WO2002046451A2 - Identification d'inhibiteurs de l'expression ou de l'activite de la cysteine synthase dans les plantes - Google Patents

Identification d'inhibiteurs de l'expression ou de l'activite de la cysteine synthase dans les plantes Download PDF

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WO2002046451A2
WO2002046451A2 PCT/US2001/050890 US0150890W WO0246451A2 WO 2002046451 A2 WO2002046451 A2 WO 2002046451A2 US 0150890 W US0150890 W US 0150890W WO 0246451 A2 WO0246451 A2 WO 0246451A2
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plant
cysteine synthase
compound
cysteine
candidate
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PCT/US2001/050890
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WO2002046451A3 (fr
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Andreas Kloti
Jeffrey Woessner
Adel Zayed
Douglas Boyes
Keith Davis
Carol Hamilton
Robert Ascenzi
Neil Hoffman
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Paradigm Genetics, Inc.
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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • 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
    • C12Q1/527—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving lyase

Definitions

  • the invention relates generally to plant molecular biology.
  • the invention relates to methods for the identification of herbicides.
  • Cysteine synthase (EC 4.2.99.8) is an enzyme involved in the final step of cysteine biosynthesis and catalyzes the formation of the amino acid L-cysteine from O-acetyl-L-serine (OAS) and inorganic sulfide. Cysteine synthase genes have been characterized from a number of plants, and in most cases consist of a small multigene family. At least ten different cysteine synthase genes have been identified in Arabidopsis thaliana.
  • the Arabidopsis cysteine synthase gene, AtcysCl encodes a mitochodrial protein having both cysteine synthase activity (EC 4.2.99.8) and ⁇ - cyanoalanine synthase activity (EC 4.4.1.9). Yamaguchi et al. (2000) Plant Cell Physiol 4P ⁇ 65-476.
  • cysteine synthase activity is essential for plant growth and development. Rather, there have been several reports that inhibition of cysteine synthase expression had no effect on plant growth.
  • constitutive antisense RNA expression of a spinach cytoplasmic cysteine synthase (CSase A) cDNA in tobacco plants results in decreased cysteine synthase activity, but did not damage transgenic plants.
  • cysteine synthase is essential for plant growth and development.
  • the present inventors have discovered that antisense expression of cysteine synthase cDNA in Arabidopsis results in the production of white seeds that fail to germinate.
  • cysteine synthase is essential for normal seed development and germination, and can be used as a target for the identification of herbicides.
  • the present invention provides methods for the identification of compounds that inhibit cysteine synthase expression or activity, comprising: contacting a compound with a cysteine synthase and detecting the presence and/or absence of binding between said compound and said cysteine synthase, or detecting a decrease in cysteine synthase expression or activity.
  • the methods of the invention are useful for the identification of herbicides.
  • 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.
  • Cysteine is described as compound No. 2850 in The Merck Index, 12 th Ed.,
  • cysteine is synonymous with L-cysteine, Cys, ⁇ -mercaptoalanine, (R)-2-amino-3- mercaptopropanoic acid, 2-amino-3-mercaptopropionic acid, -amino- ⁇ - thiolpropionic acid, half-cysteine and thioserine.
  • cysteine synthase refers to any enzyme that catalyzes the formation of amino acid L-cysteine and acetate from O-acetylserine and H 2 S.
  • cysteine synthase C Arabidopsis enzyme referred to as referred to as cysteine synthase C (AtCS-C, Csase C) and encoded by AtcysCl, which has both beta-cyanoalanine synthase activity and cysteine synthase activity, is included within the meaning of "cysteine synthase”.
  • 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.
  • inhibitor refers to a chemical substance that inactivates the enzymatic activity of cysteine synthase.
  • the inhibitor may function by interacting directly with the enzyme, a cofactor of the enzyme, the substrate of the enzyme, or any combination thereof.
  • 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.
  • 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
  • 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.
  • binding refers to an interaction between cysteine synthase and a molecule or compound, wherein' the interaction is dependent upon the primary amino acid sequence or the conformation of cysteine synthase.
  • sulfide refers to inorganic sulfide or S 2 ⁇
  • the sulfide useful in the methods of the invention may be supplied by chemicals such as, but not limited to, hydrogen sulfide, sodium sulfide, potassium sulfide, rubidium sulfide, cesium sulfide, ammonium sulfide, beryllium sulfide, magnesium sulfide, calcium sulfide, strontium sulfide, barium sulfide and the like.
  • the present inventors have discovered that inhibition of cysteine synthase gene expression strongly inhibits the development and germination of plant seeds.
  • the inventors are the first to demonstrate that cysteine synthase is a target for herbicides.
  • the invention provides methods for identifying compounds that inhibit cysteine synthase gene expression or activity. Such methods include ligand binding assays, assays for enzyme activity and assays for cysteine synthase gene expression. Any compound that is a ligand for cysteine synthase, other than its substrates, O-acetyl-L-serine and sulfide, or cofactors 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 for the modulation of plant growth and development.
  • the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) contacting a cysteine synthase with said compound; and b) detecting the presence and/or absence of binding between said compound and said a cysteine synthase; wherein binding indicates that said compound is a candidate for a herbicide.
  • cysteine synthase any enzyme that catalyzes the formation of cysteine from O-acetyl-serine and sulfide.
  • the cysteine synthase may have the amino acid sequence of a naturally occuring cysteine synthase found in a plant, animal or microorganism, or may have an amino acid sequence derived from a naturally occuring sequence.
  • the cysteine synthase is a plant cysteine synthase.
  • plant cysteine synthase is meant an enzyme that can be found in at least one plant, and which catalyzes the formation of cysteine from O-acetyl-serine and sulfide.
  • the cysteine synthase may be from any plant, including both monocots and dicots.
  • the cysteine synthase is an Arabidopsis cysteine synthase.
  • 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 wallichii.
  • the Arabidopsis cysteine synthase is from Arabidopsis thaliana, more preferably from Arabidopsis thaliana strain Columbia.
  • cysteine synthase cDNAs and genes from a variety of other plants and organisms are publicly available. See, for example, gb I BE611445, gb
  • AI822708 Mesembryanthemum crystallinum cysteine synthase cDNA
  • cysteme synthase cDNA (Chlamydomaonas reinhardtii cysteine synthase cDNA). All of the above cysteme synthase cDNA sequences may be used as probes to isolate cysteine synthase cDNAs or genes from additional organisms, and to synthesize cysteine synthase polypeptides.
  • the cysteine synthase is from barnyard grass (Echinochloa crus-gall ⁇ ), 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 LA), Brachiara plantaginea, Cassia occidentalis, Ipomoea aristolochiaefolia, Lpomoea purpurea, Euphorbia heterophylla, Setaria spp, Amaranthus retro ⁇ exus, Sida spinosa, Xanthium strumarium and the like.
  • Fragments of a plant cysteine synthase may be used in the methods of the invention.
  • the fragments comprise at least 10 consecutive amino acids of a plant cysteme synthase.
  • 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 plant cysteine synthase.
  • the fragment comprises at least 10 consecutive amino acid residues of S ⁇ Arabidopsis cysteine synthase.
  • the fragment contains an amino acid sequence conserved among plant cysteine synthases. Such conserved fragments are identified in Rolland et al. (1993) Biochem J 293:829-833. Those skilled in the art could identify additional conserved fragments using sequence comparison software.
  • Polypeptides having at least 80% sequence identity with a plant cysteine synthase 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%.
  • the polypeptide has at least 50% of the activity of a plant cysteine synthase. More preferably, the polypeptide has at least 60%, at least 70%), at least 80% or at least 90% of the activity of a plant cysteine synthase.
  • 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 'Columbia' cysteine synthase of SEQ ID NO:6.
  • the activity of the polypeptide is compared to the activity of the Arabidopsis cysteine synthase polypeptide.
  • cysteine synthase activity refers to the ability to convert O-acetyl-L-serine and sulfide to L-cysteine and acetate. Methods for measuring cysteine synthase activity are known in the art and are described herein.
  • 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 cysteine synthase, a polypeptide comprising at least ten consecutive amino acids of a plant cysteine synthase, a polypeptide having at least 85% sequence identity with a plant cysteine synthase, and a polypeptide having at least 80%) sequence identity with a plant cysteine synthase 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 cysteine synthase, a polypeptide comprising at least ten consecutive amino acids of a plant cysteine synthase, a polypeptide having at least 85% sequence identity with a plant cysteine
  • 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.
  • Polypeptides and proteins that can reduce non-specific binding such as BS A or protein extracts from cells that do not produce the target, may be included in binding assay.
  • 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 cysteine synthase protein or a fragment or variant thereof, the unbound protein is removed and the bound cysteine synthase is detected.
  • bound cysteine synthase is detected using a labeled binding partner, such as a labeled antibody.
  • cysteine synthase 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 cysteine synthase 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.
  • the herbicide candidate causes at least a 20% 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.
  • the ability of a compound to inhibit cysteine synthase activity can be detected using in vitro enzymatic assays in which the disappearance of a substrate or the appearance of a product is directly or indirectly detected.
  • Cysteine synthase converts O-acetyl-L-serine and sulfide to cysteine and acetate.
  • Methods for detection of these substrates and products such as spectrophotometry, mass spectroscopy, thin layer chromatography (TLC), reverse phase HPLC and sulfide ion selective electrode assays are known to those skilled in the art.
  • cysteine can be measured by complexing with red ninhydrin (Gaitonde (1967) Biochem J 104:627-633), derivitaization of the thiol group and identification by HPLC (Fahey et al. (1987) Meth Enzymol 143:85-96; Cooper et al. (1987) Meth Enzymol 743:141-143), determination of pyruvic acid after coupling with cysteine desulphydrase (Wedding (1987) Meth Enzymol 143:29-31) and by protein binding assays (Smith et al. (1987) Meth Enzymol 745:144-148).
  • cysteine synthase activity is measured using a ninhydrin assay in which cysteine concentration is measured by quantitating red ninhydrin complex at 550-560 nm.
  • a ninhydrin assay in which cysteine concentration is measured by quantitating red ninhydrin complex at 550-560 nm.
  • the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) contacting O-acetyl-L-serine and sulfide with cysteine synthase; b) contacting O-acetyl-L-serine and sulfide with cysteine synthase and said compound; and c) determining the concentration of cysteme, acetate, O-acetyl-L- serine and/or sulfide after the contacting of steps (a) and (b).
  • a candidate compound inhibits cysteine synthase activity, a higher concentration of the substrates (O-acetyl-L-serine and sulfide) and a lower level of the products (cysteine and acetate) will be detected in the presence of the candidate compound (step b) than in the absence of the compound (step a).
  • the cysteine synthase is a plant cysteine synthase.
  • Enzymatically active fragments of a plant cysteine synthase are also useful in the methods of the invention.
  • a polypeptide comprising at least 100 consecutive amino acid residues of a plant cysteine synthase may be used in the methods of the invention.
  • polypeptide having at least 80%, 85%, 90%, 95%, 98% or at least 99% sequence identity with a plant cysteine synthase may be used in the methods of the invention.
  • polypeptides having at least 80% sequence identity with at least 15 consecutive amino acid residues of a plant cysteine synthase are also useful in the methods of the invention.
  • the polypeptide has at least 80% sequence identity with a plant cysteine synthase 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 O-acetyl-L-serine and sulfide with a polypeptide selected from the group consisting of: a polypeptide having at least 85% sequence identity with a plant cysteine synthase, a polypeptide having at least
  • a candidate compound inhibits cysteine synthase activity, a higher concentration of the substrate (O-acetyl-L-serine and sulfide) and a lower level of the product (cysteine and acetate) will be detected in the presence of the candidate compound (step b) than in the absence of the compound (step a).
  • cysteine synthase 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.
  • these proteins are produced using a baculovirus or E. coli expression system. See, for example, Saito et al. (1992) Proc Natl Acad Sci 5P:8078-8082.
  • Methods for the purification of cysteine synthase from potato tubers, bell pepper, spinach and Salmonella typhimurium are described in Ishizawa et al. (1991) Plant and Cell Physiol 41 :200- 208; Romer 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 cysteine synthase 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 cysteine synthase in said plant or plant cell; c) comparing the expression of cysteine synthase in steps (a) and (b).
  • a reduction in cysteine synthase expression indicates that the compound is a herbicide candidate.
  • the plant or plant cell is an Arabidopsis thaliana plant or plant cell.
  • cysteine synthase can be measured by detecting cysteine synthase primary transcript or mRNA, cysteine synthase polypeptide or cysteine synthase 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- erscience, New York, 1995. The method of detection is not critical to the invention.
  • Methods for detecting cysteine synthase 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 cysteine synthase promoter fused to a reporter gene, bDNA assays and microarray assays.
  • amplification assays such as quantitative PCR
  • hybridization assays such as Northern analysis, dot blots, slot blots, in-situ hybridization, transcriptional fusions using a cysteine synthase 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 cysteine synthase protein expression.
  • a polynucleotide encoding a reporter protein is fused in frame with cysteine synthase, so as to produce a chimeric polypeptide.
  • Methods for using reporter systems are known to those skilled in the art. Examples of reporter genes include, but are not limited to, chloramphenicol acetylfransferase (Gorman et al.
  • Chemicals, compounds or compositions identified by the above methods as modulators of cysteine synthase 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 retroflexus, Sida spinosa, Xanthium strumarium and the like.
  • the plates transferred into a growth chamber with a day and night temperature of 22 and 20 °C, respectively, 65% humidity and a light intensity of A 00 ⁇ -E m "2 s "1 supplied over 16 hour day period.
  • 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 3(5:195-204.
  • 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.
  • Portions of two Arabidopsis thaliana cysteine synthase cDNAs were ligated into the Pad /Ascl sites of the E.coli/Agrobacterium binary vector PGT3.2 in the antisense orientation. This placed transcription of the cysteine synthase antisense RNAs 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. pPG237 expresses an A.
  • thaliana cysteine synthase antisense RNA that is complementary to 761/772 nucleotides of the AtcysCl mRNA (nucleotides 1348 to 578 of gb
  • pPG244 expresses an A.
  • thaliana cysteine synthase antisense RNA that is complementary to 554/556 nucleotides of the Atoacly mRNA (nucleotides 1226 to 671 of emb
  • the antisense expression cassette and a constitutive barnase 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.
  • pPG237 and pPG244 were transformed into Agrobacterium tumefaciens by electroporation. Transformed Agrobacterium colonies were isolated using Basta selection. DNA was prepared from purified Basta resistant colonies and the inserts were amplified by PCR and sequenced to confirm sequence and orientation. The clones were stored as a frozen glycerol stock.
  • the cysteine synthase target expression cassettes were 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.
  • Yeast extract 5 g L NaCl, pH 7.0 plus 25 mg/L kanamycin added prior to use
  • 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
  • the previously clipped Arabidopsis plants were dipped into the Agrobacterium suspension so that all above ground parts were immersed and agitated gently for 10 seconds. The dipped plants were then cover 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 using glufosinate treatment. 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.
  • the surface of the seeds was sterilized using the chlorine gas method. Briefly, the open vials were placed in a vacuum desiccator in a safety hood. A glass beaker containing 200 ml 5.25% sodium hypochlorite solution was placed in the desiccator. Two ml concentrated HC1 was added to the hypochlorite solution and the cover was placed on the desiccator. Vacuum was applied briefly to seal the dessicator, and the seeds were left in the desiccator overnight.
  • the cysteine synthase target plants from the transfonned 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 daily for growth and development. During this period, approximately half of seeds derived from one of the pPG237 cysteine synthase antisense target lines developed abnormal cotyledons and showed reduced root growth. This pPG237 line also showed a 76% reduction in total leaf area and a 73% reduction in root length as compared to wild-type plants.
  • cysteine synthases are essential genes for normal plant growth and development.
  • progeny derived from other lines selected from the pPG237 and pPG244 transformations did not exhibit any abnormal phenotypes is not unexpected since it well known that antisense expression does not work equally well in all independently transformed lines containing the same construct.
  • the enzymatic activity of the cysteine synthase of SEQ ID NO:6 is determined in the presence and absence of candidate inhibitors in a 0.5 ml reaction mixture containing 50 mM potassium phosphate, pH 8.0, 4 m-M sodium sulfide, 12.5 mM O- acetyl-L-serine and 10 ⁇ g cysteine synthase.
  • the reaction mixture is incubated at 30oC for thirty minutes and terminated by the addition of 0.1 ml 7.5% TCA.
  • the amount of cysteine in the resulting mixture is determined spectrophotometrically at 560 nm by the acid-ninhydrin method of Gaitonde (1967) Biochem J 104:627-633.

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Abstract

On a découvert que la cystéine synthase était essentielle à la croissance des plantes. Plus particulièrement, l'inhibition de l'expression du gène de la cystéine synthase dans les semis entraîne de graves anomalies du développement. C'est pourquoi la cystéine synthase peut être utilisée comme cible pour l'identification d'herbicides. Par voie de conséquence, la présente invention concerne des méthodes permettant d'identifier des composés qui inhibent l'expression ou l'activité de la cystéine synthase. Ces méthodes consistent à : mettre un composé en contact avec une cystéine synthase et détecter la présence et/ou l'absence de liaison entre ledit composé et ladite cystéine synthase, ou bien détecter une baisse de l'expression ou de l'activité de la cystéine synthase. Les méthodes selon l'invention conviennent bien pour l'identification d'herbicides.
PCT/US2001/050890 2000-10-26 2001-10-25 Identification d'inhibiteurs de l'expression ou de l'activite de la cysteine synthase dans les plantes WO2002046451A2 (fr)

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GB2392444A (en) * 2002-08-29 2004-03-03 Syngenta Participations Ag Nucleic acid molecules encoding proteins essential for plant growth and development and uses thereof
FR2854902A1 (fr) * 2003-05-14 2004-11-19 Metabolic Explorer Sa Microorganisme a activite cysteine synthase modifiee et procede de preparation de la cysteine
US7745195B2 (en) 2003-02-18 2010-06-29 Metabolic Explorer Method for the preparation of an evolved microorganism for the creation or the modification of metabolic pathways
CN111218525A (zh) * 2020-03-30 2020-06-02 中国农业科学院棉花研究所 棉花纤维品质相关的GhCYSb基因SNP标记及其应用

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2392444A (en) * 2002-08-29 2004-03-03 Syngenta Participations Ag Nucleic acid molecules encoding proteins essential for plant growth and development and uses thereof
US7745195B2 (en) 2003-02-18 2010-06-29 Metabolic Explorer Method for the preparation of an evolved microorganism for the creation or the modification of metabolic pathways
FR2854902A1 (fr) * 2003-05-14 2004-11-19 Metabolic Explorer Sa Microorganisme a activite cysteine synthase modifiee et procede de preparation de la cysteine
CN111218525A (zh) * 2020-03-30 2020-06-02 中国农业科学院棉花研究所 棉花纤维品质相关的GhCYSb基因SNP标记及其应用
CN111218525B (zh) * 2020-03-30 2022-08-05 中国农业科学院棉花研究所 棉花纤维品质相关的GhCYSb基因SNP标记及其应用

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