WO1989012230A1 - Analyse selective de produits agrochimiques - Google Patents

Analyse selective de produits agrochimiques Download PDF

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WO1989012230A1
WO1989012230A1 PCT/US1989/002151 US8902151W WO8912230A1 WO 1989012230 A1 WO1989012230 A1 WO 1989012230A1 US 8902151 W US8902151 W US 8902151W WO 8912230 A1 WO8912230 A1 WO 8912230A1
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plant
gene
promoter
expression
regulated
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PCT/US1989/002151
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Leona Claire Fitzmaurice
Christopher John Lamb
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The Salk Institute Biotechnology/Industrial Associ
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12N9/1037—Naringenin-chalcone synthase (2.3.1.74), i.e. chalcone synthase
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems
    • C12N15/8238—Externally regulated expression systems chemically inducible, e.g. tetracycline
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems
    • C12N15/8239—Externally regulated expression systems pathogen inducible
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters

Definitions

  • the present invention relates generally to methods and compositions for analyzing agrichemicals. More particularly, the invention relates to a novel screening method for identifying agriche ical compounds that may be useful for inducing transcription of trait- specific plant genes. In addition, the invention relates to a method for identifying trait-specific nucleic acid sequences from plants that respond to agrichemicals by regulating gene transcription. Finally, the invention relates to regulatory elements that control selective induction of plant defense genes. BACKGROUND OF THE INVENTION Agricultural chemicals, or agrichemicals, form the basis of a multi-billion dollar component of the chemical industry. Agrichemicals are used as pesticides, herbicides and plant growth regulators. Typically, a chemical company screens hundreds of thousands of chemical compounds to identify one or a few with desirable attributes or activities. These screening methodologies are labor intensive, time consuming and very costly.
  • the screening methodologies in use at the present time include everything from whole plant assays to in vitro tests on mammalian cells.
  • whole plant assays are typically used to test chemicals for herbicidal activity.
  • Flats of seedling plants are sprayed with different compounds and monitored for the effect( ⁇ ) of the chemical.
  • Pesticidal activities are identified by examining the effec (s) of the application of different compounds on, e.g., a specific insect pest.
  • pesticidal activity upon a known enzymatic pathway is hypothesized to cause a desired e ect
  • in vitro assays in heterologous systems are developed.
  • Assays for plant growth regulators can be even more time consuming and complex. For example, to * identify a plant growth regulator capable of delaying senescence in soybeans, using presently available technology, it is necessary to grow soybean plants to maturity, a process requiring three to four months, and then to expose different sets of plants to the effects of different chemicals. This is a time consuming and costly process.
  • Examples of such life processes include rooting and plant propagation, germination and dormancy, flowering, gamete production, abscission, fruit set and development, plant and organ size, production of axillary buds, self-pruning, formation of shape, tillering, resistance to and control of insects and diseases, overcoming environmental stress, uptake of minerals, plant composition, metabolic effects including ripening and yield increases, modification of sexual expression, senescence, dessication, protection against herbicide damage, and increase of herbicide absorption and translocation. Any and all of these processes that are mediated by changes in gene transcription are amenable to definition and can be used in the agrichemical screening assays that are disclosed and described herein.
  • these screening assays will provide a tremendous time and cost savings to the agrichemical industry.
  • the initial selection of promising agrichemicals, for further testing in standard seedling screens, will be shortened from months to days.
  • the actual numbers of seedling screens will be greatly reduced.
  • the assays will permit thousands of potential agrichemicals to be screened for new base compounds in a very short time because the labor and plant growth time involved is significantly less than with standard assays. In conjunction with this, significant savings on capital expenditures for new laboratories and greenhouses, as well as associated operating expenses, will be realized.
  • the key to analyzing the effect an agrichemical has on molecular events associated with a specific trait, such as growth or stress defense, is the ability to identify and isolate genetic material (i.e., the nucleic acids) associated with the trait, and then place the isolated genetic material in a strictly controlled environment where its response to the agrichemical can be directly and unambiguously measured.
  • the present invention provides a means for identifying and isolating genetic materials associated with traits of interest.
  • the invention further provides a means of using the identified and isolated genetic materials to evaluate agrichemicals 1 effects on the genetic materials, and by extrapolation, the trait of interest.
  • the present invention is based on several discoveries regarding plant regulatory elements which are associated with genes encoding protein products that influence plants in trait-specific manners.
  • the first discovery is that DNA sequences responsible for transcription of trait-specific genes can be identified by the combined means of biochemical and molecular analytical techniques. Once identified, these regulatory elements can be isolated from the plant genome and characterized.
  • a second discovery is that the isolated plant gene regulatory elements can be used to evaluate the effect a specific agrichemical has on the isolated regulatory element, and by extrapolation, the effect the agrichemical has on the trait-specific gene and the trait itself.
  • the present invention is further based on recent discoveries regarding activation of plant defense genes.
  • the first such discovery is that the reduced form of glutathione (GSH) , a small, water-soluble, non- toxic cellular metabolite, stimulates transcription of certain defense genes, including those that encode cell wall hydroxyproline-rich glycoproteins and the phenylpropanoid biosynthetic enzymes phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) . It has been found that transcriptional activation of these genes leads to marked transient accumulation of the corresponding transcripts, contributing to a massive change in the overall pattern of protein synthesis which closely resembles the change observed in response to fungal elicitor.
  • GSH glutathione
  • PAL phenylalanine ammonia-lyase
  • CHS chalcone synthase
  • Figure 1 is a photograph illustrating accumulation of plant defense gene transcripts in response to GSH.
  • Figure 2 shows two graphs that illustrate the kinetics for induction of (A) PAL and CHS transcripts and (B) PAL enzyme activity in response to GSH.
  • Figure 3 is a photograph illustrating dose response for induction of plant defense gene transcripts by GSH.
  • Figure 4 is a photograph illustrating the effect of GSH on the transcription of plant defense genes.
  • Figure 5 is a photograph illustrating the effect of GSH on the pattern of protein synthesis in plants.
  • Figure 6 is a graph illustrating the induction of PAL activity by GSSG.
  • Figure 8 is a photograph that illustrates expression of the chimeric CHS-CAT-NOS gene electroporated into protoplasts derived from suspension cultured cells of soybean.
  • Figure 9 is a photograph that illustrates the correlation between the accumulation of CAT transcripts and CAT activity in electroporated protoplasts containing the CHS-CAT-NOS gene.
  • Figure 10 is a graph illustrating glutathione induction of CHS-CAT-NOS relative to basal levels of expression as a function of the amount of the chimeric construct electroporated.
  • Figure 11 shows thin layer chromatography analysis and a graph which illustrate the effect of 5* deletions on glutathione regulation of the CHS-CAT-NOS gene electroporated into soybean protoplasts.
  • glutathione refers to 7 -L- glutamy1-L-cysteiny1-glycine.
  • peptide analogs of glutathione are substances having the formula 7 -L-glutamyl-L- cysteinyl-X where X is an amino acid other than glycine.
  • homoglutathione refers to ⁇ - L-glutamyl-L-cysteinyl- ⁇ -alanine.
  • GSH refers to the reduced form of glutathione.
  • GSSG refers to the oxidized form of glutathione.
  • PAL refers to phenylalanine ammonia-lyase. PAL catalyzes the conversion of the amino acid L-phenylalanine to ir ⁇ z «s-cinnamic acid and
  • CHS refers to chalcone synthase.
  • CHS catalyzes the condensation of 4- cou aroyl-CoA with three acetate units from malonyl-CoA to yield naringenin chalcone.
  • This reaction is the first step in a branch of phenylpropanoid metabolism specific for the synthesis of isoflavonoid phytoalexin antibiotics in legumes, and flavonoid pigments which are ubiquitous in higher plants (Dixon, et al., 1983; and Hahlbrock, el al., 1979).
  • 4CL refers to 4-coumarate:CoA ligase. 4CL synthesizes the thiol esters that are central intermediates in the synthesis of many phenylpropanoid compounds in higher plants (Douglas, et al., 1987) .
  • HRGP refers to hydroxyproline-rich glycoproteins.
  • exogenously controlled plant regulatory elements refer to nucleic acid sequences that affect transcription of functionally linked structural genes in response to exogenous stimuli.
  • exogenously controlled plant regulatory sequences include plant defense gene promoters, elicitor-regulated activator domains, upstream silencer domains, etc.
  • elicitor substances are com ⁇ pounds that can "turn on”, induce or otherwise activate exogenously-controlled plant regulatory elements such as the stress-regulated promoters for the plant genes that encode the phenylpropanoid biosynthetic enzymes phenylalanine ammonia-lyase (PAL) , chalcone synthase
  • PAL phenylalanine ammonia-lyase
  • Elicitor substances of the invention include, but are not limited to, the reduced form of glutathione; the reduced form of homoglutathione and the reduced form of other peptide analogs of glutathione; glycan elicitors such as hexa( -D-glucopyranosyl)-D-glucitols (Sharp, et al., 1984); lipid elicitors such as arachidonic acid and eicosapentaenoic acid, glycoprotein elicitors, fungal elicitors, and abiotic elicitors such as mercuric chloride HGC1 2 .
  • fungal elicitor refers to the high molecular weight material heat-released from mycelial cell walls of the bean fungal pathogen Col- letotrich m lindemulhianum (Lawton, et al., 1983) .
  • marker or reporter genes refer to genes that encode easily assayable protein products.
  • Examples of marker or reporter genes are CAT, GUS, ⁇ - galactosidase, and the firefly luciferase gene.
  • marker or reporter genes are functionally linked to plant gene regulatory elements that respond to exogenous stimuli by modulating transcription of genes (such as the CAT, GUS, ⁇ - galactosidase or luciferase reporter genes) functionally linked thereto.
  • CAT refers to chloramphenicol acetyltransferase.
  • NOS refers to nopaline synthase
  • GUS refers to beta glucuronidas .
  • lacZ refers to ⁇ - galactosidase.
  • native gene means a gene that exists in nature.
  • chimeric gene means a gene that has been constructed or engineered through the efforts of human beings.
  • CGC means a chimeric gene cassette.
  • transcription refers to syn- thesis of RNA from a DNA template.
  • promoter refers to a region of DNA involved in binding of RNA polymerase and other factors that initiate or modulate transcription.
  • transcription start site refers to the position on DNA corresponding to the first base incorporated into RNA. In the DNA sequences shown in the Figures, the transcription start site is designated as +1.
  • the TATA box refers to a con- served A-T rich septamer found about 25 base pairs upstream of the transcription start site of each eukaryotic RNA polymerase II transcription unit; the TATA box is believed to be involved in positioning the RNA polymerase enzyme for correct initiation of transcription.
  • terminator or 3' terminator refers to a sequence of DNA, represented at the 3' end of a gene or transcript, that causes the RNA polymerase to terminate transcription.
  • terminators include the 3' flanking region of the nopaline synthase (NOS) gene and the 3' flanking region of the octopine synthase (OCS) gene.
  • mho refers to a standard unit of conductivity.
  • IVT means in vitro translation.
  • suitable plant material means and expressly includes, plant protoplasts, plant cells, plant callus, plant tissues, developing plantlets, immature whole plants and mature whole plants.
  • a plant protoplast is a single plant cell that does not have a plant cell wall.
  • plant callus refers to an undiffentiated mass of plant cells.
  • substantially pure DNAs, RNAs, polypeptides and proteins are useful in ways that the non-separated, impure DNAs, RNAs, polypeptides or proteins are not.
  • operatively linked and functionally linked are equivalent terms that are used interchangeably. Both terms mean that the linked DNA sequences (e.g., the promoter(s) , the reporter gene(s) , and the terminator sequence(s) ) are operational or functional, i.e., work for their intended purposes.
  • terminator sequences refer to DNA sequences that "instruct" the RNA polymerase (i.e., the enzyme that catalyzes the synthesis of RNA from a DNA template in the process known as transcription) to stop transcribing the DNA.
  • Terminator sequences useful in the methods of the present invention include, but are not limited to, the 3' flanking region of the NOS gene and the 3' flanking region of the OCS gene.
  • stop codons refer to DNA sequences that, when transcribed into RNA stop translation of the RNA into proteins.
  • plant material engineered with human effort refers to plant material created by scientists who use the techniques of modern genetic engineering rather than traditional plant breeding techniques to generate new strains; engineered plant material does not exist "in nature", and therefore is not a product of nature.
  • elicitor-regulated activator domain and upstream silencer domain refer to two nucleotide sequence regions on exogenously-controlled plant promoters.
  • an elicitor-regulated activator domain refers to a first nucleotide sequence region on exogenously-controlled plant promoters such as promoters from the stress-regulated plant genes PAL,
  • CHS, 4CL, and the plant genes that encode the cell wall hydroxyproline-rich glycoproteins are defined by function.
  • these elicitor-regulated activator domains or regions confer on the promoter the property of being activated when protoplasts, plant cells, or plants that carry the promoter are treated with exogenous elicitors that affect transcription, e.g., reduced glutathione, reduced homoglutathione, reduced peptide analogs of glutathione, fungal elicitor preparations, etc.
  • the activator region extends from nucleotides -29 to -173; this region has substantial sequence homology to analogous activator regions in the promoters of co-ordinately regulated genes such as PAL and 4CL.
  • the upstream silencer domain refers to a second nucleotide sequence region on exogenously-controlled plant promoters such as promoters from the stress-regulated plant genes PAL, CHS, 4CL, and the plant genes that encode the cell wall hydroxyproline-rich glycoproteins. As defined by function, this silencer domain represses the activity of these promoters to the extent that, when the domain is removed and activity is analyzed by functional assays, elicitor induced expression (mediated by the remaining portions of the promoter) is enhanced several fold.
  • the silencer domain is known to contain binding site(s) for a repressor factor.
  • the silencer domain or region extends from nucleotides -173 to -326; this region has substantial sequence homology to analogous silencer regions in the promoters of co- ordinately regulated genes such as PAL and 4CL.
  • amino acids which make up the various amino acid sequences appearing herein may be identified according to the following three-letter or one-letter abbreviations:
  • nucleotides which comprise the various nucleotide sequences appearing herein have their usual single-letter designations (A, G, T, C or U) used routinely in the art.
  • the present invention discloses a method for identifying plant gene regulatory elements that respond to stimuli and affect transcription of structural genes operatively linked thereto.
  • the invention further discloses a screening assay useful for identifying substances that can be used exogenously to activate or deactivate plant trait-specific gene regulatory elements
  • plant gene regulatory sequences e.g., plant defense gene promoters, elicitor-regulated activator domains, upstream silencer domains and the like.
  • the plant gene regulatory elements are first identified and isolated, and then functionally linked to at least one reporter or marker gene whose expression can be monitored.
  • reporter or marker gene(s) constructs or cassettes are transformed into a cell and the cell is exposed to specific agrichemical(s) , the effect the agrichemical(s) has on the plant regulatory element of interest can be determined by monitoring, for expression of the reporter gene(s) .
  • the present invention comprises a method for identifying and isolating plant gene regulatory elements functionally linked to structural genes encoding protein products associated with a specific trait.
  • the method is a combination of biochemical and molecular techniques that make possible the identification of cDNAs associated with a specific trait of interest.
  • the cDNAs are then used as probes to isolate the corresponding gene regulatory element(s) from genomic DNA.
  • this method of the invention is comprised essentially of the following steps. First, RNAs are collected from two types of plant tissue: (1)
  • RNA pool is then translated in vitro and the translation products are examined in one- dimensional and two-dimensional electrophoretic gels. The data obtained from these comparative analyses are used to identify protein product(s) encoded by structural genes whose transcription is regulated in the desired trait-specific fashion.
  • a cDNA library is constructed from RNA isolated from the appropriate tissue. The cDNA library is then separately screened in a "plus-minus” fashion using RNA from a time when gene expression is "on" ("plus”) and a time when gene expression is "off” (“minus”) .
  • cDNAs that are expressed under the desired trait-specific conditions clones that hybridize to the "plus” RNA but not the "minus” RNA are selected. These "plus” or “minus” clones are then further characterized, to identify the ones that encode a protein having the characteristics of the candidate protein identified from the 1-D and 2-D electrophoretic gel analyses.
  • the chosen cDNA is then used as a probe to screen a genomic library of plant DNA to identify the gene regulatory element(s) (e.g., the promoter, elicitor-regulated activator domain, the upstream silencer domain, etc.) associated with the gene corresponding to the cDNA. Once identified and isolated, such gene regulatory element(s) can be further characterized by restriction analysis and sequencing.
  • trait-specific plant gene regulatory elements are referred to herein as trait-specific plant gene regulatory elements.
  • trait-specific promoters and other plant gene regulatory elements are operatively linked to at least one reporter or marker gene and at least one transcription terminator.
  • the chimeric regulatory elemen (s)/reporter gene(s) construct, or cassette, thus produced is transformed into cells which are then used to screen agrichemicals for their transcriptional effect on the trait-specific plant gene regulatory elements.
  • the transformed cells are preferably plant cells that can be propagated in cell culture or as whole plants.
  • the transformed cell preparation is contacted with an agrichemical and the reporter or marker gene product(s) is assayed, either directly (e.g., luciferase) or indirectly (e.g., CAT) .
  • Expression of the reporter gene(s) is an indication that the agrichemical induced transcription of the trait-specific plant gene regulatory element(s) and thus would likely induce transcription of the same regulatory element(s) if applied to a native plant. Thus, the agrichemical is considered a good candidate for influencing the trait of interest.
  • the present invention comprises substantially pure plant defense gene promoters that direct stress-regulated expression of target genes when chimeric gene fusions are introduced into plant cells.
  • promoters include, but are not limited to, promoters for the plant genes that encode the phenylpropanoid biosynthetic enzymes phenylalanine ammonia-lyase (PAL) , chalcone synthase (CHS) , and 4- coumarate:CoA ligase (4CL) , plus promoters for the plant genes that encode the cell wall hydroxyproline-rich glycoproteins.
  • the present invention comprises substantially pure functional domains of stress-regulated plant promoters, which domains include, but are not limited to, functional domains from promoters for the plant genes that encode the phenylpropanoid biosynthetic enzymes phenylalanine am onia-lyase (PAL) , chalcone synthase (CHS) , and 4- coumarate:CoA ligase (4CL) , plus promoters for the plant genes that encode the cell wall hydroxyproline-rich glycoproteins.
  • PAL phenylpropanoid biosynthetic enzymes phenylalanine am onia-lyase
  • CHS chalcone synthase
  • 4CL 4- coumarate:CoA ligase
  • Such substantially pure functional domains include: (1) an elicitor-regulated activator, located in the CHS promoter between the TATA box and nucleotide position - 173; and (2) an upstream silencer, located in the CHS promoter between nucleotides found at positions -173 and -326. (Nucleotide positions refer to those shown in Figure 7. ) There is substantial sequence homology between elements within these functional domains within the CHS promoter and the promoters for the co-ordinately regulated genes PAL and 4CL; in addition, the substantially homologous domains in the
  • PAL and 4CL promoters are organized in similar relative dispositions as those in the CHS promoter.
  • the PAL and CHS promoters have approximately 70% homology in the silencer region, while the CHS and 4CL promoters have 70% homology on 68 base pairs just upstream of the TATA box (the activator domain) , and PAL and 4CL have more than 60% homology in this same region. See Edwards, et al., (1985) and Cramer, et al., (1989).
  • the present invention comprises two substantially pure sequences shown in
  • Figure 7 as nucleotide sequences (-242 to -194) and (-74 to -52) in the 5' flanking region of the CHS promoter.
  • the two sequence elements are:
  • the present invention com ⁇ prises chimeric plasmids selected from the group con- sisting of pCHCl and pCHC2.
  • the present invention com ⁇ prises a chimeric gene cassette (CGC) comprising: (a) a promoter selected from the group consisting of promoters for the plant genes that encode the phenylpropanoid biosynthetic enzymes phenylalanine ammonia-lyase (PAL) , chalcone synthase (CHS), and 4-coumarate:CoA ligase (4CL) , plus promoters for the plant genes that encode the cell wall hydroxyproline-rich glycoproteins, wherein the promoter is operatively linked to: (b) at least one reporter gene, and (c) at least transcription termination sequence.
  • CGC chimeric gene cassette
  • useful reporter genes include, but are not limited to, CAT, GUS, lacZ and firefly luciferase; useful terminator sequences include, but are not limited to, the 3' flanking region of the NOS gene and the 3' flanking region of the OCS gene.
  • the chimeric gene cassettes of the present invention will be extremely useful to those wishing to engineer transgenic plant strains that have enhanced defense capabilities.
  • modified versions of the natural gene vector system of Agrobacter ⁇ um t mefaciens have been used successfully to create a number of "engineered dicotyledonous plants, including tobacco, potato, carrot, flax, eggplant, tomato, chili pepper, sunflower and rapeseed (cabbage) .
  • Such methods can be used by those skilled in the art of plant genetic engineering, without undue experimentation, to create new plant strains that contain, as part of their genetic makeup, the chimeric gene cassettes of the present invention.
  • those skilled in the art can use methods such as the leaf disk transformation procedure disclosed in Horsch, et al., (1985) to create transgenic plants, or the method of Rhodes, et ah, that was used recently to transform the monocotyledonous plant, maize (Rhodes, et al., (1988)).
  • the leaf disk transformation procedure has been used successfully to engineer soybean protoplasts and transgenic tobacco plants that contain the chimeric gene cassettes of the present invention.
  • the present invention com ⁇ prises use of exogenous elicitor substances to activate plant defense genes.
  • Exogenous elicitor substances useful for this purpose include, but are not limited to, the reduced form of glutathione; the reduced form of homoglutathione, and the reduced form of other peptide analogs of glutathione; glycan elicitors such as hexa(0- D-glucopyranosyl)-D-glucitols, lipid elicitors such as arachidonic acid, glycoprotein elicitors, fungal elicitors, and abiotic elicitors such as mercuric chloride HGC1 2 .
  • Plant defense genes that can be induced by exogenous substances include, but are not limited to, plant defense genes that encode the phenylpropanoid biosynthetic enzymes phenylalanine ammonia-lyase (PAL) , chalcone synthase (CHS), and 4-coumarate:CoA ligase
  • PAL phenylalanine ammonia-lyase
  • CHS chalcone synthase
  • (4CL) plus the plant genes that encode the cell wall hydroxyproline-rich glycoproteins.
  • the present invention comprises a screening assay for identifying substances capable of inducing transcription of stress-regulated plant defense genes.
  • a chimeric gene cassette (CGC) is introduced into plant material from a suitable plant (P) .
  • the CGC is comprised of: (a) at leaset one stress-regulated defense gene promoter (which is selected from the group consisting of promoters for the genes that encode the phenylpropanoid biosynthetic enzymes phenylalanine ammonia-lyase (PAL) , chalcone synthase (CHS) and 4-coumarate:CoA ligase (4CL) , plus promoters for the plant genes that encode the cell wall hydroxyproline-rich glycoproteins) , that is operatively linked to: (b) at least one reporter gene, such as CAT, lacZ, firefly luciferase or GUS; and (c) at least one transcription termination sequence such as the 3' flanking region of
  • the cultured plant material is monitored for induction (i-e. , the presence) of the reporter gene sequences.
  • Those substances that are capable of inducing expression of the reporter gene sequences are considered candidates for exogenously inducing expression of plant defense genes. Such substances are useful for exogenously inducing plant defense genes and chimeric transgenes that are operatively linked to a responsive promoter, as well as pre-activating a plant's own defense mechanisms.
  • the plant material will consist of whole transgenic plants
  • the promoter(s) will be CHS and/or PAL
  • the reporter gene(s) will be CAT, lacZ, GUS or firefly luciferase
  • the terminator sequence will be the 3' flanking region of the NOS or OCS genes.
  • Experimental Section I relates to the discovery that the reduced form of glutathione (GSH) , when supplied to suspension cultured cells of bean (Phaseolus vulgaris L.) at concentrations in the range 0.01 mM to 1.0 mM, stimulates transcription of defense genes including those that encode the phenylpropanoid biosynthetic enzymes phenylalanine ammonia-lyase (PAL) , chalcone synthase (CHS) , involved in lignin (PAL) and phytoalexin (PAL, CHS) production, plus those that encode the cell wall hydroxyproline-rich glycoproteins.
  • GSH glutathione
  • GSH causes a marked increase in extractable PAL activity, whereas the oxidized form of glutathione, the separate constituent amino acids of glutathione (glutamate, cysteine and glycine) , and strong SH reducing reagents such as cysteine, ascorbic acid and mercaptoethanol are inactive.
  • a particularly striking feature of the present invention is the massive quantitative effect of GSH.
  • induction of PAL and CHS transcripts is several fold greater and also more prolonged than with optimal concentrations of fungal elicitor.
  • PAL enzyme activities of about 200 ⁇ kat/kg protein ob ⁇ tained following GSH treatment are the highest that have been observed in cell suspension cultures or other in ⁇ duction systems (Lawton, et al., 1983).
  • GSH GSH is found at concentrations in the range of 0.05 to 1.5 mM (Bielawski, et al., 1986; Rennenberg 1982; Smith, 1975; and Smith, et al., 1985) and hence the effects on defense genes occur at physiological concentrations of GSH.
  • Experimental Section II relates to efforts to investigate the mechanisms underlying activation of plant defenses against microbial attack.
  • studies were carried out on elicitor regulation of a chimeric gene comprising the 5'-flanking region of a defense gene encoding the phytoalexin biosynthetic enzyme chalcone synthase fused to a bac ⁇ terial chloramphenicol acetyltransferase gene.
  • Glutathione or fungal elicitor caused a rapid, marked but transient expression of the chimeric gene electroporated into soybean protoplasts. The response closely resembled that of endogenous chalcone synthase genes in suspension cultured cells.
  • the responses of the chimeric CHS-CAT-NOS gene electroporated into protoplasts closely resemble that of endogenous chromosomal CHS genes in elicitor-treated cell suspension cultures with respect to the kinetics of induction and the relative potency of glutathione and fungal elicitor as inducers.
  • the whole plants system described herein provides a convenient functional assay for identifying exogenous substances that can be used to induce expression of plant defense genes, or pre-activate the plant's own defense mechanisms.
  • the system will be useful for analyzing ex ⁇ acting nucleotide sequences involved in elicitor regulation of defense genes.
  • Experimental Section III illustrates the methods of the present invention by disclosing protocols for (1) identifying trait-specific nucleic acid sequences that are likely to respond to agrichemicals by regulating gene transcription, and then for (2) identifying agrichemical compounds that may be useful for inducing transcription of these trait-specific nucleic acid sequences.
  • the test plant is tomato
  • the test traits are enhancement or delay of fruit ripening or development.
  • Glutathione ( ⁇ f-L-glutamyl-L-cysteinyl-glycine) is a low molecular weight thiol implicated in a wide range of metabolic processes (Meister, et al., 1983). Functions proposed for glutathione in higher plants in ⁇ clude: storage and transport of reduced sulfur; protein reductant; destruction of H 2 0 2 in chloroplasts and detoxification of xenobiotics including certain her ⁇ bicides and pesticides (Edwards, et al, 1986; and Ren- nenberg, 1982) .
  • Plant Material French Bean Phaseolus vulgaris L. cv Canadian Wonder
  • Plant Material French Bean Phaseolus vulgaris L. cv Canadian Wonder
  • the cultures were maintained in total darkness.
  • Experiments were conducted with seven to ten day old cell cultures, in which the growth medium- exhibited a conductivity between 2.5 and 2.8 mho. This represents the period of maximum responsiveness to elicitor during the cell culture cycle (Edwards, et al., 1985) .
  • Enzyme Extraction and Assay Extraction and assay of PAL were as previously described (Lawton, et al., 1983).
  • One unit of enzyme activity (1 kat) is defined as the amount of enzyme re ⁇ quired for the formation of 1 mol of product in 1 sec under the assay conditions.
  • RNA Total cellular RNA was isolated from samples homogenized directly in a phenol/O.l M Tris-HCl emul- sion, (pH 9.0), and purified as previously described
  • RNA was assayed spectrophotometrically at 260 nm. The yield of RNA was 150 to 250 ⁇ g/g fresh weight of tissue and the A 260 /A 280 ratio varied between 1.8 and 2.1.
  • RNA Blot Hybridization Total RNA was denatured by glyoxal and frac ⁇ tionated by electrophoresis in a 1.2% agarose gel in 10 mM phosphate buffer (pH 7.0) (McMaster, et al., 1977).
  • Nitrocellulose blots were hybridized with [ 32 P]-labeled cDNA sequences prepared by nick translation of the inserts of pPAL5 (Edwards, et al., 1985), pCHS5 (Ryder, et al, 1984), pHyp2.13 and pHyp4.1 (Corbin, et al., 1987). Following autoradiography, specific transcripts were quantitated by scanning densitometry. Several autoradiograms, exposed for different periods, were obtained for each blot to enable quantitation of each sample in the linear range of film response.
  • HI is a cDNA clone containing sequences from a constitutively transcribed gene that is unaffected by elicitor treatment. Changes in the rate of transcription of PAL, CHS and HRGP genes were measured by reference to the rate of HI transcription.
  • PAL catalyzes the first reaction in the biosynthesis from L-phenylalanine of phenylpropanoid natural products including lignin and phytoalexins.
  • CHS catalyzes the first reaction of a branch pathway of phenylpropanoid biosynthesis specific to the formation of flavonoid pigments and isoflavonoid phytoalexins.
  • GSH caused a massive but transient, co-ordinate accumulation of PAL and CHS transcripts from low basal levels in suspension-cultured bean cells (Figs. 1 and 2) . Maximum accumulation of these transcripts was observed about 6 h after addition of GSH, following which there is a decline to relatively low levels.
  • GSH also caused the accumulation of HRGP transcripts Hyp4.1 and Hyp2.13, which had previously been shown to be induced by fungal elicitor (Fig. 1) . As in elicitor treated cells, accumulation of these HRGP transcripts was less rapid but more prolonged than for PAL and CHS. GSH concentrations in the range 10 - 100 ⁇ M caused accumulation of PAL, CHS, Hyp2.13 and Hyp4.1 transcripts to levels comparable to, or greater than, those observed with optimal concentrations of fungal elicitor (Fig. 3) . Transcriptional Activation
  • GSH markedly stimulated the synthesis of four polypeptides whose levels of expression were little affected by fungal elicitor (Fig. 5) .
  • Simultaneous addition of GSH and fungal elicitor altered the pattern of protein synthesis in a similar manner to GSH alone.
  • GSH treatment caused a marked and prolonged increase in the level of extractable PAL activity (Fig- 2) .
  • the phase of most rapid increase in enzyme activity occurred between 3 and 8 h after GSH addition and hence was closely correlated with the timing of maximum accumulation of PAL transcripts.
  • the dose response for induction of PAL enzyme activity after 8 h resembled that for accumulation of PAL transcripts, with marked effects at concentrations of GSH as low as 10 ⁇ M (Table 1) .
  • GSH stimulation of PAL enzyme activity lead to increased flux through the pathway and appreciable accumulation of the phytoalexin end-product phaseolin (data not shown) .
  • GSH treatment also caused significant browning of the cells, which is characteristic of the accumulation of phenolic material.
  • GSH GSH is found at concentrations in the range of 0.05 to 1.5 mM
  • Figure 1 Accumulation of defense gene transcripts in response to GSH (1 mM) .
  • FIG. 1 Kinetics for induction of (A) PAL and CHS transcripts and (B) PAL enzyme activity in response to GSH (1 mM) .
  • the dotted line denotes changes in PAL mRNA levels.
  • Figure 4 Effect of GSH on the transcription of defense genes. Nuclei were isolated from cells 1.75 h after GSH treatment or from equivalent untreated con- trol cells.
  • FIG. 5 Effect of GSH on the pattern of protein synthesis.
  • Open arrows in panel B denote those species induced by both GSH and fungal elicitor; closed arrows denote those species induced by GSH but not fungal elicitor; "p” denotes PAL subunits; “c” denotes CHS subunits.
  • “IEF” Isoelectric focusing in the first dimension;
  • SDS PAGE SDS-polyacryla ide gel electrophoresis in the second dimension.
  • Plants respond to microbial attack by synthesis of antibiotics, stimulation of lytic enzymes and reinforcement of cell walls (Darvill, et al., 1984; Dixon, et al., 1983; Dixon, et al., 1986; and Ebel, 1986).
  • These defenses can also be induced by glycan and glycoprotein elicitors from fungal cell walls and cul ⁇ ture fluids or metabolites such as arachidonic acid and glutathione (Darvill, et al, 1984; Dixon, et al., 1983; Dixon, et al., 1986; Ebel, 1986; Experimental Section I and Wingate, et al., 1988).
  • Elicitor stimulates CHS transcription in bean cells within 5 min leading to a transient accumulation of CHS mRNA with maximum levels after 3 to 4 h, correlated with the onset of phytoalexin synthesis (Cramer, et al., 1985a; Lawton, et al., 1987; and Ryder, et al., 1984).
  • This example shows that glutathione or a fungal elicitor preparation of high molecular weight material heat-released from mycelial cell walls of the bean pathogen Collelolrichum lindemuthianum (fungal elicitor) cause a rapid, marked but transient expression of the chimeric CHS-CAT-NOS gene electroporated into soybean protoplasts.
  • the response of the CHS-CAT-NOS gene closely resembles that of endogenous CHS genes in elicitor treated cell suspension cultures.
  • the data show that the 429 bp nucleotide sequence immediately upstream of the CHS coding region is sufficient to confer regulation by elicitor substances such as glutathione or fungal elicitor.
  • Plasmid Constructions pDO400 is identical to the previously described cauliflower mosaic virus (CaMV) 35S promoter construct pD0432 (Ow, et al., 1986) except that an 883 bp
  • pCHS15 consists of a 2.1 kb Hin ⁇ lll Phaseolus vulgaris genomic fragment containing the full-length CHS 15 gene and flanking sequences subcloned into the riboprobe vector pSP64 (Ryder, et al., 1987).
  • pCHCl a 429 bp Hinfl fragment comprising 5'- untranslated sequences of CHS15 replaces the 35S transcript promoter of pDO400.
  • pCHCl was constructed by replacing the Hindlll/Xbal CaMV 35S promoter fragment of pDO400 with the Hindlll/Xbal polylinker fragment of pUC19 to create pCNIOO.
  • PCN100 was digested with Sail , filled-in with Klenow DNA polymerase and dNTPs and used for blunt-end ligation of the 429 bp Hinfl fragment of pCHS15 whose ends were similarly rendered blunt by Klenow fill-in.
  • the construct was sequenced by dideoxy chain termination (Sanger, et al.. 1977) of denatured double-stranded plasmid with an M13 reverse primer (Chen, et al., 1985).
  • Deletion mutants were constructed by digesting pCHCl with Hindlll followed by exonuclease III and mung bean nuclease treatment (Henikoff, 1984) . After Xbal digestion, deleted promoter fragments were purified on low melting agarose and ligated into Pstl (T 4 polymerase filled-in) /Xbal cut pCNIOO. Precise endpoints were determined by sequencing as described above. pHCNl was constructed by cloning a 235 bp EcoKL/Pvull fragment from the promoter region of a murine histone H 4 gene (Seiler-Tuyns, et al.. 1981) into EcoKL/Smal cut pIBI24 (a pUC-derived phagemid vector) . This construct was further cleaved with EcoRI/Xbal and sub-cloned into
  • Protoplast Isolation The origin and maintenance of bean (Phaseolus vulgaris L. ) , soybean (Glycine max L. ) and tobacco (Nicotiana labacum L.) cell suspension cultures was as described except that cells were collected by sieving (250 micron mesh) and transferred to fresh maintenance medium at 7 d intervals (Cramer, et al., 1985b; and Norman, et al., 1986).
  • protoplast isolation cells (7 g fresh weight) were collected 4 d after subculture and incubated by shaking (90 rpm) in 100 ml of protoplast isolation medium for 4 h at 27°C in darkness (From , et al., 1985) .
  • Protoplasts were separated from the cellular debris by sieving and by centrifugation at 70 x g for 5 min at room temperature. Viability was determined by staining with Evans Blue and protoplasts were adjusted to 5 x 10 6 /ml. Protoplasts were washed twice in electroporation medium (Fromm, et al., 1985) prior to manipulation.
  • Electroporation and Transient Assay Electroporation was performed as described (Fromm, et al., 1985) , 3 h after isolation of protoplasts with an optimal pulse of 250 V for 10 msec. Unless otherwise noted, 30 ⁇ g of test construct DNA was electroporated together with 50 ⁇ g of calf thymus DNA as carrier. Protoplasts were maintained without agitation in 6 ml of maintenance medium containing 0.3 M mannitol at 27°C in the dark. In the experiment depicted in Fig. 8 panel (A) , protoplasts were collected for analysis 8 h after electroporation.
  • Protoplasts were collected by centrifugation, and extracts assayed for CAT activity by radiometric measurement of the conversion of the substrate ["C] chlora phenicol as described (Fromm, et al., 1985) . Reaction products were separated by thin layer chromatography, visualized by autoradiography and guantitated by scintillation counting. Protein was assayed by the Bradford procedure (Bradford, 1976) . Typical CAT assays involved incubation of samples containing 5 ⁇ g protein for 3 hr at 37°C leading to the conversion of 1,000-5,000 cpm of the substrate into acetylated products.
  • RNA Analysis Protoplasts (3 x 10 ⁇ ) were resuspended in 100 ⁇ l 0.1 M Tris-HCl pH 9.0, containing 0.01% SDS. After extraction with phenol and chloroform, the supernatant was precipitated with 2 vol of 95% ethanol in the presence of 0.3 M sodium acetate. RNA was further processed and analyzed by Northern blot hybridization as described (Cramer, et al., 1985b) . The hybridization probe was a 0.8 b BamHI fragment comprising Escherichia coli CAT gene sequences (Alton, et al., 1979) labeled by nick- translation.
  • CHS 15 is one of 6 CHS genes in the bean genome and encodes a major elicitor-induced CHS transcript (Ryder, et al.,
  • the chimeric CHS-CAT-NOS gene construct pCHCl contains 429 bp of the 5' untranslated nucleotide sequences of CHS 15, comprising 326 bp upstream of the transcription start site and 103 bp of the transcribed leader sequence (Fig. 7) .
  • bean and soybean protoplasts respond to elicitor in a manner similar to the suspension cultured cells from which they were derived with respect to the accumulation of transcripts encoded by endogenous defense genes and the appearance of phenylpropanoid products (data not shown) .
  • the CHS-CAT-NOS gene was transiently expressed with maximum levels 3 h after addition of glutathione followed by a decay to relatively low levels after 6 hr (Fig. 8D) .
  • No induction of CAT activity was observed over this period in the absence of glutathione.
  • the chimeric gene was also regulated by glutathione when electroporated into protoplasts derived from tobacco cells, although the response was slower, with maximum CAT activity after 6 h (Fig. 8D) .
  • These induction kinetics closely resembled those for expression of endogenous defense genes in the respective suspension cultured cells from which the protoplasts were derived (Ryder, et al, 1984; Grab, et al., 1985; Hahn and Lamb, unpublished observations) .
  • deletions had similar relative effects on induction by the fungal elicitor preparation (data not shown) .
  • deletion to -173 likewise increased the response to fungal elicitor although this enhanced induction was somewhat weaker than that obtained with the same construct in response to glutathione.
  • further deletion to -136 and -72 proliferatively reduced the response to fungal elicitor.
  • the present data show that the CHS promoter is appropriately regulated by elicitor substances such as glutathione and fungal elicitor in electroporated protoplasts.
  • elicitor substances such as glutathione and fungal elicitor in electroporated protoplasts.
  • the CHS-CAT-NOS gene is not inserted into chromosomal DNA, and that our experiments monitor the expression of a plasmid-borne gene.
  • the responses of the chimeric CHS-CAT-NOS gene electroporated into protoplasts closely resembles that of endogenous chromosomal CHS genes in elicitor treated cell suspension cultures with respect to the kinetics of induction and the relative potency of glutathione and fungal elicitor as inducers.
  • a chimeric "cassette” such as an exogenously inducible plant defense gene promoter-structural gene- terminator cassette (CGC) of the invention can now be introduced into a variety of useful plants. See generally, Caplan, et al., 1984; Horsch, et al., 1985; and Rhodes, et al, 1988.
  • CGC plant defense gene promoter-structural gene- terminator cassette
  • CHS deletions suggest that there is an elicitor regulated activator element downstream from -173. Since 5' deletions to -130 and to -72 affect elicitor regulation by inhibition of induction rather than by elevation of basal expression, the activator appears to be a positive -acting element.
  • This functional analysis is consistent with the pattern of sites hypersensitive to DNase I digestion in CHS genes (M.A. Lawton and C.J. Lamb, unpublished). Three such sites, which denote local opening of chromatin structure associated with binding of regulatory proteins, are found in the proximal region of the promoter in nuclei from elicitor-treated but not control cells. In contrast, sites in the upstream region show pronounced DNase I hypersensitivity in nuclei from uninduced as well as elicited cells.
  • sequences between the TATA box and -130 are both necessary and sufficient for regulation by elicitor substances such as glutathione or fungal elicitor
  • upstream sequences appear to modulate expression, and maximum induction is obtained when sequences to -173 are present. This may reflect the existence of multiple -acting sequences that interact with the same /r ⁇ ns-acting factor(s) or an independent regulatory element between -173 and -130, that is distinct from the downstream element.
  • -130 may have an impact on gene expression not by abolition of the binding of trans-SLCt ng factors to ex ⁇ acting elements located in this region, but through indirect effects on chromatin structure that modulate binding of transcription factors to the activator element downstream of -130.
  • CAT-NOS gene electroporated into protoplasts derived from suspension cultured cells (A) Comparison of expression in bean, soybean and tobacco protoplasts; (B) Effect of glutathione on the expression of CHS-CAT-NOS and H 4 -CAT-NOS chimeric genes in soybean protoplasts; (C) Comparison of the induction by fungal cell wall elicitor and glutathione; (D) Time-course for glutathione-induced expression in soybean and tobacco protoplasts.
  • CAT authentic bacterial CAT enzyme
  • T tobacco
  • B bean
  • S soybean
  • SC soybean protoplasts without electroporated genes
  • G protoplasts 3 h after treatment with glutathione
  • E protoplasts 3 h after treatment with fungal elicitor
  • C equivalent, untreated control protoplasts.
  • Closed arrowheads denote the major CAT product 3-acetylchloramphenicol.
  • FIG. 9 Correlation between the accumula ⁇ tion of CAT transcripts and CAT activity in electroporated protoplasts containing the CHS-CAT-NOS gene.
  • Upper panel Northern blot of equal amounts of total cellular RNA from control protoplasts (C) or 3 h after treatment with glutathione (G) hybridized with CAT sequences.
  • Lower panel CAT activity from extracts of equivalent protoplasts.
  • FIG. 11 Effect of 5' deletions on glutathione regulation of the CHS-CAT-NOS gene electroporated into soybean protoplasts. Plus (+) : 3 h after addition of glutathione; minus (-) : equivalent untreated controls. The structure of 5' deletions are presented in Fig. 7A. Error bars denote standard deviation between independent replicates.
  • this example discloses protocols for (1) identifying trait-specific nucleic acid sequences that are likely to respond to agrichemicals by regulating gene transcription, and then for (2) identifying agrichemical compounds that may be useful for inducing transcription of these trait-specific nucleic acid sequences.
  • the test plant is tomato
  • the test traits are enhancement or delay of fruit ripening or development.
  • the following protocol can be used to develop recombinant constructs useful for testing an agrichemical's specificity for enhancing or delaying fruit ripening or development.
  • Root, leaf, stem, and fruit tissues from target tomato species were collected during several developmental stages. Collected tissues were quick frozen in liquid nitrogen prior to transport in dry ice and storage at -70°C; it was noted that initial freezing with dry ice does not protect the RNA from degradation.
  • RNA was extracted from the tissue powders using proteinase K treatment and phenol/chloroform extractions. A cesium chloride gradient and a minigel were run to evaluate the intact nature of the RNA. The poly A + fraction was isolated from total RNA by performing a lithium chloride precipitation, followed by passage over an oligo d(T) column.
  • RNA was isolated from the various developmental stages of tomato tissues (as described above) and subjected to an in vitro translation (IVT) procedure.
  • the translation mixture was comprised of rabbit reticulocyte lysate, salts, amino acid mix, creatine phosphate, and 35 S- met; the procedure was performed as outlined in protocols provided by Promega Biotec, Madison, Wisconsin.
  • IVT translation products were then run on 12.5% one dimensional (ID) gels using standard electrophoresis techniques.
  • the gel was exposed to 0 autoradiographic film and, after an appropriate time (about 20 hrs) , the film was developed.
  • Poly(A) RNA was selected.
  • RNA Early and late RNA was fractionated on sucrose gradients.
  • RNA fractions were translated (IVT) across the gradient to determine which fractions were enriched in mRNAs encoding the ca. 38.5 and ca.
  • RNA from gradient fractions of choice were labeled by a polynucleotide kinase reaction with
  • the cDNA library was probed with fractionated, kinase-labeled RNA from early or late stages of fruit development.
  • Clones were selected which hybridized differentially to the different RNA preparations (thus suggesting developmental regulation) 30 and clones which hybridized equally well to the different RNA preparations (thus suggesting constitutive expression) .
  • Clones were characterized as to insert size and sequence homology.
  • the clone encoding the ca. 50 KD protein was sequenced to identify the protein encoded and to further characterize the sequence.
  • the cDNA marker probe described in Section I is used to isolate their genomic counterparts by means of standard Southern hybridization and cloning techniques.
  • the developmental marker cDNA encoding the ca. 50 KD protein is used to isolate the gene whose transcription is developmentally regulated.
  • the promoter fragment of the cloned gene is identified by DNA sequencing and - isolated from the parent clone. In brief, sequences upstream of the start codon (ATG) are examined and found to contain a TATA box; potential promoter sequences can extend as far as about 200 base pairs upstream of the ATG.
  • ATG start codon
  • the promoter fragment is fused to a reporter genes, such as lacZ, GUS, firefly luciferase or CAT.
  • a reporter genes such as lacZ, GUS, firefly luciferase or CAT.
  • These expression constructs are inserted into vectors and gene expression is induced under appropriate conditions, e.g. a substance known to regulate development (i.e., a known developmental regulator) is added to the growth medium.
  • lacZ can be detected by the addition of X-gal to the medium. If lacZ is present, the medium will turn blue.
  • the promoter-reporter gene construct is inserted into an appropriate vector (e.g., c - the Agrobacterium Ti plasmid-based vector such as BIN19 or the vectors referred to in United States Patent 4,658,082 issued April 14, 1987 to Simpson, et al.
  • an appropriate vector e.g., c - the Agrobacterium Ti plasmid-based vector such as BIN19 or the vectors referred to in United States Patent 4,658,082 issued April 14, 1987 to Simpson, et al.
  • Transformation of tomato is accomplished by means of known procedures.
  • Transgenic plants containing the promoter- reporter gene chimeric gene cassette are tested for correct expression of the reporter 0 gene, i.e., for expression of the reporter gene under developmental regulation.
  • Plant material evidencing appropriate expression of the reporter gene is used to develop transgenic plant assay systems. For example, 5 such plants are grown to the early fruiting stage and treated with potential agrichemicals. Those chemicals which induce expression of the reporter gene are candidates for agrichemicals which will accelerate the 0 ripening process.
  • the presen -invention discloses a novel screening method for identifying agrichemical compounds that may be useful for inducing transcription of trait-specific plant genes.
  • the invention discloses a method for identifying trait-specific nucleic acid sequences from plants that respond to agrichemicals by regulating gene transcription.
  • the invention discloses regulatory elements that control selective induction of plant defense gene. Such elements can be used, for example, to construct transgenic plants that can be induced to exhibit plant defense responses when treated with specific agrichemicals that affect gene transcription.

Abstract

Un procédé permet d'identifier les éléments régulateurs de gènes végétaux qui réagissent à des stimuli et qui affectent la transcription de gènes structuraux auxquels ils sont fonctionnellement liés. L'analyse sélective est utile pour identifier des substances énergiquement utilisables afin d'activer des éléments régulateurs de gènes spécifiques de caractéristiques végétales (par exemple des promoteurs), provoquant l'expression des gènes structuraux naturels ou chimériques auxquels ces substances sont fonctionnellement liées. En outre, l'invention concerne des séquences régulatrices des gènes de défense de plantes (par exemple, promoteurs de gènes de défense de plantes, domaines d'activation régulés par des entraîneurs, domaines de blocage en amont et similaires) qui peuvent être ''branchées'', induites ou autrement activées ou désactivées par des entraîneurs exogènes.
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WO1993007279A1 (fr) * 1991-10-03 1993-04-15 Smart Plants International, Inc. Regions inductibles a action regulatrice sur les genes de defense des plantes, tirees de la pomme de terre et du riz, utilisations de ces regions et titrages
WO1993007257A3 (fr) * 1991-10-04 1993-08-05 Smart Plants Int Inc Sequences de transcription a specificite tissulaire et developpement regule et leur utilisations
WO1993007257A2 (fr) * 1991-10-04 1993-04-15 Smart Plants International, Inc. Sequences de transcription a specificite tissulaire et developpement regule et leur utilisations
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EP0922764A1 (fr) * 1997-12-03 1999-06-16 E.I. Dupont De Nemours And Company Facteur de ribosylation de l'ADP du Soja
EP1013767A1 (fr) * 1998-12-22 2000-06-28 American Cyanamid Company Méthode de criblage des compositions agrochimiques
WO2000037651A2 (fr) * 1998-12-22 2000-06-29 American Cyanamid Company Procede d'analyse de composes agrochimiques
WO2000037651A3 (fr) * 1998-12-22 2000-09-14 American Cyanamid Co Procede d'analyse de composes agrochimiques
EP1038965A1 (fr) * 1999-03-23 2000-09-27 American Cyanamid Company Procédé de criblage de composants chimiques capables d'induire ERS dans des plantes
WO2000071748A2 (fr) * 1999-05-21 2000-11-30 Basf Aktiengesellschaft Genes ers, procede de criblage de composes chimiques capables d'induire un etat de resistance accru dans les plantes
WO2000071748A3 (fr) * 1999-05-21 2001-05-31 American Cyanamid Co Genes ers, procede de criblage de composes chimiques capables d'induire un etat de resistance accru dans les plantes

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EP0416032A1 (fr) 1991-03-13
EP0416032A4 (en) 1992-11-25

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