WO1993007279A1 - Inducible plant defense gene regulatory regions from potato and rice, uses thereof, and assays - Google Patents

Inducible plant defense gene regulatory regions from potato and rice, uses thereof, and assays Download PDF

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WO1993007279A1
WO1993007279A1 PCT/US1992/008560 US9208560W WO9307279A1 WO 1993007279 A1 WO1993007279 A1 WO 1993007279A1 US 9208560 W US9208560 W US 9208560W WO 9307279 A1 WO9307279 A1 WO 9307279A1
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plant
promoters
elicitor
isolated
pal
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PCT/US1992/008560
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French (fr)
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Leona Claire Fitzmaurice
Elizabeth Louise Virts
Fen-Fen Lin
T. Erik Mirkov
Jana Gayvin Collier
Paula Kay Schoeneck
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Smart Plants International, Inc.
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Publication of WO1993007279A1 publication Critical patent/WO1993007279A1/en

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    • 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
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • This invention relates generally to plant biotechnology and specifically to novel inducible plant defense gene regulatory sequences or promoters from potato and rice, methods for identifying and isolating these and related sequences, plus uses thereof, especially in the construction of transgenic plants and plant compositions.
  • the invention also relates to methods for identifying agrichemicals and organism(s) which can induce plant defense genes.
  • Plant disease resistance mechanisms include physical and chemical barriers within the plant, as well as responses which are induced or activated by wounding or attack by pathogen(s).
  • One important inducible defense response is the production of phytoalexins, which have been defined as low-molecular weight, antimicrobial compounds synthesized and accumulated by the plant at the site of attempted infection.
  • Phytoalexins are predominantly phenylpropanoids, isoprenoids, and acetylenes.
  • PAL genes Transcription of PAL genes is rapidly activated by a wide range of stimuli including wounding, glycan elicitor preparations from fungal cell walls, and the reduced form of glutathione, a cellular metabolite. Not all PAL genes respond in the same manner to these stimuli. Therefore there is a need to identify novel PAL promoters which respond to fungal or bacterial elicitors, or to wounding. These novel PAL promoters can be used to create transgenic plant materials and transgenic plants, and as part of novel agrichemical screening assays.
  • the present invention provides isolated promoters (regulatory sequences) for plant genes that encode phenylalanine ammonia-lyase (PAL) in potato or rice.
  • the promoters are inducible by exogenous elicitor(s) or wounding, and are capable of regulating transcription of associated DNA sequences in transgenic plant cells, tissues and plants that contain the
  • the invention includes methods for obtaining these novel PAL promoters.
  • the invention provides the PAL promoter(s), probe sequences, chimeric potato and rice PAL promoter constructs (in which the PAL promoters are operatively linked to structural genes, e.g., reporter genes), as well as transgenic potato and rice plant cells, plant tissues and transgenic plants that contain these chimeric constructs.
  • the chimeric constructions and transgenic plant materials which contain them are useful in agrichemical assays.
  • the invention also provides a method for inducing transcription of a chimeric gene in transgenic plants.
  • Figure 1A and 1B are restriction enzyme maps of genomic clones of potato S. tuberosum PAL genes.
  • Figure IA shows limited restriction enzyme maps of six potato clones isolated from the S. tuberosum cv. Desiree genomic library.
  • Clone 1 ⁇ pPAL-1;
  • Clone 3 ⁇ pPAL-3;
  • Clone 4 ⁇ pPAL-4;
  • Clone 6 ⁇ pPAL-6;
  • Clone 7 ⁇ pPAL-7;
  • Clone 8 ⁇ pPAL-8.
  • the boxes indicate the regions of each clone that have been subcloned and that contain sequences which hybridize to S. tuberosum cv. Desiree cDNA clones pPAL-3 and pPAL-21.
  • Figure 1B also shows restriction enzyme maps of seven potato clones isolated from the S. tuberosum cv. Desiree genomic library.
  • Clone 1 ⁇ pPAL-1;
  • Clone 2 ⁇ pPAL-2;
  • Clone 3 ⁇ pPAL-3;
  • Clone 4 ⁇ pPAL-4;
  • Clone 6 ⁇ pPAL-6;
  • Clone 7 ⁇ pPAL-7;
  • Clone 8 ⁇ pPAL-8.
  • the orientation of the putative PAL gene is given below each map. Symbols: B: BamHI; Bgl: BglI; E: EcoRI; H: HindIII; P: PstI; S: SstI.
  • the parentheses around some symbols indicate that the restriction enzyme cleavage sites are present, but the exact location is not known.
  • the B ⁇ S sequence for ⁇ pPAL-1 is SEQ ID NO. 1;
  • the (E) ⁇ P sequence for ⁇ pPAL-2 is SEQ ID NO. 2;
  • the (E) ⁇ P sequence for ⁇ pPAL-3 is SEQ ID NO. 3;
  • the E ⁇ P sequence for ⁇ pPAL-4 is SEQ ID NO. 4;
  • the H ⁇ S sequence for ⁇ pPAL-6 in Figure IB is referred to as ⁇ pPAL-6(a) and is shown as SEQ ID NO. 5;
  • the S ⁇ H sequence (shown as a sense sequence, having been converted from the antisense sequence) for ⁇ pPAL-6 is referred to as ⁇ pPAL-6(b) and is shown as SEQ ID NO. 6;
  • the (E) ⁇ P fragment for ⁇ pPAL-8 is SEQ ID NO. 7.
  • ⁇ rPAL-2 in Figure 2 is SEQ ID NO. 8 and ⁇ rPAL-4 is SEQ ID NO. 9).
  • Figure 3 is a schematic drawing showing the self-sustained, sequence replication (3SRTM, SIBIA, La Jolla, CA) system.
  • PAL refers to phenylalanine ammonia-lyase. PAL catalyzes the conversion of the amino acid L-phenylalanine to transcinnamic acid and N ⁇ 4 + . This is the first reaction in the synthesis of a wide range of plant natural products based on the phenylpropane skeleton, including lignins, flavonoids, isoflavonoid, coumarins and hydroxycinnamic acid esters.
  • exogenously controlled plant gene regulatory regions or elements refer to nucleic acid sequences that affect transcription of "operatively linked, functionally linked or associated" structural genes in response to exogenous stimuli.
  • exogenously controlled plant gene regulatory sequences include inducible plant defense gene regulatory regions or promoters (e.g., PAL promoters), elicitor-regulated activator domains, upstream silencer domains, etc.
  • promoter refers to a non-coding region of DNA involved in binding of RNA polymerase and other factors that initiate or modulate transcription whereby an RNA transcript is produced. Promoters can be naturally occurring or synthetically produced. Promoters, depending upon the nature of the regulation, may be constitutive or regulated. A constitutive promoter is always turned on. A regulatable promoter requires specific signals in order for it to be turned on or off. A developmentally regulated promoter is one that is turned on or off as a function of
  • the promoters of the present invention are directly or indirectly responsive to exogenous elicitor(s) and/or wounding. Stated another way, exogenous elicitor(s) and/or wounding can induce the promoters of the present invention to initiate transcription of associated structural gene(s).
  • promoter and gene regulatory region are used interchangeably.
  • elicitors are compounds or organisms which control (e.g., initiate, terminate, increase or reduce), either directly or indirectly, the action of exogenously-controlled plant gene regulatory elements such as the inducible stress-regulated promoters for the plant genes that encode the phenylpropanoid biosynthetic enzymes phenylalanine ammonialyase (PAL).
  • PAL phenylalanine ammonialyase
  • Elicitors 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; lipid elicitors such as arachidonic acid and eicosapentaenoic acid, glycoprotein elicitors, fungal elicitors, abiotic elicitors such as mercuric chloride HGCl 2 , fungi (e.g., elicitors released by whole fungi), bacteria (e.g., elicitors released by whole bacteria), etc.
  • glycan elicitors such as hexa( ⁇ -D-glucopyranosyl)-D-glucitols
  • lipid elicitors such as arachidonic acid and eicosapentaeno
  • operatively linked, functionally linked or associated, or grammatical variations thereof are equivalent terms that are used interchangeably.
  • these terms refer to the linkage of a promoter or a non-coding gene regulatory sequence to an RNA-encoding DNA sequence, and especially to the ability of the regulatory sequence or promoter to induce production of RNA transcripts corresponding to the DNA-encoding sequence when the promoter or regulatory sequence is recognized by a suitable polymerase. All three terms mean that linked DNA sequences (e.g.,
  • promoter(s), structural gene (e.g., reporter gene(s)), terminator sequence(s), etc.) are operational or functional, i.e., work for their intended purposes.
  • operatively or functionally linked, or associated means that after the respective DNA segments are joined, upon appropriate activation of the promoter, the structural gene will be expressed.
  • suitable plant material means and expressly includes, plant protoplasts, plant cells, plant callus, plant tissues, developing plantlets, immature whole plants and mature whole plants.
  • the present invention comprises isolated or isolatable promoters (i.e., regulatory sequences) for plant defense genes that encode phenylalanine ammonia-lyase (PAL) in potato or rice wherein the promoters are capable of regulating transcription of an associated DNA sequence in suitable hosts and wherein the promoters can be induced by, or are otherwise directly or indirectly responsive to, exogenous elicitor(s) and/or wounding.
  • promoters i.e., regulatory sequences
  • PAL phenylalanine ammonia-lyase
  • promoters are exemplified herein by the potato PAL promoters associated with the PAL structural gene sequences comprised within clones ⁇ pPAL-1, ⁇ pPAL-2, ⁇ pPAL-3, ⁇ pPAL-4, ⁇ pPAL-6, ⁇ pPAL-7 ( ⁇ pPAL-6 and ⁇ pPAL-7 are identical as described in Example IIC and are referred to herein as ⁇ pPAL6/7), and ⁇ pPAL-8, plus rice PAL promoters comprised within clones ⁇ rPAL-2 and ⁇ rPAL-4.
  • the potato and rice PAL promoters of the invention are operatively linked to at least one associated DNA sequence that encodes protein(s) which directly or indirectly gives rise to a phenotypic trait.
  • the linkage can be at the level of transcription (i.e., a "transcriptional fusion") so that the protein which gives rise to the phenotypic trait is expressed as a non-fused peptide.
  • transcriptional fusion a transcriptional fusion
  • the linkage can also be at the level of translation (i.e., a "translational fusion") such that the marker protein is expressed as fusion peptide with a portion of the amino terminal end of the native PAL protein.
  • the phenotypic trait can consist of tolerance or resistance to: herbicide, fungus, virus, bacterium, insect, nematode or arachnid; production of secondary metabolites, male or female sterility, or production of an enzyme or reporter compound.
  • the associated structural gene encodes a reporter enzyme or compound, preferably the compound will be chloramphenicol acetyltransferase (CAT), neomycin phosphotransferase (NPT), nopaline synthase (NOS), octopine synthase (OCS), ⁇ -1,3-glucuronidase
  • GUS acetohydroxyacid synthase
  • AHAS acetohydroxyacid synthase
  • ⁇ -GAL ⁇ -galactosidase
  • LUX luciferase
  • the potato and rice PAL promoters - associated DNA sequence constructs are used to transform suitable hosts and to create transgenic plant compositions, e.g., plant protoplasts, plant cells, plant callus, plant tissue, developing plantlets, immature whole plants, mature whole plants, or seeds.
  • transgenic plant compositions e.g., plant protoplasts, plant cells, plant callus, plant tissue, developing plantlets, immature whole plants, mature whole plants, or seeds.
  • the invention encompasses these transformed hosts and transgenic compositions, especially transgenic potato and rice plants and seeds that contain potato and rice PAL promoters and associated DNA sequences.
  • SEQ ID NOS. 1-9 are DNA coding sequences from potato PAL clones ⁇ pPAL-1, ⁇ pPAL-2, ⁇ pPAL-3, ⁇ pPAL-4, ⁇ pPAL-6 (two sequences) and ⁇ pPAL-8, respectively.
  • SEQ ID NOS. 8 and 9 are coding and non-coding DNA sequences from rice PAL clones ⁇ rPAL-2 and ⁇ rPAL-4, respectively.
  • the DNA sequences of the invention can be used as probes to identify identical or homologous PAL sequences.
  • the probes will be at least 10 nucleotides in length, and most preferably will be from about 100 to about 500 nucleotides long.
  • the coding sequences of potato PAL clones ⁇ pPAL-1, ⁇ PAL-2, ⁇ pPAL-3, ⁇ pPALA, ⁇ pPAL-6/7 or ⁇ pPAL-8, or ⁇ rPAL-2 or ⁇ rPAL-4 can be used to probe cDNA or genomic libraries for identical or highly homologous sequences.
  • the promoters may not be functionally equivalent.
  • the invention includes a method for identifying sequences that are not only homologous to the sequences of the invention, but are also regulated by inducible PAL promoters.
  • a plant is subjected to an elicitor, and then RNA from the plant is isolated.
  • PolyA + RNA is selected, e.g., by using an oligo(dT) column.
  • a cDNA library is prepared from this RNA and cloned into a suitable vector.
  • the library is probed with a probe comprised of nucleotide sequences from the coding region(s) of elicitor inducible genes, e.g., the coding sequences of SEQ ID NOS.
  • cDNA which hybridizes with the probe i.e., positive clone(s)
  • a genomic library from the plant of interest is probed with the cDNA, and the segment of genomic DNA that hybridizes with the probe is identified.
  • An antisense RNA transcript from this genomic DNA is made and labeled (e.g., radioactively) and then used as a probe to hybridize to mRNA from elicitor-treated and non-elicitor treated plants.
  • the hybridizing mix is then subjected to RNase degradation of all single-stranded RNA. If the transcript of interest is present in total RNA, then a double-stranded RNA-RNA hybrid that is protected by RNA from elicitor-treated plants but that is not protected by RNA from non-elicitor containing no mismatches will be formed and will be unaffected by the RNase treatment. This product can be identified by size following gel electrophoresis.
  • the promoter from the genomic clone that yields the antisense RNA transcript that is protected by RNA from elicitor-treated plants but that is not protected by RNA from non-elicitor treated plants is inducible with elicitor. The promoter is isolated and used to make chimeric constructs for use in agrichemical assays, and to produce transformed cells and transgenic plants and transgenic plant compositions.
  • the invention also includes a method for identifying exogenous elicitors which are capable of inducing, either directly or indirectly, a potato or rice PAL promoter.
  • a suitable host e.g., a plant composition
  • a potato or rice PAL promoter operatively linked to a structural gene whose expression can be detected (e.g., a marker gene).
  • Putative exogenous elicitor is then applied to the transformed host.
  • Exogenous elicitor that can induce expression of the marker gene is concluded to be an elicitor that can induce the potato or rice PAL promoter.
  • whole plants are used in the assay.
  • whole transgenic plants which contain at least one chimeric DNA sequence comprised of a potato or rice PAL promoter operatively linked to a reporter structural gene.
  • Putative exogenous elicitor(s) is then applied to the plant.
  • the elicitor(s) that induces expression of the reporter gene is concluded to be elicitor(s) which can induce expression of the potato or rice PAL promoters.
  • the invention also provides a method for identifying elicitor-inducible promoters.
  • a plant is subjected to an elicitor, then RNA is isolated from the plant.
  • a cDNA library is prepared from the isolated RNA, which is then probed with a probe comprised of nucleotide sequences from the transcribed coding and/or non-coding region(s) of gene(s) of interest.
  • cDNA which hybridized with the probe is then used to probe a genomic library from the plant of interest. The segment of genomic DNA that hybridzed with the probe is identified, and a labeled antisense RNA transcript is made from it.
  • This labeled antisense RNA transcript is used as a probe to hybridize to mRNA from elicitor-treated and non-elicitor treated plants.
  • the hybridized mix is subjected to RNase, and those antisense RNA transcripts that were protected from degradation by elicitor-treated RNA but were not protected from degradation by RNA from non-elicitor treated plants are identified.
  • the promoter from the genomic clone which yielded the antisense RNA transcript that was protected by only RNA from elicitor-treated plants is inducible with elicitor.
  • the invention also discloses a related method for identifying elicitor-inducible promoters.
  • a plant genomic library is probed with a probe comprised of nucleotide sequences from the transcribed coding and/or non-coding region(s) of a gene of interest.
  • the segment of genomic DNA that hybridized with the probe is identified.
  • a labeled antisense RNA transcript is made from the genomic DNA segment that hybridized with the probe.
  • This labeled antisense RNA is used as a hybridization probe for mRNA obtained from elicitor-treated and non-elicitor treated plants.
  • the hybridization mix is subjected to RNase, and those antisense RNA transcripts that were protected from degradation by elicitor- treated RNA but were not protected from degradation by RNA from non- elicitor treated plants are identified.
  • the promoter from the genomic clone which yielded the antisense RNA transcript that was protected by only RNA from elicitor-treated plants is inducible with elicitor.
  • the invention discloses an amplification method for identifying agrichemicals which can induce expression of a plant gene.
  • RNA from plant material not exposed to a putative inducer is isolated and from within this isolated RNA, an RNA encoded by a gene of interest is identified by hybridization to a probe.
  • the RNA from this identified gene is amplified using the self-sustained sequence replication or 3SRTM (SIBIA, La Jolla, CA 92037-4641) technique for amplification of specific RNA sequences and primers specific for the inducible gene transcript of interest. See Guatelli, et al., 1990. Proc. Natl. Acad. Sci USA. 87:1874-1878.
  • RNA encoded by the gene of interest is then identified and amplified using 3SR.
  • the amplification products from unexposed and exposed plant material are compared. If the level of expected product in the amplification of RNA from exposed plant material is higher than in the amplification of RNA from unexposed plant material, it can be concluded that this agrichemical(s) can induce expression of the gene of interest.
  • the amplification method With regard to the amplification method, its sensitivity has two sigmficant advantages. Firstly, very small samples can be analyzed, thus for the first time permitting use in agrichemical screens of plants such as Arabidopsis and cells in tissue culture. Secondly, some chemicals may have a very small but very significant effect on plant genes, e.g., plant defense genes, such that the response may be difficult to detect with methods less sensitive than 3SR.
  • the 3SR amplification method of the invention makes it possible for the first time to identify these chemical elicitors.
  • Potato cell suspension cultures were initiated from potato callus cultures. The cultures were maintained and proliferated on LS2T liquid (or suspension) medium and incubated at 27°C in the dark on a gyratory shaker at a speed of 125 rpm.
  • Desiree suspension cultures were subcultured weekly. At each subculture, five grams fresh weight of cells were transferred to 100 ml fresh LS2T liquid medium. A five-fold increase in growth was observed in a seven-day growth period. Three hours prior to isolation of RNA, the culture was treated with arachidonic acid (Sigma, St. Louis, MO) to a final concentration of 0.1 mM.
  • Arachidonic acid is a fatty acid normally found in fungal cell walls and induces at least some PAL gene expression (Fritzemeier et al., 1987, Plant Physiol. 85:34-41).
  • RNA was isolated as follows: 1. Measure out 3-5 g of tissue. Grind into a fine powder using a mortar and pestle (pre-cooled with liquid N 2 ). 2. Add tissue to pre-chilled 10 ml equilibrated phenol +
  • RNA was isolated from 400 ⁇ g total RNA using the mRNA Purification Kit (Pharmacia, Piscataway, NJ; catalog #27-9258) and following the manufacturer's instructions. Approximately 3.1 ⁇ g poly A + was recovered, and used for cDNA synthesis following the BRL cDNA synthesis system protocol (BRL, Bethesda, MD; catalog #8269 SA). The cDNA was treated for 30 min with 9 units T4 DNA polymerase (BRL), following which time a 50-fold molar excess of EcoRI adapters (Pharmacia) were ligated to the cDNA (overnight at 15oC).
  • BRL cDNA synthesis system protocol BRL cDNA synthesis system protocol
  • the cDNA was treated for 30 min with 9 units T4 DNA polymerase (BRL), following which time a 50-fold molar excess of EcoRI adapters (Pharmacia) were ligated to the cDNA (overnight at 15oC).
  • the cDNA was separated from the excess adapters through a 1 ml G50 spin column (Sephadex G50 in a 1 ml syringe, according to Maniatis et al., in Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1982) and the purified cDNA was ligated to EcoRI-digested ⁇ gtlO arms (Promega, Madison, WI) according to manufacturer's instructions. The entire ligation mixture was packaged using GigapackTM11 Gold Packaging Extract (Stratagene, La Jolla,
  • the cDNA library was titered using E. coli strain C600 (Promega, Madison, WI). The titer of the library was determined to be 2.6 X 10 7 plaque forming units/ml.
  • the library was screened with a 0.9 kb fragment of plasmid pCP63.15, a pUC19-based plasmid comprising a cDNA encoding a portion of the potato PAL exon II gene sequence (Fritzemeier et al., 1987, Plant Physiol. 85:34-41).
  • the screening conditions were as follows:
  • Hybridization 42 ° C, 35% Formamide, 5X Denhardts, 5X
  • SSC 0.2% SDS, 200 ⁇ g/ml salmon sperm DNA.
  • the screening and five rounds of plaque purification identified three putative PAL-encoding cDNAs, which were called ⁇ pPAL-3, ⁇ pPAL-21, and ⁇ pPAL-25.
  • EcoRI EcoRI-digested pUC119
  • Amp R colonies were selected. Colonies carrying correct plasmid released a 450, 500, or 250 bp fragment, respectively, upon digestion of plasmid DNA with EcoRI.
  • the EcoRI inserts were sequenced using the Sequenase (U.S. Biochemical, Cleveland, OH) protocol for double-stranded DNA, and the nucleotide sequence of each was compared to the nucleotide sequence of the bean gPAL-2 gene (Cramer et al., 1989, Plant Mol. Biol. 12:367-383). The similarity was as follows:
  • a genomic library was constructed from total genomic DNA isolated from young leaves of Solanum tuberosum cv. Desiree. DNA isolation was by the procedure of Bendich, 1988, in Plant Molecular Biology Manual,
  • the genomic DNA was partially digested with Sau3AI, resulting in fragments of between 9-23 kb in size.
  • the Sau3AI ends were partially filled in using dATP and dGTP.
  • SSC 0.2% SDS, 200 ⁇ g/ml salmon sperm DNA.
  • a map for ⁇ pPAL-5 is not shown because no sequence similarity to PAL has been found in this insert.
  • the insert DNAs were sequenced according to the Sequenase protocol (U.S. Biochemical Corporation) for double-stranded
  • genes corresponding to ⁇ pPAL- 2, ⁇ pPAL-3, ⁇ pPAL-4, and ⁇ pPAL-8 are members of the same PAL gene subfamily
  • ⁇ pPAL-6 and ⁇ pPAL-7 are genomic clones of the same PAL gene
  • ⁇ pPAL-1 contains a PAL gene that is different from the PAL gene contained in ⁇ pPAL-6.
  • RNASE PROTECTION ASSAY RNase protection assays are based on the different susceptibility of single-stranded RNA and RNA-RNA hybrids to degradation by the single-stranded RNA-specific enzyme RNase.
  • total RNA is hybridized to a radioactively-labeled antisense probe complementary to the transcript of interest, followed by RNase-mediated degradation of all single-stranded RNA. If the transcript of interest is present in total RNA, then a double stranded RNA-RNA hybrid containing no mismatches will have formed and will be unaffected by the RNase treatment. This product can be identified by size following gel electrophoresis.
  • transcripts of interest were defined by the genomic subclone inserts as follows:
  • the antisense transcript is transcribed using the T7 promoter and the sense transcript is transcribed using the SP6 promoter.
  • the noted inserts were ligated into appropriately digested pGEM-7Z(-), except for the 500 bp insert of ⁇ pPAL-6 which was ligated into pGEM-11Z(+ ) (Promega).
  • the polylinker regions in both these plasmids are flanked by the SP6 and T7 RNA polymerase promoters making it possible to generate 32 P-labeled antisense RNA transcripts of the inserts using SP6 or T7 RNA polymerase.
  • the choice of which polymerase to use depends upon the orientation of the gene fragment in the polylinker.
  • RNA transcripts 32 P-labeled antisense RNA transcripts were synthesized in vitro from the subclones pPAL-1, pPAL-2EP, pPAL-3EP, pPAL-4EP, pPAL-8EP, and pPAL-6HS using the Riboprobe Gemini System II (Promega) and following manufacturer's instructions. The RNAs were then used as probes in RNase protection assays of total RNA from Solanum tuberosum cv. Desiree suspension culture cells. Suspension cells were prepared as described in Potato Example LA. and treated for three hours with freshly prepared 0.1 mM arachidonic acid (induced) or water (uninduced) prior to isolation of total RNA from the cells (RNA was isolated as described in Potato Example I.A.).
  • RNAs were separately hybridized overnight at 45°C to each of the six different antisense probes.
  • the hybridization conditions were as follows: 40 mM Pipes, pH 6.4, 1 mM EDTA, 400 mM NaCl, 50% Formamide.
  • the hybridization mixtures were then digested with a mixture of RNase A (40 ⁇ g/ml; Sigma, St. Louis, MO) and RNase Tl (2 U/ml; BRL, Bethesda, MD) at 34 ° C, and analyzed on a 6% polyacrylamide, 8M urea gel.
  • RNase A 40 ⁇ g/ml
  • RNase Tl 2 U/ml
  • BRL Bethesda, MD
  • sense RNA was transcribed in vitro from subclones pPAL-1, pPAL-2EP, pPAL-3EP, pPAL-4EP, pPAL-8EP, and pPAL-6HS and hybridized with the corresponding labeled antisense RNA. These hybrids should be a perfect match.
  • the antisense RNA transcripts were protected from RNase-mediated degradation when hybridized with sense RNA synthesized from the same subclone, but were completely degraded in the absence of sense RNA.
  • RNA from induced cells This result was not surprising as significant background levels of PAL gene expression were consistently observed in these cells.
  • SstI-BamHI fragment from ⁇ pPAL-2 was gel purified and subcloned into pGEMHZ( + ) (Promega) using standard DNA cloning procedures (Maniatis et al., supra). The ligation mixture was transformed into DH5 ⁇ and Amp R colonies were selected. A correct construct was confirmed by the presence of 6 and 3.2 kb bands in SstI-BamHI-digested DNA and was called pD2.2.
  • a 1.4 kb KpnI-SstI fragment and a 3.3 kb SstI-EcoRI fragment were gel purified from ⁇ pPAL-7. Equal molar ratios of these fragments were mixed and subcloned into pUC119 previously digested with both KpnI and EcoRI. The ligation reaction was transformed into DH5 ⁇ cells and Amp R colonies were selected. Correct construct was confirmed by the presence of 4.7 and 3.2 kb bands upon double digestion of plasmid DNA with EcoRI and KpnI, and was called 7P.
  • PAL coding regions share 80% sequence similarity at the nucleotide level.
  • ARACHIDONIC-INDUCED GENE EXPRESSION A. Construction of pUC-GUS.l, pUC-GUS.2, pUC-GUS.3 Promoter-less GUS ( ⁇ -glucuronidase) gene cassettes are available as HindIII-EcoRI inserts in plasmids pBI101, pBI101.2, and pBI101.3
  • the GUS cassettes in plasmids pBI101.2 and pBI101.3 are identical to the one in pBI101 except their reading frames are shifted one and two nucleotides, respectively, relative to the polylinker. As a result, a promoter fragment and a portion of the coding region can be inserted in all three reading frames upstream of the GUS gene creating both
  • the GUS-encoding insert was removed from each plasmid and each insert was separately ligated with HindIII-EcoRI-digested pUC119. The ligation was transformed into D ⁇ 5 ⁇ cells, and Amp R colonies were selected. Correct plasmids demonstrated bands of 2.2 and 3.2 kb in size upon digestion with EcoRI and HindIII and were called pUC-GUS.1, pUC-GUS.2, and pUC- GUS3, respectively.
  • Plasmid pD2.2 (Potato Example IX.A.) was digested with HaeIII, and the insert was separately ligated with SmaI-digested pUC-GUS.1, pUC- GUS.2, and pUC-GUS.3. After transformation into DH5 ⁇ cells, and selection of Amp R colonies, correct plasmids were identified by release of a 1.8 kb band upon digestion with HindIII. Correct plasmids were called pPAL2.1, pPAL2.2, and pPAL2.3.
  • Plasmids pPAL2.1, 2.2, and 2.3 were digested with Xbal and SstI and each insert was ligated to XbaI- and SstI-digested pBI101.
  • Plasmid pBI101 (Clontech, Palo Alto, CA) is a broad host range plasmid used in the binary vector, plant transformation system of Agrobacterium tumefaciens. The ligations were transformed into DH5 ⁇ cells and Kan R colonies were selected. Correct plasmids were identified by release of a 6 kb fragment upon digestion with Xbal and SstI, and were called pBIN-2.1, pBIN-2.2, and pBIN-2.3, respectively.
  • Plasmid 7P was digested with PstI and EcoRV and the insert was ligated with PstI- and SmaI-digested pUC-GUS.1, pUC-GUS.2, and pUC- GUS.3. After transformation into DH5 ⁇ cells and selection of Amp R colonies, correct plasmids were identified by release of a 4.7 kb band upon digestion with EcoRI and BamHI. Correct plasmids were called pGUS7.1, pGUS7.2, and pGUS7.3, respectively.
  • Plasmid pGUS7.1, 7.2, and 7.3 were digested with HindIII and EcoRI, and each insert was ligated to HindIII- and EcoRI-digested pBIN19.
  • Plasmid pBIN19 is a broad host range plasmid used in the binary vector Agrobacterium tumefaciens transformation system and is available from
  • Clontech The ligations were transformed into TB-1 cells and Kan R colonies were selected. Correct plasmids were identified by release of a 6 kb fragment upon digestion with HindIII and EcoRI and were called pBIN7.1, pBIN-7.2, and pBIN-7.3, respectively.
  • plasmids pBIN2.1, 2.2, 2.3, 7.1, 7.2 and 7.3 were transferred from the E. coli host to Agrobacterium tumefaciens strain LBA4404 by triparental mating using pRK2073 as the helper strain (Corbin, D. et al., 1982, J. Bacteriol. 149:221-228).
  • the potato tubers used in this experiment were obtained from potato plants of Solanum tuberosum cv. Desiree. Tubers that had been stored in the dark at 4 ° C for one week were washed and rinsed with deionized water to remove soil, surface sterilized one minute in 95% ethanol and rinsed in sterile distilled water. The tubers were peeled and disinfected for 15 minutes in 10% PurexTM (commercial bleach) containing two drops of Tween 20TM per 100 ml solution followed by five rinses with sterile distilled water. The proximal and distal quarter portions of the tubers were discarded. The sterilized potato tubers were immersed in MS liquid medium without hormones (Murashige and Skoog, 1962, Physiol. Plant 15:473-496) for 20 min prior to removal of the discs.
  • the explants were floated in 20 mis of MS liquid medium without hormones containing an overnight culture of separate Agrobacterium
  • Agrobacterium strains were pre-induced with 50 ⁇ M acetosyringone.
  • the tissue and Agrobacterium were incubated at room temperature on a gyratory shaker with gentle shaking (at approximately 60 rpm). After 20 minutes, the explants were blotted on sterile Whatman paper No. 1 and transferred to incubation media which were two-day-preconditioned tobacco feeder plates.
  • Feeder plates were prepared according to the method of Horsch and Jones (In Vitro, 1980, 16:103-108) with the following modification: cells from six-day-old suspension culture were filtered through a sterile 30-mesh sieve, collected on two layers of sterile kimwipes and freed of excess medium through a funnel.
  • MM medium contained MS basal medium supplemented with 0.5 mg/1 2,4-D and 0.5 mg/l BA. The suspension was stirred and 1.5 ml aliquots were pipetted onto plates containing two kinds of shoot regeneration media, 3C52R medium (Steerman and Bevan, 1988, (Plant Cell Reports 7:13- 16)) and the medium defined in Jarrett et al. (Physiol. Plant., 1980, 49:177-184).
  • infected tuber discs were transferred to selection media containing cefotaxime (500 ⁇ g/ml) and kanamycin (100 ⁇ g/ml).
  • the composition of the media was identical to incubation media, but there was no feeder layer.
  • Tissues were transferred to fresh selection media at two-week intervals, and the cefotaxime concentration was reduced to 250 ⁇ g/ml after four weeks in culture.
  • Potato shoots were regenerated after three weeks in culture. When regenerated potato shoots reached 3-5 mm in size, they were excised from tuber discs and grown on the selection media containing 250 ⁇ g/ml cefotaxime and 100 ⁇ g/ml kanamycin.
  • transgenic potato shoots When putative transgenic potato shoots reached 2 cm in size, they were transferred to rooting media containing 250 ⁇ g/ml cefotaxime and 100 ⁇ g/ml kanamycin. The rooting media were identical to shoot regeneration media except plant hormones were not added. When the shoots had rooted, the resulting plantlets were transplanted from culture into soil and grown in a plant growth chamber. At desired times, the transgenic potato plants are assayed for GUS activity.
  • plants are grown to a size and/or stage of development which is both manageable under greenhouse conditions and susceptible to a pathogen of interest, e.g., Phythophthora infestans.
  • the plants are treated either by foliage spray or soil immersion with a range of concentrations of the unknown chemical (elicitors).
  • additional plants are exposed to water as a negative control or to a known elicitor as a positive control.
  • leaf tissue is removed and assayed for GUS activity using the fluorometric assay of Jefferson et al. (Plant Mol. Biol. Rep., 1987, 5:387-405).
  • a positive result is indicated by levels of GUS activity significantly higher than those observed with the negative control.
  • a positive result is indicative of, e.g., identifies, chemicals which are capable of inducing potato PAL promoter(s).
  • a rice (Oryza sativa) genomic library (Clontech, Palo Alto, CA; average insert size of 15 kb in ⁇ EMBL3) was screened with elicitor-inducible bean PAL1 exonII cDNAs to identify clones encoding rice PAL genes.
  • the cDNAs encoding PAL1 exonII sequences were pPALl-B6 and pSPP1 (both obtained from C. Lamb, The Salk Institute, La Jolla, CA; no published reference).
  • the probe was an equal mixture of the 800 bp insert of pPAL1-B6 and the 500 bp insert of pSPP1.
  • the screening conditions were as follows:
  • Hybridization 42oC, 35% Formamide, 5X SSC, 5X Denhardts,
  • Clones ⁇ rPAL-8 and -12 were identical; clones ⁇ rPAL-4 and -10 had several fragments in common; and clone ⁇ rPAL-2 was unique. Partial restriction enzyme maps of inserts from clones ⁇ rPAL-2, -4, and -10 are shown in Rice
  • the insert DNAs were sequenced according to the Sequenase protocol (U.S Biochemical Corporation, Cleveland, OH) for double-stranded dideoxynucleotide sequencing.
  • the partial sequence of the PAL region of ⁇ rPAL-2 was found to have 84.3% identity to the published sequence of a rice (Otyza sativa cv. Nipponbare) genomic PAL gene (Minami et al., 1989, Eur. J. Biochem. 185:19-25); the partial sequences of the fragments from ⁇ rPAL-4 and ⁇ rPAL-10 were the same and showed 79.6% identity to the rice PAL genomic sequence; and the partial sequence of the ⁇ rPAL-8 fragment has 53.8% identity to the rice PAL genomic sequence.
  • These data suggested that clones ⁇ rPAL-2, ⁇ rPAL-4, and ⁇ rPAL-10 contain portions of the rice PAL genes, and that ⁇ rPAL-8 may or may not contain a rice PAL gene.
  • the orientation of the PAL genes within clones ⁇ rPAL-2, -4, and - 10 was determined from the sequence data. Based on the size and orientation of the inserts compared to the published sequence of a rice PAL gene
  • the promoter fragment from ⁇ rPAL-4 was subcloned as a 3 kb ClaI-SstI fragment into Accl-SstI digested pGEM5Z (Promega, Madison, WI). This 3 kb fragment contains about 300 bp of the right arm of EMBL3, the 1800 bp SalI fragment (black box in Rice Figure 2) and a portion of the 1850 SalI fragment (hatched box).
  • the composite sequence of ⁇ rPAL-4 and -10 is shown as SEQ ID NO. 9 ( ⁇ rPAL-4).
  • the sequence of the promoter region and a portion of the coding region of ⁇ rPAL-2 is shown in SEQ. ID NO. 8 ( ⁇ rPAL-2).
  • the sequencing data showed that the promoter regions of ⁇ rPAL-4 and ⁇ rPAL-10, as well as a portion of their coding regions, are identical, except that ⁇ rPAL-4 contains an additional 850 bp of upstream sequence compared to ⁇ rPAL-10.
  • Comparison between the published rice (Oryza sativa cv. Nipponbare) PAL genomic sequence (Minami et al., 1989, supra) and the sequences of the subclones of ⁇ rPAL-4 and ⁇ rPAL-10 showed that the translational start (ATG) of the published sequence is located five base pairs upstream of the SalI restriction site (position 1724 in SEQ ID NO. 9) in ⁇ rPAL-4 and ⁇ rPAL-10. Therefore, it is likely that the translational start site of the PAL gene encoded by ⁇ rPAL-4 and ⁇ rPAL-10 is near this SalI site.
  • a plant translational start consensus sequence (GNNATGG) is present at position 1704 of the composite sequence in (SEQ ID NO. 9) in ⁇ rPAL-4 and ⁇ rPAL-10.
  • Computer-generated translation of the nucleotide sequence starting with the ATG at position 1702 and extending to position 1828 yielded protein sequence which shows 78% similarity to the amino acid sequence predicted from the published rice PAL gene sequence (Minami et al., 1989, supra) and 73% similarity to the amino acid sequence deduced from bean gPAL-2 (Cramer et al., 1989, Plant Mol. Biol. 12:367-383).
  • the putative translational start of ⁇ rPAL-2 is located at nucleotide position 1873 of SEQ ID NO. 8.
  • Computer generated translation of the nucleotide sequence starting at this position results in protein sequence which shows 80% similarity to the amino acid sequence predicted from the published rice PAL gene sequence (Minami et al., 1989, supra), and 51% similarity to the amino acid sequence deduced from ⁇ rPAL4 and ⁇ rPAL-10.
  • Promoter-less GUS ( ⁇ -glucuronidase) gene cassettes are available as HindIII-EcoRI inserts in plasmids pBI101, pBI101.2, and pBI101.3
  • the GUS cassettes in plasmids pBI101.2 and pBI101.3 are identical to the one in pBI101 except their reading frames are shifted one and two nucleotides, respectively, relative to the polylinker.
  • inserting a promoter fragment and a portion of the gene coding region into each vector creates both translational and transcriptional fusion constructs in all three reading frames upstream of the GUS gene.
  • each of the three plasmids, pBI101, pBI101.2, and pBI101.3 was digested with HindIII and EcoRI and the GUS-encoding insert was removed and purified on a 1% TBE gel.
  • Each insert was ligated separately with HindIII-EcoRI-digested pUC119. The ligation was transformed into D ⁇ 5 ⁇ cells, and Amp R colonies were selected. Correct plasmids demonstrated bands of 2.2 and 3.2 kb in size upon digestion with EcoRI and HindIII and were called pUC-GUS.1, pUC-GUS.2, and pUC-GUS.3, respectively.
  • Plasmid pSV2hyg obtained from J. Kwoh, Baxter Healthcare, San
  • Plasmid p35S-hyg was digested with HindIII and the 2 kb fragment was purified on a 1% TBE gel. The fragment then was ligated separately into the pUC-GUS vectors (pUC-GUS.1, pUC-GUS.2, and pUC-GUS.3) which had been digested with HindIII. The ligation reaction was transformed into D ⁇ 5 ⁇ cells and Amp R colonies were selected. The correct plasmids released a 2 kb fragment upon digestion with HindIII and were called pHyg-GUS.1, pHyg-GUS.2, and pHyg-GUS.3, respectively.
  • the promoter fragments from ⁇ rPAL-10 were isolated by the following method. Plasmid pSal1000 (the 800 bp SalI fragment from black boxed portion of ⁇ rPAL-10 in Rice Figure 2, ligated into pUC119) was digested with SalI and PstI, and the 800 bp fragment was purified on a 1% TBE gel. Plasmid p4410 (see Rice Example VI.B2.) was digested with BssHII, treated with Klenow, digested with SalI and SstII (to remove the other BssHII- SalI fragment which would interfere in the ligation).
  • the 800 bp fragment and the entire SalI-SstII digestion were ligated into SmaI- and PstI-digested pHyg- GUS.1, pHyg-GUS.2, and pHyg-GUS.3 in three way ligations.
  • the ligations were transformed into DH5 ⁇ cells and Amp R colonies were selected.
  • the correct constructs were determined by linearization of an approximately 6200 bp plasmid upon digestion with EcoRI, and were called rPAL10.1, rPAL10.2, and rPAL10.3. 3. Promoter from ⁇ rPAL-2
  • the promoter fragment from ⁇ rPAL-2 was digested with EcoRI, treated with Klenow, and phosphorylated BamHI linkers (New England
  • Rice suspension cultures (Oryza sativa cv. IR54) were provided by
  • the cultures were subcultured weekly. At each subculture, 2.5 g fresh weight of rice suspension cells were transferred to 50 ml fresh medium. The cultures were incubated at 27 ° C in the dark on a gyratory shaker at a speed of approximately 125 rpm.
  • the enzyme-protoplast mixture was passed through a 300 mesh tissue sieve to remove debris, then centrifuged at approximately 147 ⁇ g for 10 minutes at room temperature. Pelleted protoplasts were washed twice by resuspending in approximately 35 ml Protoplast Wash Solution and
  • Protoplasts were purified by centrifugation through a Percoll Solution step gradient. Protoplasts were resuspended in 6 ml of 70% Percoll Solution (Rice Table III), a 50% Percoll Solution (6 ml) was layered on top of the resuspended protoplasts, and 6 ml of a 25% Percoll Solution was layered on top of the 50% Percoll Solution.
  • the Percoll-protoplast gradient was centrifuged at approximately
  • Protoplasts were collected at the interface of the 25% and 50% Percoll Solutions using a sterile pasteur pipet and transferred to 25 ml of Protoplast Wash Solution. Protoplasts were washed twice by resuspending in 25 ml Protoplast Wash Solution and centrifuging at approximately 147 ⁇ g for 10 minutes after each resuspension.
  • the protoplast-PEG mixture was heat shocked at 45 ° C for five minutes followed by chilling on ice for 20 seconds. The solution was then brought to room temperature followed by incubation at 30o C for 30 minutes. The protoplast-PEG mixture then was diluted with Krens' F solution until the PEG concentration was less than 2% according to the following time schedule as a reference:
  • the protoplasts treated with plasmid DNA were collected by centrifugation at approximately 147 ⁇ g for 10 minutes and resuspended in N6 medium (Rice Table II) containing 0.3 M mannitol and incubated in the dark at room temperature on a gyratory shaker at ⁇ 50 rpm.
  • Table IV Rice: Preparation of Fungal Elicitor Maintenance media: Corn meal agar (DifcoTM 0386- 01-3); plate.
  • Growth media Liquid culture.
  • Corn meal broth Blend 50 g corn meal in 800 ml of distilled H 2 O; refrigerate mixture overnight; then heat for about 1 h at 60o C; filter solution; bring solution to 1 liter; autoclave for 20 min. Double autoclave (120°C/20 min) all media and instruments. Leave plates/liquid media for > 1 week to check for contamination.
  • To innoculate growth media take 5 ⁇ 0.6 mm cork borer discs from edge of growing mycelia/50 ml media (in 250 ml conical flask).
  • step 1 three more times * .
  • Fungal elicitor at a concentration of approximately 60 or 80 ⁇ g/ml was added 22 (80 ⁇ g) or 36 (60 ⁇ g) hours after transformation, and incubation was continued for an additional 20 hours. Following this 42 or 56 hour incubation, control and treated samples were processed as described below.
  • the assay for GUS activity was a fluorometric assay which measures the production of 4-methyl umbelliferyl from 4-methyl umbelliferyl glucuronide (MUG), a fluorogenic substrate. Protein concentration was determined according to the Bradford Protein Assay using reagents obtained from Bio- Rad.
  • a higher level of uninduced GUS activity was measured in protoplasts transformed with plasmid DNA, but ng£ induced with elicitor, than in control protoplasts not transformed and not induced.
  • the higher GUS levels are due to a certain level of constitutive and/or induced (induction other than elicitor-mediated induction) GUS expression in the experimental transformed protoplasts and are indicative of successful transfer of the constructs into the protoplasts.
  • the difference in the background GUS levels of protoplasts transformed with rPAL2.2 and rPAL4.3 could be the result of differences in transformation efficiencies, differences in the fusion between the rice PAL promoter and GUS genes in the constructs, or, most likely, differences in the responsiveness of the two rice PAL promoters to possible other induction factors.
  • Elicitor-Induced GUS Expression Elicitor-treated protoplasts transformed with rPAL2.2 yielded GUS activities that were an average of 9.8% higher than those of untreated protoplasts transformed with the same construct. No significant increase in GUS activity was seen in elicitor-treated protoplasts transformed with rPAL4.3 relative to untreated protoplasts that had been transformed with this construct.
  • Rice suspension cultures were initiated from the rice calli on two types of suspension media, N6 medium and R2 medium. Rice calli grown on MS2 medium were initiated in R2 medium and rice calli proliferated on N6 medium were initiated in N6 medium.
  • the R2 medium is as follows:
  • Sucrose 30.0 g/l pH 5.8 The initiated cultures were incubated at 27°C in the dark on a gyratory shaker at a speed of about 50 rpm. Rice suspension cultures were subcultured weekly. At each subculture, 2.5 grams fresh weight of cells were transferred to 25 ml fresh suspension media in a 250 ml flask. A 2-fold increase in growth was observed in a seven day growth period.
  • the Percoll-protoplast gradient was centrifuged at about 200 ⁇ g for 15 minutes at room temperature. Protoplasts were collected from the top of 25% Percoll Solution and transferred to 30 ml of Protoplast Wash Solution. Protoplasts were washed twice by resuspending in 30 ml Protoplast Wash
  • Solidified agarose containing the protoplasts were cut into four blocks and transferred to a 100 mm ⁇ 150 mm sterile petri dish containing 15 ml R2 protoplast medium resuspended with about 500 mg 5-day-old Sasanishiki suspension cells which were initiated on N6 medium and sieved through a 30 mesh tissue sieve.
  • the cultures were incubated at room temperature in the dark on a gyratory shaker at a speed of about 40 rpm.
  • the cultures were incubated at room temperature in the dark on a gyratory shaker at a speed of about 30 rpm.
  • N6 soft agarose medium N6 basal medium, 2 mg/l 2,4-dichlorophenoxy acetic acid, 6% sucrose and 0.25% Sigma type I Agarose, pH 5.7
  • N6 medium containing 50 ⁇ g/ml hygromycin. This medium was the same as N6 soft agarose medium except that this medium contained 0.5% Sigma Type I Agarose.
  • Transgenic rice are propagated by germinating seeds in suitable tissue culture medium containing suitable concentrations of hygromycin to ensure that the PAL-GUS fusion construct is present in all tissues of the transgenic plant. Once the transgenic plantlets have reached a suitable size, e.g., 3-6 inches tall, they are transplanted to soil and maintained under standard greenhouse conditions.
  • the 3SR technique is illustrated in Figure 3.
  • a target RNA sequence is initially transformed into an RNA/DNA duplex by reverse transcriptase in the region specified by the first primer.
  • This duplex is attacked by the RNaseH present on the AMV reverse transcriptase.
  • the RNaseH destroys the RNA template but leaves the cDNA intact.
  • the cDNA is used as a template for the synthesis of a second strand of cDNA using reverse transcriptase and the second primer.
  • the resulting double stranded DNA template serves as a substrate for T7 RNA polymerase to produce multiple copies of antisense RNA which will hybridize to primer 2 to start a second cycle of double-stranded cDNA synthesis.
  • the target RNA sequence is initially transformed into an RNA/DNA duplex by reverse transcriptase in the region specified by the first primer.
  • This example illustrates use of the 3SR technique to identify inducible promoters from bean genomic DNA.
  • Bean (Phaseolus vulgaris cv. Canadian Wonder) suspension cultures were obtained from Dr. C. Lamb. The cultures were maintained and proliferated on modified Schenk and Hildebrandt medium (SH medium) (infra, Assay Table VII). The cultures were transferred weekly. At each subculture, six grams fresh weight of bean suspension cells were transferred to 100 ml fresh SH medium. The cultures were incubated at 27 ° C in the dark on a gyratory shaker at a speed of 125 rpm.
  • SH medium modified Schenk and Hildebrandt medium
  • Pseudomonas syringae pv tomato ATCC, Rockville, MD
  • Pseudomonas syringae pv tabaci ATCC, Rockville, MD
  • Erwinia carotovora subsp ECC, Rockville, MD
  • Total nucleic acid was isolated from 0.2 - 0.3 grams of each sample using the following protocol: (1) frozen tissue was ground to a fine powder in a mortar and pestle, (2) pulverized tissue was added to 450 ⁇ l NT buffer (0.1M NaCl, 0.01M Tris, pH9.0, lmM EDTA) and 450 ⁇ l phenokchloroform (1:1) and vortexed until thawed, (3) the slurry was centrifuged for 10 min.
  • NT buffer 0.1M NaCl, 0.01M Tris, pH9.0, lmM EDTA
  • the aqueous layer was removed and precipitated with EtOH according to standard protocols in Maniatis et al., (1982), (5) the nucleic acids were recovered by centrifugation and the concentration determined by spectrophotometry at 260nm, (6) the final concentration was adjusted to 0.6 ⁇ g/ml.
  • the 3SR reaction products were analyzed using a dot blot apparatus (Schleicher and Schuell, Keene, NH). Two microliters of the reaction was added to 100 microliters of DM5 (2.6mM Tris, pH 8.0, 0.26mM EDTA, 10 ⁇ SSC, 7.4% Formaldehyde), and the samples were heated to 55 ° C for 20 minutes, then placed on ice prior to loading onto the nitrocellulose in the dot blot apparatus according to manufacturer's instructions. The nucleic acids were fixed to the nitrocellulose by UV crosslinking (Stratagene, LaJolla, CA).
  • the blot was probed with an oligonucleotide derived from the bean PAL-1 gene (Edwards et al., 1985, supra). The sequence was identical to the mRNA (sense) strand. The probe is shown in SEQ ID NO. 13. The oligonucleotide was end-labeled with gamma 32 P-ATP using T4 kinase (BRL,
  • the blot was hybridized with 2 ⁇ 10 6 cpm/ml for 1 hour in 5 ⁇ SSPE, 4 ⁇ BP (2% BSA, 2% Polyvinylpyrrolidone-40), 1% SDS.
  • the filter was washed three times at room temperature for 5 minutes each in 1 ⁇ SSPE, 1% SDS and one time at 42°C for 1 minute in 1 ⁇ SSPE, 1% SDS.
  • PAL-1 is a gene inducible by plant pathogenic bacteria.
  • 3SRTM is a rapid and sensitive technique which can be used for the identification of inducers of plant defense genes.
  • Sorbitol (66.7% w/v) (Sigma, St. Louis, MO) 22.5 ⁇ l Primer 1 5.0 ⁇ l
  • Target nucleic acid 5.0 ⁇ l
  • Sequence ID NO. 1 pPAL-1, 5' ⁇ 3', B ⁇ S fragment; 245 bp
  • Sequence ID NO. 2 pPAL-2, 5' ⁇ 3', (E) ⁇ P fragment; 271 bp sequence
  • Sequence ID NO. 3 pPAL-3, 5' ⁇ 3', (E) ⁇ P frament; 277 bp
  • Sequence ID NO. 4 pPAL-4, 5' ⁇ 3', E ⁇ P fragment; 276 bp
  • Sequence ID NO. 5 pPAL-6(a), 5' ⁇ 3' H ⁇ S fragment; 300 bp sequence
  • Sequence ID NO. 6 pPAL-6(b), 5' ⁇ 3', H ⁇ S fragment; sequenced
  • Sequence ID NO.7 pPAL-8, 5' ⁇ 3' (E) ⁇ P fragment; 278 bp
  • Sequence ID NO.8 rPAL-2, 5' ⁇ 3'; 2338 bp sequence, sense
  • Sequence ID NO.9 rPAL-4, 5' ⁇ 3'; 1997 bp sequence, sense
  • Sequence ID NO.11 T7 RNA Polymerase Binding Site on 3SR
  • Sequence ID NO.13 Probe from bean PAL-1
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • AACTCGTCGC GCCGGCACAC CAATCCCGTG GTCAACCCAA CCGGCCACAC CGAACCCACA 1680
  • AAAGCCGGCA AGGCATGGCT CGCGGACTAC GGCCTGGCGC GGGTGGTCTC GTCCCTACGG 240
  • MOLECULE TYPE DNA (genomic)

Abstract

The present invention provides isolated regulatory sequences or promoters for plant genes that encode phenylalanine ammonia-lyase (PAL) in potato or rice. The promoters are inducible by exogenous elicitor(s) and/or wounding, and are capable of regulating transcription of associated DNA sequences in chimeric plant cells, tissues and plants that contain the chimeric constructs. The invention provides chimeric potato and rice PAL constructions, transgenic potato and rice plant cells, plant tissues and transgenic plants that contain them. The chimeric constructions and transgenic plant materials are useful in agrichemical assays. The invention also provides a method for identifying inducible plant defense genes and promoters. In addition, the invention provides a method for identifying agrichemicals that can induce plant defense genes, especially agrichemicals which induce these plant gene regulatory elements immediately or very soon after exposure. Finally, the invention provides a method for identifying organisms which can induce plant defense gene regulatory elements.

Description

INDUCIBLE PLANT DEFENSE GENE REGULATORY REGIONS
FROM POTATO AND RICE, USES THEREOF, AND ASSAYS
FIELD OF THE INVENTION
This invention relates generally to plant biotechnology and specifically to novel inducible plant defense gene regulatory sequences or promoters from potato and rice, methods for identifying and isolating these and related sequences, plus uses thereof, especially in the construction of transgenic plants and plant compositions. The invention also relates to methods for identifying agrichemicals and organism(s) which can induce plant defense genes.
BACKGROUND OF THE INVENTION
Plant disease resistance mechanisms include physical and chemical barriers within the plant, as well as responses which are induced or activated by wounding or attack by pathogen(s). One important inducible defense response is the production of phytoalexins, which have been defined as low-molecular weight, antimicrobial compounds synthesized and accumulated by the plant at the site of attempted infection. Phytoalexins are predominantly phenylpropanoids, isoprenoids, and acetylenes.
The first committed step in the biosynthesis of phenylpropenoids in higher plants is the formation of ammonia and trans-cinnamate from L-phenylalanine. This reaction is catalyzed by the enzyme L-phenylalanine ammonia-lyase (PAL). The organization of PAL genes has been most extensively studied in bean (Phaseolus vulgaris). See Cramer, et al., 1989, Plant Mol. Biol. 12:367-383. In the bean genome, there are three divergent classes of PAL genes: gPAL-1, gPAL-2 and gPAL-3.
Transcription of PAL genes is rapidly activated by a wide range of stimuli including wounding, glycan elicitor preparations from fungal cell walls, and the reduced form of glutathione, a cellular metabolite. Not all PAL genes respond in the same manner to these stimuli. Therefore there is a need to identify novel PAL promoters which respond to fungal or bacterial elicitors, or to wounding. These novel PAL promoters can be used to create transgenic plant materials and transgenic plants, and as part of novel agrichemical screening assays.
It is an object of the present invention to identify and provide novel PAL promoters which respond for example, to fungal or bacterial elicitors, or wounding. It is a further object of the invention to use these novel promoters to create transgenic plant materials and transgenic plants. It is another object to provide novel methods which will allow the new PAL promoters to be identified and isolated. It is yet another object of the invention to provide novel whole plant agrichemical screening assays and techniques useful for identifying new agrichemicals which induce the plant's normal PAL-related defense responses, and will also induce expression in transgenic plants of structural genes associated with responsive PAL
promoters. It is also an object to provide an improved method for identifying agrichemicals which can induce plant defense gene promoters or regulatory regions, especially agrichemicals which induce these plant gene regulatory regions immediately or very soon after exposure. Finally, it is an object of the invention to provide a method for identifying organisms which can induce plant defense gene regulatory regions.
SUMMARY OF THE INVENTION
The present invention provides isolated promoters (regulatory sequences) for plant genes that encode phenylalanine ammonia-lyase (PAL) in potato or rice. The promoters are inducible by exogenous elicitor(s) or wounding, and are capable of regulating transcription of associated DNA sequences in transgenic plant cells, tissues and plants that contain the
promoters as part of chimeric constructs. The invention includes methods for obtaining these novel PAL promoters. The invention provides the PAL promoter(s), probe sequences, chimeric potato and rice PAL promoter constructs (in which the PAL promoters are operatively linked to structural genes, e.g., reporter genes), as well as transgenic potato and rice plant cells, plant tissues and transgenic plants that contain these chimeric constructs. The chimeric constructions and transgenic plant materials which contain them are useful in agrichemical assays. The invention also provides a method for inducing transcription of a chimeric gene in transgenic plants. The invention further provides a method for identifying agrichemicals that can induce plant defense gene elements, especially agrichemicals which induce plant gene regulatory elements immediately or very soon after exposure. Finally, the invention provides a method for identifying organisms which can induce plant defense gene regulatory elements.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1A and 1B are restriction enzyme maps of genomic clones of potato S. tuberosum PAL genes.
Figure IA shows limited restriction enzyme maps of six potato clones isolated from the S. tuberosum cv. Desiree genomic library. Clone 1 = λpPAL-1; Clone 3 = λpPAL-3; Clone 4 = λpPAL-4; Clone 6 = λpPAL-6; Clone 7 = λpPAL-7; Clone 8 = λpPAL-8. The boxes indicate the regions of each clone that have been subcloned and that contain sequences which hybridize to S. tuberosum cv. Desiree cDNA clones pPAL-3 and pPAL-21.
The orientation of the putative PAL gene is given within the boxes. Symbols: B: BamHI; E: EcoRI; H: HindIII; K: KpnI; S: SstI. The parentheses around some symbols indicate the restriction enzyme cleavage sites are present, but the exact location is not known.
Figure 1B also shows restriction enzyme maps of seven potato clones isolated from the S. tuberosum cv. Desiree genomic library. Clone 1 = λpPAL-1; Clone 2 = λpPAL-2; Clone 3 = λpPAL-3; Clone 4 = λpPAL-4; Clone 6 = λpPAL-6; Clone 7 = λpPAL-7; Clone 8 = λpPAL-8. The orientation of the putative PAL gene is given below each map. Symbols: B: BamHI; Bgl: BglI; E: EcoRI; H: HindIII; P: PstI; S: SstI. The parentheses around some symbols indicate that the restriction enzyme cleavage sites are present, but the exact location is not known. (In the present specification, and with reference to the figures, the B→S sequence for λpPAL-1 is SEQ ID NO. 1; the (E)→P sequence for λpPAL-2 is SEQ ID NO. 2; the (E)→P sequence for λpPAL-3 is SEQ ID NO. 3; the E→P sequence for λpPAL-4 is SEQ ID NO. 4; the H→S sequence for λpPAL-6 in Figure IB is referred to as λpPAL-6(a) and is shown as SEQ ID NO. 5; the S→H sequence (shown as a sense sequence, having been converted from the antisense sequence) for λpPAL-6 is referred to as λpPAL-6(b) and is shown as SEQ ID NO. 6; and the (E)→P fragment for λpPAL-8 is SEQ ID NO. 7.)
Figure 2 shows restriction enzyme maps of genomic clones of the rice Otyza sativa PAL genes. More specifically, Figure 2 shows limited restriction enzyme maps of three rice PAL clones isolated from the Otyza sativa genomic library. Clone 2 = λrPAL-2; Clone 4 = λrPAL-4; Clone 10 = λrPAL-10. The scale is shown in the bottom left corner. The boxes indicate regions that have been subcloned into a pUC vector. The arrows above the maps show the orientation of the PAL gene within the genomic clone.
Symbols: E: EcoRI; H: HindIII; S: SstI. (In the present specification, λrPAL-2 in Figure 2 is SEQ ID NO. 8 and λrPAL-4 is SEQ ID NO. 9).
Figure 3 is a schematic drawing showing the self-sustained, sequence replication (3SR™, SIBIA, La Jolla, CA) system.
DEFINITIONS
In the present specification and claims, reference will be made to phrases and terms of art which are expressly defined for use herein as follows:
As used herein, PAL refers to phenylalanine ammonia-lyase. PAL catalyzes the conversion of the amino acid L-phenylalanine to transcinnamic acid and NΗ4 +. This is the first reaction in the synthesis of a wide range of plant natural products based on the phenylpropane skeleton, including lignins, flavonoids, isoflavonoid, coumarins and hydroxycinnamic acid esters.
As used herein, exogenously controlled plant gene regulatory regions or elements refer to nucleic acid sequences that affect transcription of "operatively linked, functionally linked or associated" structural genes in response to exogenous stimuli. Examples of exogenously controlled plant gene regulatory sequences include inducible plant defense gene regulatory regions or promoters (e.g., PAL promoters), elicitor-regulated activator domains, upstream silencer domains, etc.
As used herein, promoter refers to a non-coding region of DNA involved in binding of RNA polymerase and other factors that initiate or modulate transcription whereby an RNA transcript is produced. Promoters can be naturally occurring or synthetically produced. Promoters, depending upon the nature of the regulation, may be constitutive or regulated. A constitutive promoter is always turned on. A regulatable promoter requires specific signals in order for it to be turned on or off. A developmentally regulated promoter is one that is turned on or off as a function of
development. The promoters of the present invention are directly or indirectly responsive to exogenous elicitor(s) and/or wounding. Stated another way, exogenous elicitor(s) and/or wounding can induce the promoters of the present invention to initiate transcription of associated structural gene(s).
In the present specification and claims, the terms promoter and gene regulatory region are used interchangeably.
As used herein, elicitors are compounds or organisms which control (e.g., initiate, terminate, increase or reduce), either directly or indirectly, the action of exogenously-controlled plant gene regulatory elements such as the inducible stress-regulated promoters for the plant genes that encode the phenylpropanoid biosynthetic enzymes phenylalanine ammonialyase (PAL). Elicitors 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(β-D-glucopyranosyl)-D-glucitols; lipid elicitors such as arachidonic acid and eicosapentaenoic acid, glycoprotein elicitors, fungal elicitors, abiotic elicitors such as mercuric chloride HGCl2, fungi (e.g., elicitors released by whole fungi), bacteria (e.g., elicitors released by whole bacteria), etc.
As used herein, the terms operatively linked, functionally linked or associated, or grammatical variations thereof, are equivalent terms that are used interchangeably. In particular these terms refer to the linkage of a promoter or a non-coding gene regulatory sequence to an RNA-encoding DNA sequence, and especially to the ability of the regulatory sequence or promoter to induce production of RNA transcripts corresponding to the DNA-encoding sequence when the promoter or regulatory sequence is recognized by a suitable polymerase. All three terms mean that linked DNA sequences (e.g.,
promoter(s), structural gene (e.g., reporter gene(s)), terminator sequence(s), etc.) are operational or functional, i.e., work for their intended purposes.
Stated another way, operatively or functionally linked, or associated, means that after the respective DNA segments are joined, upon appropriate activation of the promoter, the structural gene will be expressed.
As used herein, suitable plant material means and expressly includes, plant protoplasts, plant cells, plant callus, plant tissues, developing plantlets, immature whole plants and mature whole plants.
As used herein, transgenic plants or plant compositions refer to plants or plant compositions in which heterologous or foreign DNA is expressed or in which the expression of a gene naturally present in the plant has been altered. Such DNA will be in operative linkage with plant
biochemical regulatory signals and sequences. Expression may be constitutive or may be regulatable. The DNA may be integrated into a chromosome or integrated into an episomal element, such as the chloroplast, or may remain as an episomal element. In creating transgenic plants or plant compositions, any method for introduction of such DNA known to those of skill in the art may be employed.
Use of the phrase isolated in the present specification and claims, as a modifier of DNA or RNA, means that the DNA or RNA so designated have been separated from their in vivo cellular environments through the efforts of human beings; as a result of this separation, the isolated DNAs or RNAs are useful in ways that the non-separated, impure DNAs or RNAs are not.
DESCRIPTION OF SPECIFIC EMBODIMENTS
In one aspect, the present invention comprises isolated or isolatable promoters (i.e., regulatory sequences) for plant defense genes that encode phenylalanine ammonia-lyase (PAL) in potato or rice wherein the promoters are capable of regulating transcription of an associated DNA sequence in suitable hosts and wherein the promoters can be induced by, or are otherwise directly or indirectly responsive to, exogenous elicitor(s) and/or wounding. These promoters are exemplified herein by the potato PAL promoters associated with the PAL structural gene sequences comprised within clones λpPAL-1, λpPAL-2, λpPAL-3, λpPAL-4, λpPAL-6, λpPAL-7 (λpPAL-6 and λpPAL-7 are identical as described in Example IIC and are referred to herein as λpPAL6/7), and λpPAL-8, plus rice PAL promoters comprised within clones λrPAL-2 and λrPAL-4.
In another aspect, the potato and rice PAL promoters of the invention are operatively linked to at least one associated DNA sequence that encodes protein(s) which directly or indirectly gives rise to a phenotypic trait. The linkage can be at the level of transcription ( i.e., a "transcriptional fusion") so that the protein which gives rise to the phenotypic trait is expressed as a non-fused peptide. Alternatively, and especially in the case of some marker genes, the linkage can also be at the level of translation (i.e., a "translational fusion") such that the marker protein is expressed as fusion peptide with a portion of the amino terminal end of the native PAL protein. According to the teaching of the invention, the phenotypic trait can consist of tolerance or resistance to: herbicide, fungus, virus, bacterium, insect, nematode or arachnid; production of secondary metabolites, male or female sterility, or production of an enzyme or reporter compound. When the associated structural gene encodes a reporter enzyme or compound, preferably the compound will be chloramphenicol acetyltransferase (CAT), neomycin phosphotransferase (NPT), nopaline synthase (NOS), octopine synthase (OCS), β-1,3-glucuronidase
(GUS), acetohydroxyacid synthase (AHAS), β-galactosidase (β-GAL), or luciferase (LUX).
According to the invention, the potato and rice PAL promoters - associated DNA sequence constructs are used to transform suitable hosts and to create transgenic plant compositions, e.g., plant protoplasts, plant cells, plant callus, plant tissue, developing plantlets, immature whole plants, mature whole plants, or seeds. The invention encompasses these transformed hosts and transgenic compositions, especially transgenic potato and rice plants and seeds that contain potato and rice PAL promoters and associated DNA sequences.
The present invention provides novel isolated DNA sequences identified herein as SEQ ID NOS. 1-9. SEQ ID NOS. 1-7 are DNA coding sequences from potato PAL clones λpPAL-1, λpPAL-2, λpPAL-3, λpPAL-4, λpPAL-6 (two sequences) and λpPAL-8, respectively. SEQ ID NOS. 8 and 9 are coding and non-coding DNA sequences from rice PAL clones λrPAL-2 and λrPAL-4, respectively.
The DNA sequences of the invention can be used as probes to identify identical or homologous PAL sequences. Preferably the probes will be at least 10 nucleotides in length, and most preferably will be from about 100 to about 500 nucleotides long. For example, the coding sequences of potato PAL clones λpPAL-1, λρPAL-2, λpPAL-3, λpPALA, λpPAL-6/7 or λpPAL-8, or λrPAL-2 or λrPAL-4 can be used to probe cDNA or genomic libraries for identical or highly homologous sequences. These identical or highly
homologous sequences will be associated with inducible promoters that are well within the scope of the present invention.
Even though related PAL coding sequences may have sufficient homology to hybridize with one another under moderately stringent
hybridization conditions, the promoters may not be functionally equivalent.
For that reason the invention includes a method for identifying sequences that are not only homologous to the sequences of the invention, but are also regulated by inducible PAL promoters. According to this method, a plant is subjected to an elicitor, and then RNA from the plant is isolated. PolyA+ RNA is selected, e.g., by using an oligo(dT) column. A cDNA library is prepared from this RNA and cloned into a suitable vector. The library is probed with a probe comprised of nucleotide sequences from the coding region(s) of elicitor inducible genes, e.g., the coding sequences of SEQ ID NOS. 2-7 (λpPAL-2, λpPAL-3, λpPAL-4, λpPAL-6, λpPAL-8), or SEQ ID NOS. 8 and 9 (λrPAL-2 and λrPAL-4). cDNA which hybridizes with the probe (i.e., positive clone(s)) is subcloned and sequenced. A genomic library from the plant of interest is probed with the cDNA, and the segment of genomic DNA that hybridizes with the probe is identified. An antisense RNA transcript from this genomic DNA is made and labeled (e.g., radioactively) and then used as a probe to hybridize to mRNA from elicitor-treated and non-elicitor treated plants. The hybridizing mix is then subjected to RNase degradation of all single-stranded RNA. If the transcript of interest is present in total RNA, then a double-stranded RNA-RNA hybrid that is protected by RNA from elicitor-treated plants but that is not protected by RNA from non-elicitor containing no mismatches will be formed and will be unaffected by the RNase treatment. This product can be identified by size following gel electrophoresis. The promoter from the genomic clone that yields the antisense RNA transcript that is protected by RNA from elicitor-treated plants but that is not protected by RNA from non-elicitor treated plants is inducible with elicitor. The promoter is isolated and used to make chimeric constructs for use in agrichemical assays, and to produce transformed cells and transgenic plants and transgenic plant compositions.
The invention also includes a method for identifying exogenous elicitors which are capable of inducing, either directly or indirectly, a potato or rice PAL promoter. According to the method, a suitable host (e.g., a plant composition) is transformed with a potato or rice PAL promoter operatively linked to a structural gene whose expression can be detected (e.g., a marker gene). Putative exogenous elicitor is then applied to the transformed host. Exogenous elicitor that can induce expression of the marker gene is concluded to be an elicitor that can induce the potato or rice PAL promoter. In a related method, whole plants are used in the assay. According to this method, whole transgenic plants are created which contain at least one chimeric DNA sequence comprised of a potato or rice PAL promoter operatively linked to a reporter structural gene. Putative exogenous elicitor(s) is then applied to the plant. The elicitor(s) that induces expression of the reporter gene is concluded to be elicitor(s) which can induce expression of the potato or rice PAL promoters.
The invention also provides a method for identifying elicitor-inducible promoters. According to the method, a plant is subjected to an elicitor, then RNA is isolated from the plant. A cDNA library is prepared from the isolated RNA, which is then probed with a probe comprised of nucleotide sequences from the transcribed coding and/or non-coding region(s) of gene(s) of interest. cDNA which hybridized with the probe is then used to probe a genomic library from the plant of interest. The segment of genomic DNA that hybridzed with the probe is identified, and a labeled antisense RNA transcript is made from it. This labeled antisense RNA transcript is used as a probe to hybridize to mRNA from elicitor-treated and non-elicitor treated plants. The hybridized mix is subjected to RNase, and those antisense RNA transcripts that were protected from degradation by elicitor-treated RNA but were not protected from degradation by RNA from non-elicitor treated plants are identified. The promoter from the genomic clone which yielded the antisense RNA transcript that was protected by only RNA from elicitor-treated plants is inducible with elicitor.
The invention also discloses a related method for identifying elicitor-inducible promoters. According to this method, a plant genomic library is probed with a probe comprised of nucleotide sequences from the transcribed coding and/or non-coding region(s) of a gene of interest. The segment of genomic DNA that hybridized with the probe is identified. A labeled antisense RNA transcript is made from the genomic DNA segment that hybridized with the probe. This labeled antisense RNA is used as a hybridization probe for mRNA obtained from elicitor-treated and non-elicitor treated plants. The hybridization mix is subjected to RNase, and those antisense RNA transcripts that were protected from degradation by elicitor- treated RNA but were not protected from degradation by RNA from non- elicitor treated plants are identified. The promoter from the genomic clone which yielded the antisense RNA transcript that was protected by only RNA from elicitor-treated plants is inducible with elicitor.
In another aspect, the invention discloses an amplification method for identifying agrichemicals which can induce expression of a plant gene. According to this aspect of the invention, RNA from plant material not exposed to a putative inducer is isolated and from within this isolated RNA, an RNA encoded by a gene of interest is identified by hybridization to a probe. The RNA from this identified gene is amplified using the self-sustained sequence replication or 3SR™ (SIBIA, La Jolla, CA 92037-4641) technique for amplification of specific RNA sequences and primers specific for the inducible gene transcript of interest. See Guatelli, et al., 1990. Proc. Natl. Acad. Sci USA. 87:1874-1878. The unexposed plant material from which the first RNA was obtained is then exposed to a putative chemical inducer. RNA encoded by the gene of interest is then identified and amplified using 3SR. The amplification products from unexposed and exposed plant material are compared. If the level of expected product in the amplification of RNA from exposed plant material is higher than in the amplification of RNA from unexposed plant material, it can be concluded that this agrichemical(s) can induce expression of the gene of interest.
With regard to the amplification method, its sensitivity has two sigmficant advantages. Firstly, very small samples can be analyzed, thus for the first time permitting use in agrichemical screens of plants such as Arabidopsis and cells in tissue culture. Secondly, some chemicals may have a very small but very significant effect on plant genes, e.g., plant defense genes, such that the response may be difficult to detect with methods less sensitive than 3SR. The 3SR amplification method of the invention makes it possible for the first time to identify these chemical elicitors.
In yet another aspect, the invention discloses a novel method for identifying organisms which can induce plant defense genes. According to this method, a transgenic plant containing at least one chimeric DNA sequence comprised of an inducible plant defense gene promoter operatively linked to at least one reporter structural gene is exposed to a potentially inducing organism. The organism can be living, or can be dead or otherwise disabled. By monitoring expression of the reporter gene it is possible to conclude which organisms induce transcription of the reporter gene, and thus are candidates for use as "inducers" or plant vaccines, or are potentially pathogenic to plants.
The invention will now be described in greater detail in the following examples, which are presented solely for purposes of illustration and not of limitation.
EXPERIMENTAL POTATO
EXAMPLE I: POTATO
ISOLATION of ARACHIDONIC ACID-INDUCED cDNAs from POTATO
A. Construction of cDNA Library
Potato tubers (Solanum tuberosum cv. Desirée) were obtained from the United States Dept of Agriculture. Tubers were washed with deionized water to remove soil, then soaked in 95% ethanol for one min and rinsed three times with sterile distilled water. Tubers were peeled and surface sterilized in 10% Purex™ (commercial bleach) containing two drops TWEEN 20™ per 100 ml solution for 15 min and rinsed three times with sterile distilled water. Cylinders of tissue were excised with a cork borer (approximately 8 mm in diameter) inserted through the long axis of perimedullaiy tissue of the tuber and sliced into discs of approximately 3 mm thickness. Tuber discs were transferred to potato callus initiation medium. The potato callus initiation medium contained Murashige and Skoog basal medium (MS medium,
Murashige and Skoog, 1962, Physiol. Plant. 15:493-497) supplemented with 10 mg/l 2,4-Dichlorophenoxyacetic acid.
Initiated calli were multiplied and maintained on LS2T medium. Potato Table I: LS2T Medium
Murashige and Skoog
inorganic salts
Tniamine·HCl 0.5
Pyridoxine·HCl 0.5
Nicotinic acid 5.0
Glycine 2.0
Biotin 0.05
Folic acid 0.5
trans-Zeatin 0.1
2,4-Dichlorophenoxyacetic acid 2.0
Myo-inositol - 100.0 Sucrose 30,000.0
Tissue culture agar 8 g/l
pH 5.8
Potato cell suspension cultures were initiated from potato callus cultures. The cultures were maintained and proliferated on LS2T liquid (or suspension) medium and incubated at 27°C in the dark on a gyratory shaker at a speed of 125 rpm.
Desiree suspension cultures were subcultured weekly. At each subculture, five grams fresh weight of cells were transferred to 100 ml fresh LS2T liquid medium. A five-fold increase in growth was observed in a seven-day growth period. Three hours prior to isolation of RNA, the culture was treated with arachidonic acid (Sigma, St. Louis, MO) to a final concentration of 0.1 mM. Arachidonic acid is a fatty acid normally found in fungal cell walls and induces at least some PAL gene expression (Fritzemeier et al., 1987, Plant Physiol. 85:34-41).
After induction, total RNA was isolated from the cultured cells following a modification of the procedure described in Haffner et al., 1978, Can. J. Biochem. 56:729-733.
With the modified procedure, the RNA was isolated as follows: 1. Measure out 3-5 g of tissue. Grind into a fine powder using a mortar and pestle (pre-cooled with liquid N2). 2. Add tissue to pre-chilled 10 ml equilibrated phenol +
5 ml 0.1 M Tris-Cl (pH 9). Homogenize using vortex. All the following steps should be performed at 4ºC.
3. Centrifuge 10 minutes at 3,000 × g. Transfer aqueous
phase to a 50 ml tube.
4. Back extract phenol. Add 5 ml 0.1 M Tris-Cl (pH 9), mix and re-centrifuge.
5. Combine aqueous phases, add one volume chloroform, mix and re-centrifuge. Remove lower, chloroform phase.
Repeat. 6. Transfer aqueous phase to another tube and add 0.1 volume 3 M sodium acetate (pH 5.2) + 2.5 volumes ethanol. Keep at -20ºC for 2 hours (minimum).
7. Pellet RNA 30 minutes at 10,000 × g in 30 ml Corex tubes.
8. Resuspend pellet in 6 ml H2O. When dissolved, add 2.0 ml 8 M LiCl. Mix well. Keep at +4ºC overnight.
9. Centrifuge LiCl-precipitated RNA 30 minutes at 10,000 × g. Wash pellet with 10 ml 70% ethanol and re-centrifuge. Decant and remove liquid.
10. Resuspend pellet in 5 ml H2O.
11. Add 0.5 ml 3 M sodium acetate (pH 5.2) plus 13 ml
ethanol. Keep at -20ºC for 2 hours (minimum).
12. Centrifuge RNA for 30 minutes at 10,000 × g. Wash with
5 ml 70% ethanol, re-spin, and dry.
13. Resuspend pellet in 1 ml H2O. Store at -70ºC.
Poly A* RNA was isolated from 400 μg total RNA using the mRNA Purification Kit (Pharmacia, Piscataway, NJ; catalog #27-9258) and following the manufacturer's instructions. Approximately 3.1 μg poly A+ was recovered, and used for cDNA synthesis following the BRL cDNA synthesis system protocol (BRL, Bethesda, MD; catalog #8269 SA). The cDNA was treated for 30 min with 9 units T4 DNA polymerase (BRL), following which time a 50-fold molar excess of EcoRI adapters (Pharmacia) were ligated to the cDNA (overnight at 15ºC). The cDNA was separated from the excess adapters through a 1 ml G50 spin column (Sephadex G50 in a 1 ml syringe, according to Maniatis et al., in Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1982) and the purified cDNA was ligated to EcoRI-digested λgtlO arms (Promega, Madison, WI) according to manufacturer's instructions. The entire ligation mixture was packaged using Gigapack™11 Gold Packaging Extract (Stratagene, La Jolla,
CA) following manufacturer's instructions. The cDNA library was titered using E. coli strain C600 (Promega, Madison, WI). The titer of the library was determined to be 2.6 X 107 plaque forming units/ml.
B. Library Screening
The library was screened with a 0.9 kb fragment of plasmid pCP63.15, a pUC19-based plasmid comprising a cDNA encoding a portion of the potato PAL exon II gene sequence (Fritzemeier et al., 1987, Plant Physiol. 85:34-41). The screening conditions were as follows:
Hybridization: 42 ° C, 35% Formamide, 5X Denhardts, 5X
SSC, 0.2% SDS, 200 μg/ml salmon sperm DNA.
Wash: 2X SSC, 0.1% SDS, repeated a total of four times, at one hour each, at 50 ° C.
The screening and five rounds of plaque purification identified three putative PAL-encoding cDNAs, which were called λpPAL-3, λpPAL-21, and λpPAL-25.
C. Subcloning and Characterization
All ligations were performed with a ratio of insert to vector of 5:1.
Each of the three lambda clones, λpPAL-3, λpPAL-21, and λpPAL-25, was digested with EcoRI, which released the 450, 500, and 250 bp inserts, respectively. After gel purification each insert was ligated to EcoRI-digested pUC119 (Vieira, J., and Messing, J., 1987, In Methods in Enzymology (R. Wu and L. Grossman, Eds.) Vol. 153 pp. 3-11, Academic Press, New York) and the ligations were separately transformed into DH5α cells. AmpR colonies were selected. Colonies carrying correct plasmid released a 450, 500, or 250 bp fragment, respectively, upon digestion of plasmid DNA with EcoRI.
The EcoRI inserts were sequenced using the Sequenase (U.S. Biochemical, Cleveland, OH) protocol for double-stranded DNA, and the nucleotide sequence of each was compared to the nucleotide sequence of the bean gPAL-2 gene (Cramer et al., 1989, Plant Mol. Biol. 12:367-383). The similarity was as follows:
Potato Clone # Homology to gPAL-2 3 55%
21 62%
25 no significant homology
EXAMPLE II: POTATO
ISOLATION of GENOMIC CLONES CORRESPONDING to
ARACHIDONIC ACID-INDUCED cDNA from POTATO
A. Construction of Genomic Library
A genomic library was constructed from total genomic DNA isolated from young leaves of Solanum tuberosum cv. Desiree. DNA isolation was by the procedure of Bendich, 1988, in Plant Molecular Biology Manual,
Kluwer Academic Publishers, Section A6, p. 1-10. The genomic DNA was partially digested with Sau3AI, resulting in fragments of between 9-23 kb in size. The Sau3AI ends were partially filled in using dATP and dGTP.
Approximately 0.43 μg of fragment were ligated with 1 μg of XhoI-digested plasmid λFIXII™ (Stratagene), which had the overhang partially filled in using dTTP and dCTP. The ligation reaction was packaged using the Gigapack™11 Gold packaging mix (Stratagene), following manufacturer's directions.
Four libraries were constructed; three contained approximately 3 × 105 clones and the fourth had 7 × 105 clones.
B. Library Screening
All four libraries were screened under conditions of high stringency using an equal mixture of isolated fragments from the Solanum tuberosum cv. Desiree cDNA clones pPAL-3 (450 bp EcoRI fragment) and pPAL-21 (500 bp EcoRI fragment) (see Potato Example I.B.) as probes. The screening conditions were as follows:
Hybridization: 42°C, 50% Formamide, 5X Denhardts, 5X
SSC, 0.2% SDS, 200 μg/ml salmon sperm DNA.
Wash: 0.2X SSC, 0.1% SDS, for a total of three washes at 45 °C, 15 min each wash; and 0.2X
SSC, 0.1% SDS, for one wash at 65ºC for 15 min. A total of 43 putative PAL-encoding genomic DNA clones were identified from this screening. Twelve clones were chosen and subjected to five rounds of plaque purification. Nine clones were selected for further characterization. The selected clones referred to herein are called λpPAL-1 through λpPAL-8.
C. Clone Analysis
Analysis of restriction enzyme digests of these nine clones revealed that λpPAL-6 and λpPAL-7 were overlapping clones with several restriction enzyme fragments in common, and that the remaining seven clones were unique (Potato Figure IA). (A map for λpPAL-2 is shown in Figure 1B).
A map for λpPAL-5 is not shown because no sequence similarity to PAL has been found in this insert.
Through restriction and hybridization analysis (using the inserts from pPAL-3 and pPAL-21 as probes) the open boxed segments of the genomic clones shown in Potato Figure 1A were determined to be the portion of the insert that hybridized to the probe(s). Thus, the boxed segments were excised by digestion with the appropriate restriction enzymes (see Potato Figure 1A) and ligated into appropriately digested plasmid pGEM-7Z (-) (Promega, Madison, WI) to yield the following subclone plasmids:
Genomic Clone # Insert (5'-3') Subclone plasmids λpPAL-1 BamHI/SstI pPAL-1 λpPAL-2 EcoRI/EcoRI pPAL-2 λpPAL-3 EcoRI/EcoRI pPAL-3 λpPAL-4 EcoRI/BglII pPAL-4 λpPAL-6 HindIII/EcoRI pPAL-6 λpPAL-7 BamHI/EcoRI pPAL-7 λpPAL-8 EcoRI/EcoRI pPAL-8
The insert DNAs were sequenced according to the Sequenase protocol (U.S. Biochemical Corporation) for double-stranded
dideoxynucleotide, and the sequences were analyzed using the University of
Wisconsin Genetics (UWG) comparison program, FASTA. The partial sequences of the genomic clones, except λpPAL-5, showed identity to a PAL cDNA clone isolated from Solanum tuberosum cv. Datura. In addition, partial sequences of pPAL-2, pPAL-3, pPAL-4, and pPAL-8 show greater than 90% sequence similarity to each other; the partial sequences of pPAL-6 and pPAL- 7 are identical; and the partial sequence of pPAL-1 showed 62% sequence similarity to pPAL-6 and pPAL-7.
These results suggested that the genes corresponding to λpPAL- 2, λpPAL-3, λpPAL-4, and λpPAL-8 are members of the same PAL gene subfamily, that λpPAL-6 and λpPAL-7 are genomic clones of the same PAL gene, and that λpPAL-1 contains a PAL gene that is different from the PAL gene contained in λpPAL-6.
EXAMPLE III: POTATO
RNASE PROTECTION ASSAY RNase protection assays (Winter et al., 1985, Proc. Natl. Acad. Sci. 82:7575-7579) are based on the different susceptibility of single-stranded RNA and RNA-RNA hybrids to degradation by the single-stranded RNA- specific enzyme RNase. In these assays, total RNA is hybridized to a radioactively-labeled antisense probe complementary to the transcript of interest, followed by RNase-mediated degradation of all single-stranded RNA. If the transcript of interest is present in total RNA, then a double stranded RNA-RNA hybrid containing no mismatches will have formed and will be unaffected by the RNase treatment. This product can be identified by size following gel electrophoresis.
To determine if the genes from which the genomic subclones described in Potato Example II.C. were derived are induced by arachidonic acid, RNase protection assays were performed in which labeled antisense transcripts generated from each subclone were hybridized to total RNA from arachidonic acid-treated and untreated potato (Solanum tuberosum cv.
Desiree) suspension culture cells. The hybridization mixture was then treated with RNase and analyzed by electrophoresis. Protection of a labeled antisense probe from degradation in the presence of RNA from arachidonic acid-treated cells but not in the presence of RNA from untreated cells would indicate that expression of the corresponding gene is induced by arachidonic acid. Therefore, this assay method enabled a determination of whether the genes corresponding to the genomic clones were regulated by arachidonic acid-inducible promoters.
A. Protocol
For the RNase protection assays described herein, the transcripts of interest were defined by the genomic subclone inserts as follows:
Genomic Subclone Insert Used Subclone for RNase exp.
λpPAL-1 1250 bp BamHI/SstI pPAL-1* λpPAL-2 111 bp EcoRI/PstI pPAL-2EP** λpPAL-3 276 bp EcoRI/PstI pPAL-3EP** λpPAL-4 276 bp EcoRI/PstI pPAL-4EP* λpPAL-8 278 bp EcoRI/PstI pPAL-8EP** λpPAL-6 500 bp HindIII/SstI pPAL-6HS**
* The antisense transcript is transcribed using the SP6 promoter and the sense transcript is transcribed using the T7 promoter.
** The antisense transcript is transcribed using the T7 promoter and the sense transcript is transcribed using the SP6 promoter.
The noted inserts were ligated into appropriately digested pGEM-7Z(-), except for the 500 bp insert of λpPAL-6 which was ligated into pGEM-11Z(+ ) (Promega). The polylinker regions in both these plasmids are flanked by the SP6 and T7 RNA polymerase promoters making it possible to generate 32P-labeled antisense RNA transcripts of the inserts using SP6 or T7 RNA polymerase. The choice of which polymerase to use depends upon the orientation of the gene fragment in the polylinker.
32P-labeled antisense RNA transcripts were synthesized in vitro from the subclones pPAL-1, pPAL-2EP, pPAL-3EP, pPAL-4EP, pPAL-8EP, and pPAL-6HS using the Riboprobe Gemini System II (Promega) and following manufacturer's instructions. The RNAs were then used as probes in RNase protection assays of total RNA from Solanum tuberosum cv. Desiree suspension culture cells. Suspension cells were prepared as described in Potato Example LA. and treated for three hours with freshly prepared 0.1 mM arachidonic acid (induced) or water (uninduced) prior to isolation of total RNA from the cells (RNA was isolated as described in Potato Example I.A.). The isolated RNAs were separately hybridized overnight at 45°C to each of the six different antisense probes. The hybridization conditions were as follows: 40 mM Pipes, pH 6.4, 1 mM EDTA, 400 mM NaCl, 50% Formamide.
The hybridization mixtures were then digested with a mixture of RNase A (40 μg/ml; Sigma, St. Louis, MO) and RNase Tl (2 U/ml; BRL, Bethesda, MD) at 34 ° C, and analyzed on a 6% polyacrylamide, 8M urea gel.
To provide positive controls in each of these experiments, sense RNA was transcribed in vitro from subclones pPAL-1, pPAL-2EP, pPAL-3EP, pPAL-4EP, pPAL-8EP, and pPAL-6HS and hybridized with the corresponding labeled antisense RNA. These hybrids should be a perfect match.
B. Results
As expected, the antisense RNA transcripts were protected from RNase-mediated degradation when hybridized with sense RNA synthesized from the same subclone, but were completely degraded in the absence of sense RNA.
Antisense transcripts derived from pPAL2-EP, pPAL3-EP, pPAL- 4EP, pPAL-8EP, and pPAL-6HS each were protected from degradation by RNA prepared from Solanum tuberosum cv. Desiree cells induced with freshly prepared arachidonic acid. This result confirmed that the promoter associated with the gene(s) to which these clones correspond is inducible with arachidonic acid.
Some protection also was observed with RNA prepared from cells which had not been induced, although it was much less than that observed with
RNA from induced cells. This result was not surprising as significant background levels of PAL gene expression were consistently observed in these cells.
The probe derived from pPAL-1 was protected equally well from degradation by RNA from both induced and uninduced cells. This result suggested that the gene corresponding to λpPAL-1 is not an arachidonic-acid inducible PAL gene. EXAMPLE IV: POTATO
ISOLATION of POTATO PAL PROMOTER
A. Cloning
An approximately 6 kb SstI-BamHI fragment from λpPAL-2 was gel purified and subcloned into pGEMHZ( + ) (Promega) using standard DNA cloning procedures (Maniatis et al., supra). The ligation mixture was transformed into DH5α and AmpR colonies were selected. A correct construct was confirmed by the presence of 6 and 3.2 kb bands in SstI-BamHI-digested DNA and was called pD2.2.
A 1.4 kb KpnI-SstI fragment and a 3.3 kb SstI-EcoRI fragment were gel purified from λpPAL-7. Equal molar ratios of these fragments were mixed and subcloned into pUC119 previously digested with both KpnI and EcoRI. The ligation reaction was transformed into DH5α cells and AmpR colonies were selected. Correct construct was confirmed by the presence of 4.7 and 3.2 kb bands upon double digestion of plasmid DNA with EcoRI and KpnI, and was called 7P.
B. Characterization
The complete promoter-containing region and a portion of the coding region of λpPAL-7, and the majority of the promoter-containing region and a portion of the coding region of λpPAL-2 have been sequenced. The two
PAL coding regions share 80% sequence similarity at the nucleotide level.
EXAMPLE V: POTATO
ARACHIDONIC-INDUCED GENE EXPRESSION A. Construction of pUC-GUS.l, pUC-GUS.2, pUC-GUS.3 Promoter-less GUS (β-glucuronidase) gene cassettes are available as HindIII-EcoRI inserts in plasmids pBI101, pBI101.2, and pBI101.3
(Clontech, Palo Alto, CA). The GUS cassettes in plasmids pBI101.2 and pBI101.3 are identical to the one in pBI101 except their reading frames are shifted one and two nucleotides, respectively, relative to the polylinker. As a result, a promoter fragment and a portion of the coding region can be inserted in all three reading frames upstream of the GUS gene creating both
translational and transcriptional fusion constructs. The GUS-encoding insert was removed from each plasmid and each insert was separately ligated with HindIII-EcoRI-digested pUC119. The ligation was transformed into DΗ5α cells, and AmpR colonies were selected. Correct plasmids demonstrated bands of 2.2 and 3.2 kb in size upon digestion with EcoRI and HindIII and were called pUC-GUS.1, pUC-GUS.2, and pUC- GUS3, respectively.
B. Construction of PAL-GUS Fusion Vectors
1. Promoter λpPAL-2
Plasmid pD2.2 (Potato Example IX.A.) was digested with HaeIII, and the insert was separately ligated with SmaI-digested pUC-GUS.1, pUC- GUS.2, and pUC-GUS.3. After transformation into DH5α cells, and selection of AmpR colonies, correct plasmids were identified by release of a 1.8 kb band upon digestion with HindIII. Correct plasmids were called pPAL2.1, pPAL2.2, and pPAL2.3.
Plasmids pPAL2.1, 2.2, and 2.3 were digested with Xbal and SstI and each insert was ligated to XbaI- and SstI-digested pBI101. Plasmid pBI101 (Clontech, Palo Alto, CA) is a broad host range plasmid used in the binary vector, plant transformation system of Agrobacterium tumefaciens. The ligations were transformed into DH5α cells and KanR colonies were selected. Correct plasmids were identified by release of a 6 kb fragment upon digestion with Xbal and SstI, and were called pBIN-2.1, pBIN-2.2, and pBIN-2.3, respectively.
2. Promoter λpPAL-7
Plasmid 7P was digested with PstI and EcoRV and the insert was ligated with PstI- and SmaI-digested pUC-GUS.1, pUC-GUS.2, and pUC- GUS.3. After transformation into DH5α cells and selection of AmpR colonies, correct plasmids were identified by release of a 4.7 kb band upon digestion with EcoRI and BamHI. Correct plasmids were called pGUS7.1, pGUS7.2, and pGUS7.3, respectively.
Plasmid pGUS7.1, 7.2, and 7.3 were digested with HindIII and EcoRI, and each insert was ligated to HindIII- and EcoRI-digested pBIN19. Plasmid pBIN19 is a broad host range plasmid used in the binary vector Agrobacterium tumefaciens transformation system and is available from
Clontech. The ligations were transformed into TB-1 cells and KanR colonies were selected. Correct plasmids were identified by release of a 6 kb fragment upon digestion with HindIII and EcoRI and were called pBIN7.1, pBIN-7.2, and pBIN-7.3, respectively.
Prior to transformation of potato, plasmids pBIN2.1, 2.2, 2.3, 7.1, 7.2 and 7.3 were transferred from the E. coli host to Agrobacterium tumefaciens strain LBA4404 by triparental mating using pRK2073 as the helper strain (Corbin, D. et al., 1982, J. Bacteriol. 149:221-228).
C. Transformation of Potato
The potato tubers used in this experiment were obtained from potato plants of Solanum tuberosum cv. Desiree. Tubers that had been stored in the dark at 4 ° C for one week were washed and rinsed with deionized water to remove soil, surface sterilized one minute in 95% ethanol and rinsed in sterile distilled water. The tubers were peeled and disinfected for 15 minutes in 10% Purex™ (commercial bleach) containing two drops of Tween 20™ per 100 ml solution followed by five rinses with sterile distilled water. The proximal and distal quarter portions of the tubers were discarded. The sterilized potato tubers were immersed in MS liquid medium without hormones (Murashige and Skoog, 1962, Physiol. Plant 15:473-496) for 20 min prior to removal of the discs.
The explants were floated in 20 mis of MS liquid medium without hormones containing an overnight culture of separate Agrobacterium
tumefaciens LBA 4404 (Clontech, Palo Alto, CA) strains harboring plasmids pBIN2.1, 2.2, 2.3; and pBIN7.1, 7.2, 7.3 (Potato Example IV.B.). The
Agrobacterium strains were pre-induced with 50 μM acetosyringone. The tissue and Agrobacterium were incubated at room temperature on a gyratory shaker with gentle shaking (at approximately 60 rpm). After 20 minutes, the explants were blotted on sterile Whatman paper No. 1 and transferred to incubation media which were two-day-preconditioned tobacco feeder plates. Feeder plates were prepared according to the method of Horsch and Jones (In Vitro, 1980, 16:103-108) with the following modification: cells from six-day-old suspension culture were filtered through a sterile 30-mesh sieve, collected on two layers of sterile kimwipes and freed of excess medium through a funnel. Cells were then resuspended in fresh MM medium to a final density of 0.3 g fresh weight per ml. MM medium contained MS basal medium supplemented with 0.5 mg/1 2,4-D and 0.5 mg/l BA. The suspension was stirred and 1.5 ml aliquots were pipetted onto plates containing two kinds of shoot regeneration media, 3C52R medium (Steerman and Bevan, 1988, (Plant Cell Reports 7:13- 16)) and the medium defined in Jarrett et al. (Physiol. Plant., 1980, 49:177-184).
After two days, infected tuber discs were transferred to selection media containing cefotaxime (500 μg/ml) and kanamycin (100 μg/ml). The composition of the media was identical to incubation media, but there was no feeder layer. Tissues were transferred to fresh selection media at two-week intervals, and the cefotaxime concentration was reduced to 250 μg/ml after four weeks in culture. Potato shoots were regenerated after three weeks in culture. When regenerated potato shoots reached 3-5 mm in size, they were excised from tuber discs and grown on the selection media containing 250 μg/ml cefotaxime and 100 μg/ml kanamycin.
When putative transgenic potato shoots reached 2 cm in size, they were transferred to rooting media containing 250 μg/ml cefotaxime and 100 μg/ml kanamycin. The rooting media were identical to shoot regeneration media except plant hormones were not added. When the shoots had rooted, the resulting plantlets were transplanted from culture into soil and grown in a plant growth chamber. At desired times, the transgenic potato plants are assayed for GUS activity.
The cultures were incubated at 27 °C with 16 hours of light under
4000 lux light intensity throughout the experiments.
EXAMPLE V: POTATO
USE OF TRANSGENIC POTATO PLANTS AS AN AGRICHEMICAL SCREEN
Transgenic potato plants containing PAL-GUS fusion constructs are clonally propagated from tubers derived from transgenic plants shown to contain stably-integrated PAL-GUS constructs which are induced, in this example, by arachidonic acid. These transgenic plants are maintained under standard greenhouse conditions.
For use in agrichemical screening assays, plants are grown to a size and/or stage of development which is both manageable under greenhouse conditions and susceptible to a pathogen of interest, e.g., Phythophthora infestans. The plants are treated either by foliage spray or soil immersion with a range of concentrations of the unknown chemical (elicitors). In parallel, additional plants are exposed to water as a negative control or to a known elicitor as a positive control. Six to twenty four hours following treatment, leaf tissue is removed and assayed for GUS activity using the fluorometric assay of Jefferson et al. (Plant Mol. Biol. Rep., 1987, 5:387-405). A positive result is indicated by levels of GUS activity significantly higher than those observed with the negative control. A positive result is indicative of, e.g., identifies, chemicals which are capable of inducing potato PAL promoter(s).
RICE EXAMPLE VI: RICE
ISOLATION of PAL PROMOTERS from RICE GENOMIC DNA
A. Screening Rice Genomic Library
A rice (Oryza sativa) genomic library (Clontech, Palo Alto, CA; average insert size of 15 kb in λEMBL3) was screened with elicitor-inducible bean PAL1 exonII cDNAs to identify clones encoding rice PAL genes. The cDNAs encoding PAL1 exonII sequences were pPALl-B6 and pSPP1 (both obtained from C. Lamb, The Salk Institute, La Jolla, CA; no published reference). The probe was an equal mixture of the 800 bp insert of pPAL1-B6 and the 500 bp insert of pSPP1. The screening conditions were as follows:
Hybridization: 42ºC, 35% Formamide, 5X SSC, 5X Denhardts,
0.2% SDS, 200 μg/ml salmon sperm DNA.
Wash: 2X SSC, 0.1% SDS, repeated a total of four times, one hour each, at 50º C. Five clones comprising putative rice PAL sequences were selected following six rounds of plaque purification. Clones λrPAL-2, -4, and -10 gave strong signals when hybridized to the probe, whereas clones λrPAL-8 and -12 hybridized weakly.
B. Clone Characterization
1. Restriction Mapping
The five clones were characterized by restriction enzyme analysis. Clones λrPAL-8 and -12 were identical; clones λrPAL-4 and -10 had several fragments in common; and clone λrPAL-2 was unique. Partial restriction enzyme maps of inserts from clones λrPAL-2, -4, and -10 are shown in Rice
Figure 2.
2. DNA Sequencing
Through restriction enzyme mapping and hybridization analysis using the bean PAL1 exon II DNA fragments as probe, the portions of clones λrPAL-2, -4, -8, and -10, which contained PAL-hybridizing regions were determined. These portions are shown in Rice Figure 2 by a stippled box for λrPAL-2, and cross-hatched boxes for λrPAL-4 and -10. The boxed fragments were excised from the λrPAL clones and ligated to appropriately digested pUC119 (Vieira and Messing, 1987, Meth. Enzym. 153:3-11). After
transforming the ligation reaction into DH5α cells and selecting for AmpR colonies, correct plasmids were identified by release of an appropriately sized fragment (i.e., insert size) upon digestion with the appropriate enzymes. The stippled region of λrPAL-2 was in p6, and the cross-hatched regions of λrPAL-4 and -10 were in p296 and p4410, respectively.
The insert DNAs were sequenced according to the Sequenase protocol (U.S Biochemical Corporation, Cleveland, OH) for double-stranded dideoxynucleotide sequencing. The partial sequence of the PAL region of λrPAL-2 was found to have 84.3% identity to the published sequence of a rice (Otyza sativa cv. Nipponbare) genomic PAL gene (Minami et al., 1989, Eur. J. Biochem. 185:19-25); the partial sequences of the fragments from λrPAL-4 and λrPAL-10 were the same and showed 79.6% identity to the rice PAL genomic sequence; and the partial sequence of the λrPAL-8 fragment has 53.8% identity to the rice PAL genomic sequence. These data suggested that clones λrPAL-2, λrPAL-4, and λrPAL-10 contain portions of the rice PAL genes, and that λrPAL-8 may or may not contain a rice PAL gene.
The orientation of the PAL genes within clones λrPAL-2, -4, and - 10 was determined from the sequence data. Based on the size and orientation of the inserts compared to the published sequence of a rice PAL gene
(Minami et al., 1989, supra) it appeared likely that all three clones contained rice PAL promoters. To localize the promoter regions of these clones, fragments were further subcloned and sequenced.
For clone λrPAL-2, both the HindIII-EcoRI and EcoRI-HindIII fragments (Rice Figure 2), striped boxes were separately subcloned into pUC119. Because the region of similarity between the λrPAL-4 and λrPAL- 10 clones and the 5' end of the published rice PAL genomic sequence lies at the right end of the SalI fragment (cross-hatched boxes in Rice Figure 2), the SalI fragment (black box) from λrPAL-10 was subcloned into pUC119 and sequenced. The promoter fragment from λrPAL-4 was subcloned as a 3 kb ClaI-SstI fragment into Accl-SstI digested pGEM5Z (Promega, Madison, WI). This 3 kb fragment contains about 300 bp of the right arm of EMBL3, the 1800 bp SalI fragment (black box in Rice Figure 2) and a portion of the 1850 SalI fragment (hatched box).
The composite sequence of λrPAL-4 and -10 is shown as SEQ ID NO. 9 (λrPAL-4). The sequence of the promoter region and a portion of the coding region of λrPAL-2 is shown in SEQ. ID NO. 8 (λrPAL-2).
The sequencing data showed that the promoter regions of λrPAL-4 and λrPAL-10, as well as a portion of their coding regions, are identical, except that λrPAL-4 contains an additional 850 bp of upstream sequence compared to λrPAL-10. Comparison between the published rice (Oryza sativa cv. Nipponbare) PAL genomic sequence (Minami et al., 1989, supra) and the sequences of the subclones of λrPAL-4 and λrPAL-10 showed that the translational start (ATG) of the published sequence is located five base pairs upstream of the SalI restriction site (position 1724 in SEQ ID NO. 9) in λrPAL-4 and λrPAL-10. Therefore, it is likely that the translational start site of the PAL gene encoded by λrPAL-4 and λrPAL-10 is near this SalI site.
A plant translational start consensus sequence (GNNATGG) is present at position 1704 of the composite sequence in (SEQ ID NO. 9) in λrPAL-4 and λrPAL-10. Computer-generated translation of the nucleotide sequence starting with the ATG at position 1702 and extending to position 1828 yielded protein sequence which shows 78% similarity to the amino acid sequence predicted from the published rice PAL gene sequence (Minami et al., 1989, supra) and 73% similarity to the amino acid sequence deduced from bean gPAL-2 (Cramer et al., 1989, Plant Mol. Biol. 12:367-383).
The putative translational start of λrPAL-2 is located at nucleotide position 1873 of SEQ ID NO. 8. Computer generated translation of the nucleotide sequence starting at this position results in protein sequence which shows 80% similarity to the amino acid sequence predicted from the published rice PAL gene sequence (Minami et al., 1989, supra), and 51% similarity to the amino acid sequence deduced from λrPAL4 and λrPAL-10.
EXAMPLE VII: RICE
CONSTRUCTION of RICE PAL-GUS CONSTRUCTS A. Construction of GUS Marker-Hygromycin
Selectable Base Expression Vectors
Promoter-less GUS ( β-glucuronidase) gene cassettes are available as HindIII-EcoRI inserts in plasmids pBI101, pBI101.2, and pBI101.3
(Clontech, Palo Alto, CA). The GUS cassettes in plasmids pBI101.2 and pBI101.3 are identical to the one in pBI101 except their reading frames are shifted one and two nucleotides, respectively, relative to the polylinker. As a result, inserting a promoter fragment and a portion of the gene coding region into each vector creates both translational and transcriptional fusion constructs in all three reading frames upstream of the GUS gene.
All ligations were performed with an insert to vector molar ratio of 5:1. Thus, each of the three plasmids, pBI101, pBI101.2, and pBI101.3 was digested with HindIII and EcoRI and the GUS-encoding insert was removed and purified on a 1% TBE gel. Each insert was ligated separately with HindIII-EcoRI-digested pUC119. The ligation was transformed into DΗ5α cells, and AmpR colonies were selected. Correct plasmids demonstrated bands of 2.2 and 3.2 kb in size upon digestion with EcoRI and HindIII and were called pUC-GUS.1, pUC-GUS.2, and pUC-GUS.3, respectively.
Plasmid pSV2hyg (obtained from J. Kwoh, Baxter Healthcare, San
Diego, CA) containing the E. coli hygromycin B gene, was digested with HindIII and BglII and the entire digest was treated with T4 DNA polymerase (BRL, Bethesda, MD). The fragment comprising the hygromycin gene was isolated on a 1% TBE gel and ligated to PstI-digested, T4 DNA polymerase-treated pCAMVCN DNA (Pharmacia, Piscathaway, NJ) using established cloning procedures. The ligation reaction was transformed into DH5α cells and AmpR colonies were selected. The correct plasmid released a 2 kb fragment upon digestion with HindIII and was called p35S-hyg.
Plasmid p35S-hyg was digested with HindIII and the 2 kb fragment was purified on a 1% TBE gel. The fragment then was ligated separately into the pUC-GUS vectors (pUC-GUS.1, pUC-GUS.2, and pUC-GUS.3) which had been digested with HindIII. The ligation reaction was transformed into DΗ5α cells and AmpR colonies were selected. The correct plasmids released a 2 kb fragment upon digestion with HindIII and were called pHyg-GUS.1, pHyg-GUS.2, and pHyg-GUS.3, respectively. In all three plasmids, the direction of transcription of the hygromycin resistance gene was opposite to that of the GUS gene; therefore, any GUS activity detected in plants transformed with these vectors will not be due to read-through transcription from the CaMV35S promoter.
B. Construction of Expression Vectors Having Rice PAL Promoters
1. Clone λrPAL-4
The promoter-carrying fragment from λrPAL-4 was isolated from the Clal-SstI subclone (see Rice Example VI.B.2.) following digestion of the subclone with BssHII, treatment with Klenow, and digestion with PstI. The approximately 1850 bp resultant fragment was ligated separately into pHyg-GUS.1, pHyg-GUS.2, and pHyg-GUS.3 plasmids (see Rice Example VII.A.), previously digested with SmaI and PstI. The ligations were transformed into DH5α cells and AmpR colonies were selected. The correct constructs were determined by linearization of a 7000 bp plasmid upon digestion with EcoRI, and were called plasmids rPALA.1, rPAL4.2, and rPAL4.3, respectively.
2. Promoter from λrPAL-10
The promoter fragments from λrPAL-10 were isolated by the following method. Plasmid pSal1000 (the 800 bp SalI fragment from black boxed portion of λrPAL-10 in Rice Figure 2, ligated into pUC119) was digested with SalI and PstI, and the 800 bp fragment was purified on a 1% TBE gel. Plasmid p4410 (see Rice Example VI.B2.) was digested with BssHII, treated with Klenow, digested with SalI and SstII (to remove the other BssHII- SalI fragment which would interfere in the ligation). The 800 bp fragment and the entire SalI-SstII digestion were ligated into SmaI- and PstI-digested pHyg- GUS.1, pHyg-GUS.2, and pHyg-GUS.3 in three way ligations. The ligations were transformed into DH5α cells and AmpR colonies were selected. The correct constructs were determined by linearization of an approximately 6200 bp plasmid upon digestion with EcoRI, and were called rPAL10.1, rPAL10.2, and rPAL10.3. 3. Promoter from λrPAL-2
The promoter fragment from λrPAL-2 was digested with EcoRI, treated with Klenow, and phosphorylated BamHI linkers (New England
Biolabs, Inc., Beverly, MA) were added. The DNA then was digested with NotI, treated with Klenow, and digested with BamHI. The resultant
approximately 2100 bp fragment was ligated separately into BamHI-SmaI digested pHyg-GUS.1, pHyg-GUS.2 and pHyg-GUS.3 according to established cloning procedures. The ligations were transformed into DH5α cells and the correct constructs were determined by linearization of the approximately 7 kb plasmid upon digestion with BamHI. Correct plasmids were called rPAL2.1, rPAL22, and rPAL2.3
EXAMPLE VIII: RICE INDUCIBILITY of RICE PAL PROMOTER-GUS FUSIONS A. Isolation and Transformation of Protoplasts
Rice suspension cultures (Oryza sativa cv. IR54) were provided by
C. Lamb, The Salk Institute, La Jolla, CA. The suspension cultures were maintained and proliferated on N6 medium (Rice Table II) with 2.3 g/L-proline.
Rice Table II: N6 Medium
Constituent mg/l
N6 inorganic salts (Chu et al.,
1975, Sci. Sinica 18:659-668)
MS vitamins (Murashige et al.,
1962, Physiol. Plant. 15:473-497)
2,4-Dichlorophenoxyacetic acid 2.0
L-Tryptophan 50.0
Myo-inositol 100.0
Sucrose 30,000.0 pH 5.8
The cultures were subcultured weekly. At each subculture, 2.5 g fresh weight of rice suspension cells were transferred to 50 ml fresh medium. The cultures were incubated at 27 ° C in the dark on a gyratory shaker at a speed of approximately 125 rpm.
Ten grams fresh weight of 5-day-old suspension cells (Oryza sativa cv. IR54) were incubated in 100 ml Protoplast Wash Solution (Wen et al., 1991, Plant Mol. Biol. Rep. 9(4):308-321) containing 1% Cellulase RS, 0.1% Pectolyase Y-23 (pH 6.0) at room temperature in the dark on a gyratory shaker at a speed of ~50 rpm for 4 hours.
The enzyme-protoplast mixture was passed through a 300 mesh tissue sieve to remove debris, then centrifuged at approximately 147 × g for 10 minutes at room temperature. Pelleted protoplasts were washed twice by resuspending in approximately 35 ml Protoplast Wash Solution and
centrifuging at approximately 147 × g for 10 minutes after each resuspension. Protoplasts were purified by centrifugation through a Percoll Solution step gradient. Protoplasts were resuspended in 6 ml of 70% Percoll Solution (Rice Table III), a 50% Percoll Solution (6 ml) was layered on top of the resuspended protoplasts, and 6 ml of a 25% Percoll Solution was layered on top of the 50% Percoll Solution.
Rice Table III: Percoll Solution
(1) 70% Percoll Solution
Percoll solution 70.0 ml/100 ml
Commercial Murashige and Skoog
salt base 0.43 g/100 ml
Sucrose 3.0 g/100 ml
Mannitol 0.3M
(2) 50% Percoll Solution
Percoll solution 50.0 ml/100 ml
Commercial Murashige and Skoog
salt base 0.43 g/100 ml
Sucrose 3.0 g/100 ml
Mannitol 0.3M
(3) 25% Percoll Solution
Percoll solution 25.0 ml/ 100 ml
Commercial Murashige and Skoog
salt base 0.43 g/100 ml
Sucrose 3.0 g/100 ml
Mannitol 0.3M
The Percoll-protoplast gradient was centrifuged at approximately
297 × g for 15 minutes at room temperature. Protoplasts were collected at the interface of the 25% and 50% Percoll Solutions using a sterile pasteur pipet and transferred to 25 ml of Protoplast Wash Solution. Protoplasts were washed twice by resuspending in 25 ml Protoplast Wash Solution and centrifuging at approximately 147 × g for 10 minutes after each resuspension.
The protoplasts were resuspended in Protoplast Wash Solution to a density of approximately 1 × 107 protoplasts per ml for transformation experiments. PEG-mediated transformation was conducted as follows: One-milliliter aliquots of protoplasts were separately mixed with 100 μg/ml plasmid DNA from either rPAL2.2 or rPAL4.3 (see Rice Example VII.B.l. and 3). An equal volume of polyethylene glycol (PEG 8000, 40% w/v) in medium (Krens'F solution; Krens et al., 1982, Nature 296:72-74) was added to the mixtures of protoplasts and plasmid DNAs. The protoplast-PEG mixture was heat shocked at 45 ° C for five minutes followed by chilling on ice for 20 seconds. The solution was then brought to room temperature followed by incubation at 30º C for 30 minutes. The protoplast-PEG mixture then was diluted with Krens' F solution until the PEG concentration was less than 2% according to the following time schedule as a reference:
0- 2 min two drops every 30 sec
2- 5 min five drops every 30 sec
5-10 min 0.5 ml every 30 sec 10-15 min 1 ml every 30 sec
15-30 min 2 ml every 5 min
The protoplasts treated with plasmid DNA were collected by centrifugation at approximately 147 × g for 10 minutes and resuspended in N6 medium (Rice Table II) containing 0.3 M mannitol and incubated in the dark at room temperature on a gyratory shaker at ~50 rpm.
Samples of each protoplast mixture were treated with fungal elicitor prepared from the cell walls of Pyricularia oryzae. Preparation of fungal elicitor is described in below, in Rice Table IV.
Table IV: Rice: Preparation of Fungal Elicitor Maintenance media: Corn meal agar (Difco™ 0386- 01-3); plate.
Growth media: Liquid culture.
Corn meal broth: Blend 50 g corn meal in 800 ml of distilled H2O; refrigerate mixture overnight; then heat for about 1 h at 60º C; filter solution; bring solution to 1 liter; autoclave for 20 min. Double autoclave (120°C/20 min) all media and instruments. Leave plates/liquid media for > 1 week to check for contamination.
To maintain cultures: take 1 × 0.6 mm cork borer disc and place in center of corn meal agar dish. Take the sample from the edge of dark grown mycelia.
To innoculate growth media: take 5 × 0.6 mm cork borer discs from edge of growing mycelia/50 ml media (in 250 ml conical flask).
Maintain at in the dark at 23 º C. Harvest mycelia after 1 month.
Preparation of crude elicitor from Pyricularia oryzae mycelial walls.
1. Homogenize mycelium in Waring™ blender for 60 sec (5 ml H2O per gram wet weight of mycelia). Filter homogenate through a coarse scintered glass filter*; retain the residue. (N.B., it is often necessary to clear the glass filter with cone, nitric acid and then rinse well with distilled H2O, since the pores clog up.)
2. Repeat step 1 three more times*.
3. Homogenize once with a mixture of chloroform: methanol (1:1) (1 g mycelia per 5 ml of fluid); filter*.
4. Homogenize once with acetone (1 g mycelia to 5 ml of acetone); filter.
5. Air dry at room temperature. This treatment leaves mycelial wall fraction.
6. Suspend 5 g of walls in 100 ml of water and autoclave for 1 11/2 hr at 121 °C.
7. Autoclaved suspension was filtered through a course scintered glass filter.
8. Concentrate by freeze drying (lyophilization).
9. Resuspend in sterile (approx. 10 ml) H,0.
10. Do carbohydrate and protein assay.
* This may vary. Do until filtrate is clear, at final acetone stage.
Refs: Ayers, et al., Plant Physiol., 1976, 57:760. Dixon, Planta, 1981, 157:272.
Fungal elicitor at a concentration of approximately 60 or 80 μg/ml was added 22 (80 μg) or 36 (60 μg) hours after transformation, and incubation was continued for an additional 20 hours. Following this 42 or 56 hour incubation, control and treated samples were processed as described below.
1. Transient Expression Assays
After 40 hours of incubation post-transformation, rice protoplasts that had been transformed with the rice PAL-GUS fusion constructs were collected by centrifugation in a microfuge for about 30 seconds. Protoplasts were homogenized in 50 μl GUS extraction buffer (Jefferson, 1987, Plant Mol.
Biol. Rep. 5:387-405). Another 150 μl of GUS extraction buffer was added, and protoplasts were frozen in liquid nitrogen, then thawed at room
temperature. This procedure was repeated, and the protoplast mixture was centrifuged in a microfuge at 4°C for 10 minutes. Protoplast extracts (100 μl) were incubated with 0.6 ml MUG buffer containing 20% methanol according to the procedure of Jefferson (1987, supra) to determine GUS activity. Briefly, the assay for GUS activity was a fluorometric assay which measures the production of 4-methyl umbelliferyl from 4-methyl umbelliferyl glucuronide (MUG), a fluorogenic substrate. Protein concentration was determined according to the Bradford Protein Assay using reagents obtained from Bio- Rad.
2. Results
Protoplasts were separately transformed with two rice PAL-GUS fusion constructs, rPAL2.2 and rPAL4.3, each containing a translational fusion between a rice PAL promoter and the GUS gene. The extracts of these protoplasts were assayed for GUS activity. The results of these assays are shown in Rice Table V. Rice Table V: GUS Activity in Rice Protoplasts
Transformed with Rice PAL-GUS Fusion Construct rPAL2.2
GUS Activity % Increase
(pmol/min/mg)a with elicitor
Experiment 1: No DNA control 26. - rPAL 2.2 785. - rPAL 2.2 + elicitor 862. 9.8% rPAL 4.3 86. - rPAL 4.3 + elicitor 70 -
Experiment 2: rPAL 2.2 1,542. - rPAL 2.2 + elicitor 1,707. 10.7%
Experiment 3: rPAL 2.2 2,464. - rPAL 2.2 + elicitor 2,683. 8.8% rPAL 2.2 + elicitor 2,655. 7.7% aThese values are an average of data obtained at different time points in the enzyme assay.
a. Background GUS Expression
A higher level of uninduced GUS activity was measured in protoplasts transformed with plasmid DNA, but ng£ induced with elicitor, than in control protoplasts not transformed and not induced. The higher GUS levels are due to a certain level of constitutive and/or induced (induction other than elicitor-mediated induction) GUS expression in the experimental transformed protoplasts and are indicative of successful transfer of the constructs into the protoplasts. The difference in the background GUS levels of protoplasts transformed with rPAL2.2 and rPAL4.3 could be the result of differences in transformation efficiencies, differences in the fusion between the rice PAL promoter and GUS genes in the constructs, or, most likely, differences in the responsiveness of the two rice PAL promoters to possible other induction factors.
b. Elicitor-Induced GUS Expression Elicitor-treated protoplasts transformed with rPAL2.2 yielded GUS activities that were an average of 9.8% higher than those of untreated protoplasts transformed with the same construct. No significant increase in GUS activity was seen in elicitor-treated protoplasts transformed with rPAL4.3 relative to untreated protoplasts that had been transformed with this construct.
These data suggest that the λrPAL-2 promoter is inducible by the fungal elicitor evaluated herein while the λrPAL-4 promoter is not.
EXAMPLE IX: RICE
PRODUCTION OF TRANSGENIC RICE PLANTS
A. Isolation, Transformation of Protoplasts, and Regeneration of Transgenic Rice Plants Rice seeds (Oryza sativa cv. Sasanishiki) were obtained from
National Small Grains Collection, USDA. Callus cultures were initiated according to the protocol of Kyozuka et al. (Mol. Gen. Genet., 1987, 206:408- 413). The initiated calli were maintained and proliferated on two different media: MS2 medium (Kyozuka et al. 1987) and N6 medium (Rice Table II). The formulas for these media are given supra.
Rice suspension cultures were initiated from the rice calli on two types of suspension media, N6 medium and R2 medium. Rice calli grown on MS2 medium were initiated in R2 medium and rice calli proliferated on N6 medium were initiated in N6 medium. The R2 medium is as follows:
Rice Table VI: Rice Suspension R2 Medium Constituent Amount
R2 inorganic salts (Ohira et al.,
1973, Plant Cell Physiol. 14: 113- 114).
MS vitamins (Murashige et al.
1962, Physiol. Plant. 15:473-497).
2,4-Dichlorophenoxyacetic Acid 2.0 mg/l
Myo-Inositol 100.0 mg/l
Sucrose 30.0 g/l pH 5.8 The initiated cultures were incubated at 27°C in the dark on a gyratory shaker at a speed of about 50 rpm. Rice suspension cultures were subcultured weekly. At each subculture, 2.5 grams fresh weight of cells were transferred to 25 ml fresh suspension media in a 250 ml flask. A 2-fold increase in growth was observed in a seven day growth period.
Thirteen grams fresh weight of 5-day-old suspension cells which were initiated on R2 medium (two-month-old after the initiation) was incubated in 100 ml Protoplast Wash Solution containing 2% cellulase RS, 0.2% pectolyase Y-23 (pH 6.0) at room temperature on a gyratory shaker at a speed of about 50 rpm in the dark for 3 hours and 45 minutes.
The enzyme-protoplast mixture was passed through a 300 mesh tissue sieve to remove debris and centrifuged at about 100 × g for 10 minutes at room temperature. Pelleted protoplasts were washed twice by resuspending in about 35 ml Protoplast Wash Solution and centrifuging at about 100 × g for 10 minutes after each resuspension. Protoplasts were purified as described above for Oryza sativa cv. IR-54. (See Rice Example VIIIA.)
The Percoll-protoplast gradient was centrifuged at about 200 × g for 15 minutes at room temperature. Protoplasts were collected from the top of 25% Percoll Solution and transferred to 30 ml of Protoplast Wash Solution. Protoplasts were washed twice by resuspending in 30 ml Protoplast Wash
Solution and centrifuging at about 100 × g for 10 minutes after each
resuspension. The protoplast were resuspended in Protoplast Wash Solution to a density of about 3 × 107 protoplast per ml for transformation experiments.
PEG-mediated transformation was conducted using the transformation method described above for Oryza sativa cv. IR-54.
The protoplasts treated with plasmid DNA were collected by centrifugation at approximately 100 x g for 15 minutes and resuspended in 1 ml of R2 protoplast medium and mixed with an equal volume of the R2 protoplast medium (which was identical to R2 medium except that it contained 0.4M sucrose) containing 2.5% Sea Plaque agarose and transferred to a 35 mm
× 15 mm sterile petri dish. Solidified agarose containing the protoplasts were cut into four blocks and transferred to a 100 mm × 150 mm sterile petri dish containing 15 ml R2 protoplast medium resuspended with about 500 mg 5-day-old Sasanishiki suspension cells which were initiated on N6 medium and sieved through a 30 mesh tissue sieve.
The cultures were incubated at room temperature in the dark on a gyratory shaker at a speed of about 50 rpm.
After 10 days, agarose blocks containing rice protoplasts were transferred to new plates and Sasamishiki suspension cells were completely removed by washing with the R2 protoplast medium. The agarose blocks were cultured in the 100 mm × 150 mm petri dishes containing 20 ml of R2 protoplast medium.
The cultures were incubated at room temperature in the dark on a gyratory shaker at a speed of about 40 rpm.
After 4 days, agarose blocks containing protoplast-derived colonies were transferred to R2 protoplast medium containing 50 μg/ml hygromycin B.
The cultures were incubated at room temperature in the dark on a gyratory shaker at a speed of about 30 rpm.
After 11 days, protoplast-derived rice calli were visible. The agarose blocks containing rice calli were transferred to N6 soft agarose medium (N6 basal medium, 2 mg/l 2,4-dichlorophenoxy acetic acid, 6% sucrose and 0.25% Sigma type I Agarose, pH 5.7) containing 50 μg/ml hygromycin B.
The cultures were incubated at 27 ° C with 16 hours of light under 4,000 lux light intensity.
After 10 days, rice calli on agarose blocks were about 1 mm diameter and were picked and transferred to N6 medium containing 50 μg/ml hygromycin. This medium was the same as N6 soft agarose medium except that this medium contained 0.5% Sigma Type I Agarose.
After 2 weeks, when rice calli were about 2-3 mm in diameter, they were transferred onto the regeneration medium containing 50 μg/ml hygromycin. The regeneration medium contained N6 inorganic salts,
Murashige and Skoog vitamins (Murashige et al., 1962, Physiol. Plant. 15:473- 497), 6% sucrose, 1% Sigma type I agarose and 2 mg/ml kinetin. When regenerated shoots were 3 cm or longer, they were rooted in the hormone-free medium containing 50 ug/ml hygromycin B with 1% Sigma type I agarose. The incubation conditions throughout the experiment were the same as above. When regenerated, the transgenic rice plants are used, for example, in an agrichemical screen to identify inducers of these promoters.
RICE EXAMPLE X USE OF TRANSGENIC RICE PLANTS AS AN AGRICHEMICAL SCREEN
Transgenic rice are propagated by germinating seeds in suitable tissue culture medium containing suitable concentrations of hygromycin to ensure that the PAL-GUS fusion construct is present in all tissues of the transgenic plant. Once the transgenic plantlets have reached a suitable size, e.g., 3-6 inches tall, they are transplanted to soil and maintained under standard greenhouse conditions.
For use in agrichemical screening assays, plants are grown to a size and/or stage which is manageable under greenhouse conditions and susceptible to a pathogen of interest, e.g., Pyricularia oryzae. The plants are treated either by foliage spray or soil immersion with a range of concentrations of the unknown chemical or chemicals. In parallel, additional plants are exposed to water as a negative control or to a known elicitor, e.g., probenazole (Iwata, et al. Ann. Phytopath. Soc. Japan, 1980, 46: 297-306) as a positive control. Six to twenty four hours following treatment, leaf tissue is removed and assayed for GUS activity using the fluorometric assay of Jefferson, et al., (Plant Mol. Biol. Rep., 1987, 5:387-405). A positive result is indicated by levels of GUS activity significantly higher than those observed with the negative control. A positive result is indicative of, e.g., identifies, chemicals which are capable of inducing rice PAL promoter(s). EXAMPLE XI: ASSAY
USE OF 3SR
TO IDENTIFY INDUCIBLE PROMOTER FROM RICE GENOMIC DNA
A. 3SR
The 3SR reaction (self-sustained sequence replication) is a method for in vitro amplification of specific RNA sequences (Guatelli, et al., 1990. Proc. Natl. Acad. Sci. USA. 87:1874-1878). Synthesis of the predominantly RNA product is provided by the action of AMV reverse transcriptase and T7 RNA polymerase in the presence of ribo- and deoxyribonucleotides. The region to be amplified is specified by a pair of DNA primers, one of which contains a T7 RNA polymerase binding site and is complementary (antisense) to the target mRNA. The second primer is identical to the mRNA (sense) in a region approximately 200-300 bp away from the first primer.
The 3SR technique is illustrated in Figure 3. According to the technique, a target RNA sequence is initially transformed into an RNA/DNA duplex by reverse transcriptase in the region specified by the first primer. This duplex is attacked by the RNaseH present on the AMV reverse transcriptase. The RNaseH destroys the RNA template but leaves the cDNA intact. The cDNA is used as a template for the synthesis of a second strand of cDNA using reverse transcriptase and the second primer. The resulting double stranded DNA template serves as a substrate for T7 RNA polymerase to produce multiple copies of antisense RNA which will hybridize to primer 2 to start a second cycle of double-stranded cDNA synthesis. This again results in RNA transcription, and the antisense RNA permits the recycling of the 3SR reaction until some limitation in the reaction stops the cycling. The target
RNA may be amplified 106 - 109 fold.
This example illustrates use of the 3SR technique to identify inducible promoters from bean genomic DNA.
B. Tissue Culture Conditions
Bean (Phaseolus vulgaris cv. Canadian Wonder) suspension cultures were obtained from Dr. C. Lamb. The cultures were maintained and proliferated on modified Schenk and Hildebrandt medium (SH medium) (infra, Assay Table VII). The cultures were transferred weekly. At each subculture, six grams fresh weight of bean suspension cells were transferred to 100 ml fresh SH medium. The cultures were incubated at 27 ° C in the dark on a gyratory shaker at a speed of 125 rpm.
C. Amplification of Bean PAL-1
PAL induction experiments were conducted on bean cell suspension cultures seven days after subculture. Two gram aliquots of the cultures were treated for three hours with the following conditions: (1) 20 μg/ml of fungal elicitor isolated from the walls of Colletotrichum
lindemuthianum race Alpha according to Rice Table IV; (2) Escherichia coli strain DH5a (BRL, Bethesda, MD); (3) Agrobacterium tumefaciens strain C58 (Dr. Maarten Chrispeels, UCSD, LaJolla, CA); (4) Clavibacter michiganese pv michiganese (American Type Culture Collection, Rockville, MD); (5)
Xanthomonas campestris pv malvacearum (ATCC, Rockville, MD); (6)
Pseudomonas syringae pv tomato (ATCC, Rockville, MD); (7) Pseudomonas syringae pv tabaci (ATCC, Rockville, MD); (8) Erwinia carotovora subsp.
carotovora (ATCC, Rockville, MD); (9) SH medium (negative control, t=3); and (10) no treatment, immediately frozen (t=0). All bacteria were added to a concentration of approximately 107 - 108 cells/ml.
Total nucleic acid was isolated from 0.2 - 0.3 grams of each sample using the following protocol: (1) frozen tissue was ground to a fine powder in a mortar and pestle, (2) pulverized tissue was added to 450 μl NT buffer (0.1M NaCl, 0.01M Tris, pH9.0, lmM EDTA) and 450 μl phenokchloroform (1:1) and vortexed until thawed, (3) the slurry was centrifuged for 10 min. in a microfuge, (4) the aqueous layer was removed and precipitated with EtOH according to standard protocols in Maniatis et al., (1982), (5) the nucleic acids were recovered by centrifugation and the concentration determined by spectrophotometry at 260nm, (6) the final concentration was adjusted to 0.6 μg/ml.
The total nucleic acid was subjected to 3SR amplification using primers derived from the sequence of the bean PAL-1 gene (Edwards, et al., 1985. Proc. Natl. Acad. Sci. USA. 82:6731-6735.) The sequence of the primers is given in SEQ ID NOS. 10 and 12. (The T7 RNA polymerase binding site on Primer 1 is given in SEQ ID NO. 11.) Total nucleic acid at a concentration of approximately 0.6 μg was used for the 3SR reaction, and the exact details are given in Assay Table VIII.
The 3SR reaction products were analyzed using a dot blot apparatus (Schleicher and Schuell, Keene, NH). Two microliters of the reaction was added to 100 microliters of DM5 (2.6mM Tris, pH 8.0, 0.26mM EDTA, 10 × SSC, 7.4% Formaldehyde), and the samples were heated to 55 ° C for 20 minutes, then placed on ice prior to loading onto the nitrocellulose in the dot blot apparatus according to manufacturer's instructions. The nucleic acids were fixed to the nitrocellulose by UV crosslinking (Stratagene, LaJolla, CA). The blot was probed with an oligonucleotide derived from the bean PAL-1 gene (Edwards et al., 1985, supra). The sequence was identical to the mRNA (sense) strand. The probe is shown in SEQ ID NO. 13. The oligonucleotide was end-labeled with gamma 32P-ATP using T4 kinase (BRL,
Bethesda, MD) according to Maniatis et al., 1982, supra. The blot was hybridized with 2 × 106 cpm/ml for 1 hour in 5 × SSPE, 4 × BP (2% BSA, 2% Polyvinylpyrrolidone-40), 1% SDS. The filter was washed three times at room temperature for 5 minutes each in 1 × SSPE, 1% SDS and one time at 42°C for 1 minute in 1 × SSPE, 1% SDS.
More PAL hybridizable material was detected with the induced samples than with the uninduced samples. Therefore this data suggests that PAL-1 is a gene inducible by plant pathogenic bacteria. In addition, it could be concluded that 3SR™ is a rapid and sensitive technique which can be used for the identification of inducers of plant defense genes.
Assay Table VII: Schenck and Hildebrant SH Medium Constituent Amount, mg/l
KNO3 2500.00
NaH2PO4 300.00
MgSO4· 7H2O 400.00
CaCl2 · 2H2O 225.00
Na2 -EDTA 20.00 FeSO4 · 7H2O 13.90
MnSO4 · H2O 12.50
H3BO3 5.00
ZnSO4 · 7H2O 1.00
KI 1.00
CuSO4·5H2O 0.20
Na2MoO4 -2H2O 0.10
Thiamine · HCl 15.00
Pyridoxine · HCl 1.50
Nicotinic Acid 15.00
2,4-Dichlorophenoxyacetic Acid 0.440
P-Chlorophenoxyacetic acid 2.100
Kinetin 0.105
Sucrose 34,000.00
Myo-Inositol 1,000.00 pH 5.8
Assay Table VIII
Protocol
5x Reaction Buffer 20.0 μl
200 mM Tris-HCL, pH 8.1
150 mM MgCl2
100 mM KCl
20 mM Spermidine
50 mM Dithiothreitol
dNTP's (25 mM) (Promega, Madison, WI) 4.0 μl rNTP's (25 mM) (Aldrich, Milwaukee, WI) 20.0 μl
DMSO (Sigma, St. Louis, MO) 10.0 μl
Sorbitol (66.7% w/v) (Sigma, St. Louis, MO) 22.5 μl Primer 1 5.0 μl
Primer 2 5.0 μl
Water 8.5 μl
Target nucleic acid 5.0 μl
Heat sample at 65 ° C, 1 min
Transfer to 37 °C, 1 min
Place on ice
Add 1 μl AMV Reverse Transcriptase (20 u) (Life Sciences, St.
Petersburg, FL)
1 μl T7 RNA Polymerase (50 u) (Stratagene, LaJolla, CA)
Reaction is placed at 42 ° C for 1 hour
Stop amplification by placing on ice
CONCLUSION
Without departing from the spirit and scope of this invention, one of ordinary skill can make various changes and modifications to the invention to adapt it to various usages and conditions. As such, these changes and modifications are properly, equitably, and intended to be, within the full range of equivalence of the following claims. SUMMARY OF SEQUENCES
Sequence ID NO. 1: pPAL-1, 5'→ 3', B↔ S fragment; 245 bp
sequence
Sequence ID NO. 2: pPAL-2, 5'→ 3', (E)↔ P fragment; 271 bp sequence
Sequence ID NO. 3: pPAL-3, 5'→ 3', (E)↔ P frament; 277 bp
sequence
Sequence ID NO. 4: pPAL-4, 5'→ 3', E↔ P fragment; 276 bp
sequence
Sequence ID NO. 5: pPAL-6(a), 5'→ 3' H↔ S fragment; 300 bp sequence
Sequence ID NO. 6: pPAL-6(b), 5'→ 3', H↔ S fragment; sequenced
S→ H, i.e., antisense, beginning at SstI and continuing towards HindIII site antisense sequence converted to sense sequence; 260 bp sequence
Sequence ID NO.7: pPAL-8, 5'→ 3' (E)↔ P fragment; 278 bp
sequence
Sequence ID NO.8: rPAL-2, 5'→ 3'; 2338 bp sequence, sense
strand, promoter and partial structural gene coding sequence
Sequence ID NO.9: rPAL-4, 5'→ 3'; 1997 bp sequence, sense
strand, promoter and partial structural gene coding sequence
Sequence ID NO.10: 3SR Primer 1
Sequence ID NO.11: T7 RNA Polymerase Binding Site on 3SR
Primer 1
Sequence ID NO.12: 3SR Primer 2
Sequence ID NO.13: Probe from bean PAL-1
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Fitzmaurice Ph.D., Leona C.
Virts Ph.D., Elizabeth L.
Lin, Fen-Fen
Mirkov Ph.D., T. Erik
Collier, Jana G.
Schoeneck, Paula
(ii) TITLE OF INVENTION: Inducible Plant Defense Gene Regulatory
Sequences from Potato and Rice, Uses Thereof, and Assays
(iii) NUMBER OF SEQUENCES: 13
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: McCubbrey, Bartels, Meyer, & Ward
(B) STREET: One Post St.
(C) CITY: San Francisco
(D) STATE: CA
(E) COUNTRY: USA
(F) ZIP: 94104-5231
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.24
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 635,820
(B) FILING DATE: 02-JAN-1991
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 352,658
(B) FILING DATE: 18-MAY-1989
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 343,466
(B) FILING DATE: 26-APR-1989 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 197,122
(B) FILING DATE: 20-MAY-1988
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Meyer Esq., Virginia H.
(B) REGISTRATION NUMBER: 30089
(C) REFERENCE/DOCKET NUMBER: 51633M
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (415) 391-6665
(B) TELEFAX: (415) 391-6663
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 245 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ϋi) HYPOTHETICAL: N (iv) ANTI-SENSE: N
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Solanum tuberosum
(B) STRAIN: cv. Desiree
(vii) IMMEDIATE SOURCE:
(B) CLONE: pPAL-1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
GATCCTCTTC AGAAACCAAA GCAAGCATCG TTATGCTCTC CGAACATCTC CACAATGGCT 60
TGGCCCTCAA ATTGAAGTCA TACGCGCAGC AACTAAGATG ATTGAGAGGG AGATTAACTC 120
AGTGAACGAC AATCCATTGA TCGATGTATC AAGAAACAAG GCCTTGCACG GTGGCAACTT 180
TCAAGGCACC CATATGGTGT GTCATGGATA ATACAGATTG GCCTGCATCA TAGGAATGAT 240
GTTTG 245
(2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 271 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Solanum tuberosum
(B) STRAIN: cv. Desiree
(vii) IMMEDIATE SOURCE:
(B) CLONE: pPAL-2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
CTTGGGATTA ATCTCAGCCA GGAAAACAGC TGAGGCTGTT GATATCTTGA AGCTAATGTC 60
ATCAACCTAT CTCGTGGCGC TTTGCCAAGC TATAGACTTA CGGCATTTGG AGGAGAACTT 120
GAAGAGTGCT GTCAAGAACA CAGTTAGCCA AGTAGCTAAG AGAACTTTGA CAATGGGTGC 180
TAATGGGGAA CTTCATCCAG CAAGATTCTG TGAGAAGGAA TTGCTTCGAG TCGTGGATAG 240
GGAATACTTG TTTGCCTATG CAGATGACCC C 271
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 277 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: N
(iv) ANTI-SENSE: N
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Solanum tuberosum (B) STRAIN: cv. Desiree
(vii) IMMEDIATE SOURCE:
(B) CLONE: pPAL-3
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GAATTCCTTG GGATTAATCT CAGCCAGGAA AACAGCCGAG GCTGTTGATA TCTTGAAGCT 60
AATGTCATCA ACCTATCTCG TGGCGCTTTG CCAAGCTATA GACTTANNGC ATTTGGAGGA 120
GAACTTGAAG AGTGCTGTCA AGAACACAGT TAGCCAAGTA GCTAAGAGAA CTTTGACAAT 180
GGGTGCTAAT GGTGAACTTC ATCCAGCAAG ATTCTGCGAA AAGGAATTGC TTCGAGTCGT 240
GGACAGGGAA TACTTGTTTG CCTATGCAGA TGACCCC 277
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 276 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: N
(iv) ANTI-SENSE: N
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Solanum tuberosum
(B) STRAIN: cv. Desiree
(vii) IMMEDIATE SOURCE:
(B) CLONE: pPAL-4
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
GAATTCCTTG GGCTTAATCT CAGCCAGGAA AACAGCTGAG GCTGTTGATA TCTTGAAGCT 60
AATGTCATCA ACCTATCTCG TGGCGCTTTG CCAAGCTATA GACTTACGGC ATTTGGAGGA 120
GAACTTGAAG AGTGCTGTCA AGAACACAGT TAGCCAAGTA GCTAAGAGAA CTTTGACAAT 180
GGGTGCTAAT GGTGAACTTC ATCCAGCAAG ATTTTGCGAA AAGGAATTGC TTCGAGTCGT 240
GGACAGGGAA TACTTGTTTG CCTATGCAGA TGACCC 276 (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 300 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Solanum tuberosum
(B) STRAIN: cv. Desiree
(vii) IMMEDIATE SOURCE:
(B) CLONE: pPAL-6a
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
AAGCTTGGAC TATGGTTTCA AGGGAGCTGA AATCGCGATG GCTTCTTACT GCTCGGAACT 60
TCAATTCTTG GCAAATCCAG TGACCAACCA TGTTCAGAGT GCCGAGCAAC ACAACCAAGA 120
TGTGAACTCC TTAGGCTTAA TCTCAGCAAG GAAAACAGCT GAGGCTGTCG ACATCTTAAA 180
GCTAATGTCA TCAACCTATC TCGTGGCACT TTGCCAAGCT ATAGACTTGA GGCATTTGGA 240
GGAGAACTTG AAGAGTGTTG TCAAGAACAC AGTTAGCCAA GTAGCTAAGA GACTTTGACA 300
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 260 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: N
(iv) ANTI-SENSE: N (vi) ORIGINAL SOURCE:
(A) ORGANISM: Solanum tuberosum
(B) STRAIN: cv. Desiree
(vii) IMMEDIATE SOURCE:
(B) CLONE: pPAL-6b
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
TGGTGAACTT CATCCAGCAA GATTCTGCGA GAAGGAATTG CTTCGAGTCG TAGACAGGGA 60
ATACTTGTTT ACCTATGCTG ATGACCCCTG CAGCTCCACC TATCCTTTGA TGCAGAAGCT 120
GAGACAGGTC CTTGTTGATC ATGCAATGAA GAATGGTGAA AGTGAGAAGA ATATCAACAG 180
CTCAATCTTC CAAAAGATTG GAGCTTTCGA GGACGAATTA AATGCTGTGT TGCCTAAAGA 240
AGTTGAGAGT GCAAGAGCTC 260
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 278 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: N
(iv) ANTI-SENSE: N
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Solanum tuberosum
(B) STRAIN: cv. Desiree
(vii) IMMEDIATE SOURCE:
(B) CLONE: pPAL-8
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
GAATTCCTTG GGATTAATCT CAGCCAGGAA AACAGCCGAG GCTGTCGATA TCTTGAAGCT 60
AATGTCATCA ACCTATCTCG TGGCGCTTTG CCAAGCTATA GACTTGAGGC ATTTGGAGGA 120
AAACTTGAAG AGTGCTGTCA AGAACACAGT TAGCCAAGTA GCTAAGAGAA CTTTGACAAT 180
GGGTGCTAAT GGTGAACTTC ATCCAGCAAG ATTCTGCGAA AAGGAATTGC TTCGAGTCGT 240 GGACAGGGAA TACTTGTTTG CCTATGCAGA TGATCCCC
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2338 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: N
(iv) ANTI-SENSE: N
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Oryza sativa
(vii) IMMEDIATE SOURCE:
(B) CLONE: lambda rPAL-2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
CTCTCCTTGT CCCGGCCAAG TCGGCCGAGC TCGAATTCCA ATGATAGTTA TGTTTGATTT 60
TTACAATCCC AATGACAATA AAGAGTATGC GGCGGACGAG TCGTAGAGGA GTATAGTGGC 120
AGTGTTTGAC GGGTTTTCTA AAAATTATAA AAAACATGAA ACCCAACGAG ACAATAAACT 180
CTAAAAACTA TAAGGTTCAA ATTTTAAAGG TTCGGGCTTC TAAAAAATAA AAAAATAAAC 240
CCTAATGATA ATCATATTTG ATTTTTAAAA TCTCAATGAC AATAAAGAAG GGCGGCAGCG 300
GGCGGGCCGT AGAGGAGTAC AGTGGCAAAG CAACTGACGG TTTGGCAGGA CTTCTAGAAA 360
GTAAAAAATG AACCCGAATG ATAATTATGT TCGATTTTTA AAATCCCAAT GATAATAAAT 420
AGAAAAGACA ATTGACGGGG CATAGAGGAG TATAATGACA GCGTTTGACG GGACTTATAG 480
AAATTATAAA AACGAAACCC AACGAGACAC TAAACTCTAA AAACTATAAG ATCCTATTTT 540
TAAAGGTTTC AAGGAGAATG AATAGAAATA GTGGTAGATT GAGCAAGCAA ATAAAAAATG 600
ATATGAGAAA AGTAAGACGT AGCAGCTGGT GTGACTTTAA AAACCATATA ATTAGAAATA 660
TGGAGATGAT AAGGTTTGGT CTTTCAAAGT CTTAAGACAA CGAAATAGCT ATTTAATAAA 720
TTTTAAGCAA AATCATACTT AAAAAATATA TAATTTTGTT TGTGTACTAG CCGCGCAGTT 780
GCGCGGGCCA CCAGCTAGTT GAGAGTATAA TTAACTTTTT TTCTTTAAAA TATACACAAT 840 AAACTATATT TTTTAAAAGA TTTTCTGTCG CACAGACATT ATACTAGTTC TGAGAAAAAA 900 CTGTCTATAT TTTCTCAGTC AAGTCAGGTG TGTATTGCGC AGAACGAAAG CTCGGAGAGA 960 ATACTAGCAC TTGGTATGAA CCATTAGGAC TTGCTAAAGA CAGATGAAAG GGTTATGCCA 1020 ACACCAGTTT TTGTCGGGGG ACTGGTGACC GCGCAATTGG ATTGGTACCT CTCTTGTGCC 1080 GTAGTTTCCC CCCTCATGCA CCCCCAAAAC CCCAGAAAAA TTTTGTTGTT TGTACACAGA 1140 CCGCTTGACC ATCAGCCCAT CACCTACGTG CGGAGAACCA ATGACCTATG GAATATGTAA 1200 CTAGAACACA AAACCTAAAC AAACGTGTTG CACGAAGTAG AAAGCGATAG AGAAAGATAA 1260
GCCGGGGGAC CAAGGAAATG ATATCTGGAT ATGAGTTCAC AGCCCTTCCA GATAAACGGA 1320
CGCCCGGACG AAACGAAAGA CGACGAGTCG AGGACCGTCA GCAGCCGCAG AAACGGCGAG 1380
AGACGCCCCC AAGCCAAACG TGGCTTCGTG GCGTGACGAC GCGAGGTGTT TCACGCCCCG 1440
TATCCCCCCG CGCCGCGCTG CCGCGTGCAA CTCTCTCTCT CTTCCCCCGC ATGCACTCCC 1500
GCCACTGCCC GCCGCCCGCA TCGCTCCGCT CCCCCGAGCC CAACCGCCAC AGGGCACGCC 1560
ACGACCACCA CGAAACCTCT ACGTAGCCAC ACGCCCACCC GGCCCGTAGT TGCGGTCCCA 1620
AACTCGTCGC GCCGGCACAC CAATCCCGTG GTCAACCCAA CCGGCCACAC CGAACCCACA 1680
CTCCCCACTC CCACCCATCC TGCGCCTCCT ATTTAAACTC CCCACAACTC CCTCCATTCC 1740
CCTCCAAGAG CAAAGCCACT GCAGCTTCCA TATCCCCGGC TCTTCCGCAC ACACAACTCC 1800
TCCACCTCCA TCGGGAGCAA ACCGCTCGAG CAACCACCAC TCGTTACAGC TAGACATCGA 1860
TCTCCCCTCT CGTTCGCCGT TCCGATGGAG TGCGAGAACG GGCACGTCGC CCCCGCCGCC 1920
AACGGCAGCA GCCTGTGCGT GGCTAAGCCG CGTGCCGACC CGCTCAACTG GGGGAAGGCG 1980
GCGGAGGAGC TGTCCGGGAG CCATCTGGAC GCGGTGAAGC GCATGGTGGA GGAGTACCGC 2040
AGGCCCGTGG TGACGATCGA GGGCGCCAGC CTGACCATCG CGCAGGTCGC GGCGGTGGCC 2100
TCCGCCGGCG CCGCCAGGGT GGAGCTCGAC GAGTCCGCCC GCGGCCGCGT CAAGGCCAGC 21 60
AGCGACTGGG TCATGAACAG CATGATGAAC GGCACCCACA GCTACGGCGT CACCACCGGC 2220
TTCGGCGCCA CCTCCCACCG GAGGACCAAG GAGGGCGGCG CGCTCCAGCG AGAGCTTATC 2280
CGGTAAGAAG CCGCAAGAGT TTGCTGTTCG TCTGGTGAGA GCTTGTGTGG ATCAGAGG 2338
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2001 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: N (iv) ANTI-SENSE: N
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Oryza sativa
(vii) IMMEDIATE SOURCE:
(B) CLONE: lambda rPAL-4
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
TGTCCGTCGA CCCAGATCTG GGTCGACCTG CAGGTCAACG GATCTTCTAC TGGCGTTCCA 60
CCTGCAAGTA CCTTTTTTCT AAGTAGTGCT AGCTAGCATA CATCCACCAC GTGAGCCGCC 120
GGGGCAGTGG GATGCCGACG TCGCGCACAA CAACATCAAG AGCACGAACA TACTGCTCGC 180
AAAGCCGGCA AGGCATGGCT CGCGGACTAC GGCCTGGCGC GGGTGGTCTC GTCCCTACGG 240
GCCGCCGCGA GCTCGGCGTG GTACCGGCCC CCGACGCACC CCCGGTGCCA CGGGCGTGGG 300
CGTCGAAGAA GGGCGACGTG TACGCGTTCG GCGTGGTGCT CTCGTGCCCG GGCAAGCGAG 360
CTTCCCAACG CGACGCCGCC GTGCCCACCA GCTGCGGCCA CGGTTGCGTC ATCCCCCTTG 420
GCGAAACCAG CGGGCACAGC AACAGCAGCG GGGAAAAGAT GAGCCGGGCA GCTGTGGTAG 480
CAATTGTGGC CGGGGATTTC GCCGGTACAG GTTGAGGCGA TACAGAAGAA GAGAGGAGAG 540
AGAAGGAAGA AGATGGAAAA TGAAGAAGAT GATGGAAAAC GTGATGGTAG TGGTATGATC 600
GTAGTTTTGT AAAATTTCGA TGGCACGACT ACGAATAGAT AAATTTAATT ATAATGGTAT 660
TTTTCTGAAT AGACAAATTT ACAATGGCAT GGACCAATTA ACCCTACCTC TTTCCCATGT 720
GGAGAGTATG CAAGCATGCA ACAACTAGAA AAGATACTCA TGATAGTTAA ACTCCAAATA 780
GTTTTTTCTT GCAAATTACA TATCCGATCA ACAATCCGAT TATACCATTG TATTCGTTGT 840
AATTAAATCT TATAACAAGA TCTTACAAAG ATTATATTTT GATAAAAAAA ATTATATATG 900
TTGTAATTAA TTATATATGT AAGTTACTTT ATCGTATATA TAAATTACTT CTAGATTTAA 960
TTAAATTATA TTTTGGACAT TTGAAAATTT TATTTTATTG GTTAGTTCTA TAATGTGTTC 1020
ATTAAATTTA ATTTCTAGAC AGTCTTATGT TTTTATCCCC ATAATAGATT TGTTTATAAT 1080 GTCGTGACAA GTTACTTTAT TTTTATGTTA CTTCTATACT TATATGCAAA TTACATTTAG 1140
GCTTTATTGA ATTTACTTTT ATATGTCTAA GAAGTAATTT AATGAAATCT AAAAATAATT 1200
CAGATATATT ATAAAAGTAA TTTGTATTTT TATAAAAATT ATAGCTATAA ATATTCAATT 1260
GTTACGAACA ATAGTGTTAT CGGATCGTAA ATTGGATGAG TAGTTTAATA GAAATTTTTA 1320
TTTGAATCAT AGGTGGGAGG GATATATTTT TCTACTTGCT TTATGGCCTA GTAGTATCGA 1380
GATAAACATT AAGGCTGTGT TTAGTTCACA CCAAAATTGG AAGTTTGGTT GAAATTGGAA 1440
CGATGTGACG GAAAAGTTAG AAGTTTGTGT GTGTAGGAAA GGTTTTGATG TGATGGAAAA 1500
GTTAGGAAGT TTGAAGAATT ATTTTGTAAC TAAACACGGC GTAAGAGGTC TCTTTGATTT 1560
AGATTTTGCA TAAAACAAGG GACGGTCCCG CTCTTGCTAC TTATTTAAGC ACCCCCCTCA 1620
GTAGTCCTGA CTCCAACAAG CTCCACCGCA AAGATCCTCT GTTAGCTGGA CGACCTGTGG 1680
ACTGCGGTAC GTGGCGCTGC GAGCAATGGA GTGTGAGACC GGTCTGGTCG ACCGTCCCCT 1740
CAACGGCGAC CCCTTGTACT GGGGCAAGGC GGCGGAGGGT CTTGCGGGGA GCCACCTCGA 1800
CGAGGTGAAG AGGATGGTGG TGGAGTACCG CGCGCCCGCT GGTGAAGATC GACGGCGCCA 1860
TGCTCAGCGT CGCCAAGGTG GCAGCCGTCG CTGGCGAGGC CGCCCGGGTG CAGGTGGTGC 1920
TGGACGAATC CGCACGACCC CGCCTGGAGG CTAGTCGCGA GTGGGTCTTC GACAGCACCA 1980
TGAACGGCAC CGACACGTAC A 2001
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: N
(iv) ANTI-SENSE: Y
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Phaseolus vulgaris
(vii) IMMEDIATE SOURCE:
(B) CLONE: Primer 1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
AATTTAATAC GACTCACTAT AGAAACAATA GGAAGCCATG GCAATTTCAG CTCC
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(iii) HYPOTHETICAL: N
(iv) ANTI-SENSE: N
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Synthetic Construct
(vii) IMMEDIATE SOURCE:
(B) CLONE: T7 RNA Polymerase Binding Site on Primer 1
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
AATTTAATAC GACTCACTAT AGAAA
(2) INFORM ATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: N
(iv) ANTI-SENSE: N
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Phaseolus vulgaris
(vii) IMMEDIATE SOURCE: (B) CLONE: Primer 2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: GAGAGAGATT AACTCCGTGA ATGACAACC
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: N
(iv) ANTI-SENSE: N
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Phaseolus vulgaris
(vii) IMMEDIATE SOURCE:
(B) CLONE: Probe
(xi) SEQUENCE DESCRIPΗON: SEQ ID NO:13: TCTACAACAA CGGTCTGCCT TCAA

Claims

WHAT IS CLAIMED IS:
1. Isolated promoters for plant genes that encode phenylalanine ammonia-lyase (PAL) in potato or rice wherein said promoters are capable of regulating transcription of an associated DNA sequence in suitable hosts and wherein said promoters are inducible, or are otherwise directly or indirectly responsive to, exogenous elicitor(s) and/or wounding.
2. Isolated phenylalanine ammonia-lyase (PAL) gene promoters capable of regulating transcription of an associated DNA sequence in suitable hosts, wherein said promoters are inducible, or are otherwise directly or indirectly responsive to, exogenous elicitor(s) and/or wounding, and wherein said promoters are selected from the group consisting of λpPAL-1, λpPAL-2, λpPAL-3, λpPAL-4, λpPAL-6, λpPAL-7, λpPAL-8, λrPAL-2, λrPAL-4 and λrPAL-10 promoters.
3. Isolated phenylalanine ammonia-lyase (PAL) promoters according to any of Claims 1 or 2 wherein said suitable hosts include plant protoplasts, plant cells, plant callus, plant tissue, developing plantlets, immature whole plants, mature whole plants and seeds.
4. Isolated promoters according to Claim 1 wherein said promoters are operatively linked to at least one associated DNA sequence that encodes protein(s) which directly or indirectly gives rise to a phenotypic trait.
5. Isolated promoters according to Claim 2 wherein said promoters are operatively linked to at least one associated DNA sequence that encodes protein(s) which directly or indirectly gives rise to a phenotypic trait.
6. Isolated promoters according to Claim 1 wherein said promoters are operatively linked to at least one associated DNA sequence that encodes protein(s) which directly or indirectly gives rise to a phenotypic trait wherein said phenotypic trait is selected from the group consisting of tolerance or resistance to: herbicide, fungus, virus, bacterium, insect, nematode or arachnid; production of secondary metabolites, male or female sterility, and production of an enzyme or reporter compound.
7. Isolated promoters according to Claim 2 wherein said promoters are operatively linked to at least one associated DNA sequence that encodes protein(s) which directly or indirectly gives rise to a phenotypic trait wherein said phenotypic trait is selected from the group consisting of tolerance or resistance to: herbicide, fungus, virus, bacterium, insect, nematode or arachnid; production of secondary metabolites, male or female sterility, and production of an enzyme or reporter compound.
8. Isolated promoters according to Claim 1 wherein said promoters are operatively linked to at least one associated DNA sequence that encodes a reporter protein selected from the group consisting of
chloramphenicol acetyltransferase (CAT), neomycin phosphotransferase (NPT), nopaline synthase (NOS), octopine synthase (OCS), β-1,3-glucuronidase
(GUS), acetohydroxyacid synthase (AHAS), β-galactosidase (β-GAL), and luciferase (LUX).
9. Isolated promoters according to Claim 2 wherein said
promoters are operatively linked to at least one associated DNA sequence wherein said associated DNA sequence(s) encodes reporter protein(s) selected from the group consisting of chloramphenicol acetyltransferase (CAT), neomycin phosphotransferase (NPT), nopaline synthase (NOS), octopine synthase (OCS), β-1,3-glucuronidase (GUS), acetohydroxyacid synthase
(AHAS), β-galactosidase (β-GAL), and luciferase (LUX).
10. Isolated promoters according to Claim 1 wherein said promoters are inducible by, or are otherwise directly or indirectly responsive to, exogenous elicitor(s) wherein said elicitor(s) is selected from the group consisting of the reduced form of glutathione; the reduced form of
homoglutathione; glycan elicitors; hexa(β-D-glucopyranosyl)-D-glucitols; lipid elicitors; arachidonic acid; eicosapentaenoic acid; glycoprotein elicitors; fungal elicitors; abiotic elicitors; mercuric chloride; viruses, fungi; bacteria and insects.
11. Isolated promoters according to Claim 2 wherein said promoters are inducible by, or are otherwise directly or indirectly responsive to, exogenous elicitor(s) wherein said elicitor(s) is selected from the group consisting of the reduced form of glutathione; the reduced form of
homoglutathione; glycan elicitors; hexa(β-D-glucopyranosyl)-D-glucitols; lipid elicitors; arachidonic acid; eicosapentaenoic acid; glycoprotein elicitors; fungal elicitors; abiotic elicitors; mercuric chloride; viruses, fungi; bacteria and insects.
12. Transgenic plant compositions containing any of the isolated promoters of any of Claims 1-2 or 4-10 wherein said transgenic plant compositions include plant protoplasts, plant cells, plant callus, plant tissue, developing plantlets, immature whole plants, mature whole plants, and seeds.
13. Isolated promoters for plant genes that encode phenylalanine ammonia-lyase (PAL) in potato wherein said promoters are capable of regulating transcription of an associated DNA sequence in suitable hosts and wherein said promoters are inducible, or are otherwise directly or indirectly responsive to, exogenous elicitor(s) and/or wounding.
14. Isolated phenylalanine ammonia-lyase (PAL) promoters capable of regulating transcription of an associated DNA sequence in suitable hosts, wherein said promoters are inducible or otherwise directly or indirectly responsive to exogenous elicitor(s) and/or wounding, and wherein said promoters are selected from the group consisting of λpPAL-1, λpPAL-2, λpPAL-3, λpPAL-4, λpPAL-6, λpPAL-7, and λpPAL-8.
15. Isolated phenylalanine ammonia-lyase (PAL) promoters according to any of Claims 13 or 14 wherein said suitable hosts include plant protoplasts, plant cells, plant callus, plant tissue, developing plantlets, immature whole plants, mature whole plants and seeds.
16. Isolated promoters according to Claim 13 wherein said promoters are operatively linked to at least one associated DNA sequence that encodes protein(s) which directly or indirectly gives rise to a phenotypic trait.
17. Isolated promoters according to Claim 14 wherein said promoters are operatively linked to at least one associated DNA sequence that encodes protein(s) which directly or indirectly gives rise to a phenotypic trait.
18. Isolated promoters according to Claim 13 wherein said promoters are operatively linked to at least one associated DNA sequence that encodes protein(s) which directly or indirectly gives rise to a phenotypic trait wherein said phenotypic trait is selected from the group consisting of tolerance or resistance to: herbicide, fungus, virus, bacterium, insect, nematode or arachnid; production of secondary metabolites, male or female sterility, and production of an enzyme or reporter compound.
19. Isolated promoters according to Claim 14 wherein said promoters are operatively linked to at least one associated DNA sequence that encodes protein(s) which directly or indirectly gives rise to a phenotypic trait wherein said phenotypic trait is selected from the group consisting of tolerance or resistance to: herbicide, fungus, virus, bacterium, insect, nematode or arachnid; production of secondary metabolites, male or female sterility, and production of an enzyme or reporter compound.
20. Isolated promoters according to Claim 13 wherein said promoters are operatively linked to at least one associated DNA sequence wherein said associated DNA sequence encodes reporter protein(s) selected from the group consisting of chloramphenicol acetyltransferase (CAT), neomycin phosphotransferase (NPT), nopaline synthase (NOS), octopine synthase (OCS), β-1,3-glucuronidase (GUS), acetohydroxyacid synthase
(AHAS), β-galactosidase (β-GAL), and luciferase (LUX).
21. Isolated promoters according to Claim 14 wherein said promoters are operatively linked to at least one associated DNA sequence wherein said associated DNA sequence encodes reporter protein(s) selected from the group consisting of chloramphenicol acetyltransferase (CAT), neomycin phosphotransferase (NPT), nopaline synthase (NOS), octopine synthase (OCS), β-1,3-glucuronidase (GUS), acetohydroxyacid synthase
(AHAS), β-galactosidase (β-GAL), and luciferase (LUX).
22. Isolated promoters according to Claim 13 wherein said promoters are inducible by, or are otherwise directly or indirectly responsive to, exogenous elicitor(s) wherein said elicitor(s) is selected from the group consisting of the reduced form of glutathione; the reduced form of
homoglutathione; glycan elicitors; hexa(β-D-glucopyranosyl)-D-glucitols; lipid elicitors; arachidonic acid; eicosapentaenoic acid; glycoprotein elicitors; fungal elicitors; abiotic elicitors; mercuric chloride; viruses, fungi; bacteria and insects.
23. Isolated promoters according to Claim 14 wherein said promoters are inducible by, or are otherwise directly or indirectly responsive to, exogenous elicitor(s) wherein said elicitor(s) is selected from the group consisting of the reduced form of glutathione; the reduced form of
homoglutathione; glycan elicitors; hexa(β-D-glucopyranosyl)-D-glucitols; lipid elicitors; arachidonic acid; eicosapentaenoic acid; glycoprotein elicitors; fungal elicitors; abiotic elicitors; mercuric chloride; viruses, fungi; bacteria and insects.
24. Transgenic plant compositions containing any of the isolated promoters of any of Claims 13-14 or 16-22 wherein said transgenic plant compositions include plant protoplasts, plant cells, plant callus, plant tissue, developing plantlets, immature whole plants, mature whole plants, and seeds.
25. Transgenic plant compositions containing any of the isolated promoters of any of Claims 13-14 or 16-22 wherein said transgenic plant compositions are Solanum tuberosum plant compositions.
26. Isolated promoters for plant genes that encode phenylalanine ammonia-lyase (PAL) in rice wherein said promoters are capable of regulating transcription of an associated DNA sequence in suitable hosts and wherein said promoters are inducible, or are otherwise directly or indirectly responsive to, exogenous elicitor(s) and/or wounding.
27. Isolated phenylalanine ammonia-lyase (PAL) promoters capable of regulating transcription of an associated DNA sequence in suitable hosts, wherein said promoters are inducible or otherwise directly or indirectly responsive to exogenous elicitor(s) and/or wounding, and wherein said promoters are selected from the group consisting of λrPAL-2, λrPAL-4 and λrPAL-10 promoters.
28. Isolated phenylalanine ammonia-lyase (PAL) promoters according to any of Claims 26 or 27 wherein said suitable hosts include plant protoplasts, plant cells, plant callus, plant tissue, developing plantlets,
immature whole plants, mature whole plants and seeds.
29. Isolated promoters according to Claim 26 wherein said promoters are operatively linked to at least one associated DNA sequence that encodes protein(s) which directly or indirectly gives rise to a phenotypic trait.
30. Isolated promoters according to Claim 27 wherein said promoters are operatively linked to at least one associated DNA sequence that encodes protein(s) which directly or indirectly gives rise to a phenotypic trait.
31. Isolated promoters according to Claim 26 wherein said promoters are operatively linked to at least one associated DNA sequence that encodes protein(s) which directly or indirectly gives rise to a phenotypic trait wherein said phenotypic trait is selected from the group consisting of tolerance or resistance to: herbicide, fungus, virus, bacterium, insect, nematode or arachnid; production of secondary metabolites, male or female sterility, and production of an enzyme or reporter compound.
32. Isolated promoters according to Claim 27 wherein said promoters are operatively linked to at least one associated DNA sequence that encodes protein(s) which directly or indirectly gives rise to a phenotypic trait wherein said phenotypic trait is selected from the group consisting of tolerance or resistance to: herbicide, fungus, virus, bacterium, insect, nematode or arachnid; production of secondary metabolites, male or female sterility, and production of an enzyme or reporter compound.
33. Isolated promoters according to Claim 26 wherein said promoters are operatively linked to at least one associated DNA sequence wherein said associated DNA sequence encodes reporter protein(s) selected from the group consisting of chloramphenicol acetyltransferase (CAT), neomycin phosphotransferase (NPT), nopaline synthase (NOS), octopine synthase (OCS), β-1,3-glucuronidase (GUS), acetohydroxyacid synthase
(AHAS), β-galactosidase (β-GAL), and luciferase (LUX).
34. Isolated promoters according to Claim 27 wherein said promoters are operatively linked to at least one associated DNA sequence wherein said associated DNA sequence encodes reporter protein(s) selected from the group consisting of chloramphenicol acetyltransferase (CAT),
neomycin phosphotransferase (NPT), nopaline synthase (NOS), octopine synthase (OCS), β-1,3-glucuronidase (GUS), acetohydroxyacid synthase
(AHAS), β-galactosidase (β-GAL), and luciferase (LUX).
35. Isolated promoters according to Claim 26 wherein said promoters are inducible by, or are otherwise directly or indirectly responsive to, exogenous elicitor(s) wherein said elicitor(s) is selected from the group consisting of the reduced form of glutathione; the reduced form of
homoglutathione; glycan elicitors; hexa(β-D-glucopyranosyl)-D-glucitols; lipid elicitors; arachidonic acid; eicosapentaenoic acid; glycoprotein elicitors; fungal elicitors; abiotic elicitors; mercuric chloride; viruses, fungi; bacteria and insects.
36. Isolated promoters according to Claim 27 wherein said promoters are inducible by, or are otherwise directly or indirectly responsive to, exogenous elicitor(s) wherein said elicitor(s) is selected from the group consisting of the reduced form of glutathione; the reduced form of
homoglutathione; glycan elicitors; hexa(β-D-glucopyranosyl)-D-glucitols; lipid elicitors; arachidonic acid; eicosapentaenoic acid; glycoprotein elicitors; fungal elicitors; abiotic elicitors; mercuric chloride; viruses, fungi; bacteria and insects.
37. Transgenic plant compositions containing any of the isolated promoters of any of Claims 26-27 or 29-35 wherein said transgenic plant compositions include plant protoplasts, plant cells, plant callus, plant tissue, developing plantlets, immature whole plants, mature whole plants, and seeds.
38. Transgenic plant compositions according to any of Claims 26-27 or 29-35 wherein said transgenic plant compositions are Oryza sativa or
Oryza indica plant compositions.
39. A chimeric composition which comprises a first DNA component sequence which is, or has substantial sequence homology to, a non-coding DNA sequence of a naturally occurring PAL gene, wherein said non-coding sequence is capable of regulating transcription of an associated DNA sequence in suitable hosts and is inducible by exogenous elicitor(s) and/or wounding and wherein said non-coding DNA sequence is any of the non-coding DNA sequences of λpPAL-1, λpPAL-2, λpPAL3, λpPAL-4, λpPAL-6/7, λpPAL-8, λrPAL-2, λrPAL-4 or λrPAL-10, and a second DNA component which is, or has substantial sequence homology to, part but not all of a transcribable PAL-encoding DNA sequence with which said first component is associated in the naturally occurring gene.
40. A chimeric composition according to Claim 39 wherein at least one structural gene is contained in frame within said second DNA component such that the expression product is a fused PAL peptide.
41. A chimeric composition according to Claim 40 wherein said structural gene(s) encodes a reporter protein selected from the group consisting of chloramphenicol acetyltransferase (CAT), neomycin
phosphotransferase (NPT), nopaline synthase (NOS), octopine synthase (OCS), β-1,3-glucuronidase (GUS), acetohydroxyacid synthase (AHAS), β- galactosidase (β-GAL), and luciferase (LUX).
42. Plant tissue, plant or seed transformed with any of the chimeric compositions of any of Claims 39-41.
43. Plant tissue, plant or seed transformed with any of the chimeric compositions of any of Claims 39-41 wherein said compositions contain potato PAL promoter(s) and said plant tissue, plant or seed is Solanum.
44. Plant tissue, plant or seed transformed with any of the chimeric compositions of any of Claims 39-41 wherein said compositions contain potato PAL promoter(s) and said plant tissue, plant or seed is Solanum tuberosum.
45. Plant tissue, plant or seed transformed with any of the chimeric compositions of any of Claims 39-41 wherein said compositions contain rice PAL promoter(s) and said plant tissue, plant or seed is Oryza.
46. Plant tissue, plant or seed transformed with any of the chimeric compositions of any of Claims 39-41 wherein said compositions contain rice PAL promoter(s) and said plant tissue, plant or seed is Oriza sativa or Oriza indica.
47. A probe comprising at least 10 nucleotides wherein said probe is comprised of deoxyribonucleotides from any of SEQ ID NOS. 1-9, or mRNA corresponding thereto.
48. An antisense probe comprising at least 10 nucleotides wherein said probe is comprised of deoxyribonucleotides complementary to any of SEQ ID NOS. 1-9, or mRNA corresponding thereto.
49. An antisense probe comprising at least 10 nucleotides wherein said probe is comprised of deoxyribonucleotides complementary to any of SEQ ID NOS. 1-9, or mRNA corresponding thereto.
50. A method for initiating transcription of a chimeric composition comprised of at least one PAL promoter from potato or rice wherein said promoter is operatively linked to at least one structural gene, wherein said promoter is inducible by, or is otherwise directly or indirectly responsive to, exogenous elicitor(s), said method comprising: introducing said chimeric composition into a suitable host and subjecting said host to exogenous elicitor(s) capable of directly or indirectly inducing a response by said promoter.
51. A method for initiating transcription of a chimeric composition contained within a transgenic plant wherein said composition is comprised of at least one PAL promoter from potato or rice wherein said promoter is operatively linked to at least one structural gene, and wherein said promoter is inducible by, or is otherwise directly or indirectly responsive to, exogenous elicitor(s), said method comprising: subjecting said transgenic plant to exogenous elicitor(s) capable of directly or indirectly inducing a response by said promoter.
52. A method for identifying elicitor-inducible promoters, said method comprising: a), subjecting a plant to elicitor; b). isolating RNA from said plant from step a) after said plant has been subjected to said elicitor; c). preparing a cDNA library from said RNA from step b); d). probing said library with a probe comprised of nucleotide sequences from the transcribed coding and/or non-coding region of gene(s) of interest; e). using said probe described in step d) or the cDNA which hybridized with said probe from step d) to probe a genomic library from a plant of interest; f). identifying the segment of genomic DNA that hybridized with said probe from step e); g). making and labeling an antisense RNA transcript from the genomic DNA segment of step f); h). using said labeled antisense RNA transcript from step g) as a probe to hybridize to mRNA from elicitor-treated and non-elicitor treated plants; i). subjecting the hybridized mix to RNase; j). identifying those antisense RNA transcripts that were protected from degradation by elicitor-treated RNA but were not protected from degradation by RNA from non-elicitor treated plants; k). concluding that the promoter from the genomic clone that yielded the antisense RNA transcript that was protected by only RNA from elicitor-treated plants is inducible with elicitor.
53. A method for identifying elicitor-inducible promoters, said method comprising: a), probing a plant genomic library with a probe comprised of nucleotide sequences from the transcribed coding and/or non- coding region(s) of a gene of interest; b). identifying the segment of genomic DNA that hybridized with said probe from step a); c). making and labeling an antisense RNA transcript from the genomic DNA segment of step b); d). using said labeled antisense RNA transcript from step c) as a probe to hybridize to mRNA from elicitor-treated and non-elicitor treated plants; e). subjecting the hybridized mix to RNase; f). identifying those antisense RNA transcripts that were protected from degradation by elicitor-treated RNA but were not protected from degradation by RNA from non-elicitor treated plants; g).
concluding that the promoter from the genomic clone that yielded the antisense RNA transcript that was protected by only RNA from elicitor-treated plants is inducible with elicitor.
54. Isolated DNA sequences comprised of any of the DNA sequences of any of SEQ ID NOS. 1-9.
55. Isolated DNA sequences according to Claim 53 wherein said sequences are operatively linked to at least one structural gene encoding sequence and at least one terminator sequence.
56. Isolated DNA sequences according to Claim 54 wherein said structural gene encodes protein(s) which directly or indirectly gives rise to a phenotypic trait wherein said phenotypic trait is selected from the group consisting of tolerance or resistance to: herbicide, fungus, virus, bacterium, insect, nematode or arachnid; production of secondary metabolites, male or female sterility, and production of an enzyme or reporter compound.
57. Isolated DNA sequences according to Claim 54 wherein said structural gene(s) encodes at least one reporter protein selected from the group consisting of chloramphenicol acetyltransferase (CAT), neomycin phosphotransferase (NPT), nopaline synthase (NOS), octopine synthase (OCS), β-1,3-glucuronidase (GUS), acetohydroxyacid synthase (AHAS), β- galactosidase (β-GAL), and luciferase (LUX).
58. A method for identifying exogenous elicitors which are capable of inducing, either directly or indirectly, a PAL promoter, said method comprising transforming a suitable host with a chimeric DNA sequence of any of Claims 39-41 or a vector containing any of said chimeric DNA sequences, applying putative exogenous elicitor to said transformed host, and identifying as an exogenous elicitor those exogenous elicitor(s) which cause expression of said second DNA component sequence.
59. A whole plant assay system for identifying exogenous elicitors which are capable of inducing, either directly or indirectly, a PAL promoter from potato or rice, said method comprising creating a transgenic plant that contains at least one chimeric DNA sequence comprised of a potato or rice
PAL promoter operatively linked to a reporter structural gene, applying putative exogenous elicitor to said transgenic plant, and identifying as an exogenous elicitor those exogenous elicitor(s) which cause expression of said reporter structural gene.
60. A method for identifying organisms which can induce transcription of defense genes, said method comprising exposing a transgenic plant that contains a plant defense gene promoter operatively linked to a reporter gene to an organism of interest, monitoring expression of said reporter gene, and concluding that an organism of interest which can induce transcription of said reporter gene can induce expresssion of plant defense genes, or chimeric constructs that contain regulatory regions or promoters for such plant defense genes.
61. A method for according to Claim 60 wherein said orgainsim is selected from living organisms, non-living organisms, or living but non-viral or otherwise disabled organisms.
62. A method according to Claim 60 wherein said organism is a virus, a bacteria, a fungus, or an insect.
63. A method for identifying agrichemicals which can induce expression of a plant gene, said method comprising (a) isolating RNA from plant material not exposed to a putative inducer; (b) identifying from within the RNA of step (a) an RNA encoded by a plant gene of interest; (c) amplifying said identified RNA from step (b) using 3SR techniques; (d) exposing to a putative chemical inducer plant material that is identical to the plant material from which the RNA of step (a) was isolated; (e) isolating RNA from said exposed plant material of step (d); (f) identifying from within the RNA of step (e) RNA encoded by same plant gene of interest used to identify the RNA of step (b); (g) amplifying said identified RNA from step (e) using 3SR techniques; (h) comparing the amplification products from steps (c) and
(g); (i) identifying as an agrichemical that can induce expression of the gene of interest those agrichemicals which induce a stronger amplification in step (g) than was obtained in step (c).
PCT/US1992/008560 1991-10-03 1992-10-02 Inducible plant defense gene regulatory regions from potato and rice, uses thereof, and assays WO1993007279A1 (en)

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US6355468B1 (en) 2000-07-24 2002-03-12 Pcbu Services, Inc., Phenylalanine ammonia lyase polypeptide and polynucleotide sequences and methods of obtaining and using same
US6368837B1 (en) 1999-08-06 2002-04-09 E. I. Du Pont Nemours And Company Bioproduction of para-hydroxycinnamic acid
US6521748B2 (en) 1999-08-06 2003-02-18 E. I. Du Pont De Nemours And Company Polynucleotide encoding a mutant Rhodotorula glutinis tyrosine ammonia lyase polypeptide
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WO2007039317A2 (en) 2005-10-05 2007-04-12 Bayer Cropscience Ag Plants having an increased content of amino sugars
DE19860313B4 (en) * 1998-12-24 2007-10-11 Felsenstein, Friedrich, Dr. Method for the detection and characterization of active substances against plant pathogens
US7390937B2 (en) 1996-02-14 2008-06-24 The Governors Of The University Of Alberta Plants with enhanced levels of nitrogen utilization proteins in their root epidermis and uses thereof
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US7714186B2 (en) 2002-12-19 2010-05-11 Bayer Cropscience Ag Plant cells and plants which synthesize a starch with an increased final viscosity
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WO2011064224A1 (en) * 2009-11-24 2011-06-03 Katholieke Universiteit Leuven, K.U.Leuven R&D Banana promoters
US8148517B2 (en) 2005-04-01 2012-04-03 Bayer Cropscience Ag Phosphorylated waxy potato starch
US8288611B2 (en) 2005-12-23 2012-10-16 Arcadia Biosciences, Inc. Nitrogen-efficient monocot plants
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Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989012230A1 (en) * 1988-05-20 1989-12-14 The Salk Institute Biotechnology/Industrial Associ Agrichemical screens

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989012230A1 (en) * 1988-05-20 1989-12-14 The Salk Institute Biotechnology/Industrial Associ Agrichemical screens

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
AGRIC. BIOL. CHEM. vol. 52, no. 4, 1988, pages 943 - 950 HAGA, M., ET AL. 'Dependence on ethylene of the induction of phenylalanine ammonia-lyase activity in rice leaf infected with blast fungus' *
BIOLOGICAL ABSTRACTS vol. 85 , 1988, Philadelphia, PA, US; abstract no. 20305, FRITZEMEIER, K.-H., ET AL. 'Transient induction of phenylalanine ammonia-lyase and 4-coumarate: coenzyme A ligase messenger RNA in potato leaves infected with virulent or avirulent races of Phytophtora infestans' *
BIOTECHNOLOGY vol. 8, no. 9, September 1990, NEW YORK US pages 845 - 848 DOERNER, P.W., ET AL. 'Plant defense gene promoter-reporter gene fusions in transgenic plants: tools for identification of novel inducers' *
EMBO JOURNAL vol. 8, no. 6, 1989, EYNSHAM, OXFORD GB pages 1641 - 1648 LOIS, R., ET AL. 'A phenylalanine ammonia-lyase gene from parsley: structure, regulation and identification of elicitor and light responsive cis-acting elements' *
EUR. J. BIOCHEM. vol. 185, no. 1, 1989, pages 19 - 25 MINAMI, E., ET AL. 'Structure and some characterization of the gene for phenylalanine ammonia-lyase from rice plants' cited in the application *
JOURNAL OF BIOLOGICAL CHEMISTRY vol. 264, no. 24, 25 August 1989, BALTIMORE, MD US pages 14486 - 14492 LIANG, X., ET AL. 'Differential regulation of phenylalanine ammonia-lyase genes during plant development and by environmental cues' *
PLANT MOLECULAR BIOLOGY. vol. 12, 1989, DORDRECHT, THE NETHERLANDS. pages 367 - 383 CRAMER, C.L., ET AL. 'Phenylalanine ammonia-lyase gene organization and structure' cited in the application *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA vol. 86, December 1989, WASHINGTON US pages 9284 - 9288 LIANG, X., ET AL. 'Developmental and environmental regulation of a phenylalanine ammonia-lyase-Beta-glucuronidase gene fusion in transgenic tobacco plants' *
THE PLANT CELL. vol. 2, December 1990, ROCKVILLE, MD, USA. pages 1131 - 1143 CASTRESENA, C., ET AL. 'Tissue-specific and pathogen-induced regulation of a Nicotiana plumbaginifolia Beta-1,3-glucanase gene' *
THE PLANT CELL. vol. 2, no. 9, 1990, ROCKVILLE, MD, USA. pages 837 - 848 OHL, S., ET AL. 'Functional properties of a phenylalanine ammonia-lyase promoter from Arabidopsis' *

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