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WO2003014401A1 - Methodes permettant d'identifier des inhibiteurs d'expression ou d'activite d'oxydoreductase de ferredoxine nadp chez des plantes - Google Patents

Methodes permettant d'identifier des inhibiteurs d'expression ou d'activite d'oxydoreductase de ferredoxine nadp chez des plantes Download PDF

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WO2003014401A1
WO2003014401A1 PCT/US2002/025111 US0225111W WO03014401A1 WO 2003014401 A1 WO2003014401 A1 WO 2003014401A1 US 0225111 W US0225111 W US 0225111W WO 03014401 A1 WO03014401 A1 WO 03014401A1
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Prior art keywords
fnr
plant
seq
compound
candidate
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PCT/US2002/025111
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English (en)
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Adel Zayed
Robert Ascenzi
Douglas Boyes
Rao Mulpuri
Neil Hoffman
Susanne Kjemtrup
Keith Davis
Carol Hamilton
Jeffrey Woessner
Jorn Gorlach
Kenneth Phillips
Veeresh Sevala
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Paradigm Genetics, Inc.
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Priority to EP02750449A priority Critical patent/EP1414998A4/fr
Publication of WO2003014401A1 publication Critical patent/WO2003014401A1/fr
Priority to US10/770,755 priority patent/US20040248228A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0095—Oxidoreductases (1.) acting on iron-sulfur proteins as donor (1.18)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5097—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving plant cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/415—Assays involving biological materials from specific organisms or of a specific nature from plants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
    • G01N2430/20—Herbicides, e.g. DDT
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention relates generally to plant molecular biology.
  • the invention relates to methods for the identification of herbicides.
  • PSII Photosystem I
  • PSIII contains electron carriers similar to those in the R. viridis complex (pheophytin, quinones), whereas PSI contains bound Fe-S centers as stable electron acceptors. Electrons from PSI are transferred to the 2Fe-2S Fe-S protein ferredoxin, located in the chloroplast stroma. This electron carrier does not transfer electrons directly to NADP + , but rather by way of an intermediate enzyme called ferredoxin-NADP + reductase (FNR).
  • FNR ferredoxin-NADP + reductase
  • FNR is a FAD-containing enzyme that can be reduced in two single-electron steps. The first electron reduces FNR to the flavin semiquinone state; the second, to the fully reduced state, FADH 2 . FNR then transfers the two electrons to NADP + . FNR is loosely associated with the thylakoid membrane and is easily dissociated. To date there do not appear to be any publications describing lethal effects of over-expression, antisense expression or knock-out of this gene in Arabidopsis. Thus, the prior art has not suggested that FNR is essential for plant growth and development. It would be desirable to determine the utility of this enzyme for evaluating plant growth regulators, especially herbicide compounds.
  • the present inventors have discovered that antisense expression of a FNR cDNA in Arabidopsis causes developmental abnormalities, resulting in seedlings that looked pale and very stunted.
  • FNR is essential for normal seed development and growth, and can be used as a target for the identification of herbicides.
  • the present invention provides methods for the identification of compounds that inhibit FNR expression or activity, comprising: contacting a candidate compound with a FNR and detecting the presence or absence of binding between said compound and said FNR, or detecting a decrease in FNR expression or activity. The methods of the invention are useful for the identification of herbicides.
  • Fig. 1 shows the ferredoxin NADP oxidoreductase reaction.
  • binding refers to a noncovalent interaction that holds two molecules together.
  • two such molecules could be an enzyme and an inhibitor of that enzyme.
  • Noncovalent interactions include hydrogen bonding, ionic interactions among charged groups, van der Waals interactions and hydrophobic interactions among nonpolar groups. One or more of these interactions can mediate the binding of two molecules to each other.
  • cDNA means complementary deoxyribonucleic acid.
  • DCPIP refers to 2,6-dichlorophenol-indophenol.
  • dl means deionized.
  • DNA means deoxyribonucleic acid
  • ELISA means enzyme-linked immunosorbent assay.
  • FAD flavin adenine dinucleotide, a coenzyme important in various biochemical reactions. It comprises a phosphorylated vitamin B2 (riboflavin) molecule linked to the nucleotide adenine monophosphate (AMP).
  • AMP nucleotide adenine monophosphate
  • FAD is usually tightly bound to the enzyme forming a flavoprotein. It functions as a hydrogen acceptor in dehydrogenation reactions, being reduced to FADH 2 . This in turn is oxidized to FAD by the electron transport chain, thereby generating ATP (two molecules of ATP per molecule of FADH 2 ).
  • ferredoxin NADP oxidoreductase (EC 1.1.18.1) is synonymous with “FNR” and refers to an enzyme that catalyses the conversion of reduced ferredoxin and NADP to oxidized ferredoxin and NADPH, as shown in Fig. 1.
  • Fe-S refers to an iron-sulfur group.
  • herbicide refers to a compound that may be used to kill or suppress the growth of at least one plant, plant cell, plant tissue or seed.
  • GUS means ⁇ -glucouronidase.
  • HPLC high pressure liquid chromatography.
  • inhibitor refers to a chemical substance that inactivates the enzymatic activity of FNR.
  • the inhibitor may function by interacting directly with the enzyme, a cofactor of the enzyme, the substrate of the enzyme, or any combination thereof.
  • LB means Luria-Bertani media.
  • mRNA messenger ribonucleic acid
  • NADP and “NADPH” refer to nicotinamide adenine dinucleotide phosphate, a coenzyme which participates in redox reactions during the light reaction of photosynthesis.
  • High-energy reactions cause the photolysis of water, in which the hydrogen reduces NADP+ to NADPH and generates the oxygen released during photosynthesis.
  • the reduced NADPH is used in the conversion of carbon dioxide to carbohydrate during the dark reaction of photosynthesis.
  • Ni refers to nickel.
  • Ni-NTA refers to nickel sepharose.
  • PCR means polymerase chain reaction
  • the "percent (%) sequence identity" between two polynucleotide or two polypeptide sequences is determined according to the either the BLAST program (Basic Local Alignment Search Tool; Altschul and Gish (1996) Meth Enzymol 266:460-480 and Altschul (1990) J Mol Biol 275:403-410) in the Wisconsin Genetics Software Package (Devererreux et al. (1984) Nucl Acid Res 72:387), Genetics Computer Group (GCG), Madison, Wisconsin.
  • PKI plant growth inhibition
  • Plant refers to whole plants, plant organs and tissues (e.g., stems, roots, ovules, stamens, leaves, embryos, meristematic regions, callus tissue, gametophytes, sporophytes, pollen, microspores and the like) seeds, plant cells and the progeny thereof.
  • plant organs and tissues e.g., stems, roots, ovules, stamens, leaves, embryos, meristematic regions, callus tissue, gametophytes, sporophytes, pollen, microspores and the like
  • a polynucleotide may be "introduced" into a plant cell by any means, including transfection, transformation or transduction, electroporation, particle bombardment, agroinfection and the like.
  • the introduced polynucleotide may be maintained in the cell stably if it is incorporated into a non-chromosomal autonomous replicon or integrated into the plant chromosome.
  • the introduced polynucleotide may be present on an extra-chromosomal non-replicating vector and be transiently expressed or transiently active.
  • polypeptide is meant a chain of at least four amino acids joined by peptide bonds.
  • the chain may be linear, branched, circular or combinations thereof.
  • the polypeptides may contain amino acid analogs and other modifications, including, but not limited to glycosylated or phosphorylated residues.
  • PSDI photosystem II
  • RNA means ribonucleic acid.
  • SDS sodium dodecyl sulfate.
  • SDS-PAGE means sodium dodecyl sulfate - polyacrylimide gel electrophoresis.
  • binding refers to an interaction between FNR and a molecule or compound, wherein the interaction is dependent upon the primary amino acid sequence or the conformation of FNR.
  • TATA box refers to a sequence of nucleotides that serves as the main recognition site for the attachment of RNA polymerase in the promoter region of eukaryotic genes. Located at around 25 nucleotides before the start of transcription, it consists of the seven-base consensus sequence TATAAAA, and is analogous to the Pribnow box in prokaryotic promoters.
  • TLC thin layer chromatography
  • Embodiments of the Invention The present inventors have discovered that inhibition of FNR gene expression strongly inhibits the growth and development of plant seedlings. Thus, the inventors are the first to demonstrate that FNR is a target for herbicides.
  • the invention provides methods for identifying compounds that inhibit FNR gene expression or activity. Such methods include ligand binding assays, assays for enzyme activity and assays for FNR gene expression. Any compound that is a ligand for FNR, other than its substrates, reduced ferredoxin and NADP, may have herbicidal activity.
  • ligand refers to a molecule that will bind to a site on a polypeptide. The compounds identified by the methods of the invention are useful as herbicides.
  • the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) contacting a FNR with said compound; and b) detecting the presence and/or absence of binding between said compound and said FNR; wherein binding indicates that said compound is a candidate for a herbicide.
  • FNR is meant any enzyme that catalyzes the interconversion of reduced ferredoxin and NADP with oxidized ferredoxin and NADPH.
  • the FNR may have the amino acid sequence of a naturally occurring FNR found in a plant, animal or microorganism, or may have an amino acid sequence derived from a naturally occurring sequence.
  • the FNR is a plant FNR.
  • the cDNA (SEQ ID NO: 1) encoding the FNR protein or polypeptide (SEQ ID NO:2) can be found herein as well as in the TIGR database at locus Atlg30510.
  • plant FNR is meant an enzyme that can be found in at least one plant, and which catalyzes the interconversion of reduced ferredoxin and NADP with oxidized ferredoxin and NADPH.
  • the FNR may be from any plant, including both monocots and dicots.
  • the FNR is an Arabidopsis FNR.
  • Arabidopsis species include, but are not limited to, Arabidopsis arenosa, Arabidopsis bursifolia, Arabidopsis cebennensis, Arabidopsis croatica, Arabidopsis griffithiana, Arabidopsis halleri, Arabidopsis himalaica, Arabidopsis korshinskyi, Arabidopsis lyrata, Arabidopsis neglecta, Arabidopsis pumila, Arabidopsis suecica, Arabidopsis thaliana and Arabidopsis xvallichii.
  • the Arabidopsis FNR is from Arabidopsis thaliana.
  • the FNR can be from barnyard grass (Echinochloa crus-galli), crabgrass (Digitaria sanguinalis), green foxtail (Setana viridis), perennial ryegrass (Lolium perenne), hairy beggarticks (Bidens pilosa), nightshade (Solanum nigrum), smartweed (Polygonum lapathifolium), velvetleaf (Abutilon theophrasti), common lambsquarters (Chenopodium album L.), Brachiara plantaginea, Cassia occidentalis, Ipomoea aristolochiaefolia, Ipomoea purpurea, Euphorbia heterophylla,
  • Fragments of a FNR polypeptide may be used in the methods of the invention.
  • the fragments comprise at least 10 consecutive amino acids of a FNR.
  • the fragment comprises at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or at least 100 consecutive amino acids residues of a FNR.
  • the fragment is from an Arabidopsis FNR.
  • the fragment contains an amino acid sequence conserved among plant ferredoxin NADP oxidoreductases. Such conserved fragments are identified in Grima-Pettenuti et al. (1993) Plant Mol Biol 27:1085-1095 and Taveres et al. (2000), supra. Those skilled in the art could identify additional conserved fragments using sequence comparison software.
  • Polypeptides having at least 80% sequence identity with a plant FNR are also useful in the methods of the invention.
  • the sequence identity is at least 85%, more preferably the identity is at least 90%, most preferably the sequence identity is at least 95% or 99%.
  • the related FNR proteins or polypeptides (SEQ ID NO: 4, 5, 6, 8, 9, 10, 11), which have 99%, 85%, 85%, 84%, 80%, 80%, and 80% sequence identity with FNR, respectively, and their encoding cDNAs (SEQ ID NO: 3 (for AAF19753.1) and 7 (for CAB81081)), can be found herein as well as in the Genbank database as Accession numbers AAF197553.1, JS0728, S53305, CAB81081, Q41014, 023877, and T06773 for the proteins, and AC009917.2, AL161503.2 for the cDNAs, respectively.
  • the polypeptide has at least 50% of the activity of a plant FNR. More preferably, the polypeptide has at least 60%, at least 70%, at least 80% or at least 90% of the activity of a plant FNR. Most preferably, the polypeptide has at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the activity of the A. thaliana FNR protein.
  • the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) contacting said compound with at least one polypeptide selected from the group consisting of: a plant FNR, a polypeptide comprising at least ten consecutive amino acids of a plant FNR, a polypeptide having at least 85% sequence identity with a plant FNR, and a polypeptide having at least 80% sequence identity with a plant FNR and at least 50% of the activity thereof; and b) detecting the presence and/or absence of binding between said compound and said polypeptide; wherein binding indicates that said compound is a candidate for a herbicide.
  • at least one polypeptide selected from the group consisting of: a plant FNR, a polypeptide comprising at least ten consecutive amino acids of a plant FNR, a polypeptide having at least 85% sequence identity with a plant FNR, and a polypeptide having at least 80% sequence identity with a plant FNR and at least 50% of the activity thereof.
  • any technique for detecting the binding of a ligand to its target may be used in the methods of the invention.
  • the ligand and target are combined in a buffer.
  • Many methods for detecting the binding of a ligand to its target are known in the art, and include, but are not limited to the detection of an immobilized ligand- target complex or the detection of a change in the properties of a target when it is bound to a ligand.
  • an array of immobilized candidate ligands is provided. The immobilized ligands are contacted with a FNR protein or a fragment or variant thereof, the unbound protein is removed and the bound FNR is detected.
  • bound FNR is detected using a labeled binding partner, such as a labeled antibody.
  • FNR is labeled prior to contacting the immobilized candidate ligands.
  • Preferred labels include fluorescent or radioactive moieties.
  • Preferred detection methods include fluorescence correlation spectroscopy (FCS) and FCS-related confocal nanofluorimetric methods. See http://www.evotec.de/technology.
  • a compound Once a compound is identified as a candidate for a herbicide, it can be tested for the ability to inhibit FNR enzyme activity.
  • the compounds can be tested using either in vitro or cell based enzyme assays.
  • a compound can be tested by applying it directly to a plant or plant cell, or expressing it therein, and monitoring the plant or plant cell for changes or decreases in growth, development, viability or alterations in gene expression.
  • the invention provides a method for determining whether a compound identified as a herbicide candidate by an above method has herbicidal activity, comprising: contacting a plant or plant cells with said herbicide candidate and detecting the presence or absence of a decrease in the growth or viability of said plant or plant cells.
  • decrease in growth is meant that the herbicide candidate causes at least a 10% decrease in the growth of the plant or plant cells, as compared to the growth of the plants or plant cells in the absence of the herbicide candidate.
  • a decrease in viability is meant that at least 20% of the plants cells, or portion of the plant contacted with the herbicide candidate are nonviable.
  • the growth or viability will be at decreased by at least 40%. More preferably, the growth or viability will be decreased by at least 50%, 75% or at least 90% or more. Methods for measuring plant growth and cell viability are known to those skilled in the art. It is possible that a candidate compound may have herbicidal activity only for certain plants or certain plant species.
  • FNR catalyzes the irreversible or reversible reaction of reduced ferredoxin and NADP to oxidized ferredoxin and NADPH.
  • Methods for detection of reduced ferredoxin and NADP, and/or oxidized ferredoxin and NADPH, include spectrophotometry, mass spectroscopy, thin layer chromatography (TLC) and reverse phase HPLC.
  • the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) contacting a reduced ferredoxin and NADP with FNR; b) contacting said reduced ferredoxin and NADP with FNR and said candidate compound; and c) determining the concentration of oxidized ferredoxin or NADPH after the contacting of steps (a) and (b).
  • a candidate compound inhibits FNR activity, a higher concentration of the substrates (reduced ferredoxin or NADP) and a lower level of the product (oxidized ferredoxin or NADPH) will be detected in the presence of the candidate compound (step b) than in the absence of the compound (step a).
  • the FNR is a plant FNR.
  • Enzymatically active fragments of a plant FNR are also useful in the methods of the invention.
  • a polypeptide comprising at least 100 consecutive amino acid residues of a plant FNR may be used in the methods of the invention.
  • a polypeptide having at least 80%, 85%, 90%, 95%, 98% or at least 99% sequence identity with a plant FNR may be used in the methods of the invention.
  • the polypeptide has at least 80% sequence identity with a plant FNR and at least 50%, 75%, 90% or at least 95% of the activity thereof.
  • the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) contacting reduced ferredoxin and NADP with a polypeptide selected from the group consisting of: a polypeptide having at least 85% sequence identity with a plant FNR, a polypeptide having at least 80% sequence identity with a plant FNR and at least 50% of the activity thereof, and a polypeptide comprising at least 100 consecutive amino acids of a plant FNR; b) contacting said reduced ferredoxin and NADP with said polypeptide and said compound; and c) determining the concentration of oxidized ferredoxin or NADPH after the contacting of steps (a) and (b).
  • a polypeptide selected from the group consisting of: a polypeptide having at least 85% sequence identity with a plant FNR, a polypeptide having at least 80% sequence identity with a plant FNR and at least 50% of the activity thereof, and a polypeptide comprising at least 100 consecutive amino acids of a plant
  • FNR protein and derivatives thereof may be purified from a plant or may be recombinantly produced in and purified from a plant, bacteria, or eukaryotic cell culture. Preferably these proteins are produced using a baculovirus or E. coli expression system. Methods for purifying FNR may be described in Jin et al.
  • the invention also provides plant and plant cell based assays.
  • the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) measuring the expression of FNR in a plant or plant cell in the absence of said compound; b) contacting a plant or plant cell with said compound and measuring the expression of FNR in said plant or plant cell; c) comparing the expression of FNR in steps (a) and (b).
  • a reduction in FNR expression indicates that the compound is a herbicide candidate.
  • the plant or plant cell is an Arabidopsis thaliana plant or plant cell.
  • FNR FNR primary transcript or mRNA, FNR polypeptide or FNR enzymatic activity.
  • Methods for detecting the expression of RNA and proteins are known to those skilled in the art. See, for example, Current Protocols in Molecular Biology Ausubel et al., eds., Greene Publishing and Wiley-Interscience, New York, 1995. The method of detection is not critical to the invention.
  • Methods for detecting FNR RNA include, but are not limited to amplification assays such as quantitative PCR, and/or hybridization assays such as Northern analysis, dot blots, slot blots, in-situ hybridization, transcriptional fusions using a FNR promoter fused to a reporter gene, bDNA assays and microarray assays.
  • Methods for detecting protein expression include, but are not limited to, immunodetection methods such as Western blots, His Tag and ELISA assays, polyacrylamide gel electrophoresis, mass spectroscopy and enzymatic assays.
  • any reporter gene system may be used to detect FNR protein expression.
  • reporter genes include, but are not limited to, chloramphenicol acetyltransferase (Gorman et al. (1982) Mol Cell Biol 2:1104; Prost et al. (1986) Gene 45:107-111), ⁇ -galactosidase (Nolan et al. (1988) Proc Natl Acad Sci USA 55:2603-2607), alkaline phosphatase (Berger et al.
  • Chemicals, compounds or compositions identified by the above methods as modulators of FNR expression or activity can then be used to control plant growth.
  • compounds that inhibit plant growth can be applied to a plant or expressed in a plant, in order to prevent plant growth.
  • the invention provides a method for inhibiting plant growth, comprising contacting a plant with a compound identified by the methods of the invention as having herbicidal activity.
  • Herbicides and herbicide candidates identified by the methods of the invention can be used to control the growth of undesired plants, including both monocots and dicots.
  • undesired plants include, but are not limited to barnyard grass (Echinochloa crus-galli), crabgrass (Digitaria sanguinalis), green foxtail (Setana viridis), perennial ryegrass (Lolium perenne), hairy beggarticks (Bidens pilosa), nightshade (Solanum nigrum), smartweed (Polygonum lapathifolium), velvetleaf (Abutilon theophrasti), common lambsquarters (Chenopodium album L.), Brachiara plantaginea, Cassia occidentalis, Ipomoea aristolochiaefolia, Ipomoea purpurea, Euphorbia heterophylla, Setaria spp, Amaranthus retrqflexus, Sida spinosa, Xanthium strumarium and the like.
  • barnyard grass Echinochloa crus-galli
  • crabgrass Digitaria sanguinalis
  • green foxtail Setana vi
  • the "Driver” is an artificial transcription factor comprising a chimera of the DNA-binding domain of the yeast GAL4 protein (amino acid residues 147) fused to two tandem activation domains of herpes simplex virus protein VP16 (amino acid residues 413-490). Schwechheimer et al. (1998) Plant Mol Biol 36:195-204.
  • This chimeric driver is a transcriptional activator specific for promoters having GAL4 binding sites. Expression of the driver is controlled by two tandem copies of the constitutive CaMV 35S promoter.
  • the driver expression cassette was introduced into Arabidopsis thaliana by agroinfection. Transgenic plants that stably expressed the driver transcription factor were obtained.
  • a fragment, fragment or variant of an Arabidopsis thaliana cDNA corresponding to SEQ ID NO:l was ligated into the Pacl/Ascl sites of an E.coli/Agrobacterium binary vector in the antisense orientation. This placed transcription of the antisense RNA under the control of an artificial promoter that is active only in the presence of the driver transcription factor described above.
  • the artificial promoter contains four contiguous binding sites for the GAL4 transcriptional activator upstream of a minimal promoter comprising a TATA box.
  • the ligated DNA was transformed into E.coli. Kanamycin resistant clones were selected and purified. DNA was isolated from each clone and characterized by PCR and sequence analysis. The DNA was inserted in a vector that expresses the A. thaliana antisense RNA, which is complementary to a portion of the DNA of SEQ ID NO: 1. This antisense RNA is complementary to the cDNA sequence found in the TIGR database at locus Atlg30510. The coding sequence for this locus is shown as SEQ ID NO: 1. The protein encoded by these mRNAs is shown as SEQ ID NO: 2.
  • the antisense expression cassette and a constitutive chemical resistance expression cassette are located between right and left T-DNA borders.
  • the antisense expression cassettes can be transferred into a recipient plant cell by agroinfection.
  • the vector was transformed into Agrobacterium tumefaciens by electroporation. Transformed Agrobacterium colonies were isolated using chemical selection. DNA was prepared from purified resistant colonies and the inserts were amplified by PCR and sequenced to confirm sequence and orientation.
  • the antisense expression cassette was introduced into Arabidopsis thaliana wild-type plants by the following method. Five days prior to agroinfection, the primary inflorescence of Arabidopsis thaliana plants grown in 2.5 inch pots were clipped in order enhance the emergence of secondary bolts.
  • 5 ml LB broth (10 g/L Peptone, 5 g/L Yeast extract, 5 g/L NaCI, pH 7.0 plus 25 mg/L kanamycin added prior to use) was inoculated with a clonal glycerol stock of Agrobacterium carrying the desired DNA.
  • the cultures were incubated overnight at 28°C at 250 rpm until the cells reached stationary phase.
  • 200 ml LB in a 500 ml flask was inoculated with 500 ⁇ l of the overnight culture and the cells were grown to stationary phase by overnight incubation at 28°C at 250 rpm.
  • the cells were pelleted by centrifugation at 8000 rpm for 5 minutes.
  • the supernatant was removed and excess media was removed by setting the centrifuge bottles upside down on a paper towel for several minutes.
  • the cells were then resuspended in 500 ml infiltration medium (autoclaved 5% sucrose) and 250 ⁇ l/L Silwet L-77TM (84% polyalkyleneoxide modified heptamethyltrisiloxane and 16% allyloxypolyethyleneglycol methyl ether), and transferred to a one liter beaker.
  • Agrobacterium suspension so that all above ground parts were immersed and agitated gently for 10 seconds.
  • the dipped plants were then covered with a tall clear plastic dome in order to maintain the humidity, and returned to the growth room. The following day, the dome was removed and the plants were grown under normal light conditions until mature seeds were produced. Mature seeds were collected and stored desiccated at 4 °C.
  • Transgenic Arabidopsis Tl seedlings were selected. Approximately 70 mg seeds from an agrotransformed plant were mixed approximately 4: 1 with sand and placed in a 2 ml screw cap cryo vial.
  • One vial of seeds was then sown in a cell of an 8 cell flat.
  • the flat was covered with a dome, stored at 4°C for 3 days, and then transferred to a growth room.
  • the domes were removed when the seedlings first emerged.
  • the flat was sprayed uniformly with a herbicide corresponding to the chemical resistance marker plus 0.005% Silwet (50 ⁇ l/L) until the leaves were completely wetted.
  • the spraying was repeated for the following two days. Ten days after the first spraying resistant plants were transplanted to 2.5 inch round pots containing moistened sterile potting soil. The transplants were then sprayed with herbicide and returned to the growth room. These herbicide resistant plants represented stably transformed Tl plants.
  • the Tl antisense target plants from the transformed plant lines obtained in Example 4 were crossed with the Arabidopsis transgenic driver line described above.
  • the resulting FI seeds were then subjected to a PGI plate assay to observe seedling growth over a 2-week period. Seedlings were inspected for growth and development.
  • the transgenic plant lines containing the antisense construct exhibited significant developmental abnormalities during early development.
  • the antisense expression of this gene resulted in significantly impaired growth in the two antisense lines examined, indicating that this gene represents an essential gene for normal plant growth and development.
  • the transgenic lines containing the antisense construct for ferredoxin NADP oxidoreductase exhibited significant seedling abnormalities. Two of ten seedlings from the first transgenic line and one of ten seedlings from the second transgenic line were pale and very stunted in growth. Thus, ferredoxin NADP oxidoreductase is essential for normal plant growth and development.
  • the following protocol may be employed to obtain the purified FNR protein.
  • a FNR gene can be cloned into E. coli (pET vectors-Novagen), Baculovirus (Pharmingen) and Yeast (Invitrogen) expression vectors containing His/fusion protein tags. Evaluate the expression of recombinant protein by SDS-PAGE and Western blot analysis.
  • Purification Purify recombinant protein by Ni-NTA affinity chromatography (Qiagen).
  • Example 7 Assays for Testing Inhibitors or Candidates for Inhibition of FNR Activity
  • the enzymatic activity of FNR may be determined in the presence and absence of candidate inhibitors in a suitable reaction mixture, such as described by any of the following known assay protocols:
  • the FNR diaphorase activity measured with DCPIP as an electron acceptor, can be taken as a measure of the ability of the enzyme to be reduced by the pyridine nucleotide, which acts as electron donor, as described in Martinez -Julvez et al. (2001) JBiol Chem 276: 27498 - 510 (PMID: 11342548).
  • the enzymatic activity of this enzyme can be monitored by the change in absorbance at 340nm or change in fluorescence at ex. 340/em. 460 due to the formation of NADPH.

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Abstract

Les inventeurs ont découvert que l'oxydoréductase de ferredoxine NADP (FNR) est essentielle pour la croissance des plantes. Plus particulièrement, l'inhibition de l'expression génique de FNR chez des semis a produit des plantules d'aspect pâle et rabrougri. Ainsi FNR peut être utilisée comme cible pour l'identification d'herbicides. L'invention présente donc des méthodes d'identification de composés qui inhibent l'expression ou l'activité de FNR. Ces méthodes consistent à placer un composé au contact d'une FNR afin de détecter la présence et/ou l'absence de liaison entre ledit composé et ladite FNR; ou de détecter une réduction de l'expression ou de l'activité de FNR. Les méthodes de l'invention sont utiles pour l'identification d'herbicides.
PCT/US2002/025111 2001-08-06 2002-08-06 Methodes permettant d'identifier des inhibiteurs d'expression ou d'activite d'oxydoreductase de ferredoxine nadp chez des plantes WO2003014401A1 (fr)

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EP02750449A EP1414998A4 (fr) 2001-08-06 2002-08-06 Methodes permettant d'identifier des inhibiteurs d'expression ou d'activite d'oxydoreductase de ferredoxine nadp chez des plantes
US10/770,755 US20040248228A1 (en) 2002-08-06 2004-02-03 Methods for the identification of inhibitors of ferredoxin NADP oxidoreductase expression or activity in plants

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6124242A (en) * 1998-06-26 2000-09-26 Basf Aktiengesellschaft Herbicidal compositions and processes based on ferrodoxin:NADP reductase inhibitors

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6124242A (en) * 1998-06-26 2000-09-26 Basf Aktiengesellschaft Herbicidal compositions and processes based on ferrodoxin:NADP reductase inhibitors

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [online] April 1999 (1999-04-01), BOWSHER: "Ferredoxin-NADP+ reductase (EC 1.18.1.2) - garden pea (fragment)", XP002959280, Database accession no. (TO6773) *
DATABASE GENBANK [online] March 2001 (2001-03-01), HUNTER ET AL.: "Hypothetical protein F26G16.13 - arabidopsis thaliana", XP002959274, Database accession no. (B86430) *
DATABASE GENBANK [online] May 2000 (2000-05-01), AOKI ET AL.: "Ferredoxin NADP+ reductase enzyme", XP002959275, accession no. EMBL Database accession no. (Q9SB29) *
DATABASE GENBANK [online] November 1996 (1996-11-01), RITCHIE ET AL.: "Ferredoxin NADP+ reductase precursor (fragment)", XP002959276, accession no. EMBL Database accession no. (Q41736) *
DATABASE GENBANK [online] October 2000 (2000-10-01), SPIEGEL ET AL.: "Ferredoxin NADP+ reductase-like protein", XP002959277, accession no. EMBL Database accession no. (Q9MOV6) *
DATABASE SWISSPROT_40 [online] December 1998 (1998-12-01), AOKI ET AL.: "Ferredoxin-NADP+ reductase, embryo isozyme, chloroplast precursor", XP002959279, Database accession no. (O23877) *
DATABASE SWISSPROT_40 [online] December 1998 (1998-12-01), BOWSHER ET AL.: "Ferredoxin-NADP+ reductase, root isozyme, chloroplast precursor", XP002959278, Database accession no. (Q41014) *
See also references of EP1414998A4 *

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