Methods for the identification of inhibitors of thioredoxin expression or activity in plants

FIELD OF THE INVENTION

[0002] The invention relates generally to plant molecular biology. In particular, the invention relates to methods for the identification of herbicides.

BACKGROUND OF THE INVENTION

[0003] Thioredoxins are small proteins of approximately one hundred amino-acid residues, which participate in various oxidation-reduction reactions via the reversible oxidation of an active center disulfide bond. They exist in either a reduced form, or an oxidized form where the two cysteine residues are linked in an intramolecular disulfide bond. The disulfide bridge of the oxidized (—S—S—) form of thioredoxin can be reduced to the sulfhydryl (—SH) level by either reduced ferredoxin or NADPH via one of two specific enzymes. The reduced form is an excellent catalyst for the reduction of disulfide bonds that are, at best, sluggishly reduced by glutathione (Holmgren (1985) Annu Rev Biochem 54: 237-71 (PMID: 3896121); Holmgren (1989) J Biol Chem 264: 13963-6 (PMID: 2668278); Eklund et al. (1991) Proteins 11: 13-28 (PMID: 1961698)).

[0004] Thioredoxin is present in prokaryotes and eukaryotes and the sequence around the redox-active disulfide bond is well conserved. Bacteriophage T4 also encodes for a thioredoxin but its primary structure is not homologous to bacterial, plant and vertebrate thioredoxins.

[0005] While only one type of thioredoxin has been detected in E. coli or animal cells, three well characterized variants exist in photosynthetic cells. Two of the three (m and f) are located in chloroplasts and can be distinguished from one another on the basis of their primary structure and specificity for target enzymes. The two chloroplast thioredoxins are members of the ferredoxin/thioredoxin system, a regulatory system in oxygenic photosynthesis. Electrons provided by the excitation of chlorophyll are transferred via ferredoxin and an iron-sulfur enzyme, ferredoxin-thioredoxin reductase (FTR) to either of the two types of plastid thioredoxins, which, in turn, selectively activate photosynthetic enzymes by reduction of well defined regulatory sites (see Buchanan (1980) Annu Rev Plant Physiol 31: 341-374; Buchanan (1991) Arch Biochem Biophys 288: 1-9 (PMID: 1910303); Buchanan (1992) Photosynth Res 33: 147-162; Buchanan et al. (1994a) In A. R. Grossman (ed.), Seminars in Cell Biology. Vol. 5, Academic Press, London, pp. 285-293; Scheibe (1991) Plant Physiol 26: 1-3; and Wolosiuk et al. (1993) FASEB J. 7: 622-37 (PMID: 8500687) for reviews on the ferredoxin/thioredoxin system). Studies with the unicellular alga Chlamydomonas reinhardtii have extended the role of chloroplast thioredoxins to the control of mRNA translation (Danon and Mayfield (1994) Science 266: 1717-9 (PMID: 7992056)).

[0006] Plants contain a second thioredoxin system composed of NADPH, a flavin enzyme called NADP-thioredoxin reductase (NTR), and an associated thioredoxin of yet another type (Suske et al. (1979) Z. Naturforsch 34c: 214-221; Berstermann et al. (1983) Eur J Biochem 131: 339-44 (PMID: 6682037)). Named thioredoxin h (for heterotrophic) as it was first identified in cultured cells, seeds and roots (Johnson et al. (1987a) Planta 171: 321-31 (PMID: 11539727); Johnson et al. (1987b) Plant Physiol 85: 446-451), h-type thioredoxins are also present in leaves and eukaryotic algae (Wagner et al. (1978) Z Naturforsch [C] 33: 517-20 (PMID: 212888); Wolosiuk et al. (1979) J Biol Chem 254: 1627-32 (PMID: 216700); Florencio et al. (1988) Arch Biochem Biophys 266: 496-507 (PMID: 3190242); Marcus et al. (1991) Arch Biochem Biophys 287: 195-8 (PMID: 1897989); Schürmann (1993) Plant thioredoxins. In De Kok, L. J. (Ed.). Sulfur nutrition and assimilation in higher plants: regulatory agricultural and environmental aspects. Second Workshop on Sulfur Metabolism in Higher Plants, Garmisch-Partenkirchen, Germany. SPB Academic Publishing bv: The Hague, Netherlands, pp. 153-162). The NADP/thioredoxin system is widely distributed among organisms and is thought to be ubiquitous in aerobes. The elucidation of the biological role of NADP-linked thioredoxin is currently an area of extensive investigation in plants as well as animals where it functions in a growing array of critical processes.

[0007] To date there do not appear to be any publications describing lethal effects of over-expression, antisense expression or knock-out of this thioredoxin gene in plants. Thus, the prior art has not suggested that TRX is essential for plant growth and development. It would be desirable to determine the utility of this enzyme for evaluating plant growth regulators, especially herbicide compounds.

SUMMARY OF THE INVENTION

[0008] Surprisingly, the present inventors have discovered that antisense expression of two TRX cDNAs in Arabidopsis causes developmental abnormalities, seedling deformities, reduced and severely stunted growth, and chlorosis. Thus, the present inventors have discovered that TRX is essential for normal seed development and growth, and can be used as a target for the identification of herbicides. Accordingly, the present invention provides methods for the identification of compounds that inhibit TRX expression or activity, comprising: contacting a candidate compound with a TRX and detecting the presence or absence of binding between said compound and said TRX, or detecting a decrease in TRX expression or activity. The methods of the invention are useful for the identification of herbicides.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]
FIG. 1 shows a Thioredoxin reaction.

DETAILED DESCRIPTION OF THE INVENTION

[0010] Definitions

[0011] The term “binding” refers to a noncovalent interaction that holds two molecules together. For example, 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.

[0012] As used herein, the term “cDNA” means complementary deoxyribonucleic acid.

[0013] As used herein, the term “DNA” means deoxyribonucleic acid.

[0014] As used herein, the term “dI” means deionized.

[0015] As used herein, the term “ELISA” means enzyme-linked immunosorbent assay.

[0016] As used herein, the term “GUS” means β-glucouronidase.

[0017] The term “herbicide”, as used herein, 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.

[0018] As used herein, the term “HPLC” means high pressure liquid chromatography.

[0019] The term “inhibitor”, as used herein, refers to a chemical substance that inactivates or decreases the enzymatic activity of TRX. The inhibitor may function by interacting directly with the enzyme, a cofactor of the enzyme, the substrate of the enzyme, or any combination thereof.

[0020] 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. Alternatively, the introduced polynucleotide may be present on an extra-chromosomal non-replicating vector and be transiently expressed or transiently active.

[0021] As used herein, the term “LB” means Luria-Bertani media.

[0022] As used herein, the term “mRNA” means messenger ribonucleic acid.

[0023] As used herein, the terms “NADP” and “NADPH” refer to nicotinamide adenine dinucleotide phosphate, a coenzyme which participates in redox reactions during the light reaction of photosynthesis. High-energy reactions cause the photolysis of water, in which the hydrogen reduces NADP+ to NADPH and generates the oxygen released during photosynthesis. The reduced NADPH is used in the conversion of carbon dioxide to carbohydrate during the dark reaction of photosynthesis. The term “NADP+” or “NADP” refers to nicotinamide adenine dinucleotide phosphate, oxidized form. The term “NADPH” refers to nicotinamide adenine dinucleotide phosphate, reduced form.

[0024] As used herein, the term “NADP-thioredoxin reductase” (EC 1.6.4.5) or “NTR” refers to a flavin enzyme that catalyses the conversion of NADPH and oxidized thioredoxin to NADP and reduced thioredoxin.

[0025] As used herein, the term “Ni” refers to nickel.

[0026] As used herein, the term “Ni-NTA” refers to nickel sepharose.

[0027] As used herein, the term “NTR” or “NADP-thioredoxin reductase” (EC 1.6.4.5) refers to a flavin enzyme that catalyses the conversion of NADPH and oxidized thioredoxin to NADP and reduced thioredoxin.

[0028] As used herein, “oxidized molecule” refers to a molecule, which has a relative oxidation state described in the art as “oxidized”. “Oxidized target protein” refers to a target protein, which has a relative oxidation state described in the art as “oxidized”. “Oxidized”/“oxidation” refers to a loss of electrons. NADPH is oxidized to become NADP+, for example.

[0029] As used herein, the term “PCR” means polymerase chain reaction.

[0030] The “percent (%) sequence identity” between two polynucleotide or two polypeptide sequences can be determined according to the either the BLAST program (Basic Local Alignment Search Tool, Altschul and Gish (1996) Meth Enzymol 266: 460-480; Altschul (1990) J Mol Biol 215: 403-410) in the Wisconsin Genetics Software Package (Devererreux et al. (1984) Nucl Acid Res 12: 387), Genetics Computer Group (GCG), Madison, Wis. (NCBI, Version 2.0.11, default settings) or using Smith Waterman Alignment (Smith and Waterman (1981) Adv Appl Math 2: 482) as incorporated into GeneMatcher Plus™ (Paracel, Inc., using the default settings and the version current at the time of filing). 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 a uracil nucleotide.

[0031] As used herein, the term “PGI” means plant growth inhibition.

[0032] “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.

[0033] By “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.

[0034] As used herein, “reduced molecule” refers to a molecule, which has a relative oxidation state described in the art as “reduced”. “Reduced target protein” refers to a target protein, which has a relative oxidation state described in the art as “reduced”. “Reduced”/“reduction” refers to a gain of electrons. NADP+ is reduced to become NADPH, for example.

[0035] As used herein, the term “RNA” means ribonucleic acid.

[0036] As used herein, the term “SDS” means sodium dodecyl sulfate.

[0037] As used herein, the term “SDS-PAGE” means sodium dodecyl sulfate-polyacrylimide gel electrophoresis.

[0038] The term “specific binding” refers to an interaction between TRX and a molecule or compound, wherein the interaction is dependent upon the primary amino acid sequence or the conformation of TRX.

[0039] As used herein, the term “target protein” or “target proteins” refers to a protein or proteins with intramolecular disulfide bonds that thioredoxin reduces or takes part in reducing, such as thiocalsin, peroxiredoxins, gliadins, and glutenins.

[0040] As used herein, the term “TATA box” refers to a sequence of nucleotides that serves as the main recognition site for the attachment of RNA polymerase in the promoter region of eukaryotic genes. Located at around 25 nucleotides before the start of transcription, it consists of the seven-base consensus sequence TATAAAA, and is analogous to the Pribnow box in prokaryotic promoters.

[0041] As used herein, the term “Thioredoxin” is synonymous with “TRX” and refers to a protein that may reduce target proteins through the reduction of intramolecular disulfide bonds, as shown in FIG. 1, and is included herein as the proteins of SEQ ID NO: 2 and SEQ ID NO: 4 and/or the respective encoding cDNAs, SEQ ID NO: 1 and SEQ ID NO: 3.

[0042] As used herein, the term “TLC” means thin layer chromatography.

[0043] Embodiments of the Invention

[0044] The present inventors have discovered that inhibition of TRX gene expression strongly inhibits the growth and development of plant seedlings. Thus, the inventors are the first to demonstrate that TRX is a target for herbicides.

[0045] Accordingly, the invention provides methods for identifying compounds that inhibit TRX gene expression or activity. Such methods include ligand binding assays, assays for enzyme activity and assays for TRX gene expression. Any compound that is a ligand for TRX, other than its substrates, may have herbicidal activity. For the purposes of the invention, “ligand” refers to a molecule that will bind to a site on a polypeptide. The compounds identified by the methods of the invention are useful as herbicides.

[0046] Thus, in one embodiment, the invention provides a method for identifying a compound as a candidate for a herbicide, comprising:

[0047] a) contacting a TRX with a compound; and

[0048] b) detecting the presence and/or absence of binding between said compound and said TRX;

[0049] wherein binding indicates that said compound is a candidate for a herbicide.

[0050] By “TRX” is meant any protein that catalyzes the reduction target proteins through the reduction of intramolecular disulfide bonds, as shown in FIG. 1. The TRX may have the amino acid sequence of a naturally occurring TRX found in a plant, animal or microorganism, or may have an amino acid sequence derived from a naturally occurring sequence. In one instance, the TRX is a plant TRX. The cDNA (SEQ ID NO: 1) encoding the TRX protein or polypeptide (SEQ ID NO: 2) can be found herein as well as in the TIGR database at locus At5g42980. In another instance, the TRX is a plant TRX, with the cDNA (SEQ ID NO: 3) encoding the TRX protein or polypeptide (SEQ ID NO: 4) found herein as well as in the TIGR database at locus At1g03680.

[0051] By “plant TRX” is meant a protein that can be found in at least one plant, and which that catalyzes the reduction target proteins through the reduction of intramolecular disulfide bonds, as shown in FIG. 1. The TRX may be from any plant, including monocots and dicots.

[0052] In one embodiment, the TRX is an Arabidopsis TRX. 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. Preferably, the Arabidopsis TRX is from Arabidopsis thaliana.

[0053] In various embodiments, the TRX can be from barnyard grass (Echinochloa crus-galli), crabgrass (Digitaria sanguinalis), green foxtail (Setana viridis), perennial ryegrass (Lolium perenne), hairy beggarticks (Bidens pilosa), nightshade (Solanum nigrum), smartweed (Polygonum lapathifolium), velvetleaf (Abutilon theophrasti), common lambsquarters (Chenopodium album L.), Brachiara plantaginea, Cassia occidentalis, Ipomoea aristolochiaefolia, Ipomoea purpurea, Euphorbia heterophylla, Setaria spp, Amaranthus retroflexus, Sida spinosa, Xanthium strumarium and the like.

[0054] Fragments of a TRX polypeptide may be used in the methods of the invention. The fragments comprise at least 10 consecutive amino acids of a TRX. Preferably, the fragment comprises at least 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100 or at least 110 consecutive amino acids residues of a TRX. In one embodiment, the fragment is from an Arabidopsis TRX. Preferably, the fragment contains an amino acid sequence conserved among plant Thioredoxins. Such conserved fragments are identified in Grima-Pettenuti et al. (1993) Plant Mol Biol 21: 1085-1095 and Taveres et al. (2000), supra. Those skilled in the art could identify additional conserved fragments using sequence comparison software.

[0055] Polypeptides having at least 80% sequence identity with a plant TRX are also useful in the methods of the invention. Preferably, the sequence identity is at least 85%, more preferably the identity is at least 90%, most preferably the sequence identity is at least 95% or 99%.

[0056] In addition, it is preferred that the polypeptide has at least 50% of the activity of a plant TRX. More preferably, the polypeptide has at least 60%, at least 70%, at least 80% or at least 90% of the activity of a plant TRX. Most preferably, the polypeptide has at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the activity of the A. thaliana TRX protein.

[0057] Thus, in another embodiment, the invention provides a method for identifying a compound as a candidate for a herbicide, comprising:

[0058] a) contacting a compound with at least one polypeptide selected from the group consisting of:

[0059] i) the polypeptide set forth in SEQ ID NO: 2 or 4; and

[0060] ii) a polypeptide have at least 80% sequence identity with the polypeptide set forth in SEQ ID NO: 2 or 4; and

[0061] 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.

[0062] Any technique for detecting the binding of a ligand to its target may be used in the methods of the invention. For example, the ligand and target are combined in a buffer. Many methods for detecting the binding of a ligand to its target are known in the art, and include, but are not limited to the detection of an immobilized ligand-target complex or the detection of a change in the properties of a target when it is bound to a ligand. For example, in one embodiment, an array of immobilized candidate ligands is provided. The immobilized ligands are contacted with a TRX protein or a fragment or variant thereof, the unbound protein is removed and the bound TRX is detected. In a preferred embodiment, bound TRX is detected using a labeled binding partner, such as a labeled antibody. In a variation of this assay, TRX 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.

[0063] Once a compound is identified as a candidate for a herbicide, it can be tested for the ability to inhibit TRX enzyme activity. The compounds can be tested using either in vitro or cell based enzyme assays. Alternatively, 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.

[0064] Thus, in one embodiment, 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 a change in the growth or viability of said plant or plant cells. The change detected may be a decrease in growth or viability.

[0065] A decrease in growth occurs where the herbicide candidate causes at least a 10% decrease in the growth of the plant or plant cells, as compared to the growth of the plants or plant cells in the absence of the herbicide candidate. A decrease in viability occurs where at least 20% of the plants cells, or portions of the plant contacted with the herbicide candidate, are nonviable. Preferably, 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.

[0066] The ability of a compound to inhibit TRX 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. TRX catalyzes the irreversible or reversible reduction target proteins through the reduction of intramolecular disulfide bonds. Methods for detection of oxidized or reduced target proteins, include spectrophotometry, mass spectroscopy, thin layer chromatography (TLC) and reverse phase HPLC.

[0067] Thus, the invention provides a method for identifying a compound as a candidate for a herbicide, comprising:

[0068] a) contacting an oxidized target protein with TRX;

[0069] b) contacting said oxidized target protein with TRX and said candidate compound; and

[0070] c) determining the concentration of reduced target protein after the contacting of steps (a) and (b).

[0071] If a candidate compound inhibits TRX activity, a higher concentration of the substrates (oxidized target protein) and a lower level of the product (reduced target protein) will be detected in the presence of the candidate compound (step b) than that detected in the absence of the compound (step a).

[0072] Preferably the TRX is a plant TRX. Enzymatically active fragments of a plant TRX are also useful in the methods of the invention. For example, a polypeptide comprising at least 100 consecutive amino acid residues of a plant TRX may be used in the methods of the invention. In addition, a polypeptide having at least 80%, 85%, 90%, 95%, 98% or at least 99% sequence identity with a plant TRX may be used in the methods of the invention. Preferably, the polypeptide has at least 80% sequence identity with a plant TRX and at least 50%, 75%, 90% or at least 95% of the activity thereof.

[0073] Thus, the invention provides a method for identifying a compound as a candidate for a herbicide, comprising:

[0074] a) contacting oxidized target protein with a polypeptide selected from the group consisting of:

[0075] i) the polypeptide set forth in SEQ ID NO: 2 or 4; and

[0076] ii) a polypeptide have at least 80% sequence identity with the polypeptide set forth in SEQ ID NO: 2 or 4; and

[0077] b) contacting said oxidized target protein with said polypeptide and said compound; and

[0078] c) determining the concentration of reduced target protein after the contacting of steps (a) and (b).

[0079] Again, if a candidate compound inhibits TRX activity, a higher concentration of the substrate (oxidized target protein) and a lower level of the product (reduced target protein) will be detected in the presence of the candidate compound (step b) than that detected in the absence of the compound (step a).

[0080] For the in vitro enzymatic assays, TRX protein and derivatives thereof may be purified from a plant or may be recombinantly produced in and purified from a plant, bacteria, or eukaryotic cell culture. Preferably TRX proteins are produced using a baculovirus or E. coli expression system. Methods for purifying TRX may be found in Florencio et al. (1988) Arch Biochem Biophys 266: 496-507 (PMID: 3190242) or Gautier et al. (1998) Eur J Biochem 252: 314-24 (PMID: 9523703). Other methods for the purification of TRX proteins and polypeptides are known to those skilled in the art.

[0081] As an alternative to in vitro assays, the invention also provides plant and plant cell based assays. In one embodiment, the invention provides a method for identifying a compound as a candidate for a herbicide, comprising:

[0082] a) measuring the expression of TRX in a plant or plant cell in the absence of said compound;

[0083] b) contacting a plant or plant cell with said compound and measuring the expression of TRX in said plant or plant cell; and

[0084] c) comparing the expression of TRX in steps (a) and (b).

[0085] A change in TRX expression indicates that the compound is a herbicide candidate. In one embodiment, the plant or plant cell is an Arabidopsis thaliana plant or plant cell.

[0086] Expression of TRX can be measured by detecting the TRX primary transcript or mRNA, TRX polypeptide or TRX enzymatic activity. Methods for detecting the expression of RNA and proteins are known to those skilled in the art. (See, for example, Current Protocols in Molecular Biology, Ausubel et al., eds., Greene Publishing and Wiley-Interscience, New York, 1995). However, the method of detection is not critical to the invention. Methods for detecting TRX 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 TRX promoter fused to a reporter gene, bDNA assays, and microarray assays.

[0087] 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. Also, any reporter gene system may be used to detect TRX protein expression. For detection using gene reporter systems, a polynucleotide encoding a reporter protein is fused in frame with TRX, 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 acetyltransferase (Gorman et al. (1982) Mol Cell Biol 2: 1104; Prost et al. (1986) Gene 45: 107-111), β-galactosidase (Nolan et al. (1988) Proc Natl Acad Sci USA 85: 2603-2607), alkaline phosphatase (Berger et al. (1988) Gene 66: 10), luciferase (De Wet et al. (1987) Mol Cell Biol 7: 725-737), β-glucuronidase (GUS), fluorescent proteins, chromogenic proteins and the like. Methods for detecting TRX activity are described above.

[0088] Chemicals, compounds or compositions identified by the above methods as modulators of TRX expression or activity can be used to control plant growth. For example, compounds that inhibit plant growth can be applied to a plant or expressed in a plant to prevent plant growth. Thus, 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.

[0089] Herbicides and herbicide candidates identified by the methods of the invention can be used to control the growth of undesired plants, including monocots and dicots. Examples of 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.

EXPERIMENTAL

[0090] Plant Growth Conditions

[0091] Unless, otherwise indicated, all plants are grown in Scotts Metro-Mix™ soil (the Scotts Company) or a similar soil mixture in an environmental growth room at 22° C., 65% humidity, 65% humidity and a light intensity of ˜100 μ-E m−2 s−1 supplied over 16 hour day period.

[0092] Seed Sterilization

[0093] All seeds are surface sterilized before sowing onto Phytagel plates using the following protocol.

[0094] 1. Place approximately 20-30 seeds into a labeled 1.5 ml conical screw cap tube. Perform all remaining steps in a sterile hood using sterile technique.

[0095] 2. Fill each tube with 1 ml 70% ethanol and place on rotisserie for 5 minutes.

[0096] 3. Carefully remove ethanol from each tube using a sterile plastic dropper; avoid removing any seeds.

[0097] 4. Fill each tube with 1 ml of 30% Clorox and 0.5% SDS solution and place on rotisserie for 10 minutes.

[0098] 5. Carefully remove bleach/SDS solution.

[0099] 6. Fill each tube with 1 ml sterile dI H2O; seeds should be stirred up by pipetting of water into tube. Carefully remove water. Repeat 3 to 5 times to ensure removal of Clorox/SDS solution.

[0100] 7. Fill each tube with enough sterile dI H2O for seed plating (˜200-400 μl). Cap tube until ready to begin seed plating.

[0101] Plate Growth Assays

[0102] Surface sterilized seeds are sown onto plate containing 40 ml half strength sterile MS (Murashige and Skoog, no sucrose) medium and 1% Phytagel using the following protocol:

[0103] 1. Using pipette man and 200 μl tip, carefully fill tip with seed solution. Place 10 seeds across the top of the plate, about ¼ inch down from the top edge of the plate.

[0104] 2. Place plate lid ¾ of the way over the plate and allow to dry for 10 minutes.

[0105] 3. Using sterile micropore tape, seal the edge of the plate where the top and bottom meet.

[0106] 4. Place plates stored in a vertical rack in the dark at 4° C. for three days.

[0107] 5. Three days after sowing, 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 ˜100 μ-E m−2 s−1 supplied over 16 hour day period.

[0108] 6. Beginning on day 3, daily measurements are carried out to track the seedlings development until day 14. Seedlings are harvested on day 14 (or when root length reaches 6 cm) for root and rosette analysis.

EXAMPLE 1

Construction of a Transgenic Plant Expressing the Driver

[0109] The “Driver” is an artificial transcription factor comprising a chimera of the DNA-binding domain of the 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. 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.

[0110] The driver expression cassette was introduced into Arabidopsis thaliana by agroinfection. Transgenic plants that stably expressed the driver transcription factor were obtained.

EXAMPLE 2

Construction of Antisense Expression Cassettes in a Binary Vector

[0111] A fragment or variant of an Arabidopsis thaliana cDNA corresponding to SEQ ID NO: 1 or SEQ ID NO: 3 was ligated into the PacI/AscI sites of an E.coli/Agrobacterium binary vector in the antisense orientation. This placed transcription of the antisense RNA under the control of an artificial promoter that is active only in the presence of the driver transcription factor described above. The artificial promoter contains four contiguous binding sites for the GAL4 transcriptional activator upstream of a minimal promoter comprising a TATA box.

[0112] The ligated DNA was transformed into E.coli. Kanamycin resistant clones were selected and purified. DNA was isolated from each clone and characterized by PCR and sequence analysis. The DNA was inserted in a vector that expresses the A. thaliana antisense RNA, which is complementary to a portion of the DNA of SEQ ID NO: 1 or SEQ ID NO: 3. In one example, this antisense RNA is complementary to the cDNA sequence found in the TIGR database at locus At5g42980. The coding sequence for this locus is shown as SEQ ID NO: 1. The protein encoded by these mRNAs is shown as SEQ ID NO: 2. In another example, this antisense RNA is complementary to the cDNA sequence found in the TIGR database at locus At1g03680. The coding sequence for this locus is shown as SEQ ID NO: 3. The protein encoded by these mRNAs is shown as SEQ ID NO: 4.

[0113] The antisense expression cassette and a constitutive chemical resistance expression cassette are located between right and left T-DNA borders. Thus, the antisense expression cassettes can be transferred into a recipient plant cell by agroinfection.

EXAMPLE 3

Transformation of Agrobacterium with the Antisense Expression Cassette

[0114] The vector was transformed into Agrobacterium tumefaciens by electroporation. Transformed Agrobacterium colonies were isolated using chemical selection. DNA was prepared from purified resistant colonies and the inserts were amplified by PCR and sequenced to confirm sequence and orientation.

EXAMPLE 4

Construction of an Arabidopsis Antisense Target Plants

[0115] 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.

[0116] At two days prior to agroinfection, 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 the desired DNA. The cultures were incubated overnight at 28° C. at 250 rpm until the cells reached stationary phase. The following morning, 200 ml LB in a 500 ml flask was inoculated with 500 μl of the overnight culture and the cells were grown to stationary phase by overnight incubation at 28° C. at 250 rpm. The cells were pelleted by centrifugation at 8000 rpm for 5 minutes. The supernatant was removed and excess media was removed by setting the centrifuge bottles upside down on a paper towel for several minutes. The cells were then resuspended in 500 ml infiltration medium (autoclaved 5% sucrose) and 250 μl/L Silwet L-77™ (84% polyalkyleneoxide modified heptamethyltrisiloxane and 16% allyloxypolyethyleneglycol methyl ether), and transferred to a one liter beaker.

[0117] 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 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.

[0118] Transgenic Arabidopsis T1 seedlings were selected. Approximately 70 mg seeds from an agrotransformed plant were mixed approximately 4:1 with sand and placed in a 2 ml screw cap cryo vial.

[0119] 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 μl/L) until the leaves were completely wetted. The spraying was repeated for the following two days.

[0120] Ten days after the first spraying resistant plants were transplanted to 2.5 inch round pots containing moistened sterile potting soil. The transplants were then sprayed with herbicide and returned to the growth room. These herbicide resistant plants represented stably transformed T1 plants.

EXAMPLE 5

Effect of Antisense Expression in Arabidopsis Seedlings

[0121] The T1 antisense target plants from the transformed plant lines obtained in Example 4 were crossed with the Arabidopsis transgenic driver line described above. The resulting F1 seeds were then subjected to a PGI plate assay to observe seedling growth over a 2-week period. Seedlings were inspected for growth and development. The antisense expression of these genes resulted in significantly impaired growth, indicating that each of these thioredoxin genes represents an essential gene for normal plant growth and development. Each of the transgenic lines containing one of the two antisense constructs for Thioredoxin exhibited significant seedling abnormalities. Seedlings showed deformities, reduced and severely stunted growth, and chlorosis.

EXAMPLE 6

Cloning and Expression Strategies, Extraction and Purfication of the TRX Protein

[0122] The following protocol may be employed to obtain the purified TRX protein.

[0123] Cloning and expression strategies:

[0124] A TRX gene can be cloned into E. coli (pET vectors-Novagen), Baculovirus (Pharmingen) and Yeast (Invitrogen) expression vectors containing His/fusion protein tags, and the expression of recombinant protein can be evaluated by SDS-PAGE and Western blot analysis.

[0125] Extraction:

[0126] Extract recombinant protein from 250 ml cell pellet in 3 mL of extraction buffer by sonicating 6 times, with 6 sec pulses at 4° C. Centrifuge extract at 15000×g for 10 min and collect supernatant. Assess biological activity of the recombinant protein by activity assay.

[0127] Purification:

[0128] Purify recombinant protein by Ni-NTA affinity chromatography (Qiagen). Purification protocol: perform all steps at 4° C.:

[0129] Use 3 ml Ni-beads (Qiagen)

[0130] Equilibrate column with the buffer

[0131] Load protein extract

[0132] Wash with the equilibration buffer

[0133] Elute bound protein with 0.5 M imidazole

EXAMPLE 7

Assays for Testing Inhibitors or Candidates for Inhibition of TRX Activity

[0134] The activity of TRX may be determined in the presence and absence of candidate inhibitors in a suitable reaction mixture, such as described by any of the following known assay protocols:

[0135] A. Fluorescent assay for reduction of target proteins:

[0136] This assay is based on monobromobimane (a fluorescent probe) revelation. Monobromobimane labels sulfhydryl groups permitting identification of reduced target proteins or Trx, as described by Yano el al. (2001) Proc Natl Acad Sci U S A 98: 4794-9 (PMID: 11274350).

[0137] B. Protein-Disulfide Reductase (Trx) Activity:

[0138] The activity of Trx as protein-disulfide reductase is assessed in the presence of an oxidized protein (e.g. insulin, di-FTC-insulin) as described in Holmgren and Bjornstedt (1995) Methods Enzymol 252: 199-208 (PMID: 7476354).

[0139] C. Coupled NADP-malate dehydrogenase assay:

[0140] The initial rate of activation or inactivation of NADP-malate dehydrogenase has been shown to be proportional to the concentration of reduced or oxidized thioredoxin, respectively, as described in Rebeille and Hatch (1986) Arch Biochem Biophys 249:164-70 (PMID: 3740849).

[0141] D. Standard NADP-Thioredoxin Reductase/NADPH coupled assay:

[0142] The standard assay for the reaction in FIG. 1 is described in Lunn et al. (1986) Biochim Biophys Acta 871: 257-67 (PMID: 3707971).

[0143] While the foregoing describes certain embodiments of the invention, it will be understood by those skilled in the art that variations and modifications may be made and still fall within the scope of the invention.

	Number	Date	Country
	60342182	Dec 2001	US
	60342184	Dec 2001	US

Methods for the identification of inhibitors of thioredoxin expression or activity in plants

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