race 2, Leptosphaerulina briosiana, Stemphylium botryosum, Stagonospora meliloti, Sclerotinia trifoliorum, Alfalfa Mosaic Virus, Verticillium albo-atrum, Xanthomonas campestris p. v.
  • Puccinia helianthi Verticillium dahliae, Erwinia carotovorum pv. carotovora, Cephalosporium acremonium, Phytophthora cryptogea, Albugo tragopogonis; Corn: Fusarium moniliforme var.
  • Stenocarpella maydi (Diplodia maydis), Pythium irregulare, Pythium debaryanum, Pythium graminicola, Pythium splendens, Pythium ultimum, Pythium aphanidermatum, Aspergillus flavus, Bipolaris maydis O, T (Cochliobolus heterostrophus) , Helminthosporium carbonum I, II & III (Cochliobolus carbonum), Exsewhilum turcicum I, II & III, Helminthosporium pedicellatum, Physoderma maydis, Phyllosticta maydis, Kabatiella zeae, Colletotrichum graminicola, Cercospora zeae-maydis, Cercospora so
  • nebraskense Trichoderma viride, Maize Dwarf Mosaic Virus A & B, Wheat Streak Mosaic Virus, Maize Chlorotic Dwarf Virus, Claviceps sorghi, Pseudonomas avenae, Erwinia chrysanthemi pv.
  • zea Erwinia corotovora, Cornstunt spiroplasma, Diplodia macrospora, Sclerophthora macrospora, Peronosclerospora sorghi, Peronosclerospora philippinensis , Peronosclerospora maydis, Peronosclerospora sacchari, Spacelotheca reiliana, Physopella zeae, Cephalosporium maydis, Caphalosporium acremonium, Maize Chlorotic Mottle Virus, High Plains Virus, Maize Mosaic Virus, Maize Rayado Fino Virus,
  • Maize Streak Virus Maize Stripe Virus, Maize Rough Dwarf Virus
  • Sorghum Exsewhilum turcicum, Colletotrichum graminicola (Glomerella graminicola) , Cercospora sorghi, Gloeocercospora sorghi, Ascochyta sorghina, Pseudomonas syringae p. v. syringae, Xanthomonas campestris p. v.
  • holcicola Pseudomonas andropogonis, Puccinia purpurea, Macrophomina phaseolina, Perconia circinata, Fusarium moniliforme, Alternaria alternate, Bipolaris sorghicola, Helminthosporium sorghicola, Curvularia lunata, Phoma insidiosa, Pseudomonas avenae (Pseudomonas alboprecipitans) , Ramulispora sorghi, Ramulispora sorghicola, Phyllachara sacchari, Sporisorium reilianum (Sphacelotheca reiliana), Sphacelotheca cruenta, Sporisorium sorghi, Sugarcane mosaic H, Maize Dwarf Mosaic Virus A & B, Claviceps sorghi, Rhizoctonia solani, Acremonium strictum, Sclerophthona macrospora, Peronos
  • Plant tissue cultures and recombinant plant cells containing the proteins and nucleotide sequences, or the purified protein, of the invention may also be used in an assay to screen chemicals for potential as herbicidal compounds. Such an assay is useful as a general screen to identify chemicals which are herbicide candidates.
  • DIMBOA has been shown to be important in the resistance of maize to first brood European com borer (Ostrinia nubilalis), northern corn leaf blight (Helminithosporium turcicum), maize plant louse (Thophalosiphum maydis) and stalk rot (Diplodia maydis), as well as to the herbicide atrzine, H.M. Niemeyer (1988) Phytochemistry 27:3349.
  • DIBOA is the main hydroxamic acid in rye
  • DIMBOA is the predominant form in wheat and maize H.M. Niemeyer (1988) Phytochemistry 27:3349.
  • Bxl was mapped with the Recombinant inbred system, B. Burr et al. (1991) Trends Genet 7:55, and proved to be located on the short arm of chromosome 4 and, within the limits of the method, at exactly the same map position as Bx2 (15, Fig. 1).
  • the Bxl gene has been mapped to the same region by classical genetic means, K. D. Simcox et al. (1985) Crop. Sci.
  • CYP71C1, Bx5 CYP71C3
  • CYP71C1 CYP71C3
  • CYP71C1 CYP71C3
  • CYP71C1 CYP71C3
  • Fig. 1 The developmental expression pattern of the genes in the young maize plants, occurring predominantly in tissues that are exposed to the environment, M. Frey et al. (1995) Mol. Gen.
  • ATR1 galactose inducible Arabidopsis thaliana microsomal NADPH-P450 reductase
  • Microsomes were isolated from the transgenic yeast strains and tested for enzymatic activity G. Truan et al. (1993) Gene 125:49; D. Pompon et al. (1996) Methods Enzymol. 272:51.
  • indole was converted to DIBOA by the stepwise action of the four cytochrome P450 enzymes (Fig. 2C- F).
  • [3- 13 C] indole was incubated with yeast microsomes containing Bx2 protein, [3- 13 C]indolin-2-one was produced in the reaction assay. A sufficient amount of [3- 13 C]indolin-2-one was produced by this enzyme catalyzed reaction in order to test for subsequent enzymatic conversions.
  • cytochrome P450 enzymes are homologous proteins, they are substrate specific. Only one substrate was converted by each respective P450 enzyme to a specific product. No detectable conversions occurred in other enzyme/substrate combinations (data not shown). Enzymatic reactions, identical to the ones with the different yeast microsomal preparations, could be performed with maize microsomes, indicating that these reactions occur natively in maize. These findings suggest a similar in vivo reaction sequence from indole to HBOA as outlined in Fig. 3. According to this scheme, benzoxazinone would not be a natural intermediate for DIMBOA synthesis, as proposed earlier on the basis of feeding experiments P. Kumar et al. (1994) Phytochemistry 36:893. Whether alternative routes for DIMBOA synthesis exist remains to be determined.
  • the four cytochrome P450 genes represent a sufficient set of genes for the conversion of indole-3-glycerol phosphate to the secondary metabolite DIBOA (Fig. 3). Indole-3-glycerol phosphate appears to be the real branchpoint from the tryptophan pathway. DIMBOA is the 7-methoxy derivative of DIBOA. The conversion of DIBOA to DIMBOA most likely requires two further enzymatic reactions. Since the O atom at C-7 is incorporated from molecular O 2 , E. Glawischnig et al. , Phytochemistry (in press), hyroxylation by another cytochrome P450 enzyme followed by a methyltransferase reaction would be expected. These enzymes which are probably present only in some Gramineae, H. M. Niemeyer (1988) Phytochemistry 27:3349, remain to be isolated.
  • Indole-3 -glycerol phosphate was also proposed as a branchpoint from the tryptophan pathway for the synthesis of the indolic phytoalexin camalexin (3-thiazol-2'yl-indole) in Arabidopsis thaliana.
  • a coordinate regulation of gene expression of the tryptophan pathway and camalexin synthesis was established, J. Zhao et al. (1996) Plant Cell 8:2235.
  • the regulation of gene expression of "primary" and "secondary" metabolic genes can now be studied in maize in a developmental and tissue specific manner.
  • the 13 C label from [3- 13 C]indole has been incorporated into position 3 of indolin-2-one and 3-hydroxy-indolin-2-one, and into position 2 of DIMBOA (determined by 13 C-decoupled 'H-NMR spectroscopy).
  • the signal assignments are based on two-dimensional NMR analysis (GRASP-HMQC,GRASP-HMBC, GRASP-DQF-COSY, data not shown).

Abstract

Cette invention se rapporte à des plantes, cellules végétales et graines transformées possédant des concentrations en acide hydroxamique modifiées. Ces plantes, cellules végétales et graines sont transformées au moyen d'au moins un gène de biosynthèse de l'acide hydroxamique. Ces procédés s'avèrent utiles d'une part, à la modification des concentrations de benzoxazinones spécifiques dans des plantes, ce qui confère à ces dernières un mécanisme de défense se traduisant par une résistance aux insectes, à la maladie, et d'autre part à l'accroissement de la tolérance de ces plantes aux herbicides. Ces plantes transformées peuvent en outre constituer une source de tryptophane.
PCT/US1998/004165 1997-03-12 1998-03-05 Procedes de modification des concentrations en benzoxazinone de plantes WO1998040504A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001073091A2 (fr) * 2000-03-24 2001-10-04 Pioneer Hi-Bred International, Inc. Procede de selection et de mise au point de vegetaux de meilleure qualite racinaire et de meilleure resistance a la verse racinaire
US6331660B1 (en) 1997-03-13 2001-12-18 Dekalb Genetics Corporation Maize DIMBOA biosynthesis genes
CZ301096B6 (cs) * 2004-06-24 2009-11-04 Konfršt@Václav Elektrický rotacní stroj

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993022441A1 (fr) * 1992-04-23 1993-11-11 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Sequence d'adn d'un gene de resistance de plante
WO1995006128A2 (fr) * 1993-08-25 1995-03-02 Dekalb Genetics Corporation Plantes de mais transgeniques fertiles et leurs procedes de production

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993022441A1 (fr) * 1992-04-23 1993-11-11 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Sequence d'adn d'un gene de resistance de plante
WO1995006128A2 (fr) * 1993-08-25 1995-03-02 Dekalb Genetics Corporation Plantes de mais transgeniques fertiles et leurs procedes de production

Non-Patent Citations (13)

* Cited by examiner, † Cited by third party
Title
DATABASE BIOSIS BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; GIERL, A., ET AL.: "Genetic analysis of the DIMBOA biosynthetic pathway of Zea mays", XP002070467 *
FREY M ET AL: "Analysis of a chemical plant defense mechanism in grasses.", SCIENCE (WASHINGTON D C) 277 (5326). 1997. 696-699. ISSN: 0036-8075, 1 August 1997 (1997-08-01), XP002070321 *
FREY, M., ET AL.: "Expression of a cytchrome P450 gene family in maize", MOLECULAR AND GENERAL GENETICS, vol. 246, 1995, pages 100 - 109, XP002070314 *
FREY, M., ET AL.: "Z. mays CYP71C1 gene for cytochrome P-450", EMBL SEQUENCE DATABASE, ACCESSION NO. X81828, 26 September 1994 (1994-09-26), XP002070318 *
FREY, M., ET AL.: "Z.mays CYP71C1 mRNA for cytochrome P-450", EMBL SEQUENCE DATABASE, ACCESSION NO. X81827, 26 September 1994 (1994-09-26) *
FREY, M., ET AL.: "Z.mays CYP71C2 mRNA for cytochrome P-450", EMBL SEQUENCE DATABASE, ACCESSION NO. X81829, 26 September 1994 (1994-09-26) *
FREY, M., ET AL.: "Z.mays CYP71C3 mRNA for cytochrome P-450", EMBL SEQUENCE DATABASE, ACCESSION NO. X81830, 26 September 1994 (1994-09-26) *
FREY, M., ET AL.: "Z.mays CYP71C4 mRNA for cytochrome-P450", EMBL SEQUENCE DATABASE ACCESSION NO. X81831, 26 September 1994 (1994-09-26) *
GIERL, A.: "Z. mays cyp71c4 gene", EMBL SEQUENCE DATABASE, ACCESSION NO. Y11368, 24 February 1997 (1997-02-24), XP002070316 *
GIERL,A., ET AL.: "Z. mays cyp71c2 gene", EMBL SEQUENCE DATABASE, ACCESSION NO. Y11404, 3 March 1997 (1997-03-03), XP002070317 *
GIERL,A.: "Z. mays cyp71c3 gene", EMBL SEQUENCE DATABASE, ACCESSION NO. Y11403, 3 March 1997 (1997-03-03), XP002070319 *
INTERNATIONAL CONFERENCE ON PERSPECTIVES IN PLANT GENETICS, WARSAW, POLAND, SEPTEMBER 16-17, 1996. JOURNAL OF APPLIED GENETICS 37A (0).1996. 50-51. ISSN: 1234-1983 *
KRAMER, V.C., ET AL.: "Z.mays (C6000237) trpA gene", EMBL SEQUENCE DATABASE, ACCESSION NO. X76713, 10 January 1994 (1994-01-10), XP002070320 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6331660B1 (en) 1997-03-13 2001-12-18 Dekalb Genetics Corporation Maize DIMBOA biosynthesis genes
WO2001073091A2 (fr) * 2000-03-24 2001-10-04 Pioneer Hi-Bred International, Inc. Procede de selection et de mise au point de vegetaux de meilleure qualite racinaire et de meilleure resistance a la verse racinaire
WO2001073091A3 (fr) * 2000-03-24 2002-06-27 Pioneer Hi Bred Int Procede de selection et de mise au point de vegetaux de meilleure qualite racinaire et de meilleure resistance a la verse racinaire
CZ301096B6 (cs) * 2004-06-24 2009-11-04 Konfršt@Václav Elektrický rotacní stroj

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