WO2002068606A2 - Procedes d'identification d'herbicides et modulation de la croissance des vegetaux - Google Patents

Procedes d'identification d'herbicides et modulation de la croissance des vegetaux Download PDF

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WO2002068606A2
WO2002068606A2 PCT/US2002/006162 US0206162W WO02068606A2 WO 2002068606 A2 WO2002068606 A2 WO 2002068606A2 US 0206162 W US0206162 W US 0206162W WO 02068606 A2 WO02068606 A2 WO 02068606A2
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seq
polypeptide
plant
set forth
compound
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PCT/US2002/006162
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WO2002068606A3 (fr
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Keith Davis
Robert Ascenzi
Douglas Boyes
Neil Hoffman
Adel Zayed
Kenneth Phillips
Jeffrey Woessner
Jorn Gorlach
Carol Hamilton
Rao Mulpuri
Susanne Kjemtrup
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Paradigm Genetics, Inc.
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Priority to AU2002240557A priority Critical patent/AU2002240557A1/en
Publication of WO2002068606A2 publication Critical patent/WO2002068606A2/fr
Publication of WO2002068606A3 publication Critical patent/WO2002068606A3/fr

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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
    • G01N33/5097—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving plant cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • 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
    • 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
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • C12N15/8289—Male sterility
    • 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 generally to plant molecular biology.
  • the invention relates to methods for the identification ofherbicides.
  • the present inventors have discovered that antisense expression of a portion of the cDNAs of SEQ ID NO:3, 5, 7, and 9 individually in Arabidopsis causes failure to germinate, antisense expression of a portion ofthe cDNA of SEQ ID NO:13 causes significant seed and seedling germination abnormalities, and antisense expression of a portion ofthe cDNAs of SEQ ID NO:l and 11 individually causes developmental abnormalities and reduced growth.
  • the polypeptides encoded by the cDNAs of SEQ ID NO:l, 3, 5, 7, 9, 11 and 13 are essential for normal plant development and growth, and can each be used as a target for the identification ofherbicides.
  • the present invention provides a method for the identification of herbicide candidates, comprising: contacting a candidate compound with a polypeptide comprising the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14 or a polypeptide having at least 80% sequence identity with the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14 and detecting the presence or absence of binding between said compound and said polypeptide, wherein binding indicates that the candidate compound is a herbicide candidate.
  • the invention provides a method for the identification of herbicide candidates, comprising: contacting a plant cell with a candidate compound and detecting an increase or a decrease in the amount of a protein or mRNA selected from the group consisting of: the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14, or a polypeptide having at least 80% sequence identity with the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14, and an mRNA encoding a polypeptide having at least 80% sequence identity with the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14, wherein an alteration in the amount of protein or mRNA indicates that the candidate compound is a herbicide candidate.
  • Herbicide candidates identified by these methods can be confirmed as having herbicidal activity using conventional herbicide assays. The methods ofthe invention are useful for the identification ofherbicides.
  • the invention provides a method for identifying a compound as a herbicide, comprising: a) selecting a compound that binds to the polypeptide selected from the group consisting of: the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14 and a polypeptide having at least 80% sequence identity with the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14; and b) contacting a plant with said compound to confirm herbicidal activity.
  • the invention provides a method for the inhibition of plant growth or the modulation of plant development, comprising expressing antisense RNA specific for a polynucleotide encoding a polypeptide having at least 80% sequence identity with SEQ ID NO:2, 4, 6, 8, 10, 12 or 14 in a plant or plant tissue.
  • Antisense molecules, expression vectors, transformed plant cells and transgenic plants are also provided.
  • antisense refers to a nucleic acid comprising a polynucleotide which is sufficiently complementary to all or a portion of a gene, primary transcript or processed mRNA, so as to interfere with expression of the endogenous gene.
  • binding refers to a covalent or 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.
  • “Complementary" polynucleotides are those which are capable of base pairing according to the standard Watson-Crick complementarity rules. Specifically, purines will base pair with pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. It is understood that two polynucleotides may hybridize to each other even if they are not completely complementary to each other, provided that each has at least one region that is substantially complementary to the other.
  • 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.
  • herbicidally effective amount is meant an amount of a chemical or composition sufficient to kill a plant or decrease plant growth and/or viability by at least 10%. More preferably, the growth or viability will be decreased by 25%, 50%, 75%, 80%, 90%, or more.
  • high stringency hybridization conditions refers to hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37°C, and a final wash in 0.1 x SSC at 60°C.
  • Methods for nucleic acid hybridizations are described in Meinkoth and Wahl (1984) Anal Biochem 138: 267 - 284 (PMID: 6204550); Current Protocols in Molecular Biology, Chapter 2, Ausubel et al. Eds., Greene Publishing and Wiley-Interscience, New York, 1995; and Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization with Nucleic Acid Probes, Part I, Chapter 2, Elsevier, New York, 1993.
  • inhibitor refers to a chemical substance that inactivates or decreases the expression or the activity of he polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14, or a polypeptide having at least 80% sequence identity with the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14.
  • 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.
  • an "isolated polynucleotide” is a polynucleotide that is substantially free ofthe nucleic acid sequences that normally flank the polynucleotide in its naturally occurring replicon.
  • a cloned polynucleotide is considered isolated.
  • a polynucleotide is considered isolated if it has been altered by human intervention, or placed in a locus or location that is not its natural site, or if it is introduced into cell by agroinfection.
  • isolated are: naturally-occurring chromosomes (such as chromosome spreads), artificial chromosome libraries, genomic libraries, and cDNA libraries that exist either as an in vitro nucleic acid preparation or as a transfected transformed host cell preparation, wherein the host cells are either an in vitro heterogeneous preparation or plated as a heterogeneous population of single colonies. Also specifically excluded are the above libraries wherein a specified polynucleotide makes up less than 5% ofthe number of nucleic acid inserts in the vector molecules. Further specifically excluded are whole cell genomic DNA or whole cell RNA preparations (including said whole cell preparations which are mechanically sheared or enzymatically digested).
  • the above whole cell preparations as either an in vitro preparation or as a heterogeneous mixture separated by electrophoresis (including blot transfers ofthe same) wherein the polynucleotide ofthe invention has not further been separated from the heterologous polynucleotides in the electrophoresis medium (e.g., further separating by excising a single band from a heterogeneous band population in an agarose gel or nylon blot).
  • male tissue is meant the tissues of a plant that are directly involved or supportive ofthe reproduction ofthe male gametes. Such tissues include pollen tapetum, anther, tassel, pollen mother cells and microspores.
  • a "male tissue- preferred” or “male tissue-specific” promoter will be expressed predominantly in one or more male tissues. It is possible that a male tissue preferred promoter will be expressed in non-male tissues, however, expression will usually be at a lower level than in male tissues.
  • nucleic acid and “polynucleotide” refer to RNA or DNA that is linear or branched, single or double stranded, or a hybrid thereof. The term also encompasses RNA/DNA hybrids. Less common bases, such as inosine, 5- methylcytosine, 6-methyladenine, hypoxanthine and others can also be used for antisense, dsRNA and ribozyme pairing. For example, polynucleotides which contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression.
  • the antisense polynucleotides and ribozymes can consist entirely of ribonucleotides, or can contain mixed ribonucleotides and deoxyribonucleotides.
  • the polynucleotides ofthe invention may be produced by any means, including genomic preparations, cDNA preparations, in vitro synthesis, RT-PCR and in vitro or in vivo transcription.
  • operably linked is meant that a polynucleotide is functionally linked to a promoter, so that the transcription ofthe polynucleotide can be initiated from the promoter.
  • the "percent (%) sequence identity" between two polynucleotide or two polypeptide sequences is determined according to the BLAST program (Basic Local Alignment Search Tool, Altschul and Gish (1996) Meth Enzymol 266: 460 - 480 (PMID: 8743700); Altschul (1990) J Mol Biol 215: 403 - 410 (PMID: 2231712)) in the Wisconsin Genetics Software Package (Devereux et al. (1984) Nucl Acid Res 12: 387-95 (PMID: 6546423)), Genetics Computer Group (GCG), Madison, Wisconsin (NCBI, Version 2.0.11, default settings). It is understood that for the purposes of determining sequence identity when comparing a DNA sequence to an RNA sequence, a thymine nucleotide is equivalent to an uracil nucleotide.
  • 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.
  • probe can have no more than an additional 10 nucleic acid residues at either end of a polynucleotide having a defined sequence.
  • purified does not require absolute purity; rather, it is intended as a relative definition. As an example, purification from 0.1 % concentration to 10 % concentration is two orders of magnitude.
  • cDNA clones isolated from a cDNA library have been conventionally purified to electrophoretic homogeneity.
  • the sequences obtained from these clones could not be obtained directly either from the library or from total human DNA.
  • the cDNA clones are not naturally occurring as such, but rather are obtained via manipulation of a partially purified naturally occurring substance (messenger RNA).
  • the conversion of mRNA into a cDNA library involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection.
  • cDNA synthetic substance
  • purified is further used herein to describe a polypeptide or polynucleotide ofthe invention which has been separated from other compounds including, but not limited to, polypeptides or polynucleotides, carbohydrates, lipids, etc.
  • purified may be used to specify the separation of monomeric polypeptides ofthe invention from oligomeric forms such as homo- or hetero- dimers, trimers, etc.
  • purified may also be used to specify the separation of covalently closed polynucleotides from linear polynucleotides.
  • Polypeptide and polynucleotide purity, or homogeneity is indicated by a number of means well known in the art, such as agarose or polyacrylamide gel electrophoresis of a sample, followed by visualizing a single band upon staining the gel. For certain purposes higher resolution can be provided by using HPLC or other means well known in the art.
  • recombinant polynucleotide refers to a polynucleotide that has been altered, rearranged or modified by genetic engineering. Examples include any cloned polynucleotide, and polynucleotides that are linked or joined to heterologous sequences. Two polynucleotide sequences are heterologous if they are not naturally found joined together. The term recombinant does not refer to alterations to polynucleotides that result from naturally occurring events, such as spontaneous mutations.
  • Ribozyme is meant a catalytic RNA-based enzyme capable of targeting and cleaving particular base sequences in both DNA and RNA.
  • Ribozymes comprise a polynucleotide sequence that is complementary to a portion of a target nucleic acid and a catalytic region that cleaves the target nucleic acid. Ribozymes can be designed that specifically pair with and inactivate a target RNA by catalytically cleaving the RNA at a targeted phosphodiester bond. Methods for making and using ribozymes are known to those skilled in the art. See, for example, U.S. patents 6,025,167; 5,773,260 and 5,496,698, the contents of which are incorporated by reference, and Haseloff and Gerlach (1988) Nature 334: 586 - 591 (PMID: 2457170).
  • binding refers to an interaction between the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14, a polypeptide having at least 80% sequence identity with the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14, or a polypeptide comprising at least 10 consecutive amino acid residues ofthe polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14, and a molecule or compound, wherein the interaction is dependent upon the primary amino acid sequence or the conformation of said polypeptide.
  • Transform refers to the introduction of a polynucleotide
  • Transformation may be accomplished by a variety of methods, including, but not limited to, agroinfection, electroporation, particle bombardment, and the like. This process may result in transient or stable (constitutive or regulated) expression of the transformed polynucleotide.
  • stably transformed is meant that the sequence of interest is integrated into a replicon in the cell, such as a chromosome or episome. Transformed cells, tissues and plants encompass not only the end product of a transformation process, but also the progeny thereof which retain the polynucleotide of interest.
  • transgenic refers to any plant, plant cell, callus, plant tissue or plant part, that contains all or part of at least one recombinant polynucleotide. In many cases, all or part ofthe recombinant polynucleotide is stably integrated into a chromosome or stable extra-chromosomal element, so that it is passed on to successive generations.
  • the present inventors have discovered that antisense expression of an RNA complementary to a portion ofthe cDNA of SEQ ID NO:l, 3, 5, 7, 9, 11 or 13 strongly inhibits the growth and development of Arabidopsis seedlings.
  • the cDNA of SEQ ID NO:l, 3, 5, 7, 9, 11 and 13 encode the polypeptide of, respectively, SEQ ID NO:2, 4, 6, 8, 10, 12 and 14.
  • SEQ ID NO:2, 4, 6, 8, 10, 12 and 14 have been reported in the prior art as the following database accession numbers.
  • SEQ ID NO:2 can be found in the TIGR database at At5g48920 (also found at TIGR K19E20_3).
  • SEQ ID NO:4 can be found in the TIGR database at Atlg50660 (also found at GenBank AC079279).
  • SEQ ID NO:6 can be found in the TIGR database as At4g22640 (also found at GenBank 7269071).
  • SEQ ID NO:8 can be found in the TIGR database at At5g04850 (also found at GenBank 2618605).
  • Two proteins from Oryza sativa SEQ ID NO: 16 (GenBank accession No. AAG03104), encoded by SEQ ID NO: 15, and SEQ ID NO:18 (GenBank accession No. BAB16321), encoded by SEQ ID NO:17 are highly homologous to SEQ ID NO: 8 and can also be used in the methods and compositions ofthe invention.
  • SEQ ID NO: 10 can be found in the TIGR database at At5g28150 (also found at TIGR T24G3_80).
  • SEQ ID NO: 12 can be found in the
  • TIGR database at At3g46890 also found at TIGR F13I12_30.
  • SEQ ID NO:14 can be found in the TIGR database at At5g55510 (also found at TIGRMTE17_23). However, heretofore, no function had been ascribed to SEQ ID NO:l, 3, 5, 7, 9, 11 or
  • the invention provides methods for identifying compounds that inhibit the expression or activity ofthe polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14. Such methods include ligand binding assays, and assays for RNA or protein expression. Any compound that is a ligand for the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14 may have herbicidal activity. Polypeptides having at least 80% sequence identity with the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14 can also be used in the methods ofthe invention to identify herbicide candidates.
  • the sequence identity with SEQ ID NO:2, 4, 6, 8, 10, 12 or 14 is at least 85%, 90%, or 93%, more preferably the identity is at least 95%, most preferably the sequence identity is at least 96%, 97%, 98%, or 99%.
  • the invention provides a method for identifying a compound as a herbicide, comprising: a) selecting a compound that binds to the polypeptide selected from the group consisting of: the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14 and a polypeptide having at least 80% sequence identity with the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14; and b) contacting a plant with said compound to confirm herbicidal activity.
  • the invention provides a method for identifying herbicide candidates, comprising: a) contacting a compound with a polypeptide selected from the group consisting of: i) the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14, and ii) a polypeptide having at least 80% sequence identity with the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14; and b) detecting the presence and/or absence of binding between said compound and said polypeptide, wherein binding indicates that said compound is a herbicide candidate.
  • a polypeptide selected from the group consisting of: i) the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14, and ii) a polypeptide having at least 80% sequence identity with the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14
  • binding indicates that said compound is a herbicide candidate.
  • polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14 is contacted with a test compound in the ligand-binding assay described above.
  • the polypeptides of SEQ ID NO:2, 4, 6, 8, 10, 12 and 14 are from Arabidopsis thaliana and are reported in the TIGR database at, respectively, accession numbers At5g48920,
  • the polypeptide of SEQ ID NO:2 is encoded by the cDNA of SEQ ID NO:l
  • the polypeptide of SEQ ID NO:4 is encoded by the cDNA of SEQ ID NO:3
  • the polypeptide of SEQ ID NO:6 is encoded by the cDNA of SEQ ID NO:5
  • the polypeptide of SEQ ID NO:8 is encoded by the cDNA of SEQ ID NO:7
  • the polypeptide of SEQ ID NO:10 is encoded by the cDNA of SEQ ID NO:9
  • the polypeptide of SEQ ID NO: 12 is encoded by the cDNA of SEQ ID NO:l
  • the polypeptide of SEQ ID NO:14 is encoded by the cDNA of SEQ ID NO:13.
  • polypeptides of SEQ ID NO:2 4, 6, 8, 10, 12 and 14.
  • polynucleotide of SEQ ID NO:l, 3, 5, 7, 9, 11 or 13 can be used as a probe to isolate cDNAs or genes that encode a polypeptide having at least 80% sequence identity with the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14.
  • polypeptide ID NO:2, 4, 6, 8, 10, 12 or 14 can correspond to naturally occurring polypeptides from any organism, or can be synthetic or recombinant variants of naturally occurring polypeptides.
  • the polypeptide is from a plant or a microorganism, such as bacteria or fungi. Most preferably the polypeptide is from a plant. In one embodiment, the polypeptide is from Arabidopsis.
  • Arabidopsis species include, but are not limited to, Arabidopsis arenosa, Arabidopsis bursifolia, Arabidopsis cebennensis, Arabidopsis croatica, Arabidopsis griffithiafia, Arabidopsis halleri, Arabidopsis himalaica, Arabidopsis korshinskyi, Arabidopsis lyrata, Arabidopsis neglecta, Arabidopsis pumila, Arabidopsis suecica, Arabidopsis thaliana and Arabidopsis wallichii.
  • the polypeptide is from a weed.
  • the polypeptide having at least 80% sequence identity with the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 of 14 can be from weeds including, but 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 L7), Brachiara plantaginea, Cassia occidentalis, Ipomoea aristolochiaefolia, Ipomoea purpurea, Euphorbia heterophylla, Setaria spp, Amaranthus retroflexus, Sida spinosa, Xanthium
  • Fragments ofthe polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14 may be used in the methods ofthe invention.
  • a fragment comprises at least 10 consecutive amino acids ofthe polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14.
  • Preferably, a fragment comprises at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or at least 100 or more consecutive amino acid residues of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14.
  • the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14 and polypeptides having at least 80% sequence identity with the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14, and fragments 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. Methods for protein expression and purification using these and other systems are well known to those skilled in the art.
  • Any compound may be screened for herbicidal activity using the methods of the invention.
  • compounds that could be screened include inorganic and organic compounds such as, but not limited to, amino acids, peptides, proteins, nucleotides, nucleic acids, glyco-conjugates, oligosaccharides, lipids, alcohols, thiols, aldehydes, alkylators, carbonic ethers, hydrazides, hydrazines, ketones, nitrils, amines, sulfochlorides, triazines, piperizines, sulphonamides and the like.
  • compound libraries are screened in the assays ofthe invention.
  • Any technique for detecting the binding of a ligand to its target may be used in the methods ofthe invention.
  • Polypeptides and proteins that can reduce non-specific binding, such as BSA, or protein extracts from cells that do not produce the target, may be included in the 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 the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14, a polypeptide having at least 80% sequence identity with the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14, or a fragment or variant thereof, the unbound protein is then removed and the bound polypeptide is detected.
  • bound polypeptide is detected using a labeled binding partner, such as a labeled antibody.
  • the polypeptide of SEQ LD NO:2, 4, 6, 8, 10, 12 or 14, or a fragment or variant thereof 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 Allen et al. (2000) Biomol Screen 5:63-69; Kowski and Wu (2000) Comb Chem High Throughput Screen 3:431-444; Ohmi et al. (2000) J Biomol Screen 5:463-470; Cheung and Zhang (2000) Anal Biochem 252:24-28).
  • ligand binding is detected using mass spectroscopy.
  • MALDI-TOF is capable of detecting and identifying the binding of ligands such as, but not limited to, peptides, proteins, nucleic acids, glyco-conjugates, oligosaccharides, organic polymers and the like.
  • a compound Once a compound is identified as a candidate for a herbicide or has been selected as binding to the polyeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14, or variants thereof, it can be tested for herbicidal activity 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 a method ofthe invention 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.
  • a decrease in growth occurs where the herbicide candidate causes at least a 10% decrease in the growth ofthe plant or plant cells, as compared to the growth of the plants or plant cells in the absence ofthe herbicide candidate.
  • a decrease in viability occurs where at least 20% ofthe plants cells, or portions ofthe plant contacted with the herbicide candidate are nonviable.
  • the growth or viability will be 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 invention also provides plant and plant cell based assays for detecting the amount of target RNA or protein in the presence and absence of a test compound.
  • the target RNA may be a primary RNA transcript or a processed mRNA.
  • the mRNA corresponds to the cDNA ofSEQ ID NO:l, 3, 5, 7, 9, 11, or 13.
  • an RNA sequence corresponds to a DNA sequence when the sequences are the same, except that the thymine nucleotides ofthe DNA are replaced by uracil nucleotides in the RNA.
  • the mRNA has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% sequence identity with the SEQ ID NO:l, 3, 5, 7, 9, 11, or 13.
  • the mRNA measured encodes the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14 or a polypeptide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% sequence identity with the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14.
  • the invention provides a method for identifying a compound that affects the amount of an RNA in a plant, plant cell, or plant cells, comprising: a) measuring the amount of an RNA in a plant, plant cells or plant cell in the presence and absence of said compound, wherein said RNA is selected from the group consisting of: i) an RNA corresponding to the cDNA of SEQ ID NO: 1, 3, 5, 7,
  • a change in the amount of said RNA in the presence of said compound indicates that said compound is a herbicide candidate
  • 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). However, the method of detection is not critical to the invention. Such methods 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, bDNA assays, and microarray assays.
  • the invention provides a method for identifying a compound that affects the amount of a protein in a plant, plant cells, or plant cell, comprising: a) measuring the amount of a protein in a plant or plant cell in the presence and absence of said compound, wherein said protein is selected from the group consisting of: i) the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14, and ii) a polypeptide having at least 80% sequence identity with the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14; and b) comparing the amount of said protein in the presence and absence of said compound.
  • a change in the amount of said protein in the presence of said compound indicates that said compound is a herbicide candidate.
  • the polypeptide is the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14.
  • the polypeptide has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% sequence identity with the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14.
  • 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 protein expression.
  • a polynucleotide encoding a reporter protein is fused in frame with a polynucleotide encoding the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14, or a variant or fragment thereof, so as to produce a chimeric polypeptide.
  • expression ofthe chimeric polypeptide is under the control ofthe cognate promoter that regulates expression of an mRNA in Arabidopsis corresponding to SEQ ID NO:l, 3, 5, 7, 9, 11 or 13.
  • This promoter could be obtained by using SEQ ID NO:l, 3, 5, 7, 9, 11 or 13 as a probe to identify a clone in an Arabidopsis genomic library containing at least the 5' portion ofthe gene encoding SEQ ID NO:2, 4, 6, «&, 10, 12 or 14.
  • Methods for using reporter systems are known to those skilled in the art. Examples of reporter genes include, but are not limited to, chloramphenicol acetyltransferase (Gorman et al.
  • herbicidal activity of compounds identified as herbicide candidates by the RNA and protein expression methods described above can be confirmed by contacting a plant or plant cells with the herbicide candidate and detecting the presence or absence of a decrease in growth or viability of said plant or plant cells.
  • the invention provides a method for inhibiting plant growth, comprising contacting a plant with a compound identified by the methods ofthe invention as having herbicidal activity.
  • Herbicides and herbicide candidates identified by the methods ofthe invention can be used to control the growth of undesired plants, including 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 pilos ⁇ ), nightshade (Solanum nigrum), smartweed (Polygonum lapathifolium), velvetleaf (Abutilon theophrasti), common lambsquarters (Chenopodium album L7), Brachiara plantaginea, Cassia occidentalis, Ipomoea aristolochiaefolia, Ipomoea purpurea, Euphorbia heterophylla, Setaria spp, Amaranthus retroflexiis, Sida spinosa, Xanthium strumarium, and the
  • the invention provides compounds for the inhibition and modulation of plant growth.
  • antisense expression of a portion of an RNA complementary to the cDNA of SEQ ID NO:l, 3, 5, 7, 9, 11 or 13 in plant seedlings results in developmental abnormalities and/or reduced growth.
  • the invention provides polynucleotides that specifically inhibit the expression ofthe polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14 and related polypeptides.
  • polynucleotides ofthe invention are capable of specifically inhibiting transcription or translation, or decreasing the stability of a polynucleotide encoding the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14 and polypeptides having at least 80% sequence identity with SEQ ID NO:2, 4, 6, 8, 10, 12 or 14.
  • polynucleotides include, but are not limited to, antisense molecules, ribozymes, sense molecules, interfering double-stranded RNA (dsRNA) and the like.
  • polynucleotides on plant growth and development will depend upon many factors, such as the specificity and activity ofthe polynucleotide, the level of expression ofthe polynucleotide and the expression pattern ofthe promoter driving the expression of a polynucleotide ofthe invention.
  • inducible expression of such polynucleotides can result in plant death, decreased plant size or decreased growth at the time of induction.
  • developmentally regulated expression could result in a reduction of growth or plant death at a particular stage of development.
  • the polynucleotides ofthe invention are operably linked to a tissue-specific or tissue-preferred promoter. In one embodiment, the polynucleotides ofthe invention are operably linked to a male-tissue-preferred promoter.
  • Male-tissue-preferred expression of a polynucleotide ofthe invention can result in male-sterile plants.
  • Female-tissue-preferred expression of a polynucleotide ofthe invention can result in seedless plants, or in plants having reduced seed size.
  • polynucleotides ofthe invention are not limited to a particular mechanism of action, reduction in gene expression can be mediated at the DNA level and at transcriptional, post-transcriptional, or translational levels.
  • dsRNA suppresses gene expression by both a posttranscriptional process and by DNA methylation (Sharp and Zamore (2000) Science 287: 2431 - 33 (PMID: 10766620)).
  • Ribozymes specifically bind and catalytically cleave RNA. Gene specific inhibition of expression in plants by an introduced sense polynucleotide is termed "cosuppression".
  • Antisense polynucleotides when introduced into a plant cell, are thought to specifically bind to their target polynucleotide and inhibit gene expression by interfering with transcription, splicing, transport, translation and/or stability. Reported mechanisms of antisense action include RNase H-mediated cleavage, activation or inhibition of splicing, inhibition of 5'-cap formation, translation arrest and activation of double strand RNases. (See, for example, Crooke (1999) Biochim Biophys Acta 1489: 31 - 44 (PMID: 10806995)). Antisense polynucleotides can be targeted to chromosomal DNA, to a primary RNA transcript or to a processed mRNA. Preferred target regions include splice sites and translation initiation and termination codons, and other sequences within the open reading frame.
  • the invention provides an isolated antisense RNA for modulating plant growth, comprising, an RNA selected from the group consisting of: a) an RNA complementary to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 or 17, b) an RNA complementary to at least 20 consecutive nucleotides of SEQ ID NO:l, 3, 5, 7, 9, 11, 13, 15 or 17, c) an RNA complementary to a polynucleotide having at least 80% sequence identity with SEQ ID NO:l, 3, 5, 7, 9, 11, 13, 15 or 17, d) an RNA complementary to at least 30 consecutive nucleotides of a polynucleotide encoding SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16 or 18, and e) an RNA complementary to a polynucleotide encoding a polypeptide having at least 80% sequence identity with SEQ ID NO:2, 4, 6, 8, 10, 12 or 14.
  • the polynucleotide is complementary to a plant mRNA.
  • the antisense RNA is complementary to at least 20, 30, 40, 50, 75, 100, 150 or 200 consecutive nucleotides of SEQ ID NO:l, 3, 5, 7, 9, 11, 13, 15 or 17 or another polynucleotide encoding SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16 or 18.
  • the antisense RNA is complementary to a polynucleotide having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% sequence identity with SEQ ID NO:l, 3, 5, 7, 9, 11 or 13 or another polynucleotide encoding SEQ ID NO:2, 4, 6, 8, 10, 12 or 14.
  • the invention provides antisense molecules that specifically hybridize under high stringency conditions to SEQ ID NO:l, 3, 5, 7, 9, 11, 13, 15 or 17 or a polynucleotide encoding SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16 or 18.
  • specifically hybridize is meant that the polynucleotide will hybridize to the target gene or RNA at a level of at least two-fold over background under conditions of high stringency.
  • the specificity ofthe hybridization will depend upon many factors, including the length and degree of complementarity between the antisense molecule and the target sequence, the length ofthe antisense molecule, the temperature ofthe hybridizations and washes, and the salt, detergent and formamide concentrations of the hybridization and wash buffers.
  • the antisense polynucleotides ofthe invention need not be completely complementary to the target gene or RNA, nor that they hybridize to each other along their entire length, in order to modulate expression or to form specific hybrids. Furthermore, the antisense polynucleotides ofthe invention need not be full length with respect to the target gene or RNA. In general, greater homology can compensate for shorter polynucleotide length.
  • such antisense molecules will comprise an RNA having 60-100 % sequence identity with at least 14, 15, 16, 17, 18, 19, 20, 25, 30, 50, 75 or at least 100 consecutive nucleotides of to SEQ ID NO:l, 3, 5, 7, 9, 11, 13, 15 or 17 or a polynucleotide encoding SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16 or 18.
  • the sequence identity will be at least 70%, more preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and most preferably at least 99%98%, or even 99%.
  • the active antisense molecules ofthe invention are single-stranded RNA molecules.
  • active antisense molecule is meant that the antisense RNA is capable of selectively hybridizing with a primary transcript or mRNA encoding a polypeptide having at least 80% sequence identity with the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12 or 14.
  • the term antisense molecule includes double-stranded DNA expression cassettes that can be transcribed to produce an antisense RNA.
  • the antisense polynucleotides ofthe invention are at least 8, 10, 12, 14, 16, 18, 20, 25, 30, 5 ⁇ 0, 45, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 nucleotides or more.
  • Antisense polynucleotides can be selected based on complementarity to plant genes or RNAs.
  • the complementarity may be to all or a portion ofthe gene or RNA.
  • the complementarity need not be exact, so long as the antisense molecule is specific for the target RNA.
  • the degree of complementarity necessary or antisense inhibition is related to the length ofthe hybridizing sequences.
  • the complementarity is at least 90%, more preferably 95%, even more preferably at least 98%, and most preferably 100%.
  • Antisense polynucleotides may be designed to bind to exons, introns, exon-intron boundaries, the promoter and other control regions, such as the transcription and translational initiation sites.
  • ribozymes, sense polynucleotides or dsRNA may be used to reduce expression of a polypeptide having at least 80% sequence identity with SEQ ID NO:2, 4, 6, 8, 10, 12 or 14.
  • a ribozyme, or catalytic RNA can catalyze the hydrolysis of RNA phosphodi ester bonds in trans, and thus can cleave other RNA molecules. Cleavage of a target RNA can decrease stability ofthe RNA and prevent translation of a full length protein encoded by that RNA.
  • Ribozymes contain a first RNA sequence that is complementary to a target RNA linked to a second enzymatic RNA sequence that catalytically cleaves the target RNA. Thus, the ribozyme first binds a target RNA through complementary base- pairing, and then acts enzymatically to cut the target RNA. Ribozymes may be designed to bind to exons, introns, exon-intron boundaries and control regions, such as the translational initiation sites.
  • RNAs At least six types of naturally occurring enzymatic RNAs, including hairpin ribozymes and hammerhead ribozymes, have been described.
  • the hairpin ribozyme can be assembled in various combinations to catalyze a unimolecular, bimolecular or a trimolecular cleavage/ligation reaction (Berzal-Herranz et al. (1992) Genes &
  • the invention provides ribozymes that are specific for at least one RNA encoding a polypeptide having at least 80% sequence identity with SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16 or 18.
  • a ribozyme that is "specific for at least one plant RNA encoding a polypeptide having at least 80% sequence identity with SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16 or 18" will contain a polynucleotide sequence that specifically hybridizes to a target plant primary transcript or mRNA (the "target") encoding a polypeptide having at least 80% sequence identity with SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16 or 18 and cleaves that target.
  • the portion ofthe ribozyme that hybridizes to the transcript or RNA is typically at least 7 nucleotides in length. Preferably, this portion is at least 8, 9, 10, 12, 14, 16, 18, or 20 or more nucleotides in length.
  • the portion ofthe ribozyme that hybridizes to the target need not be completely complementary to the target, as long as the hybridization is specific for the target.
  • the ribozyme will contain a portion having at least 7 or 8 nucleotides that have 100% complementarity to a portion ofthe target RNA.
  • the target RNA corresponds to the cDNA of SEQ ID NO: 1 , 3, 5, 7, 9, 11, 13, 15 or 17.
  • the invention provides a double-stranded RNA (dsRNA) that is specific for a polynucleotide encoding either the polypeptide of SEQ ID NO:2,
  • dsRNA refers to RNA hybrids comprising two strands of RNA.
  • the dsRNAs ofthe invention may be linear or circular in structure.
  • the hybridizing RNAs may be substantially or completely complementary. By substantially complementary, is meant that when the two hybridizing RNAs are optimally aligned using the BLAST program as described above, the hybridizing portions are at least 95% complementary.
  • the dsRNA will be at least 100 base pairs in length.
  • the hybridizing RNAs of will be of identical length with no over hanging 5' or 3' ends and no gaps. However, dsRNAs having 5' or 3' overhangs of up to 100 nucleotides may be used in the methods ofthe invention.
  • the invention provides a dsRNA, comprising: a first ribonucleic acid having at least 80% sequence identity with at least 100 consecutive nucleotides of a polynucleotide encoding either the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16 or 18 or a polypeptide having at least 80% sequence identity with SEQ ID NO:2, 4, 6, 8, 10, 12 or 14; and a second ribonucleic acid that is substantially complementary to said first ribonucleic acid.
  • the first ribonucleic acid ofthe dsRNA ofthe invention has at least
  • the second ribonucleic hybridizes to SEQ ID NO:l, 3, 5, 7, 9, 11, 13, 15 or 17 under high stringency conditions.
  • the dsRNA may comprise ribonucleotides or ribonucleotide analogs, such as 2'-O-methyl ribosyl residues or combinations thereof. See U.S. patents 4,130,641 and 4,024,222. A dsRNA polyriboinosinic acid:polyribocytidylic acid is described in U.S. patent 4,283,393.
  • dsRNA can be introduced into a plant or plant cell directly by standard transformation procedures. Alternatively, dsRNA can be expressed in a plant cell by transcribing two complementary RNAs.
  • the Plant Cell 2: 291 - 9 (PMID: 2152117), Smith et al (1990) Mol Gen Genetics 224: 477 - 81 (PMID: 2266949) and Napoli et al. (1990) The Plant Cell 2: 279 - 89).
  • introduction of a sense polynucleotide blocks transcription ofthe corresponding target gene.
  • the sense polynucleotide will have at least 65% sequence identity with the target plant gene or RNA.
  • the percent identity is at least 80%, 90%, 95%, or more.
  • the introduced sense polynucleotide need not be full length relative to the target gene or transcript.
  • the sense polynucleotide will have at least 65% sequence identity with at least 100 consecutive nucleotides of SEQ ID NO:l, 3, 5, 7, 9, 11, 13, 15 or 17.
  • the regions of identity can comprise introns and and/or exons and untranslated regions.
  • the introduced sense polynucleotide may be present in the plant cell transiently, or may be stably integrated into a plant chromosome or extrachromosomal replicon. Expression ofthe polynucleotides ofthe invention in a plant, plant cell or plant tissue will result in the modulation of plant growth or development.
  • the invention provides recombinant expression cassettes, comprising the antisense, sense, dsRNA or ribozyme polynucleotides ofthe invention, wherein said polynucleotide is operably linked to a promoter that can be active in a plant cell.
  • the expression cassettes ofthe invention contain 5' and 3' regulatory sequences necessary for transcription and termination ofthe polynucleotide of interest.
  • the expression cassettes will include a promoter and a transcriptional terminator.
  • Other functional sequences may be included in the expression cassettes of the inventions. Such functional sequences include, but are not limited to, introns, enhancers and translational initiation and termination sites and polyadenylation sites.
  • the control sequences can be those that can function in at least one plant, plant cell or plant tissue. These sequences may be derived form one or more genes, or can be created using recombinant technology. Promoters useful in the expression cassettes ofthe invention include any promoter that is capable of initiating transcription in a plant cell. Such promoters include, but are not limited to those that can be obtained from plants, plant viruses and bacteria that contain genes that are expressed in plants, such as Agrobacterium and Rhizobium. The promoter may be constitutive, inducible, developmental stage-preferred, cell type-preferred, tissue-preferred or organ-preferred. Constitutive promoters are active under most conditions. Examples of constitutive promoters include the CaMV 19S and 35 S promoters (Odell et al.
  • Theor Appl Genet 81: 581 - 8 the figwort mosaic virus 35S promoter, the Smas promoter (Velten et al. (1984) EMBO J 3: 2723 - 30), the GRP1-8 promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. patent 5,683,439), promoters from the T-DNA of Agrobacterium, such as mannopine synthase, nopaline synthase, and octopine synthase, the small subunit of ribulose biphosphate carboxylase (ssuRUBISCO) promoter, and the like.
  • ssuRUBISCO small subunit of ribulose biphosphate carboxylase
  • Inducible promoters are active under certain environmental conditions, such as the presence or absence of a nutrient or metabolite, heat or cold, light, pathogen attack, anaerobic conditions, and the like.
  • the hsp80 promoter from Brassica is induced by heat shock
  • the PPDK promoter is induced by light
  • the PR-1 promoters from tobacco, Arabidopsis and maize are inducible by infection with a pathogen
  • the Adhl promoter is induced by hypoxia and cold stress.
  • tissue and organ preferred promoters include those that are preferentially expressed in certain tissues or organs, such as leaves, roots, seeds, or xylem.
  • tissue preferred and organ preferred promoters include, but are not limited to, fruit-preferred, ovule-preferred, male-tissue-preferred, seed- preferred, integument-preferred, tuber-preferred, stalk-preferred, pericarp-preferred, and leaf-preferred, stigma-preferred, pollen-preferred, anther-preferred, petal- preferred, sepal-preferred, pedicel-preferred, silique-preferred, stem-preferred, root- preferred promoters, and the like.
  • the promoter is a male-tissue-preferred promoter.
  • Male tissues include pollen, tapetum, anther, tassel, pollen mother cells, and microspores.
  • Ms45 is an example of a male-preferred promoter (U.S. patent 6,037,523).
  • tissue-preferred, developmental stage-preferred and/or inducible promoters include, but are not limited to Prha (expressed in root, seedling, lateral root, shoot apex, cotyledon, petiol, inflorescence stem, flower, stigma, anthers, and silique, and auxin-inducible in roots); VSP2 (expressed in flower buds, flowers, and leaves, and wound inducible); SUC2 (expressed in vascular tissue of cotyledons, leaves and hypocotyl phloem, flower buds, sepals and ovaries); AAP2 (silique-preferred); SUCl (Anther- and pistil-preferred); AAP1 (seed-preferred); Saur-ACl (auxin-inducible in cotyledons, hypocotyl and flower); Enod 40 (expressed in root, stipule, cotyledon, hypocotyl and flower); amd VSP1 (expressed in young siliques
  • Seed-preferred promoters are preferentially expressed during seed development and/or germination.
  • seed-preferred promoters can be embryo-preferred, endosperm-preferred and seed coat-preferred. (See, Thompson and Larkins (1989) BioEssays 10: 108 - 113 (PMID: 2658986)).
  • seed- preferred promoters include, but are not limited to cellulose synthase (celA), Ciml, gamma-zein, globulin- 1, maize 19 kD zein (cZ19Bl), and the like.
  • promoters useful in the expression cassettes ofthe invention include, but are not limited to, the major chlorophyll a/b binding protein promoter, histone promoters, the prolifera promoter, the Ap3 promoter, the ⁇ -conglycin promoter, the phaseolin promoter, the napin promoter, the soy bean lectin promoter, the maize 15kD zein promoter; the 22 kD zein promoter, the 27 kD zein promoter, the g-zein promoter, the waxy, shrunken 1, shrunken 2 and bronze promoters, the Zml3 promoter .(U.S.
  • heterologous DNA-binding domains include the Lex A and GAL4 DNA-binding domains.
  • the LexA DNA-binding domain is part ofthe repressor protein LexA from Escherichia coli (E. coli) (Brent and Ptashne (1985) Cell 43: 729 - 36 (PMID: 3907859)).
  • the promoter comprises a minimal promoter operably linked to an upstream activation site comprising four DNA-binding domains ofthe yeast transcriptional activator GAL4. Schwechheimer et al. (1998) Plant Mol Biol 36: 195 - 204 (PMID: 9484432).
  • Polyadenlation signals include, but are not limited to, the Agrobacterium octopine synthase signal (Gielen et al. (1984) EMBO J 3: 835 - 46 (PMID: 6327292)) and the nopaline synthase signal (Depicker et al. (1982) Mol and Appl Genet 1: 561 — 73 (PMID: 7153689)).
  • Transcriptional termination regions include, but are not limited to, the terminators of the A. tumefaciens Ti plasmid octopine synthase and nopaline synthase genes.
  • A. tumefaciens Ti plasmid octopine synthase and nopaline synthase genes See, Ballas et al. (1989) Nuc Acid Res 17: 7891 - 903 (PMID: 2798133), Guerineau et al. (1991) Mol Gen Genet 262: 141 - 4 (PMID: 1709718), Joshi (1987) Nuc Acid Res 15: 9627 - 39 (PMID: 3697078), Mogen et al.
  • the expression cassettes ofthe invention may be covalently linked to a polynucleotide encoding a selectable or screenable marker.
  • markers include genes encoding drug or herbicide resistance, such as hygromycin resistance (hygromycin phosphotransferase (HPT)), spectinomycin (encoded by the aada gene), kanamycin and gentamycin resistance (neomycin phosphotransferase (nptll)), streptomycin resistance (streptomycin phosphotransferase gene (SPT)), phosphinothricin or basta resistance (barnase (bar)), chlorsulfuron reistance (acetolactase synthase (ALS)), chloramphenicol resistance (chloramphenicol acetyl transferase (CAT)), G418 resistance, lincomycin resistance, methotrexate resistance, glyphosate resistance, and the like.
  • HPT
  • the expression cassettes ofthe invention may be covalently linked to genes encoding enzymes that are easily assayed, for example, luciferase, alkaline phosphatase, ⁇ -galactosidase ( ⁇ -gal), ⁇ - glucuronidase (GUS) and the like.
  • the invention provides an expression cassette, comprising a polynucleotide encoding an antisense RNA that is specific for a polynucleotide encoding either the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16 or 18, or a polypeptide having at least 80% sequence identity to SEQ ID NO:2, 4, 6, 8, 10, 12 or 14, wherein said polynucleotide is operably linked to a promoter that can be active in a plant cell.
  • the antisense RNA comprises the complement of SEQ ID NO:l, 3, 5, 7, 9, 11, 13, 15 or 17. In another embodiment, the antisense RNA has at least 80% sequence identity with at least 20 consecutive nucleotides of SEQ ID NO:l, 3, 5, 7, 9, 11, 13, 15 or 17. In still another embodiment, the antisense RNA hybridizes under high stringency conditions to the polynucleotide of SEQ ID NO:l, 3, 5, 7, 9, 11, 13, 15 or 17.
  • the invention provides vectors containing the expression cassettes ofthe invention.
  • vector is intended a polynucleotide sequence that is able to replicate in a host cell.
  • the vector contains genes that serve as markers useful in the identification and/or selection of transformed cells. Such markers include, but are not limited to barnase (bar), G418, hygromycin, kanamycin, bleomycin, gentamicin and the like.
  • the vector can comprise DNA or RNA and can be single- or double-stranded, and linear or circular.
  • Various plant expression vectors and reporter genes are described in Gruber et al.
  • the vector is an E. coli/ A. tumefaciens binary vector.
  • the expression cassette is inserted between the right and left borders of a T-DNA from an Agrobacterium Ti plasmid.
  • the invention provides plants, plant cells and plant tissues transformed with at least one polynucleotide, expression cassette or vector ofthe invention.
  • transformation is meant the introduction of a polynucleotide into a target plant cell or plant tissue.
  • Antisense polynucleotides, dsRNA and ribozymes can be introduced directly into plant cells, in the form of RNA.
  • the antisense polynucleotides, dsRNA and ribozymes ofthe present invention may be provided as RNA via transcription in plant cells transformed with expression constructs encoding such RNAs.
  • a plant or plant cell is transformed with an expression cassette, comprising a polynucleotide encoding an antisense RNA that is specific for a polynucleotide encoding either the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16 or 18, or a polypeptide having at least 80% sequence identity to SEQ ID NO:2, 4, 6, 8, 10, 12 or 14, wherein said polynucleotide is operably linked to a promoter that can be active in a plant cell.
  • the polynucleotides ofthe invention may be introduced into any plant or plant cell.
  • plants is meant angiosperms (monocotyledons and dicotyledons) and gymnosperms, and the cells, organs and tissues thereof.
  • Methods for the introduction of polynucleotides into plants and for generating transgenic plants are known to those skilled in the art. (See, for example, Weissbach & Weissbach (1988) Methods for Plant Molecular Biology, Academic Press, N.Y.; Grierson & Corey (1988) Plant Molecular Biology, 2 nd Ed., Blackie, London; and Miki et al. (1993) Procedures for Introducing Foreign DNA into Plants, CRC Press, Inc. pp.67 - 80).
  • Such methods include, but are not limited to, electroporation (Fromm et al. (1985) Proc Natl Acad Sci 82: 5824 - 5828 (PMID: 3862099) and Riggs et al (1986) Proc Natl Acad Sci USA 83: 5602 - 5606 (PMJD: 3016708)), particle bombardment (US patents 4,945,050 and 5,204,253, the contents of which are incorporated by reference, Klein et al (1987) Nature 327: 70 - 73, McCabe et al. (1988) Biotechnology 6: 923 - 926), microinjection (Crossway (1985) Mol Gen Genet 202: 179 - 185 and Crossway et al.
  • the polynucleotides ofthe invention are introduced into a plant cell by agroinfection.
  • a DNA construct comprising a polynucleotide ofthe invention is inserted between the right and left T-DNA borders in an Agrobacterium tumefaciens vector.
  • the virulence proteins ofthe A. tumefaciens host cell will mediate the transfer ofthe inserted DNA into a plant cell infected with the bacterium.
  • Agrobacterium rhizogenes- mediated transformation may be used as an alternative to the A. plasmid system. (See, Lichtenstein and Fuller in: Genetic
  • transgenic seeds and plants can be produced directly.
  • a preferred method of producing transgenic Arabidopsis seeds and plants involves agroinfection ofthe flowers and collection of the transgenic seeds produced from the agroinfected flowers.
  • transformed plant cells can be regenerated into plants by methods known to those skilled in the art. (See, for example, Evans et al, Handbook of Plant Cell Cultures, Vol I, MacMollan Publishing Co. New York, 1983; and Vasil, Cell Culture and Somatic Cell Genetics of Plants, Acad Press, Orlando, Vol II, 1986).
  • transgenic plant Once a transgenic plant has been obtained, it may be used as a parent to produce progeny plants and plant lines.
  • Conventional plant breeding methods can be used, including, but not limited to, crossing and backcrossing, self-pollination and vegetative propagation. Techniques for breeding plants are known to those skilled in the art.
  • the progeny of a transgenic plant are included within the scope ofthe invention, provided that the progeny contain all or part ofthe transgenic construct.
  • the transformed plants and plant cells ofthe invention include the progeny of said plant or plant cell, as long as the progeny plants or plant cells still contain the antisense expression cassette.
  • Progeny may be generated by both asexual and sexual methods.
  • Progeny of a plant include seeds, subsequent generations ofthe plant and the seeds thereof.
  • the polynucleotides ofthe invention into a plant, plant cell or plant tissue will result in the modulation of plant growth or development. In most cases, the modulation will be a decrease or cessation of growth or development ofthe plant cells or tissues where the polynucleotides ofthe invention are expressed.
  • the antisense, ribozymes, dsRNA and sense polynucleotides ofthe invention may be directly transformed into a plant cell.
  • the expression cassettes or vectors ofthe invention may be introduced into a plant cell. Once in the cell, expression ofthe antisense, ribozymes, dsRNA and sense polynucleotides ofthe invention may be transient or stable. Stable expression requires that all or a part of the polynucleotide, expression cassette or vector is integrated into a plant chromosome or a stable extra-chromosomal replicon.
  • the invention provides a method for modulating plant growth, comprising: introducing into a plant cell at least one polynucleotide specific for a nucleic acid encoding either the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16 or 18 or a polypeptide having at least 80% sequence identity with SEQ ID NO:2, 4, 6, 8, 10, 12, or 14, wherein said polynucleotide is selected from the group consisting of: a ribozyme, an antisense molecule, and a dsRNA.
  • the invention provides a method for modulating the growth of a plant, plant cell or plant tissue, comprising: transforming said plant, plant cell or plant tissue with an expression cassette, comprising a polynucleotide encoding an antisense RNA that is specific for a polynucleotide encoding either the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16 or 18, or a polypeptide having at least 80% sequence identity to SEQ ID NO:2, 4, 6, 8, 10, 12, or 14, wherein said polynucleotide is operably linked to a promoter that can be active in a plant cell.
  • the promoter is a tissue specific promoter.
  • RNAs in one or more male tissues will result in a male sterile plant.
  • the plant progeny obtained by cross- pollination show more vigor than the progeny obtained through self-pollination.
  • the invention provides a method for the generation of plants that are male sterile, the method comprising: a) transforming plant cells with an expression cassette comprising an antisense polynucleotide operably linked to a male organ-preferredpromoter, wherein the antisense polynucleotide consists of at least 30 contiguous nucleotides complementary to the nucleotide sequence set forth in SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:15 or SEQ ID NO:17; or at least 30 contiguous nucleotides complementary to a nucleotide sequence having at least 80% homology to the nucleotide sequence set forth in SEQ ID NO: 1 , SEQ ID NO:3,
  • the promoter driving the expression ofthe antisense polynucleotide ofthe invention is selected from the group consisting of anther-specific, tapetum-specific, and pollen-specific. Also encompassed by the invention are male sterile plants obtainable by the methods ofthe invention, as well as plant cells and reproduction material ofthe male sterile plants.
  • Ovule-preferred expression ofthe antisense polynucleotides ofthe invention will result in a reduction of seed size.
  • reduced seed size is meant that the seed is reduced by at least 10%.
  • the seed is reduced in size by 25%, 50%, 75%, 90%, or is absent.
  • the seed of any plant may be reduced in size, however preferred plants include cucumbers, tomatoes, melons, cherries, grapes, pomegranates, and the like.
  • the invention provides a method for generating a plant with reduced seed size, comprising: a) transforming plant cells with an expression cassette comprising a polynucleotide encoding an antisense polynucleotide consisting of at least 30 contiguous nucleotides complementary to a nucleic acid encoding the polypeptide of SEQ ID NO:2, 4, 6, 8, 10, 12, or 14 or a nucleic acid encoding a polypeptide having at least 80% sequence identity with SEQ ID NO:2, 4, 6, 8, 10, 12, or 14; wherein said antisense polynucleotide is operably linked to an ovule-preferred promoter; b) regenerating plants from said transformed plant cells, and c) selecting from the regenerated plants, plants that contain reduced seed size.
  • the methods ofthe present invention provide for the generation of plants with improved agronomic traits such as increased yield.
  • plant expression of at least one ofthe polynucleotides ofthe invention is increased to confer on the plant enhanced growth and/or vigor.
  • the invention provides a method for improving at least one agronomic property of a plant by increasing the expression of a particular plant gene, the method comprising: a) transforming plant cells with an expression cassette comprising a nucleotide sequence operably linked to a promoter that can be active in the plant cells, wherein said nucleotide sequence is selected from the group consisting of: i) a nucleotide sequence set forth in SEQ ID NO: 1 , SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:l l, or SEQ ID NO:13; and ii) a nucleotide sequence having at least 80% homology to the nucleotide sequence set forth in SEQ ID NO:l, S
  • the promoter driving expression of the polynucleotide ofthe invention is selected from the group consisting of constitutive, inducible, developmental stage-preferred, cell type-preferred, tissue- preferred, and organ-preferred.
  • transgenic plants obtainable by the methods ofthe invention, as well as plant cells and reproduction material from the transgenic plants.
  • the plates are transferred into a growth chamber with a day and night temperature of 22 and 20°C, respectively, 65% humidity and a light intensity of -100 ⁇ -E m "2 s "1 supplied over 16 hour day period.
  • the "Driver” is an artificial transcription factor comprising a chimera ofthe DNA-binding domain ofthe yeast GAL4 protein (amino acid residues 1 - 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 (PMID: 9484432).
  • This chimeric driver is a transcriptional activator specific for promoters having GAL4 binding sites. Expression ofthe driver is controlled by two tandem copies ofthe 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 or variant of an Arabidopsis thaliana cDNA corresponding to SESEQ ID NO:l, 3, 5, 7, 9, 11, or 13 was ligated into the Pacl/Ascl sites of an E.coli/Agrobacterium binary vector in the antisense orientation. This placed transcription ofthe antisense RNA under the control of an artificial promoter that is active only in the presence ofthe 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. pPG1856 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 At5g48920.
  • the coding sequence for this locus is shown as SEQ ID NO: 1.
  • the protein encoded by this mRNA is shown as SEQ ID NO:2.
  • pPG845 expresses the A. thaliana antisense RNA which is complementary to a portion ofthe DNA of SEQ ID NO: 3.
  • This antisense RNA is complementary to the cDNA sequence found in the TIGR database at locus Atlg50660.
  • the coding sequence for this locus is shown as SEQ ID NO:3.
  • the protein encoded by this mRNA is shown as SEQ ID NO:4.
  • pPG844 expresses the A. thaliana antisense RNA which is complementary to a portion ofthe DNA of SEQ ID NO: 5. This antisense RNA is complementary to the cDNA sequence found in the TIGR database at locus At4g22640. The coding sequence for this locus is shown as SEQ ID NO:5. The protein encoded by this mRNA is shown as SEQ ID NO:6.
  • pPG747 expresses the A. thaliana antisense RNA which is complementary to a portion ofthe DNA of SEQ ID NO: 7. This antisense RNA is complementary to the cDNA sequence found in the TIGR database at locus At5g04850. The coding sequence for this locus is shown as SEQ ID NO:7.
  • the protein encoded by this mRNA is shown as SEQ ID NO:8.
  • pPG745 expresses the A. thaliana antisense RNA which is complementary to a portion ofthe DNA of SEQ ID NO: 9. This antisense RNA is complementary to the cDNA sequence found in the TIGR database at locus At5g28150. The coding sequence for this locus is shown as SEQ ID NO:9.
  • the protein encoded by this mRNA is shown as SEQ ID NO: 10.
  • pPG855 expresses the A. thaliana antisense RNA which is complementary to a portion ofthe DNA of SEQ ID NO: 11. This antisense RNA is complementary to the cDNA sequence found in the TIGR database at locus At3g46980.
  • the coding sequence for this locus is shown as SEQ JD NO: 11.
  • the protein encoded by this mRNA is shown as SEQ ID NO: 12.
  • pPG929 expresses the A. thaliana antisense RNA which is complementary to a portion ofthe DNA of SEQ ID NO: 13. This antisense RNA is complementary to the cDNA sequence found in the TIGR database at locus At5g55510.
  • the coding sequence for this locus is shown as SEQ ID NO:13.
  • the protein encoded by this mRNA is shown as SEQ ID NO: 14.
  • 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.
  • Transformation of Agrobacterium with the Antisense Expression Cassette pPG1856, pPG845, pPG844, pPG747, pPG745, pPG855, and pPG929 were 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 to enhance the emergence of secondary bolts.
  • 5 ml LB broth (10 g/L Peptone, 5 g/L Yeast extract, 5 g/L NaCl, pH 7.0 plus 25 mg/L kanamycin added prior to use) was inoculated with a clonal glycerol stock of Agrobacterium carrying pPG1856, pPG845, pPG844, pPG747, pPG745, pPG855, or pPG929.
  • the cultures were incubated overnight at 28°C at 250 rpm until the cells reached stationary phase.
  • 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.
  • 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 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 of 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. After the emergence of the first primary leaves, 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.
  • 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 Fl 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. Three of nine seedlings examined from the transgenic plant line containing the pPG1856 antisense construct exhibited developmental abnomialities and significantly reduced growth of seedlings. Thus, sense sequence corresponding to pPG1856 and protein encoded by this sequence is essential for normal plant growth and development. Five often seeds/seedlings examined from the transgenic plant line containing the pPG845 antisense construct exhibited failure to germinate.
  • sense sequence corresponding to pPG845 and protein encoded by this sequence is essential for normal plant growth and development.
  • Five often seeds/seedlings examined from the transgenic plant line containing the pPG844 antisense construct exhibited failure to germinate.
  • sense sequence corresponding to pPG844 and protein encoded by this sequence is essential for normal plant growth and development.
  • Three often seeds/seedlings examined from the transgenic plant line containing the pPG747 antisense construct exhibited failure to germinate.
  • sense sequence corresponding to pPG747 and protein encoded by this sequence is essential for normal plant growth and development.
  • Five often seeds/seedlings examined from the transgenic plant line containing the pPG745 antisense construct exhibited failure to germinate.
  • sense sequence corresponding to pPG745 and protein encoded by this sequence is essential for normal plant growth and development.
  • Seven often seedlings examined from the transgenic plant line containing the pPG855 antisense construct exhibited developmental abnormalities and significantly reduced growth of seedlings.
  • sense sequence corresponding to pPG855 and protein encoded by this sequence is essential for normal plant growth and development.
  • Four often seedlings examined from the transgenic plant line containing the pPG929 antisense construct exhibited significant seed and seedling abnormalities.
  • sense sequence corresponding to pPG929 and protein encoded by this sequence is essential for normal plant growth and development.
  • a polynucleotide ofthe invention is cloned into E. coli (pET vectors-
  • High throughput screening assays are established to identify compounds that bind to the polypeptides ofthe invention according to one or more ofthe following fluorescence and scintillation proximity assays described in Allen et al. (2000) Biomol Screen 5:63-69; Kowski and Wu (2000) Comb Chem High Throughput Screen 3:431-444; Ohmi et al. (2000) J Biomol Screen 5:463-470; and Cheung and Zhang (2000) Anal Biochem 252:24-28.

Abstract

La présente invention concerne des polypeptides inclus dans SEQ ID NO:2, 4, 6, 8, 10, 12, et 14 qui jouent un rôle essentiel dans la croissance des végétaux. Il en résulte que ces polypeptides peuvent donc être utilisés comme cibles pour l'identification d'herbicides. Cette invention concerne donc des procédés permettant d'identifier des composés qui inhibent l'expression des poylpeptides codés par l'ADNc inclus dans SEQ ID NO:1, 3, 5, 7,9, 11 et 13. Ces composés conviennent comme herbicides. Sont également décrites des méthodes et des compositions qui modulent la croissance et le développement des végétaux.
PCT/US2002/006162 2001-02-28 2002-02-28 Procedes d'identification d'herbicides et modulation de la croissance des vegetaux WO2002068606A2 (fr)

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DATABASE SPTREMBL [Online] 01 March 2001 KANEKO ET AL., XP002956146 Database accession no. (Q9FI79) *

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Publication number Priority date Publication date Assignee Title
WO2012107451A2 (fr) 2011-02-08 2012-08-16 Vib Vzw Procédé pour le criblage de composés influençant la croissance et la production de cellules végétales

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