WO2002071841A2 - Methods for the identification of inhibitors of argininosuccinate synthase expression or activity in plants - Google Patents

Abstract

The present inventors have discovered that Argininosuccinate synthase (AS) is essential for plant growth. Specifically, the inhibition of AS gene expression in plant seedlings results in severe chlorosis and reduced growth. Thus, AS can be used as a target for the identification of herbicides. Accordingly, the present invention provides methods for the identification of compounds that inhibit AS expression or activity, comprising: contacting a compound with an AS and detecting the presence or absence of binding between said compound and said AS, or detecting a change in AS expression or activity. The methods of the invention are useful for the identification of herbicides. Figure (1) shows the argininosuccinate synthase reaction.

Description

METHODS FOR THE IDENTIFICATION OF INHIBITORS OF ARGININOSUCCINATE SYNTHASE EXPRESSION OR ACTIVITY IN PLANTS

This application claims the benefit of U. S. Provisional Application No. 60/274,538 filed March 9, 2001.

FIELD OF THE INVENTION

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

BACKGROUND OF THE INVENTION

Arginine is an essential amino acid for human development. Argininosuccinate synthase (EC 6.3.4.5) (AS) is a urea cycle enzyme that catalyzes the penultimate step in arginine biosynthesis in vertebrates: the ATP-dependent ligation of citrulline to aspartate to form argininosuccinate, AMP and pyrophosphate. Very little literature is available on arginine biosynthesis in plants. AS is localized in soybean cells by Shargool et al. (1978). AS is a homotetrameric enzyme of chains of about 400 amino-acid residues. An arginine seems to be important for the enzyme's catalytic mechanism. The sequences of AS from various prokaryotes, archaebacteria and eukaryotes show significant similarity. There are two signature patterns for AS. The first is a highly conserved stretch of nine residues located in the N-terminal extremity of these enzymes. The second is derived from a conserved region which contains one of the conserved arginine residues. To date there do not appear to be any publications describing lethal effects of over-expression, antisense expression or knock-out of this gene in plants. Thus, the prior art has not suggested that AS 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

Surprisingly, the present inventors have discovered that antisense expression of an AS cDNA in Arabidopsis causes developmental abnormalities, arrested growth and chlorosis in plant seedlings. Thus, the present inventors have discovered that AS 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 AS expression or activity, comprising: contacting a candidate compound with a AS and detecting the presence or absence of binding between said compound and said AS, or detecting a decrease in AS expression or activity. The methods of the invention are useful for the identification of herbicides.

BRIEF DESCRIPTION OF THE DRAWING

Fig. 1 shows the argininosuccinate synthase reaction.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

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.

As used herein, the term "Argininosuccinate synthase (EC 6.3.4.5)" is synonymous with "AS" and refers to an enzyme that catalyses the conversion of L-asparate, L-citrulline and ATP to L-argininosuccinate, AMP (adenosine monophosphate) and diphosphate, as shown in Fig. 1.

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. The term "inhibitor", as used herein, refers to a chemical substance that inactivates the enzymatic activity of AS. The inhibitor may function by interacting directly with the enzyme, a cofactor of the enzyme, the substrate of the enzyme, or any combination thereof. A polynucleotide may be "introduced" into a plant cell by any means, including transfection, transformation or transduction, electroporation, particle bombardment, agroinfection and the like. The introduced polynucleotide may be maintained in the cell stably if it is incorporated into a non-chromosomal autonomous replicon or integrated into the plant chromosome. Alternatively, the introduced polynucleotide may be present on an extra- chromosomal non-replicating vector and be transiently expressed or transiently active. The "percent (%) sequence identity" between two polynucleotide or two polypeptide sequences is determined according to the either the BLAST program (Basic Local Alignment Search Tool; Altschul and Gish (1996) Meth Enzymol 255:460-480 and Altschul (1990) J Mol Biol 275:403-410) in the Wisconsin Genetics Software Package (Devererreux et al. (1984) Nucl Acid Res 12:387), Genetics Computer Group (GCG), Madison, Wisconsin. (NCBI, Version 2.0.11, default settings) or using Smith Waterman Alignment (Smith and Waterman (1981) AdvApplMath 2:482) as incorporated into GeneMatcher Plus™ (Paracel, Inc., http://www.paracel.com/html/genematcher.html; 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.

"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.

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.

The term "specific binding" refers to an interaction between AS and a molecule or compound, wherein the interaction is dependent upon the primary amino acid sequence or the conformation of AS .

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

Accordingly, the invention provides methods for identifying compounds that inhibit AS gene expression or activity. Such methods include ligand binding assays, assays for enzyme activity and assays for AS gene expression. Any compound that is a ligand for AS, other than its substrates, L-asparate, L-citrulline and ATP, or the cofactor zinc 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.

Thus, in one embodiment, the invention provides a method for identifying a compound that binds to an AS polypeptide, comprising: a) contacting an AS with said compound; and b) detecting the presence or absence of binding between said compound and said AS.

In a preferred embodiment of the invention, the binding between said compound and said AS indicates that said compound is a candidate for a herbicide.

By "AS" is meant any enzyme that catalyzes the interconversion of L-asparate and L- citralline with L-argininosuccinate. The AS may have the amino acid sequence of a naturally occuring AS found in a plant, animal or microorganism, or may have an amino acid sequence derived from a naturally occuring sequence. Preferably the AS is a plant AS. By "plant AS" is meant an enzyme that can be found in at least one plant, and which catalyzes the interconversion of L-asparate and L-citrulline with L-argininosuccinate. The AS may be from any plant, including both monocots and dicots.

In one embodiment, the AS is an Arabidopsis AS. 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 pumϊla, Arabidopsis suecica, Arabidopsis thaliana and Arabidopsis wallichii. Preferably, the Arabidopsis AS is from Arabidopsis thaliana.

In various embodiments, the AS 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, Jpomoea aristolochiaefolia, Jpomoea purpurea, Euphorbia heterophylla, Setaria spp, Amaranthus retroflexus, Sida spinosa, Xanthium strumarium and the like. Fragments of an AS polypeptide may be used in the methods of the invention. The fragments comprise at least 10 consecutive amino acids of an AS. Preferably, the fragment comprises at least 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or at least 100 consecutive amino acids residues of an AS. In one embodiment, the fragment is from an Arabidopsis AS. Preferably, the fragment contains an amino acid sequence conserved among plant argininosuccinate synthase. Such conserved fragments are identified in Grima-Pettenuti et al. (1993) Plant Mol Biol 27:1085-1095 and Taveres et al. (2000), supra.. Those skilled in the art could identify additional conserved fragments using sequence comparison software.

Polypeptides having at least 80% sequence identity with a plant AS 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%.

In addition, it is preferred that the polypeptide has at least 50% of the activity of a plant AS. More preferably, the polypeptide has at least 60%, at least 70%, at least 80% or at least 90% of the activity of a plant AS. 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 AS protein.

Thus, in another embodiment, the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) contacting said compound with at least one polypeptide selected from the group consisting of: a plant AS, a polypeptide comprising at least ten consecutive amino acids of a plant AS, a polypeptide having at least 85% sequence identity with a plant AS, and a polypeptide having at least 80% sequence identity with a plant AS and at least 50% of the activity thereof; and b) detecting the presence or absence of binding between said compound and said polypeptide; wherein binding indicates that said compound is a candidate for a herbicide. 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 AS protein or a fragment or variant thereof, the unbound protein is removed and the bound AS is detected. In a preferred embodiment, bound AS is detected using a labeled binding partner, such as a labeled antibody. In a variation of this assay, AS is labeled prior to contacting the immobilized candidate ligands. Preferred labels include fluorescent or radioactive moieties. Preferred detection methods include fluorescence correlation spectroscopy (FCS) and FCS-related confocal nanofluorimetric methods. See http://www.evotec.de/technology.

Once a compound is identified as a candidate for a herbicide, it can be tested for the ability to inhibit AS 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.

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 the presence or absence of a decrease in the growth or viability of said plant or plant cells.

By decrease in growth, is meant that the herbicide candidate causes at least a 10% decrease in the growth of the plant or plant cells, as compared to the growth of the plants or plant cells in the absence of the herbicide candidate. By a decrease in viability is meant that at least 20% of the plants cells, or portion of the plant contacted with the herbicide candidate are nonviable. Preferably, the growth or viability will be at decreased by at least 40%. More preferably, the growth or viability will be decreased by at least 50%, 75% or at least 90% or more. Methods for measuring plant growth and cell viability are known to those skilled in the art. It is possible that a candidate compound may have herbicidal activity only for certain plants or certain plant species. The ability of a compound to inhibit AS 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. AS catalyzes the irreversible or reversible reaction of L- asparate, L-citrulline and ATP to the corresponding L-argininosucciηate, AMP and diphosphate. Methods for detection of L-asparate, L-citrulline, ATP, L-argininosuccinate, AMP and/or diphosphate, include spectrophotometry, mass spectroscopy, thin layer chromatography (TLC) and reverse phase HPLC.

Thus, the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) contacting an L-asparate, L-citrulline and ATP with AS; b) contacting said L-asparate, L-citrulline and ATP with AS and said candidate compound; and c) determining the concentration of at least one of L-asparate, L- citrulline, ATP, L-argininosuccinate, AMP and/or diphosphate after the contacting of steps (a) and (b).

If a candidate compound inhibits AS activity, a higher concentration of the substrates

(L-asparate, L-citrulline and/or ATP) and a lower level of the products (L-argininosuccinate, AMP and/or diphosphate) will be detected in the presence of the candidate compound (step b) than in the absence of the compound (step a).

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

Thus, the invention provides a method for identifying a compound as a candidate for a herbicide, comprising: a) contacting L-asparate, L-citrulline and ATP with a polypeptide selected from the group consisting of: a polypeptide having at least 85% sequence identity with a plant AS, a polypeptide having at least 80% sequence identity with a plant AS and at least 50% of the activity thereof, and a polypeptide comprising at least 100 consecutive amino acids of a plant AS; b) contacting said L-asparate, L-citrulline and ATP with said polypeptide and said compound; and c) determining the concentration of at least one of L-asparate, L- citrulline, ATP, L-argininosuccinate, AMP and/or diphosphate after the contacting of steps (a) and (b).

Again, if a candidate compound inhibits AS activity, a higher concentration of the substrate (L-asparate, L-citrulline and or ATP) and a lower level of the product (L- argininosuccinate, AMP and/or diphosphate) will be detected in the presence of the candidate compound (step b) than in the absence of the compound (step a).

For the in vitro enzymatic assays, AS protein and derivatives thereof may be purified from a plant or may be recombinantly produced in and purified from a plant, bacteria, or eukaryotic cell culture. Preferably these proteins are produced using a baculovirus or E. coli expression system. Methods for the purification of arginosuccinate synthase may be described in Shargool PD, Steeves T, Weaver M, Russell M, The localization within plant cells of enzymes involved in arginine biosynthesis. Can J Biochem. 1978 Apr;56(4):273-9; (PMID: 565667) and in U.S. patents 6,077,835; 6,054,265; 5,972,001; 5,869,241 and 5,726,014. Other methods for the purification of AS proteins and polypeptides are known to those skilled in the art. 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: a) measuring the amount of an AS in a plant, plant cells or plant cell in the absence of said compound; b) contacting a plant, plant cells or plant cell with said compound and measuring the amount of said AS in said plant, plant cells or plant cell; c) comparing the amount of AS in steps (a) and (b).

A change in AS amount indicates that the compound is a herbicide candidate. In one embodiment, the plant, plant cells or plant cell is an Arabidopsis thaliana plant, plant cells or plant cell.

Expression of AS can be measured by detecting AS primary transcript or mRNA, AS polypeptide or AS 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-l terscience, New York, 1995. The method of detection is not critical to the invention. Methods for detecting AS 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 AS promoter fused to a reporter gene, bDNA assays and microarray assays. Methods for detecting protein expression include, but are not limited to, immunodetection methods such as Western blots, His Tag and ELISA assays, polyacrylamide gel electrophoresis, mass spectroscopy and enzymatic assays. Also, any reporter gene system may be used to detect AS protein expression. For detection using gene reporter systems, a polynucleotide encoding a reporter protein is fused in frame with AS, 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 55:2603-2607), 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 AS activity are described above.

Chemicals, compounds or compositions identified by the above methods as modulators of AS expression or activity can then be used to control plant growth. For example, compounds that inhibit plant growth can be applied to a plant or expressed in a plant, in order 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.

Herbicides and herbicide candidates identified by the methods of the invention can be used to control the growth of undesired plants, including both monocots and dicots. 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 per enne), hairy beggarticks (Bidens pϊlosa), nightshade (Solanum nigrum), smartweed (Polygonum lapathifolium), velvetleaf (Abutilon theophrasti), common lambsquarters (Chenopodium album L.), Brachiara plantaginea, Cassia occidentalis, Jpomoea aristolochiaefolia, Jpomoea purpurea, Euphorbia heterophylla, Setaria spp, Amaranthus retrqflexus, Sida spinosa, Xanthium strumarium and the like. EXPERIMENTAL

Plant Growth Conditions Unless, otherwise indicated, all plants are grown 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.

Seed Sterilization All seeds are surface sterilized before sowing onto phytagel plates using the following protocol.

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.

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

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

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

5. Carefully remove bleach/SDS solution.

6. Fill each tube with 1ml sterile dl 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. 7. Fill each tube with enough sterile dl H₂O for seed plating (~200-400 μl). Cap tube until ready to begin seed plating.

Plate Growth Assays

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:

1. Using pipette man and 200 μl tip, carefully fill tip with seed solution. Place 10 seeds across the top of the plate, about % in down from the top edge of the plate.

2. Place plate lid ³/₄ of the way over the plate and allow to dry for 10 minutes. 3. Using sterile micropore tape, seal the edge of the plate where the top and bottom meet.

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

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^" s^" supplied over 16 hour day period.

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

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 55:195-204. This chimeric driver is a rranscriptional activator specific for promoters having GAL4 binding sites. Expression of the driver is controlled by two tandem copies of the constitutive CaMV 35S promoter. The driver expression cassette is introduced into Arabidopsis thaliana by agroinfection. Transgenic plants that stably expressed the driver transcription factor are obtained.

Example 2 Construction of Antisense Expression Cassettes in a Binary Vector

A fragment, fragment or variant of ^'an Arabidopsis thaliana cDNA corresponding to SEQ ID NO:2 is ligated into the Pacl/Ascl sites of aa E.coli/Agrobacterium binary vector in the antisense orientation. This places transcription of the antisense RNA under the control of an artificial promoter that is active only in the presence of the driver transcription factor described above. The artificial promoter contains four contiguous binding sites for the GAL4 transcriptional activator upstream of a minimal promoter comprising a TATA box.

The ligated DNA is transformed into E.coli. Kanamycin resistant clones are selected and purified. DNA is isolated from each clone and characterized by PCR and sequence analysis. The DNA is inserted in a vector that expresses the A. thaliana antisense RNA, which is complementary to a portion of the DNA of SEQ ID NO:2. This antisense RNA is complementary to the cDNA sequence found in the TIGR database at locus F6I7 40, F6I7, or At4g24830. The coding sequence for this locus is shown as SEQ ID NO:2. The protein encoded by this mRNA is shown as SEQ ID NO: 1.

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

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

Example 4 Construction of aa Arabidopsis Antisense Target Plants

The antisense expression cassette is 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 are clipped in order enhance the emergence of secondary bolts. 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) is inoculated with a clonal glycerol stock of Agrobacterium carrying the desired DNA. The cultures are 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 is inoculated with 500 μl of the overnight culture and the cells are grown to stationary phase by overnight incubation at 28°C at 250 φm. The cells are pelleted by centrifugation at 8000 rpm for 5 minutes. The supernatant is removed and excess media is removed by setting the centrifuge bottles upside down on a paper towel for several minutes. The cells are 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.

The previously clipped Arabidopsis plants are dipped into the Agrobacterium suspension so that all above ground parts are immersed and agitated gently for 10 seconds.

The dipped plants are then cover with a tall clear plastic dome in order to maintain the humidity, and returned to the growth room. The following day, the dome is removed and the plants are grown under normal light conditions until mature seeds are produced. Mature seeds are collected and stored desiccated at 4 °C. Transgenic Arabidopsis TI seedlings are selected. Approximately 70 mg seeds from an agrotransformed plant are mixed approximately 4:1 with sand and placed in a 2 ml screw cap cryo vial.

One vial of seeds is then sown in a cell of an 8 cell flat. The flat is covered with a dome, stored at 4°C for 3 days, and then transferred to a growth room. The domes are removed when the seedlings first emerged. After the emergence of the first primary leaves, the flat is sprayed uniformly with a herbicide corresponding to the chemical resistance marker plus 0.005% Silwet (50 μl/L) until the leaves are completely wetted. The spraying is repeated for the following two days.

Ten days after the first spraying resistant plants are transplanted to 2.5 inch round pots containing moistened sterile potting soil. The transplants are then sprayed with herbicide and returned to the growth room. These herbicide resistant plants represent stably transformed TI plants.

Example 5 Effect of Antisense Expression in Arabidopsis Seedlings

The TI antisense target plants from the transformed plant lines obtained in Example 4 are crossed with the Arabidopsis transgenic driver line described above. The resulting Fl seeds are then subjected to a PGI plate assay to observe seedling growth over a 2-week period. Seedlings are inspected for growth and development. The two transgenic plant lines containing the antisense construct exhibited significant developmental abnormalities during early development. Four of nine seedlings from a first transgenic line and five often seedlings from a second independent transgenic line examined had chlorotic cotyledons, and red patches on the lower surfaces of the cotyledons. These data demonstrate that the antisense expression of this gene results in significantly impaired growth and indicate that this is an essential gene for normal plant growth and development

Example 6 Cloning and Expression Strategies, Extraction and Purification of the AS protein.

The following protocol may be employed to obtain the purified AS protein. Cloning and expression strategies:

AS gene can be cloned into E. coli (pET vectors-No vagen), Baculovirus (Pharmingen) and Yeast (Invitrogen) expression vectors containing His/fusion protein tags. Evaluate the expression of recombinant protein by SDS-PAGE and Western blot analysis.

Extraction:

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 15000xg for 10 min and collect supernatant. Assess biological activity of the recombinant protein by activity assay.

Purification:

Purify recombinant protein by Ni-NTA affinity chromatography (Quiagen). Purification protocol: perform all steps at 4oC: • Use 3 ml Ni-beads (Quiagen)

• Equilibrate column with the buffer

• Load protein extract

• Wash with the equilibration buffer

• Elute bound protein with 0.5 M imidazole

Example 7

Assays for Testing Inhibitors or Candidates for Inhibition of AS Activity

The enzymatic activity of AS 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:

A. Radiochemical assay:

This assay is based on the conversion of [³H] aspartate to [³H]argininosuccinate as described by O'Brein, W.E. (1979) Biochemistry 18, 5353-5356.

B. Luciferase assay: Measure the amount of ATP remaining in the reaction mix by using Luciferase (Packard Luclite).

C. Malachite green assay: Couple assay with pyrophoshatase reaction to generate orthophosphate. Measure amount of Pi formed by malachite green assay.

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.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method for identifying a compound that binds to an AS polypeptide, comprising: a) contacting an AS with a compound; and d) detecting the presence or absence of binding between said compound and said AS.

2. The method of claim 1, wherein the binding between said compound and said AS indicates that said compound is a candidate for a herbicide.

3. The method of claim 1, wherein said AS is a plant AS.

4. The method of claim 1, wherein said AS is an Arabidopsis AS.

5. The method of claim 1 , wherein said AS is SEQ ID NO : 1.

6. A method for determining whether a compound identified as a herbicide candidate by the method of claim 2 has herbicidal activity, comprising: contacting a plant, plant cells or plant cell with said herbicide candidate and detecting the presence or absence of a decrease in growth or viability of said plant, plant cells or plant cell.

7. A method for identifying a compound as a candidate for a herbicide, comprising: a) contacting a compound with at least one polypeptide selected from the group consisting of: an amino acid sequence comprising at least ten consecutive amino acids of a plant AS, an amino acid sequence having at least 85% sequence identity with a plant AS, and an amino acid sequence having at least 80% sequence identity with a plant AS and at least 50% of the activity thereof; and b) detecting the presence or absence of binding between said compound and said polypeptide; wherein binding indicates that said compound is a candidate for a herbicide.

8. A method for determining whether a compound identified as a herbicide candidate by the method of claim 7 has herbicidal activity, comprising: contacting a plant, plant cells or plant cell with said herbicide candidate and detecting the presence or absence of a decrease in growth or viability of said plant, plant cells or plant cell.

9. A method for identifying a compound as a candidate for a herbicide, comprising: a) contacting an L-asparate, L-citrulline and ATP with an AS; b) contacting said L-asparate, L-citrulline and ATP with said AS and said candidate compound; and c) determining the concentration of at least one of L-asparate, L- citrulline, ATP, L-argininosuccinate, AMP and/or diphosphate after the contacting of steps (a) and (b).

10. The method of claim 9, wherein said AS is a plant AS.

11. The method of claim 9, wherein said AS is an Arabidopsis AS.

12. The method of claim 9, wherein said AS is SEQ ID NO: 1.

13. A method for identifying a compound as a candidate for a herbicide, comprising: a) contacting L-asparate, L-citrulline and ATP with a polypeptide selected from the group consisting of: a polypeptide having at least 85% sequence identity with a plant AS, a polypeptide having at least 80% sequence identity with a plant AS and at least 50% of the activity thereof, and a polypeptide comprising at least 100 consecutive amino acids of a plant AS; b) contacting said L-asparate, L-citrulline and ATP with said polypeptide and said compound; and c) determining the concentration of at least one of L-asparate, L- citrulline, ATP, L-argininosuccinate, AMP and/or diphosphate after the contacting of steps (a) and (b).

14. A method for identifying a compound as a candidate for a herbicide, comprising: a) measuring the amount of an AS in a plant, plant cells or plant cell in the absence of a compound; b) contacting a plant, plant cells or plant cell with said compound and measuring the amount of said AS in said plant, plant cells or plant cell; c) comparing the amount of said AS in steps (a) and (b).

15. The method of claim 14 wherein said plant, plant cells or plant cell is an Arabidopsis plant, plant cells or plant cell.

16. The method of claim 14, wherein said AS is SEQ ID NO: 1.

17. The method of claim 14, wherein the amount of said AS is measured by detecting AS mRNA.

18. The method of claim 14, wherein the amount of said AS is measured by detecting AS polypeptide.