WO2002012483A1 - Promoters of plant defence-associated genes - Google Patents

Promoters of plant defence-associated genes Download PDF

Info

Publication number
WO2002012483A1
WO2002012483A1 PCT/AU2001/000961 AU0100961W WO0212483A1 WO 2002012483 A1 WO2002012483 A1 WO 2002012483A1 AU 0100961 W AU0100961 W AU 0100961W WO 0212483 A1 WO0212483 A1 WO 0212483A1
Authority
WO
WIPO (PCT)
Prior art keywords
dna
plant
promoter
gene
artificial sequence
Prior art date
Application number
PCT/AU2001/000961
Other languages
French (fr)
Other versions
WO2002012483B1 (en
Inventor
Peer Martin Philipp Schenk
Kemal Kazan
John Michael Manners
Original Assignee
Commonwealth Scientific And Industrial Research Organisation
The State Of Queensland Acting Through Its Department Of Primary Industries
The University Of Queensland
Bureau Of Sugar Experiment Stations
Grains Research & Development Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Commonwealth Scientific And Industrial Research Organisation, The State Of Queensland Acting Through Its Department Of Primary Industries, The University Of Queensland, Bureau Of Sugar Experiment Stations, Grains Research & Development Corporation filed Critical Commonwealth Scientific And Industrial Research Organisation
Priority to AU2001277407A priority Critical patent/AU2001277407A1/en
Publication of WO2002012483A1 publication Critical patent/WO2002012483A1/en
Publication of WO2002012483B1 publication Critical patent/WO2002012483B1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems

Definitions

  • This invention relates to plant promoters that are associated with and regulated by mechanisms occurring during plant defence and that confer gene expression on transgenic plants harbouring the promoters.
  • the invention also relates to utilisation of the promoters in the construction of recombinant genes for plant transformation to enable expression of the product of the gene at a certain time, in a certain tissue, at a certain rate or during a certain physiological state, hi particular, the invention relates to promoters isolated from regulatory upstream sequences of different genes of Arabidopsis thaliana that are directly or indirectly associated with plant defence mechanisms and that are potentially induceable by regulatory processes occurring during plant defence.
  • Plant genetic manipulation focuses on the cellular level of organisation and involves the interfacing of all aspects of cell biology, molecular biology and gene transfer procedures (Sharp et al. 1984, Food Technology, (Feb.) 112-119; Martin 1998, Curr. Opin. Biotechnol. 9, 220-226).
  • the genetic engineering tools of tissue culture, somaclonal and gametoclonal variation, cellular selection procedures and recombinant DNA are either indirectly or directly concerned with the enhanced expression and transfer of genes.
  • a moderate or strongly regulated promoter is required to ensure that a sufficient amount of gene product is produced at the right time, in the right tissue and during the right physiological state of the plant.
  • These applications include genetic manipulation of plants to obtain disease resistance or tolerance against plant-infecting viruses, bacteria, fungi or nematodes, to obtain resistance against herbivores, to obtain resistance against herbicides and selectable marker reagents, to obtain resistance against abiotic factors (e.g.
  • genes and gene products for research to conduct functional analyses of genes and gene products for research, to confer silencing or enhancement of genes and gene products (modulation of gene expression), to modify the composition of macromolecules and secondary metabolites (e.g. to increase nutritional value or to alter structural composition), to modify plant development, to improve fruit or crop quality (e.g. post harvest shelf life or disease resistance), to obtain industrial plants (e.g. plants producing biodegradable plastics, industrial enzymes, antibodies) and to induce or modify regulatory processes occurring during plant defence.
  • industrial plants e.g. plants producing biodegradable plastics, industrial enzymes, antibodies
  • promoters have been studied extensively in both monocot and dicot plants.
  • reporter genes such as the uidA gene encoding for ⁇ -glucuronidase (GUS, Jefferson et al. 1987, EMBO J. 6, 3901-3907) or genes encoding anfhocyanin production or the jellyfish green fluorescent protein (GFP, Chalfie et al. 1994, Science 263, 802-805) or luciferase (Himes et al. 2000, Methods Mol. Biol 130:165-174) are used to assay promoter activity in transient or stable gene expression systems. Highly regulated expression patterns have been demonstrated for several promoters, even though there is no absolute specificity in most instances.
  • a hypersensitive response which results in localised cell death at the site of infection.
  • Other defence responses may include structural alterations and the production of a wide range of plant defence molecules such as antimicrobial proteins (see Broekaert et al. 1997, Crit. Rev. Plant Set 16, 297-323; Yun et al. 1997, Plant Breeding Reviews 14, 39-88; Grant & Mansfield 1999, Curr. Opin. Plant Biol. 2, 312-319 for recent reviews).
  • plant responses to necrotrophic pathogens can lead to systemic acquired resistance (SAR), which immunises against subsequent infection.
  • SAR systemic acquired resistance
  • any promoter that can be regulated to some extend during plant defence has the potential to be used as a tool towards resistance against pathogens or herbivores.
  • These include the use of the promoter to drive expression of genes increasing resistance (e.g. genes encoding anti-microbial peptides, genes encoding insecticidal proteins, genes encoding proteins with recognition specificities such as natural resistance (R) genes, genes encoding pathogenesis related PR protein, genes involved in apoptosis, genes involved in phytoalexin production, and regulatory genes that induce local and/or systemic , resistance), the use of the promoter to screen for plant defence-inducing chemicals, and the use of the promoter operatively-linked with another gene regulatory mechanism or gene expression system.
  • genes increasing resistance e.g. genes encoding anti-microbial peptides, genes encoding insecticidal proteins, genes encoding proteins with recognition specificities such as natural resistance (R) genes, genes encoding pathogenesis related PR protein, genes involved in apoptos
  • a promoter operative in a plant cell comprising: 1) isolated DNA from Arabidopsis thaliana having a sequence as defined in any one of Figures 1 to 6;
  • a DNA construct comprising at least one gene having at least one promoter according to the first embodiment operatively linked to a coding sequence, which coding sequence encodes a desired product or products.
  • a DNA construct comprising: 1) a first gene having one or more promoters according to the first embodiment operatively linked to a coding sequence of interest; and
  • a second gene having a promoter operatively linked to a coding sequence wherein the expression product of said coding sequence modulates activity of the expression product of said first gene coding sequence.
  • a method of expressing a product in a plant cell comprising introducing a DNA construct according to the second or third embodiments or an RNA transcript of said construct into cells of a plant, wherein said DNA construct or RNA transcript coding sequence encodes said product.
  • a plant cell wherein the genome of said plant cell includes a DNA construct according to the second or third embodiments.
  • a plant, plant tissue or reproductive material of a plant wherein said plant, plant tissue or reproductive material comprises cells according to the fifth embodiment.
  • a method of identifying a chemical or regulatory molecule which modulates the activity of a promoter according to the first embodiment comprising the steps of: introducing a DNA construct according to the second embodiment into a plant cell; contacting said plant cell with said chemical or regulatory molecule; and determining the effect of said chemical or regulatory molecule on the activity of said promoter.
  • the invention includes within its scope the DNAs of the first to third embodiments as single-stranded DNA, double-stranded DNA, or as the RNA corresponding to either strands of the foregoing DNA.
  • Figure 1 comprises schematic representations of promoter-reporter gene constructs used in following examples.
  • Figure 2 comprises photographs of transient promoter activity assays in Arabidopsis thaliana plants using promoter-reporter gene constructs p498GUS, pl075GUS, pll77GUS, pl593GUS, p2007GUS > p2149GUS, p528GUS, pl865GUS or p2339GUS.
  • Figure 3 comprises photographs of transgenic Arabidopsis thaliana plants expressing a plant defence-associated promoter fused to reporter genes. Here the expression is regulated by the promoter sequence 498 of promoter reporter gene constructs pAOV498GUS or P G498GFP.
  • Figure 4 comprises photographs which show tissue specific expression in transgenic Arabidopsis thaliana plants.
  • Figure 5 comprises photographs of a reporter plant used for screening of plant defence-associated gene products.
  • a transgenic Arabidopsis thaliana plant containing the plant defence-associated PDFl.2 promoter fused to the uidA reporter gene was subsequently transformed with p35SERFl in a transient biolistic assay.
  • Figures 6 to 14 are sequences of putative promoter elements in parts of the promoter sequences shown in SEQ ID NO: 1 to SEQ JD NO: 9, respectively.
  • Coding sequence Nucleic acids sequence that encodes a functional RNA transcript which may or may not be subsequently translated into a polypeptide.
  • Homologue Nucleic acid sequences from other organisms that show a sequence identity (homology) of 75% or more to the sequences or parts thereof that are longer than 300 bp of SEQ ID NO: 1 to SEQ ID NO: 9 and have substantially the same function.
  • Plant defence A number of physiological and molecular events (e.g. hyper-sensitive response, synthesis of antimicrobial compounds, cell wall modification etc.) initiated in the host as a result of interaction with a pathogenic organism.
  • physiological and molecular events e.g. hyper-sensitive response, synthesis of antimicrobial compounds, cell wall modification etc.
  • Plant defence-associated signalling molecule Molecules such as salicylates (e.g. SA), jasmonates (e.g. JA), ethylene etc. that are capable of initiating signal transduction events that lead to a subset of specifically induced defence responses.
  • Plant genome-derived variant DNA that is present in the plant genome and has a sequence identity (homology) of 75% or more to the promoter sequences shown in Figure 1- 6 or parts thereof that are longer than 300 bp.
  • Promoter A DNA sequence flanking a coding sequence of a gene at the 5' end thereof which includes an element or elements involved in the initiation of transcription of the coding sequence.
  • Regulated promoter A promoter which mediates higher or lower transcriptional activity upon direct or indirect interaction with a regulatory molecule (e.g. transcription factors, signalling chemicals).
  • a regulatory molecule e.g. transcription factors, signalling chemicals.
  • Signal transduction pathway The process by which the information contained in an extracellular physical, chemical or biological signal is received at the cell by the activation of specific receptors and conveyed across the plasma membrane, and along and intracellular chain of signalling molecules, to stimulate the appropriate cellular responses.
  • the present inventors have identified nine promoter sequences in PCR-amplified genomic DNA sequences of Arabidopsis thaliana Columbia.
  • promoters as well as homologues comprising plant genome-derived variants, can be used separately or in combination with appropriate coding sequences to prepare transgenic plant cells and plants capable of expression of the gene(s) of interest at a suitable level.
  • DNA comprising the nine promoters according to the invention can be obtained by cloning genomic DNA from Arabidopsis thaliana Columbia. Genomic DNA isolated from Arabidopsis can be fragmented with restriction enzymes and fragments can be subcloned into plasmids that can be multiplied in Escherichia coli. Alternatively, these promoter sequences can be generated by direct polymerase chain reaction (PCR) amplification of genomic DNA. The required primers can be designed from the sequence data presented in SEQ ID NO: 1 to SEQ ID NO: 9 or equivalent sequence information. Yet another method of producing promoters having sequences such as those present in SEQ ID NO: 1 to SEQ ID NO: 9 is by DNA synthesis.
  • the invention comprises not only promoters having the sequences of SEQ ID NO: 1 to SEQ ID NO: 9 but also homologues from plant genome-derived variants (e.g.
  • DNA constructs according to the second and third embodiment can include more than one promoter operatively-linked to the coding sequence. These additional promoters can be identical or derivatives of the same promoter or heterologous promoters, hi addition, operatively-linked enhancer or silencer elements can be included in DNA constructs.
  • the coding sequence to which the promoter(s) are operatively linked can encode an RNA which functions as antisense RNA or a ribozyme or as a stractural component, or is translated into a polypeptide which functions as an enzyme, a stractural component or has some other physiological effect. Examples of fransgene products which can be usefully expressed in transgenic plants using the promoters described in the invention are products that help:-
  • industrial substances e.g. biodegradable plastics, industrial enzymes, antibodies
  • the first gene of the DNA construct includes all the variations and options of the gene comprising the DNA constract of the second embodiment.
  • the second gene of the DNA constract can have an expression product which either: -
  • the invention includes transgenic plant cells with genetically engineered genomes including the DNA constructs of the second and third embodiment.
  • DNA constructs can also comprise recombinant viral sequences with one or more coding sequences of interest that either stably or transiently express in plant cells.
  • RNA transcripts can be made from these constructs that can be used for transformation of plant cells.
  • Techniques for introducing DNA into the genome of a plant are well known in the art and are described, for example by Sagi et al. (1995, Bio/Technology 13; 481-485), May et al. (1995, Bio/Technology 13; 485-492) and Zhong et al. (1996, Plant Physiol. 110; 1097- 1107), the entire contents of which are incorporated herein by cross-reference.
  • DNA constructs according to the invention are advantageously introduced into the genome of target plant cells using methods including Agrobacterium-mediate ⁇ transformation, biolistic bombardment with DNA-coated tungsten or gold particles, electroporated or polyethylenglycol (PEG)-mediated DNA transformation of protoplasts and other mechanical DNA transfer techniques.
  • Transgenic plant cells including the DNA constructs of the invention can be propagated using conditions appropriate to the particular plant. Similarly, whole plants, or propagating material of the plant, can be prepared from the initial transgenic cells using known methods and conditions. Alternatively, RNA can also be used for transformation using the above methods.
  • methods can be used for screening of chemicals or regulatory molecules that interact directly or indirectly, in vivo or in vitro with a DNA sequence according to the first embodiment.
  • Different chemicals such as salicylic acid, jasmonic acid, oxalic acid, abscisic acid, ethylene, 2,6-dichloroisinicotinic acid (BSfA), benzothiadiazole (BTH), nitric oxide, polyacrylic acid, ⁇ -aminobutryic acid (BABA) and the like have been associated with plant defence and are known to induce certain biochemical signal transduction pathways (Malamy et ⁇ l.
  • Nucleic acid sequences with reference to the first embodiment of the invention comprise the promoters of genes that are likely to be associated with plant defence signalling. They can be used for screening of a large number of different chemicals (including proteins such as transcription factors or plant defence elicitors) that interact directly or indirectly with these sequences and may lead to a modification, induction or repression of different plant defence pathways that could have important applications in disease management and plant protection. High-throughput in vitro binding assays can be used to screen for chemicals or proteins that interact directly with the sequences according to the first embodiment. Different plant cell extracts can be used for initial screenings followed by more defined analyses.
  • yeast two-hybrid system offers an effective method to search for interacting proteins in vivo (Luban and Goff 1995, Curr. Opin. Biotechnol. 6, 59-64).
  • Other in vivo systems such as reporter plants, can be used to identify chemicals and proteins that interact directly or indirectly with the promoter sequences with reference to the first embodiment of the invention (see the examples below).
  • Transgenic reporter plants contain a reporter gene (e.g. uidA or gfp) which is controlled by a promoter according to the first embodiment.
  • Tissue extracts, chemicals, proteins, RNA or DNA can be applied to the plants using different methods that are well-established in the art such as spray application, vacuum- infiltration, Agrobacterium-m.edia.ted transformation, agro-infection or coated particle bombardment (Feldmann and Marks 1987, Mol. Gen. Genet. 208, 1-9; Finer et al. 1992, Plant Cell Rep. 11, 323-328; Bechtold and Pelletier 1998, Meth. Mol. Biol. 82, 259-266). These substances can then directly or indirectly interact with the promoter sequences of the first embodiment and will result in increased or reduced production of the reporter gene constract.
  • pBI221 contains the 35S promoter from cauliflower mosaic virus (Odell et al, 1985, Nature 313; 810-812), the uidA reporter gene (Jefferson et al. 1987, EMBO J. 6, 3901-3907) and the nopaline synthase (nos) terminator sequence from Agrobacterium tumefaciens in pUCl 18.
  • the plasmids p498GUS, pl075GUS, pll77GUS, pl593GUS, p2007GUS, p2149GUS, p528GUS, pl865GUS and ⁇ 2339GUS contain the putative promoter sequences shown in SEQ ID NO: 1 to SEQ ID NO: 9, respectively, (putative TATA boxes at 3' end) instead of the cauliflower mosaic virus promoter ( Figure 1).
  • the plasmids p498GUS, pl075GUS, pl593GUS, p2007GUS, p2149GUS, p528GUS, pl865GUS and p2339GUS were constructed by ligating the HindllT/Xbal-cut putative promoter fragments that were previously cloned into pCR-Blunt into the HindltT/Xbal-cut 5.1 kb fragment of pBI221.
  • the plasmid pl l77GUS was constracted by ligating the blunt-ended MluI/Notl-cut putative promoter fragment that was previously cloned into pCR-Blunt into the blunt-ended dephosphorylated BamHI-cut 5.1kb fragment of ⁇ 498GUS.
  • the plasmids ⁇ 528GUS, ⁇ l865GUS and p2339GUS were constracted by ligating the blunt-ended EcoRI-cut putative promoter fragments that were previously cloned into pCR-Blunt into the blunt-ended dephosphorylated BamHI-cut 5.1kb fragment of p498GUS.
  • the binary vector pAOV Mylne and Botella, 1998, Plant Mol. Biol. Rep. 16, 257-262
  • the pGreenH derivative pGREEN0229 Hellens et al. 2000, Plant Mol. Biol. 42:819-832
  • Figure 1 The plasmid pAOV498GUS was constracted by ligating the HindHI SacI-cut promoter-reporter cassettes from p498GUS into the HindJJI/SacI-cut 27kb fragment of pAOV.
  • the plasmids pG35SGUS and pG35SGFP were used as the basis for the construction of pGreenJJ-derived plasmids containing the uidA reporter gene or the sgfp(S65T) reporter gene (Chui et ⁇ l. 1996, Curr. Biol. 6:325-330), respectively.
  • the plasmid pG35SGUS was constructed by ligating the blunt-ended HindlJJ EcoRI-cut 35S-GUS-nos cassette from pBI221 into the EcoRV-cut 4.5kb fragment of ⁇ GREEN0229.
  • the plasmid pG35SGFP was constracted by ligating the blunt-ended Pstl-cut GFP fragment from pUBIGFP (Elliott et ⁇ l. 1999, Plant Cell Rep. 18:707-714) into the EcoRV-cut 4.5kb fragment of pGREEN0229. Subsequently, the plasmids pG498GUS, pG1075GUS, pG1177GUS, pG2007GUS, pG1865GUS and pG2339GUS ( Figure 1) were constracted by replacing the 35S promoter fragment of pG35SGUS with the corresponding PCR-amplified putative promoter fragments from Arabidopsis (see above).
  • PCR-amplified putative promoter fragments were phosphorilated (polynucleotide kinase, Roche) and ligated into the blunt-ended Xbal-cut 6.6kb fragment of pG35SGUS.
  • the plasmids pG498GFP, pG1075GFP, pG1177GFP, pG1593GFP, pG2007GFP, pG528GFP and pG1865GFP were constracted by replacing the 35S promoter fragment of pG35SGFP with the corresponding PCR-amplified putative promoter fragments from Arabidopsis (see above). Therefore the PCR-amplified putative promoter fragments were phosphorilated (polynucleotide kinase, Roche) and ligated into the blunt-ended Xbal- cut 5.5kb fragment of pG35SGFP.
  • All plasmid DNA was prepared from Escherichia coli DH5 ⁇ or Escherichia coli TOP 10 (Invitrogen) using the Qiaprep Spin Miniprep Kit (Qiagen). These chimaeric gene constructs are suitable to assess the promoter activity using in vivo transient and stable expression systems that were developed and optimised for this purpose.
  • Expression patterns of the native genes driven by each promoter sequence described in EXAMPLE 1 and EXAMPLE 2 were measured during plant defence responses in Arabidopsis to determine their transcriptional regulation by using quantitative real-time reverse transcriptase PCR.
  • Arabidopsis thaliana plants were grown to 8-12 leaf stage in controlled enviromnent rooms and treated with either defence-inducing chemical signal compounds or an incompatible fungal pathogen.
  • SA salicylic acid
  • Methyl jasmonate treatments were carried out by taping a cotton ball containing 400 ul of a 0.5% solution in ethanol onto the wall of a 20- liter container wrapped in plastic bags.
  • 10 ml of ethylene was injected into the air of plants kept in a sealed 20-liter container.
  • the control plants were treated in the same way but without the addition of SA, MJ or ethylene.
  • 5- ⁇ l drops of a spore suspension (5xl0 5 spores/ml in water) of Alternaria brassisicola (isolate UQ4273) were pipetted onto two to four leaves per plant. Control plants were not inoculated, but were otherwise treated the same way.
  • the plants were then placed in a 20-liter container with a clear polystyrene lid and kept at high humidity for 24 h (72 h for fungal inoculation). Local leaves used for fungal inoculation were collected separately from the remaining (systemic) leaves.
  • the primer pairs used— RT498A/RT498B, RT1075A RT1075B, RT1177A/RT1177B, RT1593A/RT1593B, RT2007A RT2007B, RT2149A/RT2149B, RT528A/RT528B, RT1865A/RT1865B and RT2339A/RT2339B— are listed in SEQ ID NO: 28 to SEQ ID NO: 45 in that order. These were used to amplify 80-100 bp for each of the Arabidopsis gene transcripts downstream of the previously used promoter sequences listed in SEQ ID NO: 1 to SEQ ID NO: 9, respectively.
  • the primers used for beta-actin amplification are listed as SEQ ID NO: 46 to SEQ ID NO: 49.
  • the data of Table 1 below is a summary of the following table summarises the expression profiles as relative induction ratios for each treatment and each gene (marked with the corresponding promoter sequence).
  • Leaves were cut from plants (Arabidopsis thaliana cv. Columbia or Brassica napus cv. Westar) that were cultivated in controlled environment rooms at 24-20 °C day and night temperature and a photoperiod of 16 h light. These were placed upside down on petridishes containing moistened 3MM filter paper (Whatman). Gold particles (Biorad) with a diameter of 1.6 ⁇ m or tungsten particles (Biorad) with a diameter of 0.7 ⁇ m were used as the carrier for DNA, that were prepared by washing in 70% ethanol, vortexing for 3 min, incubating for 15 min and removing the liquid after 30 s of centrifugation.
  • Gold particles Biorad
  • tungsten particles Biorad
  • plasmid constract used for particle bombardment (p498GUS, pl075GUS, pll77GUS, pl593GUS, p2007GUS, p2149GUS, p528GUS, pl865GUS or p2339GUS)
  • 50 ⁇ l of the gold or tungsten particle suspension were transferred into a sterile 1.5 ml centrifuge tube and vortexed thoroughly for 1 min.
  • the mixture was then vortexed for 1 min, incubated for 5-10 min at room temperature and pelleted by centrifugation for 10 s.
  • the pellet was resuspended in 12 ⁇ l of the supernatant for 1 min, and for each bombardment 3 ⁇ l portions were transferred onto grids of 3 mm
  • Swinney plastic syringe filter holders (Gelmann Sciences). For each DNA construct three bombardments were carried out on 8-14 leaves (2-4 leaves for canola or tobacco cells of 20 ml suspension concentrated on a circular filter paper) per bombardment at a distance of 17 cm using a Helium-driven pressure of 7 bar and a negative pressure of -0.85 bar in the chamber.
  • Plant material was then kept at room temperature (tobacco cells on MS medium- containing agar plates) with 16 hrs of illumination for 24 hrs before transferring the leaves to X-gluc-solution (1.25 g/1 5-bromo-4-chloro-3-indolyl- ⁇ -D glucuronic acid (dissolved in 50 ml/1 DMSO), 5 mM ferricyanide, 5 mM ferrocyanide, 0.3 % (v/v) Triton X-100, 0.1 M sodium phosphate buffer pH 7.0) and incubation at 37°C for 12 hrs. GUS activity measured by the number and size of blue spots was used to assay promoter activity. Three sets of experiments were carried out for each promoter-reporter gene constract.
  • FIG. 1 depicts typical photographs or scans of Arabidopsis leaves that were bombarded with p498GUS, pl075GUS, pl l77GUS, pl593GUS, p2007GUS, p2149GUS, p528GUS, pl865GUS or p2339GUS.
  • the results of EXAMPLE 4 demonstrate that the promoter sequences shown in SEQ ID No: 1 to SEQ ID NO: 9 are functional as biologically active promoters with different expression profiles. The results of EXAMPLE 4 further demonstrate that these sequences can provide valuable tools for gene expression in plant cells and genetic engineering.
  • Agrobacterium tumefaciens Genetic transformation of Arabidopsis thaliana plants with Agrobacterium tumefaciens was performed using the "floral dip" transformation method (Clough and Bent 1998, Plant J., 16:735-743). Briefly, A. thaliana plants (ecotype Columbia) were grown in pots under long days until flowering. Agrobacterium harbouring each of the promoter- reporter fusion constract was grown in a large culture at 28 °C in liquid LB medium with 50 mg/L kanamycin and 5 mg/L tetracycline.
  • Transgenic Arabidopsis plants expressing the P498 promoter fragment constructs pAOV498GUS and pG498GFP
  • the P2007 promoter fragment consisttracts pG2007GUS and pG2007GFP
  • Transgenic plants containing the promoter fragment P498 showed inducibility following inoculation with Alternaria brasssicicola spores ( Figure 3 A). Reporter gene expression was localised in root, stem and leaf tissue. The strongest expression was observed in mature and senescing parts of the leaves and in root tips and vascular root tissue.
  • Figure 3B shows GUS expression in senescing parts of a leaf.
  • a method using an in vivo reporter gene system was established that can be used to screen for gene products that interact directly or indirectly with plant defence-associated promoter sequences.
  • transgenic Arabidopsis thaliana lines were used (Manners et al. 1998, Plant Mol. Biol. 38:1071-1080) that express the uidA reporter gene controlled by the promoter of the PDFl.2 gene from Arabidopsis thaliana.
  • genes were cloned from genomic DNA of Arabidopsis thaliana. These included ERF1, COI1 and NPRl with the cDNA sequence Genbank accession numbers, AF076278, AF036340, U76707, respectively (Solano et al. 1998, Genes and Development 12:3703-3714; Xie et al. 1998, Science 280:1091-1094, Cao et al.
  • Genomic DNA was isolated and purified from Arabidopsis thaliana cv. Columbia leaf tissue as described in EXAMPLE 1. DNA fragments were amplified using the above primer and the Expand High Fidelity PCR System (Roche) with 10 ng of genomic DNA as template according to the manufacturer's instructions. After migration on DNA agarose gels, PCR bands were purified, ligated into the vector pCR-Blunt (Invitrogen) using a Rapid DNA Ligation Kit (Roche) and transformed into competent Escherichia coli OneShot Top 10 cells (Invitrogen). Colonies were screened for correct inserts and orientations using an internal PCR product-specific primer and an external vector-based primer.
  • the plasmid pBI221 (see EXAMPLE 1) was used as the basis for the construction of promoter-reporter cassettes that can be used for plant cell transformation using biolistic or PEG-mediated transformation techniques.
  • the plasmids p35SERFl, p35SCOIl and p35SNPRl contain the cauliflower mosaic virus 35S promoter fused to the genes ERF1, COI1 and NPRl, respectively, instead of the uidA reporter gene.
  • the plasmids p35SERFl and p35SNPRl were constracted by ligating the EcoRV/SacI-cut gene fragments that were previously cloned into pCR-Blunt into the SmaT/SacI-cut 3.9 kb fragment of pBI221.
  • the plasmid p35SCOIl was constructed by ligating the blunt-ended Hindlll/EcoRV-cut COI1 fragment that was previously cloned into pCR-Blunt into the blunt-ended dephosphorylated BamHI-cut 3.9 kb fragment of p35SNPRl.
  • Particle bombardment was carried out on leaves and whole plants from the above described transgenic Arabidopsis thaliana plants containing the PDF1.2 promoter and the uidA reporter gene that were cultivated in controlled environment rooms as described above.
  • Preparation of gold or tungsten particles, coating DNA to gold or tungsten particles and particle bombardments were carried out using the experimental procedure described in EXAMPLE 4.
  • p35SERFl, p35SCOIl or p35SNPRl For each plasmid constract that was used for particle bombardment (p35SERFl, p35SCOIl or p35SNPRl), a set of three DNA deliveries were carried out on 8- 14 leaves or whole plants per bombardment at a distance of 17 cm using a Helium-driven pressure of 7 bar and a negative pressure of -0.85 bar in the chamber.
  • FIG. 5 shows a photograph of a transgenic Arabidopsis thaliana plants containing the PDF1.2 promoter and the uidA reporter gene that was subsequently transformed with p35SERFl using a biolistic transient expression assay.
  • EXAMPLE 6 demonstrate that it was possible to screen for genes and their products that induce a plant defence-associated promoter in a transient assay. One out of the three genes that were screened for using this assay was found to positively induce the plant defence-associated promoter.
  • This method allows the identification of genes and gene products that interact with a promoter of choice, such as the promoters described in EXAMPLES 1-4 fused to a reporter gene, using transgenic reporter plants.
  • a rapid high- throughput screening could also be carried out on a large scale where hundreds or possibly thousands of genes and gene products could be screened with regard to their direct or indirect interaction with a plant defence-associated promoter.
  • the promoter sequences included in SEQ ID NO: 1 to SEQ ID NO: 9 were analysed for putative cis-acting elements to obtain some insight in the possible regulation mechanisms of these promoters.
  • Putative cts-acting promoter elements were identified by comparison with other promoter sequences or by using the PLACE database which describes elements from vascular plants (Higo et al 1999, Nucl Acids Res. 27, 297-300; Preshidge 1991, CABIOS 7, 203-206). Putative elements were checked for relevance (e.g. necessity of a repeat, distance between repeats, flanking sequences, distance from TATA-box) by comparison with literature description of the elements. Direct repeats and inverted repeats were identified using the "EREPEAT” and the "INVERTED” program, respectively, provided by WebANGIS (Australian National Genomic Information Service).
  • Figures 7-14 depict putative promoter elements in the core promoter region (approximately 800 bp of the right border of the promoter sequences included in SEQ ID
  • GT-1 consensus sequence (GT1) (Terzaghi and Cashmore 1995, Annu. Rev.
  • NtBBFl binding site (NTBBF1) (Baumann et al. 1999, Plant Cell

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Botany (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Plant Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

The invention provides plant promoters that are associated with and regulated by mechanisms occurring during plant defence. Representative sequences can be isolated from Arabidopsis thaliana and have sequences that are included in SEQ ID NO: 1 to SEQ ID NO: 9. The invention also provides DNA constructs comprising a gene under the operation of the subject promoters. Further provided are plant cells having a construct according to the invention as part of the genomes thereof and plants comprising such cells. Also provided is a method of identifying compounds that modulate the activity of promoters according to the invention.

Description

PROMOTERS OF PLANT DEFENCE- ASSOCIATED GENES
TECHNICAL FIELD
This invention relates to plant promoters that are associated with and regulated by mechanisms occurring during plant defence and that confer gene expression on transgenic plants harbouring the promoters. The invention also relates to utilisation of the promoters in the construction of recombinant genes for plant transformation to enable expression of the product of the gene at a certain time, in a certain tissue, at a certain rate or during a certain physiological state, hi particular, the invention relates to promoters isolated from regulatory upstream sequences of different genes of Arabidopsis thaliana that are directly or indirectly associated with plant defence mechanisms and that are potentially induceable by regulatory processes occurring during plant defence.
BACKGROUND ART
Genetic engineering of plants has proven to be an alternative method for plant breeding and for the introduction of new desirable traits that are reflected in altered phenotypes. In addition, it provides a valuable tool for biological research.
Plant genetic manipulation focuses on the cellular level of organisation and involves the interfacing of all aspects of cell biology, molecular biology and gene transfer procedures (Sharp et al. 1984, Food Technology, (Feb.) 112-119; Martin 1998, Curr. Opin. Biotechnol. 9, 220-226). The genetic engineering tools of tissue culture, somaclonal and gametoclonal variation, cellular selection procedures and recombinant DNA are either indirectly or directly concerned with the enhanced expression and transfer of genes. An essential prerequisite for this is the choice of a suitable promoter that results in the desired rate, location and time of gene expression, hi many applications of plant genetic engineering a moderate or strongly regulated promoter is required to ensure that a sufficient amount of gene product is produced at the right time, in the right tissue and during the right physiological state of the plant. These applications include genetic manipulation of plants to obtain disease resistance or tolerance against plant-infecting viruses, bacteria, fungi or nematodes, to obtain resistance against herbivores, to obtain resistance against herbicides and selectable marker reagents, to obtain resistance against abiotic factors (e.g. drought, salt, cold, heavy metals, chemicals, anaerobic conditions), to conduct functional analyses of genes and gene products for research, to confer silencing or enhancement of genes and gene products (modulation of gene expression), to modify the composition of macromolecules and secondary metabolites (e.g. to increase nutritional value or to alter structural composition), to modify plant development, to improve fruit or crop quality (e.g. post harvest shelf life or disease resistance), to obtain industrial plants (e.g. plants producing biodegradable plastics, industrial enzymes, antibodies) and to induce or modify regulatory processes occurring during plant defence.
The function and mode of action of promoters have been studied extensively in both monocot and dicot plants. In most cases reporter genes such as the uidA gene encoding for β-glucuronidase (GUS, Jefferson et al. 1987, EMBO J. 6, 3901-3907) or genes encoding anfhocyanin production or the jellyfish green fluorescent protein (GFP, Chalfie et al. 1994, Science 263, 802-805) or luciferase (Himes et al. 2000, Methods Mol. Biol 130:165-174) are used to assay promoter activity in transient or stable gene expression systems. Highly regulated expression patterns have been demonstrated for several promoters, even though there is no absolute specificity in most instances. These include light-inducible and leaf- specific promoters, seed-specific promoters, meristem-specific promoters, root-specific promoters, flower-specific promoters, horrnone-inducible promoters, pathogen-inducible promoters, chemically induced promoters and promoters of regulatory genes involved in signal transduction processes. For many purposes in plant genetic engineering a promoter that is regulated to some extent is required to ensure the desired expression pattern. Regulatory mechanisms of gene expression can lead to complex signal transduction processes. Some of these processes have begun to be addressed in plant defence signalling studies (Yang et al. 1997, Genes & Development 11, 1621-1639). Active disease resistance in plants is dependent on the ability of the host to recognize pathogens and initiate defence mechanisms that limit infection. Resistance in the host is often manifested by a hypersensitive response (HR), which results in localised cell death at the site of infection. Other defence responses may include structural alterations and the production of a wide range of plant defence molecules such as antimicrobial proteins (see Broekaert et al. 1997, Crit. Rev. Plant Set 16, 297-323; Yun et al. 1997, Plant Breeding Reviews 14, 39-88; Grant & Mansfield 1999, Curr. Opin. Plant Biol. 2, 312-319 for recent reviews). In addition, plant responses to necrotrophic pathogens can lead to systemic acquired resistance (SAR), which immunises against subsequent infection. Endogenous signal molecules, such as salicylic acid play a key role in signalling for resistance (Delaney et al. 1994, Science 266, 1247-1250; Cao et al. 1997, Cell 88, 57-63). Recently, signal transduction pathways that involve jasmonates and ethylene as regulators of several defence-related genes have also been identified (Penninckx et al. 1996, Plant Cell 8, 2309-2323; Penninckx et al. 1998, Plant Cell 10, 2103-2114; Pieterse et al. 1998, Plant Cell 10, 1571-1580; Thomma et al. 1998, Proc. Natl. Acad. Sci. USA 95, 15107-15111; Clarke et al. 1998, Plant Cell 10, 557-569; Reymond & Farmer 1998, Curr. Opin. Plant Biol. 1, 404- 411). Cross-talk between defence pathways mediated by salicylates, jasmonates, ethylene and pathogen infection has been proposed (Penninckx et al. 1998, Plant Cell 10, 2103-2114; Thomma et al. 1998, Proc. Natl. Acad. Sci. USA 95, 15107-15). Several inducible promoters of genes associated with plant defence have been patented for the purpose of plant genetic manipulation (e.g. the promoter of PDF1.2 plant defensin gene, WO 98/00023, "Plant Protection Method"). However, any promoter that can be regulated to some extend during plant defence has the potential to be used as a tool towards resistance against pathogens or herbivores. These include the use of the promoter to drive expression of genes increasing resistance (e.g. genes encoding anti-microbial peptides, genes encoding insecticidal proteins, genes encoding proteins with recognition specificities such as natural resistance (R) genes, genes encoding pathogenesis related PR protein, genes involved in apoptosis, genes involved in phytoalexin production, and regulatory genes that induce local and/or systemic , resistance), the use of the promoter to screen for plant defence-inducing chemicals, and the use of the promoter operatively-linked with another gene regulatory mechanism or gene expression system.
SUMMARY OF THE INVENTION It is an object of the present invention to provide promoters operative in plant cells which can be used in genetic engineering for regulable gene expression.
It is a further object of the invention to provide at least part of a chimaeric gene comprising one or more of the described promoters operatively linked to DNA encoding an RNA and/or polypeptide. According to a first embodiment of this invention, there is provided a promoter operative in a plant cell, said promoter comprising: 1) isolated DNA from Arabidopsis thaliana having a sequence as defined in any one of Figures 1 to 6;
2) isolated DNA which is a plant genome-derived variant of the DNA of ( 1 ) ;
3) a promoter-active portion of the isolated DNA of ( 1 ) or (2); 4) a regulatory cw-acting sequence element of the isolated DNA of (1) or (2);
5) isolated DNA which hybridizes under stringent conditions to the DNA of (1) or (2); or
6) a promoter-active portion of the isolated DNA of (5) .
According to a second embodiment of this invention, there is provided a DNA construct comprising at least one gene having at least one promoter according to the first embodiment operatively linked to a coding sequence, which coding sequence encodes a desired product or products.
According to a third embodiment of the invention, there is provided a DNA construct comprising: 1) a first gene having one or more promoters according to the first embodiment operatively linked to a coding sequence of interest; and
2) a second gene having a promoter operatively linked to a coding sequence, wherein the expression product of said coding sequence modulates activity of the expression product of said first gene coding sequence. According to a fourth embodiment of the invention, there is provided a method of expressing a product in a plant cell, said method comprising introducing a DNA construct according to the second or third embodiments or an RNA transcript of said construct into cells of a plant, wherein said DNA construct or RNA transcript coding sequence encodes said product. According to a fifth embodiment of the invention, there is provided a plant cell, wherein the genome of said plant cell includes a DNA construct according to the second or third embodiments. According to a sixth embodiment of the invention, there is provided a plant, plant tissue or reproductive material of a plant, wherein said plant, plant tissue or reproductive material comprises cells according to the fifth embodiment.
According to a seventh embodiment of the invention, there is provided a method of identifying a chemical or regulatory molecule which modulates the activity of a promoter according to the first embodiment, the method comprising the steps of: introducing a DNA construct according to the second embodiment into a plant cell; contacting said plant cell with said chemical or regulatory molecule; and determining the effect of said chemical or regulatory molecule on the activity of said promoter.
In yet another embodiment, there is provided a chemical or regulatory molecule identified by the method of the seventh embodiment.
The invention includes within its scope the DNAs of the first to third embodiments as single-stranded DNA, double-stranded DNA, or as the RNA corresponding to either strands of the foregoing DNA.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 comprises schematic representations of promoter-reporter gene constructs used in following examples.
Figure 2 comprises photographs of transient promoter activity assays in Arabidopsis thaliana plants using promoter-reporter gene constructs p498GUS, pl075GUS, pll77GUS, pl593GUS, p2007GUS> p2149GUS, p528GUS, pl865GUS or p2339GUS.
Figure 3 comprises photographs of transgenic Arabidopsis thaliana plants expressing a plant defence-associated promoter fused to reporter genes. Here the expression is regulated by the promoter sequence 498 of promoter reporter gene constructs pAOV498GUS or PG498GFP.
Figure 4 comprises photographs which show tissue specific expression in transgenic Arabidopsis thaliana plants. Figure 5 comprises photographs of a reporter plant used for screening of plant defence-associated gene products. Here a transgenic Arabidopsis thaliana plant containing the plant defence-associated PDFl.2 promoter fused to the uidA reporter gene was subsequently transformed with p35SERFl in a transient biolistic assay. Figures 6 to 14 are sequences of putative promoter elements in parts of the promoter sequences shown in SEQ ID NO: 1 to SEQ JD NO: 9, respectively.
BEST MODES AND OTHER MODES OF PERFORMING THE INVENTION
The following abbreviations are used throughout this specification:
GUS β-glucuronidase GFP green fluorescent protein
PCR polymerase chain reaction
PEG polyethylene glycol
SDS sodium dodecyl sulphate
EDTA ethylenediaminetetraacetic acid P498 DNA promoter region according to SEQ ID NO: 1
P 1075 DNA promoter region according to SEQ ID NO : 2
PI 177 DNA promoter region according to SEQ ID NO: 3
PI 593 DNA promoter region according to SEQ ID NO: 4
P2007 DNA promoter region according to SEQ ID NO: 5 P2149 DNA promoter region according to SEQ ID NO: 6
P528 DNA promoter region according to SEQ ID NO: 7
P 1865 DNA promoter region according to SEQ ID NO: 8
P2339 DNA promoter region according to SEQ ID NO: 9
Nos Terminator of the nopaline synthase gene of Agrobacterium tumefaciens
So that terms used throughout the description will have a clear and consistent meaning, the following definitions are provided:
Coding sequence: Nucleic acids sequence that encodes a functional RNA transcript which may or may not be subsequently translated into a polypeptide. Homologue: Nucleic acid sequences from other organisms that show a sequence identity (homology) of 75% or more to the sequences or parts thereof that are longer than 300 bp of SEQ ID NO: 1 to SEQ ID NO: 9 and have substantially the same function.
Plant defence: A number of physiological and molecular events (e.g. hyper-sensitive response, synthesis of antimicrobial compounds, cell wall modification etc.) initiated in the host as a result of interaction with a pathogenic organism.
Plant defence-associated signalling molecule: Molecules such as salicylates (e.g. SA), jasmonates (e.g. JA), ethylene etc. that are capable of initiating signal transduction events that lead to a subset of specifically induced defence responses. Plant genome-derived variant: DNA that is present in the plant genome and has a sequence identity (homology) of 75% or more to the promoter sequences shown in Figure 1- 6 or parts thereof that are longer than 300 bp.
Promoter: A DNA sequence flanking a coding sequence of a gene at the 5' end thereof which includes an element or elements involved in the initiation of transcription of the coding sequence.
Regulated promoter: A promoter which mediates higher or lower transcriptional activity upon direct or indirect interaction with a regulatory molecule (e.g. transcription factors, signalling chemicals).
Signal transduction pathway: The process by which the information contained in an extracellular physical, chemical or biological signal is received at the cell by the activation of specific receptors and conveyed across the plasma membrane, and along and intracellular chain of signalling molecules, to stimulate the appropriate cellular responses.
The one letter code for nucleotides in DNA conforms to the IUPAC-IUB standard described in The Biochemical Journal 219, 345-373 (1984). Percentages in the examples are given in weight/volume (w/v) unless otherwise stated.
The present inventors have identified nine promoter sequences in PCR-amplified genomic DNA sequences of Arabidopsis thaliana Columbia.
These promoters, as well as homologues comprising plant genome-derived variants, can be used separately or in combination with appropriate coding sequences to prepare transgenic plant cells and plants capable of expression of the gene(s) of interest at a suitable level.
DNA comprising the nine promoters according to the invention can be obtained by cloning genomic DNA from Arabidopsis thaliana Columbia. Genomic DNA isolated from Arabidopsis can be fragmented with restriction enzymes and fragments can be subcloned into plasmids that can be multiplied in Escherichia coli. Alternatively, these promoter sequences can be generated by direct polymerase chain reaction (PCR) amplification of genomic DNA. The required primers can be designed from the sequence data presented in SEQ ID NO: 1 to SEQ ID NO: 9 or equivalent sequence information. Yet another method of producing promoters having sequences such as those present in SEQ ID NO: 1 to SEQ ID NO: 9 is by DNA synthesis. This is particularly the case if a promoter active portion of a larger promoter sequence is desired where oligonucleotides from 10 to 100 nucleotides can be conveniently synthesised. Complementary oligonucleotides can also be synthesised to form a double-stranded molecule of the desired nucleotide sequence. As indicated above, the invention comprises not only promoters having the sequences of SEQ ID NO: 1 to SEQ ID NO: 9 but also homologues from plant genome-derived variants (e.g. of other members of a gene family or from related species) of the sequences of SEQ ID NO: 1 to SEQ ID NO: 9, as well as DNAs which hybridize with the DNA sequences of SEQ ID NO: 1 to SEQ ID NO: 9 under stringent conditions. Homologues and allelic variants can have identity with the DNA sequences of SEQ ID NO: 1 to SEQ ID NO: 9 as low as 75%. The stringent conditions under which a promoter according to the invention will hybridize with the DNA sequence of SEQ ID NO: 1 to SEQ ID NO: 9 can be defined as follows:
Wash solution 0.1 x SSPE*, 0.1 % SDS Wash temperature 65 °C
Number of washes two *(1 x SSPE is 180 mM NaCl, 10 mM NaH2PO4, 1 mM EDTA (pH 7.4)).
DNA constructs according to the second and third embodiment can include more than one promoter operatively-linked to the coding sequence. These additional promoters can be identical or derivatives of the same promoter or heterologous promoters, hi addition, operatively-linked enhancer or silencer elements can be included in DNA constructs. In the DNA constructs of the second and third embodiments, the coding sequence to which the promoter(s) are operatively linked can encode an RNA which functions as antisense RNA or a ribozyme or as a stractural component, or is translated into a polypeptide which functions as an enzyme, a stractural component or has some other physiological effect. Examples of fransgene products which can be usefully expressed in transgenic plants using the promoters described in the invention are products that help:-
1) to obtain disease resistance or tolerance against plant-infecting viruses, bacteria, fungi or nematodes;
2) to obtain resistance against herbivores (e.g. insects);
3) to obtain resistance against herbicides and selectable marker reagents; to obtain resistance against abiotic factors (e.g. drought, salt, cold, heavy metals, chemicals, anaerobic conditions);
5) to conduct functional analyses of genes and gene products for research;
6) to confer silencing or enhancement of genes and gene products (modulation of gene expression);
7 to modify the composition of macromolecules and secondary metabolites (e.g. to increase nutritional value or to alter stractural composition);
8) to modify plant development;
9) to improve fruit or crop quality (e.g. post harvest shelf life or disease resistance);
10) to produce industrial substances (e.g. biodegradable plastics, industrial enzymes, antibodies); and
11) to induce or modify regulatory processes occurring during plant defence .
With reference to the third embodiment of the invention, the first gene of the DNA construct includes all the variations and options of the gene comprising the DNA constract of the second embodiment. The second gene of the DNA constract can have an expression product which either: -
1) complements or enhances the effect of the expression product of the first gene;
2) counteracts the expression product of the first gene; or 3) modifies the activity of the first gene promoter(s).
These options allow a high regulation of gene expression by linkage of a second gene with the strong expression mediated by the promoter(s) of the first gene.
It will be noted from the Summary of the Invention that the invention includes transgenic plant cells with genetically engineered genomes including the DNA constructs of the second and third embodiment. These DNA constructs can also comprise recombinant viral sequences with one or more coding sequences of interest that either stably or transiently express in plant cells. Alternatively, RNA transcripts can be made from these constructs that can be used for transformation of plant cells. Techniques for introducing DNA into the genome of a plant are well known in the art and are described, for example by Sagi et al. (1995, Bio/Technology 13; 481-485), May et al. (1995, Bio/Technology 13; 485-492) and Zhong et al. (1996, Plant Physiol. 110; 1097- 1107), the entire contents of which are incorporated herein by cross-reference.
DNA constructs according to the invention are advantageously introduced into the genome of target plant cells using methods including Agrobacterium-mediateά transformation, biolistic bombardment with DNA-coated tungsten or gold particles, electroporated or polyethylenglycol (PEG)-mediated DNA transformation of protoplasts and other mechanical DNA transfer techniques. Transgenic plant cells including the DNA constructs of the invention can be propagated using conditions appropriate to the particular plant. Similarly, whole plants, or propagating material of the plant, can be prepared from the initial transgenic cells using known methods and conditions. Alternatively, RNA can also be used for transformation using the above methods.
With reference to the seventh embodiment of the invention, methods can be used for screening of chemicals or regulatory molecules that interact directly or indirectly, in vivo or in vitro with a DNA sequence according to the first embodiment. Different chemicals, such as salicylic acid, jasmonic acid, oxalic acid, abscisic acid, ethylene, 2,6-dichloroisinicotinic acid (BSfA), benzothiadiazole (BTH), nitric oxide, polyacrylic acid, β-aminobutryic acid (BABA) and the like have been associated with plant defence and are known to induce certain biochemical signal transduction pathways (Malamy et αl. 1990, Science 250:1002- 1004, Reymond and Farmer 1998, Curr Opin Plant Biol 1 :404-411; Dong 1998, Curr. Opin. Plant Biol. 1:316-323; Hammond-Kosack and Jones 1996, Plant Cell 8:1773-1791; Enyedi et al. 1992, Cell 70:879-886; Lawton et al. 1996, Plant J. 10:71-82; Delaney et al. 1995, Proced. Natl. Acad. Sci, USA, 92:6602-6606; Delledonne, et al. 1998, Nature 394:585-588; Zimmerli et al. 2001, Plant Physiol. 126:517-523). Using these chemicals for external applications on plants has previously been shown to increase disease resistance (Lawton et al. 1996, Plant J. 10:71-82; Delaney et al. 1995, Proced. Natl. Acad. Sci, USA, 92:6602- 6606; Zimmerli et al. 2001, Plant Physiol. 126:517-523).
Nucleic acid sequences with reference to the first embodiment of the invention comprise the promoters of genes that are likely to be associated with plant defence signalling. They can be used for screening of a large number of different chemicals (including proteins such as transcription factors or plant defence elicitors) that interact directly or indirectly with these sequences and may lead to a modification, induction or repression of different plant defence pathways that could have important applications in disease management and plant protection. High-throughput in vitro binding assays can be used to screen for chemicals or proteins that interact directly with the sequences according to the first embodiment. Different plant cell extracts can be used for initial screenings followed by more defined analyses. The well-established yeast two-hybrid system offers an effective method to search for interacting proteins in vivo (Luban and Goff 1995, Curr. Opin. Biotechnol. 6, 59-64). Other in vivo systems, such as reporter plants, can be used to identify chemicals and proteins that interact directly or indirectly with the promoter sequences with reference to the first embodiment of the invention (see the examples below). Transgenic reporter plants contain a reporter gene (e.g. uidA or gfp) which is controlled by a promoter according to the first embodiment.
Tissue extracts, chemicals, proteins, RNA or DNA can be applied to the plants using different methods that are well-established in the art such as spray application, vacuum- infiltration, Agrobacterium-m.edia.ted transformation, agro-infection or coated particle bombardment (Feldmann and Marks 1987, Mol. Gen. Genet. 208, 1-9; Finer et al. 1992, Plant Cell Rep. 11, 323-328; Bechtold and Pelletier 1998, Meth. Mol. Biol. 82, 259-266). These substances can then directly or indirectly interact with the promoter sequences of the first embodiment and will result in increased or reduced production of the reporter gene constract. This can be monitored visually, histochemically and fluorometrically using quantification methods that are well-established in the art (Jefferson 1987, Plant Mol. Biol. Rep. 387-405; Schledzewski and Mendel 1994, Transgen. Res. 3, 249-255; Schenk et al. 1998, Plant Mol. Biol. Rep. 16, 313-322; Remans et al. 1999, Plant Mol. Biol. Rep. 17, 385- 395). So that the invention may be better understood, several non-limiting examples follow.
General Methods
Manipulation of DNA was carried out using known methods such as those described by Sambrook et al. (1989, Molecular Cloning: a Laboratory Manual, 2nd Ed., Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY). Reagents and other materials were obtained from commercial sources or as otherwise indicated.
EXAMPLE 1
Cloning of the novel promoter sequences
Nine genes were selected from Arabidopsis thaliana on the basis that they could be associated with plant defence. The following cDNA clones listed as Genbank accession numbers were chosen: R65416, N38380, T04295, N65692, R30178, N38552, H36431, T45846, T42042. Database searches using Arabidopsis thaliana genomic sequences led to the identification of the corresponding putative genes and their putative upstream promoter sequences. Based on this sequence, specific primers were designed for amplifying upstream putative promoter sequences of approximately 2 kb. These primer pairs, relative to the above Genbank accessions, were: P498A/P498B, P1075A/P1075B, P1177A/P1177B, P1593A/P1593B, P2007A/P2007B, P2149A/P2149B, P528A/P528B, P1865A/P1865B, P2339A/P2339B. The sequences of these primers are listed in SEQ ID NO: 10 to SEQ ID NO: 27. The complete putative promoter sequences with the corresponding primer sequences on both ends are shown in SEQ ID NO: 1 to SEQ ID NO: 9. Genomic DNA was isolated and purified from Arabidopsis thaliana cultivar
Columbia leaf tissue according to the method described by Graham et al. (1994, Biotechniques 16: 48-50). DNA fragments of the expected size were amplified using the above primer pairs and the Expand High Fidelity PCR System (Roche) with 10 ng of genomic DNA as template according to the manufacturer's instructions. After migration of DNA on agarose gels, PCR bands were purified, hgated into the vector pCR-Blunt (Invitrogen) using a Rapid DNA Ligation Kit (Roche) and transformed into competent Escherichia coli OneShot ToplO cells (Invitrogen). Colonies were screened for correct inserts and orientations using an internal PCR product-specific primer and an external vector-based primer. Sequencing was carried out to confirm the identity of the cloned PCR products using primers and primer sites present in the vectors and later in the obtained sequence using the BigDye Terminator Premix (PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit, Applied Biosystems) and an Automated DNA (377) Sequencer (Applied Biosystems). The complete cloned genomic DNA sequences of all nine PCR products comprise SEQ ID NO: 1 to SEQ ID NO: 9. EXAMPLE 2
Construction of chimaeric genes
Several constructs were made using the above-described PCR products as promoters in fusion with the uidA reporter gene as shown in Figure 1.
The plasmid pBI221 (Stratagene) was used as the basis for the construction of promoter-reporter cassettes that can be used for plant cell transformation using biolistic or PEG-mediated transformation techniques. pBI221 contains the 35S promoter from cauliflower mosaic virus (Odell et al, 1985, Nature 313; 810-812), the uidA reporter gene (Jefferson et al. 1987, EMBO J. 6, 3901-3907) and the nopaline synthase (nos) terminator sequence from Agrobacterium tumefaciens in pUCl 18. The plasmids p498GUS, pl075GUS, pll77GUS, pl593GUS, p2007GUS, p2149GUS, p528GUS, pl865GUS and ρ2339GUS contain the putative promoter sequences shown in SEQ ID NO: 1 to SEQ ID NO: 9, respectively, (putative TATA boxes at 3' end) instead of the cauliflower mosaic virus promoter (Figure 1). The plasmids p498GUS, pl075GUS, pl593GUS, p2007GUS, p2149GUS, p528GUS, pl865GUS and p2339GUS were constructed by ligating the HindllT/Xbal-cut putative promoter fragments that were previously cloned into pCR-Blunt into the HindltT/Xbal-cut 5.1 kb fragment of pBI221. The plasmid pl l77GUS was constracted by ligating the blunt-ended MluI/Notl-cut putative promoter fragment that was previously cloned into pCR-Blunt into the blunt-ended dephosphorylated BamHI-cut 5.1kb fragment of ρ498GUS. The plasmids ρ528GUS, ρl865GUS and p2339GUS were constracted by ligating the blunt-ended EcoRI-cut putative promoter fragments that were previously cloned into pCR-Blunt into the blunt-ended dephosphorylated BamHI-cut 5.1kb fragment of p498GUS.
Furthermore, the binary vector pAOV (Mylne and Botella, 1998, Plant Mol. Biol. Rep. 16, 257-262) and the pGreenH derivative pGREEN0229 (Hellens et al. 2000, Plant Mol. Biol. 42:819-832) were used as the basis for the construction of promoter-reporter cassettes that can be used for Agrobacterium-mediated plant transformation (Figure 1). The plasmid pAOV498GUS was constracted by ligating the HindHI SacI-cut promoter-reporter cassettes from p498GUS into the HindJJI/SacI-cut 27kb fragment of pAOV. The plasmids pG35SGUS and pG35SGFP were used as the basis for the construction of pGreenJJ-derived plasmids containing the uidA reporter gene or the sgfp(S65T) reporter gene (Chui et αl. 1996, Curr. Biol. 6:325-330), respectively. The plasmid pG35SGUS was constructed by ligating the blunt-ended HindlJJ EcoRI-cut 35S-GUS-nos cassette from pBI221 into the EcoRV-cut 4.5kb fragment of ρGREEN0229. The plasmid pG35SGFP was constracted by ligating the blunt-ended Pstl-cut GFP fragment from pUBIGFP (Elliott et αl. 1999, Plant Cell Rep. 18:707-714) into the EcoRV-cut 4.5kb fragment of pGREEN0229. Subsequently, the plasmids pG498GUS, pG1075GUS, pG1177GUS, pG2007GUS, pG1865GUS and pG2339GUS (Figure 1) were constracted by replacing the 35S promoter fragment of pG35SGUS with the corresponding PCR-amplified putative promoter fragments from Arabidopsis (see above). Therefore the PCR-amplified putative promoter fragments were phosphorilated (polynucleotide kinase, Roche) and ligated into the blunt-ended Xbal-cut 6.6kb fragment of pG35SGUS.
The plasmids pG498GFP, pG1075GFP, pG1177GFP, pG1593GFP, pG2007GFP, pG528GFP and pG1865GFP (Figure 1) were constracted by replacing the 35S promoter fragment of pG35SGFP with the corresponding PCR-amplified putative promoter fragments from Arabidopsis (see above). Therefore the PCR-amplified putative promoter fragments were phosphorilated (polynucleotide kinase, Roche) and ligated into the blunt-ended Xbal- cut 5.5kb fragment of pG35SGFP.
All plasmid DNA was prepared from Escherichia coli DH5α or Escherichia coli TOP 10 (Invitrogen) using the Qiaprep Spin Miniprep Kit (Qiagen). These chimaeric gene constructs are suitable to assess the promoter activity using in vivo transient and stable expression systems that were developed and optimised for this purpose.
EXAMPLE 3 Plant defence-associated gene expression
Expression patterns of the native genes driven by each promoter sequence described in EXAMPLE 1 and EXAMPLE 2 (SEQ ID NO: 1 to SEQ ID NO: 9) were measured during plant defence responses in Arabidopsis to determine their transcriptional regulation by using quantitative real-time reverse transcriptase PCR. For this purpose, Arabidopsis thaliana plants were grown to 8-12 leaf stage in controlled enviromnent rooms and treated with either defence-inducing chemical signal compounds or an incompatible fungal pathogen. For treatments with salicylic acid (SA), plants were sprayed with a 5 mM solution. Methyl jasmonate treatments were carried out by taping a cotton ball containing 400 ul of a 0.5% solution in ethanol onto the wall of a 20- liter container wrapped in plastic bags. For ethylene treatments, 10 ml of ethylene was injected into the air of plants kept in a sealed 20-liter container. The control plants were treated in the same way but without the addition of SA, MJ or ethylene. For fungal inoculations, 5-μl drops of a spore suspension (5xl05 spores/ml in water) of Alternaria brassisicola (isolate UQ4273) were pipetted onto two to four leaves per plant. Control plants were not inoculated, but were otherwise treated the same way. The plants were then placed in a 20-liter container with a clear polystyrene lid and kept at high humidity for 24 h (72 h for fungal inoculation). Local leaves used for fungal inoculation were collected separately from the remaining (systemic) leaves.
To measure mRNA expression driven by each promoter, 1 μg of total RNA was purified from the leaves of treated and control Arabidopsis plants (Chirgwin et al. 1979, Biochemistry, 18:5294-5299) and was subsequently reverse-transcribed using Superscript II reverse transcriptase (Life Technologies) and an oligo(dT)23mer or the Multiscribe Reverse Transcription kit (Applied Biosystems) as recommended by the manufacturers. The resulting cDNA was subsequently taken up in a volume of 250 μl and SYBR green labelled PCR fragments were amplified by using primers designed from the coding sequence and over an RNA splice junction (if available) of each gene (Primer Express 1.5).
The primer pairs used— RT498A/RT498B, RT1075A RT1075B, RT1177A/RT1177B, RT1593A/RT1593B, RT2007A RT2007B, RT2149A/RT2149B, RT528A/RT528B, RT1865A/RT1865B and RT2339A/RT2339B— are listed in SEQ ID NO: 28 to SEQ ID NO: 45 in that order. These were used to amplify 80-100 bp for each of the Arabidopsis gene transcripts downstream of the previously used promoter sequences listed in SEQ ID NO: 1 to SEQ ID NO: 9, respectively. Real-time PCR using the ABI PRISM 7700 Sequence Detector and SYBR Green Master Mix (Applied Biosystems) was carried out using 2 μl (the equivalent of 4 ng total RNA) cDNA as template. PCR cycling conditions comprised an initial polymerase activation step at 95 C for 10 min followed by 45 cycles at 95 C for 15 sec and 59 C for 1 min. Real-time DNA amplification was monitored and analysed using the Sequence Detector 1.7 program. Differences in cycle numbers during the linear amplification phase between samples containing cDNA from treated and untreated plants were used to determine differential gene expression. Three beta-actine genes, actin-2, actin-7 and actin-8, from Arabidopsis were used as an internal standard to normalise differences in template amounts. The primers used for beta-actin amplification are listed as SEQ ID NO: 46 to SEQ ID NO: 49. The data of Table 1 below is a summary of the following table summarises the expression profiles as relative induction ratios for each treatment and each gene (marked with the corresponding promoter sequence).
Table 1
Treatment
Fungal inoculation Salicylic Methyl Ethylene
Promoter (Alternaria brassicicola) acid jasmonate sequence local leaf systemic leaves
P498 4x 64x 8x
P1075 2x
PI 177 2x 4x 4x
P1593 2x 4x
P2007 8x 4x
P2149 4x 2x 2x
P528 2x
P1865 2x 2x 2x
P2339 2x 16x
The highest induction ratio (64-fold) was observed following treatment with salicylic acid for the gene regulated by promoter sequence P498 which encodes a senescence- associated protein. The results of EXAMPLE 3 demonstrate that the expression of genes driven by promoters P498, P1075, PI 177, P1593, P2007, P2149, P528, P1865 and P2339 is associated with plant defence and plant defence signalling compounds.
EXAMPLE 4 Assaying promoter activity in plant cells under transient conditions
An in vivo test system to monitor transient reporter gene expression was available for assaying the promoter-reporter gene constracts of EXAMPLE 2. This was achieved using a method for biolistic gene transfer on leaves of different plant species (Arabidopsis thaliana and canola) or tobacco cell suspensions and subsequent histochemical analyses of reporter gene products (GUS assays).
Preparation of micro particles, coating DNA to micro particles and particle bombardments were carried out according to a modified procedure of Finer et al. (1992, Plant Cell Rep. 11, 323-328) using a custom-made Helium pressure-driven particle inflow gun.
Leaves were cut from plants (Arabidopsis thaliana cv. Columbia or Brassica napus cv. Westar) that were cultivated in controlled environment rooms at 24-20 °C day and night temperature and a photoperiod of 16 h light. These were placed upside down on petridishes containing moistened 3MM filter paper (Whatman). Gold particles (Biorad) with a diameter of 1.6 μm or tungsten particles (Biorad) with a diameter of 0.7 μm were used as the carrier for DNA, that were prepared by washing in 70% ethanol, vortexing for 3 min, incubating for 15 min and removing the liquid after 30 s of centrifugation. The following step was repeated three times: particles were resuspended in deionised sterile water, vortexed for 1 min, incubated for 1 min and pelleted down by spinning for 30 s in a microfuge. Subsequently particles were resuspended in sterile 50% (v/v) glycerol at a concentration of 60mg/ml for gold and lOOmg/ml for tungsten particles and vortexed for 1 min prior to use.
For each plasmid constract used for particle bombardment (p498GUS, pl075GUS, pll77GUS, pl593GUS, p2007GUS, p2149GUS, p528GUS, pl865GUS or p2339GUS), a set of three DNA deliveries was prepared: 50 μl of the gold or tungsten particle suspension were transferred into a sterile 1.5 ml centrifuge tube and vortexed thoroughly for 1 min.
While continuously vortexing, 8-15 μl DNA (0.2-0.6 μg/μl; freshly prepared using a
Qiaprep Mini spin kit), 50 μl sterile 2.5 M CaCl2 solution and 20 μl 0.1 M spermidine solution (sterile and stored in 20 μl aliquots at -80 °C prior to use) were added in that order.
The mixture was then vortexed for 1 min, incubated for 5-10 min at room temperature and pelleted by centrifugation for 10 s. The pellet was resuspended in 12 μl of the supernatant for 1 min, and for each bombardment 3 μl portions were transferred onto grids of 3 mm
Swinney plastic syringe filter holders (Gelmann Sciences). For each DNA construct three bombardments were carried out on 8-14 leaves (2-4 leaves for canola or tobacco cells of 20 ml suspension concentrated on a circular filter paper) per bombardment at a distance of 17 cm using a Helium-driven pressure of 7 bar and a negative pressure of -0.85 bar in the chamber.
Plant material was then kept at room temperature (tobacco cells on MS medium- containing agar plates) with 16 hrs of illumination for 24 hrs before transferring the leaves to X-gluc-solution (1.25 g/1 5-bromo-4-chloro-3-indolyl-β-D glucuronic acid (dissolved in 50 ml/1 DMSO), 5 mM ferricyanide, 5 mM ferrocyanide, 0.3 % (v/v) Triton X-100, 0.1 M sodium phosphate buffer pH 7.0) and incubation at 37°C for 12 hrs. GUS activity measured by the number and size of blue spots was used to assay promoter activity. Three sets of experiments were carried out for each promoter-reporter gene constract. Visualisation of GUS expression in planta after bombardment was achieved using a Nikon SMZ-2T stereo microscope. Photography was conducted with an Ektachrome 160-T slide film or Kodak Gold 100 ASA film or were scanned directly using a HP ScanJet 4c. All nine plasmids used, led to the observation of blue foci on the leaves while control experiments using no DNA showed no activity. Figure 2 depicts typical photographs or scans of Arabidopsis leaves that were bombarded with p498GUS, pl075GUS, pl l77GUS, pl593GUS, p2007GUS, p2149GUS, p528GUS, pl865GUS or p2339GUS.
Blue spots were monitored for all constracts used, demonstrating the functionality of the sequences listed in SEQ ID NO: 1 to SEQ ID NO: 9 as biologically active promoters. The strongest expression, indicated by the number and intensity of blue foci, was observed for the promoter constract p498GUS.
To evaluate inducibility of promoter constructs p498GUS, pl l77GUS, p2149GUS and p2339GUS, leaves were treated immediately after the bombardment with either salicylic acid, methyl jasmonate or ethylene using the above-mentioned concentrations. The plasmid p35SsGFP was co-bombarded at approximately one quarter of the promoter-reporter constract concentration and used as an internal standard as described previously (Schenk et al. 1998, Plant Mol, Biol. Rep. 16:313-322). The number of GFP expressing cells and GUS expressing foci was deteimined using a fluorescent microscope and the histochemical assay described above, respectively. Relative induction ratios were obtained by dividing the number of GUS producing foci by the number of GFP producing cells and subsequent normalisation. The results obtained are presented in Table 2. These results show that the highest induction ratio measured was for p498GUS. Table 2
Treatments Salicylic acid Methyl Ethylene jasmonate
P498 1.6x 4.9x
PI 177 ~ 1.2x 1.3x
P2149 l.Ox 1.9x
P2339 2.0x
The results of EXAMPLE 4 demonstrate that the promoter sequences shown in SEQ ID No: 1 to SEQ ID NO: 9 are functional as biologically active promoters with different expression profiles. The results of EXAMPLE 4 further demonstrate that these sequences can provide valuable tools for gene expression in plant cells and genetic engineering.
EXAMPLE 5
Expression and regulation pattern of reporter genes using plant defence-associated promoters in stably transformed plants Arabidopsis thaliana plants stably expressing the reporter genes uidA or sg ?(S65T) under the control of promoter regions, listed in SEQ ID NO: 1 to SEQ ID NO: 9 were generated using the Agrobacterium-mediated floral dip transformation method. The following binary DNA constracts described above and shown in Figure 1 were used: pAOV498GUS, pG498GUS, pG1075GUS, pG1177GUS, pG2007GUS, pG1865GUS, pG2339GUS pG498GFP, pG1075GFP, pG1177GFP, pG1593GFP, pG2007GFP, pG528GFP and pG1865GFP. Competent cells of Agrobαcterium tumefαciens strain AGL1 previously transformed with plasmid vector pSoup (Hellens et αl. 2000, Plant Mol. Biol. 42:819-832) were transformed by electroporation (Nagel et al. 1990, FEMS Microbiol Lett. 67:325-328) with each plasmid. Transformed Agrobacterium colonies were selected on LB- agar plates containing 50 mg/L kanamycin and 5 mg/L tetracycline. Colonies were screened for correct inserts and orientations using an internal PCR product-specific primer and an external vector-based primer.
Genetic transformation of Arabidopsis thaliana plants with Agrobacterium tumefaciens was performed using the "floral dip" transformation method (Clough and Bent 1998, Plant J., 16:735-743). Briefly, A. thaliana plants (ecotype Columbia) were grown in pots under long days until flowering. Agrobacterium harbouring each of the promoter- reporter fusion constract was grown in a large culture at 28 °C in liquid LB medium with 50 mg/L kanamycin and 5 mg/L tetracycline. Bacterial cultures were centrifuged and resuspended in 5 % (w/v) sucrose solution (OD600=0.8) containing 0.03% Silwet L-77 (Lehle Seed, IO). Aboveground parts of Arabidopsis plants were dipped into Agrobacterium solution for 2-3 sec with gentle agitation. Dipped plants were kept under a cover for 24 h after dipping and grown normally thereafter. The seeds collected from the dipped plants were planted to soil and two weeks after planting sprayed with Basta (2 ml L) to select for the transformed plants .
Transgenic Arabidopsis plants expressing the P498 promoter fragment (constructs pAOV498GUS and pG498GFP) or the P2007 promoter fragment (constracts pG2007GUS and pG2007GFP) were used as examples to analyse tissue specificity and pathogen inducibility. Transgenic plants containing the promoter fragment P498 showed inducibility following inoculation with Alternaria brasssicicola spores (Figure 3 A). Reporter gene expression was localised in root, stem and leaf tissue. The strongest expression was observed in mature and senescing parts of the leaves and in root tips and vascular root tissue. Figure 3B shows GUS expression in senescing parts of a leaf. This often includes the leaf tip and was visualised by GFP accumulation as seen under blue fluorescing light (showing GFP) in comparison with normal white light conditions where the whole can be seen (Figure 3E and F, respectively). Expression of sg ?(S65T) was not observed or at a very low level in the youngest leaf. Shown in Figure 3C and D is a comparison of a transgenic plant under normal white light condition and under blue light fluorescence, respectively. Figure 3G demonstrates GFP expression in the roots with highest expression in the root tip and vascular tissue.
Expression of the gfp reporter gene (constract pG2007GFP) was predominant in the roots, the hypocotyl and the two cotyledons (Figure 4). No expression was observed under the fluorescent microscope in any of the other leaves. Shown in Figure 4A and B is a comparison of a transgenic plant under normal white light condition and under blue light fluorescence, respectively. Figures 4C and 4D demonstrate GFP expression in the roots with highest expression in the root tip and vascular tissue. The results of EXAMPLE 5 show that the isolated DNA sequences are also active as promoters in transgenic plants under stable conditions. The results further demonstrate that these promoters are regulated and show tissue specificity. These results confirm that these promoters provide valuable tools for gene expression in plants and for genetic engineering. EXAMPLE 6
Establishment of a method to screen for gene products that interact directly or indirectly with plant defence-associated promoter sequences
A method using an in vivo reporter gene system was established that can be used to screen for gene products that interact directly or indirectly with plant defence-associated promoter sequences.
For the establishment of this method, transgenic Arabidopsis thaliana lines were used (Manners et al. 1998, Plant Mol. Biol. 38:1071-1080) that express the uidA reporter gene controlled by the promoter of the PDFl.2 gene from Arabidopsis thaliana. Several genes were cloned from genomic DNA of Arabidopsis thaliana. These included ERF1, COI1 and NPRl with the cDNA sequence Genbank accession numbers, AF076278, AF036340, U76707, respectively (Solano et al. 1998, Genes and Development 12:3703-3714; Xie et al. 1998, Science 280:1091-1094, Cao et al. 1997, Cell 88:57-63). Primer pairs were designed at the start and end of the coding sequences for each gene. These were called ERF-A/ERF- B, CoilAl/CoilBl and NPRIAI/NPRIBI, and comprise the sequences given as SEQ ID NO: 50 to SEQ ID NO: 55, respectively.
Genomic DNA was isolated and purified from Arabidopsis thaliana cv. Columbia leaf tissue as described in EXAMPLE 1. DNA fragments were amplified using the above primer and the Expand High Fidelity PCR System (Roche) with 10 ng of genomic DNA as template according to the manufacturer's instructions. After migration on DNA agarose gels, PCR bands were purified, ligated into the vector pCR-Blunt (Invitrogen) using a Rapid DNA Ligation Kit (Roche) and transformed into competent Escherichia coli OneShot Top 10 cells (Invitrogen). Colonies were screened for correct inserts and orientations using an internal PCR product-specific primer and an external vector-based primer. Sequencing was carried out to confirm the identity of the cloned PCR products using primers and primer sites present in the vectors and later in the obtained sequence using the BigDye Terminator Premix (PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit, Applied Biosystems) and an Automated DNA (377) Sequencer (Applied Biosystems).
The plasmid pBI221 (see EXAMPLE 1) was used as the basis for the construction of promoter-reporter cassettes that can be used for plant cell transformation using biolistic or PEG-mediated transformation techniques. The plasmids p35SERFl, p35SCOIl and p35SNPRl contain the cauliflower mosaic virus 35S promoter fused to the genes ERF1, COI1 and NPRl, respectively, instead of the uidA reporter gene. The plasmids p35SERFl and p35SNPRl were constracted by ligating the EcoRV/SacI-cut gene fragments that were previously cloned into pCR-Blunt into the SmaT/SacI-cut 3.9 kb fragment of pBI221. The plasmid p35SCOIl was constructed by ligating the blunt-ended Hindlll/EcoRV-cut COI1 fragment that was previously cloned into pCR-Blunt into the blunt-ended dephosphorylated BamHI-cut 3.9 kb fragment of p35SNPRl.
Particle bombardment was carried out on leaves and whole plants from the above described transgenic Arabidopsis thaliana plants containing the PDF1.2 promoter and the uidA reporter gene that were cultivated in controlled environment rooms as described above. Preparation of gold or tungsten particles, coating DNA to gold or tungsten particles and particle bombardments were carried out using the experimental procedure described in EXAMPLE 4. For each plasmid constract that was used for particle bombardment (p35SERFl, p35SCOIl or p35SNPRl), a set of three DNA deliveries were carried out on 8- 14 leaves or whole plants per bombardment at a distance of 17 cm using a Helium-driven pressure of 7 bar and a negative pressure of -0.85 bar in the chamber. Plant material was transferred to X-gluc-solution at 48h after bombardment for histochemical analysis as described in EXAMPLE 4. Three sets of experiments were carried out for each gene construct. Each time blue foci were observed for tissue that was bombarded with p35SERFl but not with p35SCOIl or p35SNPRl. Figure 5 shows a photograph of a transgenic Arabidopsis thaliana plants containing the PDF1.2 promoter and the uidA reporter gene that was subsequently transformed with p35SERFl using a biolistic transient expression assay.
The results of EXAMPLE 6 demonstrate that it was possible to screen for genes and their products that induce a plant defence-associated promoter in a transient assay. One out of the three genes that were screened for using this assay was found to positively induce the plant defence-associated promoter. This method allows the identification of genes and gene products that interact with a promoter of choice, such as the promoters described in EXAMPLES 1-4 fused to a reporter gene, using transgenic reporter plants. A rapid high- throughput screening could also be carried out on a large scale where hundreds or possibly thousands of genes and gene products could be screened with regard to their direct or indirect interaction with a plant defence-associated promoter.
EXAMPLE 7
Identification of putative promoter elements
The promoter sequences included in SEQ ID NO: 1 to SEQ ID NO: 9 were analysed for putative cis-acting elements to obtain some insight in the possible regulation mechanisms of these promoters.
Putative cts-acting promoter elements were identified by comparison with other promoter sequences or by using the PLACE database which describes elements from vascular plants (Higo et al 1999, Nucl Acids Res. 27, 297-300; Preshidge 1991, CABIOS 7, 203-206). Putative elements were checked for relevance (e.g. necessity of a repeat, distance between repeats, flanking sequences, distance from TATA-box) by comparison with literature description of the elements. Direct repeats and inverted repeats were identified using the "EREPEAT" and the "INVERTED" program, respectively, provided by WebANGIS (Australian National Genomic Information Service).
Figures 7-14 depict putative promoter elements in the core promoter region (approximately 800 bp of the right border of the promoter sequences included in SEQ ID
NO: 1 to SEQ ID NO: 9, respectively), such as the TATA box (Breathnach and Chambon
1981, Ann. Rev. Biochem. 50; 349-383), CAAT box (Shirsat et al. 1989, Mol. Gen. Genet.
215:326-331), GT-1 consensus sequence (GT1) (Terzaghi and Cashmore 1995, Annu. Rev.
Plant Physiol. Plant Mol. Biol 46:445-474), BOXCPSAS1 (Ngai et al. 1997, Plant J. 12:1021-1234), the nuclear factor SP8BF binding site (Ishiguro and Nakamura 1992, Plant
Mol. Biol. 18:97-108), nuclear factor SEF4 binding site (SEF4 motif) (Allen et al. 1989,
Plant Cell 1989), NtBBFl binding site (NTBBF1) (Baumann et al. 1999, Plant Cell
11:323-333), C-Box (ACGTCBOX) (Izawa et al. 1995, Plant Cell 6:1277-1287), Q-element
(Hamilton et al 1998, Plant Mol. Biol. 38:663-669), ASF-1 binding site (ASF1 motif) (Lam et al 1989, Proc. Natl. Acad. Sci, USA 86:7890-7897), API binding site (AP1SV40) (Mitchel et al 1987, Cell 50:847-861), elicitor responsive element (EIRE) (Rushton et al. 1996, EMBO J. 15:5690-5700), GATA box (Lam and Chua 1989, Plant Cell 1:1147-1156), I-box (Terzaghi and Cashmore 1995, Annu Rev. Plant Physiol. Plant Mol. Biol. 46:445- 474), Dof protein binding site (Dof-core) (Yanagisawa and Schmidt 1999, Plant J. 17:209- 214), MybStl binding site (MybSTl) (Baranowskij et al. 1994 EMBO J. 13:5383-5392), ACGT motif (ACGT OSGLUBl) Washida et al. 1999, Plant Mol. Biol. 40:1-12) and direct and inverted repeat sequences (R).
It will be appreciated by those of skill in the art that many changes can be made to the embodiments of the invention as exemplified above without departing from the broad ambit and scope of the invention.
SEQUENCE LISTING
<110> Commonwealth Scientific and Industrial Research Or The State of Queensland acting through it Departme The University of Queensland Bureau of Sugar Experiment Stations Grains Research & Development Corporation
<120> Promoters of Plant Defence-Associated Genes
<130> CRCTPP and GRDC PCT application
<140> (not known) <141> 2001-08-06
<160> 55
<170> Patentln Ver. 2.1
<210> 1
<211> 2069
<212> DNA
<213> Arabidopsis thaliana
<400> 1 tagttcagtt cttttgccgc ttctcttcca aacatctgta gatggatgga actataatga 60 acatataata aaagcataaa actgattctt ggcaaacagg tgatgaatga actaactacc 120 tctttttgct ctaagcagtg agaacaccag aatgccccat acattttagc tccaatggag 180 tttagatgtt ttgctaaagc aatagcataa gggctcgatg atgaactgat ctctgttcta 240 aaatatggca gttcaatgtc accagagctg gaaggagtga aacaaaaaac gatcgggatt 300 aaaagagacc acctgataag actcatacat ctttcggaag ctatttgttc actactcgtt 360 acaaatgact aaaatttcta tcgggactcc ccaaagaaaa ctatgaactt tgattaccgt 420 gaagggattg gttgagcagt actgtaagaa gcagtcaagg aggcaactac tatgattgct 480 aagcatatct gcaatcctac aacttgctgt atttcttgca acttcacatc ctaccacatg 540 catggagcac attatcataa tccgaagttt agaagttatc ttgagtcaac atcaggaaaa 600 aaaaagaaat ggaaataaag ggataccttt acagagagga aaaatagact aaatgataag 660 aaagcagaca ccagacaata caggcatgat gatcctgaga gttttgtgct aaggatatac 720 aggaaatatg cactagcaga tgccattgca gtcgtgatcc caaataacgc gaaacgccca 780 ttgctcttac tgattccaaa aggtagattc ccttccccaa gctctgcact tagagcggtt 840 actacgccat acataacaaa tccaatcact ggaagaggaa cacctggcaa aatgtaaaaa 900 aggattttca ttgagttcaa cttacctgca atgcaatgtt ggacaaacaa cgaagacaaa 960 gcactacata gagtggagtg aagcatgcta tttaactcta acaattctat gcatactaag 1020 taccaaagct cctaaatgat atagcttcaa gcaaagattc acagagacta tcacatgcga 1080 gtatttaatc atgcctacta taaaagagag gacaattagc tacataaatc aaacgctata 1140 atcaataaag cgatggataa taccgaaaac gacggcataa tcgctgttga ggacatcacc 1200 gcaagtgcca ccaccaatag ggcaaaacgc atcggagcca gtgaccttga ggtaagtcaa 1260 atacgcagtg tctaacatcc caataccgcc gattccggtg taccagttgt atgtcgaact 1320 gttactagta gatacttcac ttgtagacga tgaggaggag gaagacagcg acggtgccga 1380 atcctcaccg ttttccggct cagatgaaga acatttaatc ggaatcgcag gaaatctccg 1440 atcagagacc taattcagaa catctgactt caaatgaccg atcacacact actgtcactg 1500 aagaaaacac atgacaagaa tttacctcga atcgtctagg ataagaagtt accgacggag 1560 gagaaacctc acggaagccg aaatgaaact gacaagacga cacagaaaca aacctcgcca 1620 tcatcgtcgt cgaagaatcg ctattccacc attgttaact tcatcatctt ctctcctccg 1680 ctccactatc tcagccactt gtttcaatat tgggcctaaa cgaggcccaa atacgataat 1740 ttggatccat aattagttta tcacaattgg gccttacggt cttcttatcc gtccgagtca 1800 gcaagttatc ctcttcaaca cgactcacga ggcaccgagt tggacttatc atgattcatt 1860 atctacgcat tgtttacatt ttggaatcga ctttggtaca taattgaatt tatcatagaa 1920 atgatagaga ctcgaagatc ggacataaaa cttgctgacg tgacacccat acctttgtgt 1980 ctgtatgatt atataaacgc tatagaacta agagaagaag tatctaagta gcatcaaagc 2040 attctaaaag aagtttcaaa gaagaagtg 2069
<210> 2 <211> 1854 <212> DNA
<213> Arabidopsis thaliana
<400> 2 aaactgaacc aaactgaacc gatgcttaaa ctattaattg acatatccaa aaccctagaa 60 aatacccaaa aataatatcc aaactataat tttaaactca aaatactaat tatttttaga 120 atcaaaacaa tcaagtactt taattaatca acatattcaa atcttgttaa tatttatcaa 180 agtcaaatac tctattacaa aactgaacca aaatttaata ttctttataa atttttcctt 240 ttttgtattt tctttccgta tttttttggt taatttcttt tagtttcggt taattttggt 300 tattttgata aaacctaacc aaatcgaaaa gtaaccgaac agaaccaaat cgaaattcct 360 aaaatatctg aatggttatt atattaacat atccgaacta accgatacta aacgcaaacg 420 aaccaaaacc gactggttaa tcgaatgcca tgcctaccct gacaaaacaa caagattgaa 480 accctatata tttttttagt taatatatat aagaaataaa cctatatttt cttaatgtat 540 ttaaatatga ataaccaata aaaaaatgta atggtaacga aaatatgact gttttcagat 600 gaaaagcacc tccaattatt ttgaaacgat taaaaagtgc aaaaaaagtt tttccttttc 660 ttttggagga cgagtctctg gtttcatgtg ataaaagcat cgtcgagcgg ccacaatcca 720 taggatatgt gatgtcagcg gtgaggttcg tgatgaattt acttgttttt cttaacgaac 780 gcattgtaac tctaaattaa acagtcgaaa atatatctat aaacgttttc aataactctc 840 tggtgttaat tcaaaactct atgctcaaca tttttttttt ttggataact aggagataat 900 ccggcttttt accatctcac ttcatcaaga tgagtataac catcaatggc caattcaccg 960 gattttctaa taagcgtaaa acatacgact atttcgacta accaaatgaa ctgtacgtcc 1020 aatttaatta agaggctttg atgagcataa tctactacaa ggaatctttg cccaaaacac 1080 aaaaaaaaac aaacaaaatc tactacaagg aatcttgatc catatccttc tagttttgcg 1140 caaacctgcg atgcatgcca tgctaatccc caatccaacc caaattgacg cggcggcata 1200 ggcacaagaa gagattaggg gtggcatttg atcccataaa attattaaaa tttcgcgtac 1260 atttttgtca aaaaaaaaaa aaagtggtgt aaattcaatt gaccagaaaa ttagcaaaat 1320 ttaccttttc ccaccaataa atatttgttt gaaaaggtaa atattcatat ttatttattt 1380 gtatatttta accatgatga atgtttttat tattattccc gtattccggc actgcttggc 1440 atccgtgtgg tgttgatttg gtagatgtca cacaacccat ttctctttac caaataaact 1500 ctaaaaatag aaataatcaa cggttttttt ttctaattta tatatcaaga gtatatttta 1560 attttatttt taatcaacag ttcacagttg agagcttgag ataaagaaat tttcattata 1620 tcaaatataa tctaacccat tctcaacgtg ggaaaaagaa ttagtatcgg tgacactgag 1680 atagagtacg tgtacgtgaa cccttgaacc cacgggattc atgtcaccta aacctccgga 1740 aaataataag cacaatgtaa tgcataggtc agtactcttc tttacaaagg ctatataaac 1800 cctagattgg cacaaggctt ccatgcaaca caacacatac atctacatat taac 1854
<210> 3
<211> 2034
<212> DNA
< 13> Arabidopsis thaliana
<400> 3 ccctagttcc gatataaaca gagctttttc cttcactaag ctctgtaact tcttcgattc 60 aaactccatt ttctcttcat cttcacatag taacaagtag gccgttttct catgaggaaa 120 ctgcactagt ctctaaatga gagtagcaaa taaatgtaaa caaagatgat acctttctct 180 agcattgtat gcacagcatg aaacggaatt gcagcaaaaa gatcagctcc tgggagcatc 240 atccaggtgg gttcactaga gtcatgggat ccacatgtgg agcaaacttc ttggaccaaa 300 ggtttcgttg agctcccatg gatatccttc atcatagaat caaatggaga cataacacta 360 gtctttgttg tccgagctct cttccttagt gctgtaagag cctctcggtt tgcattcctc 420 tccttgtcat tctccaccat ctattacaca tattacaagc tccattttgg ttaaaaaaac 480 ttattgacct aacaacaata catatcatat aatatctgtt ttctcacaag acaaaataag 540 ttgcagacaa caatagattg aaaaatcttg tgctttcctc aatctgtcaa tcacatataa 600 attacgaagt agtgaatatt tccaactaga tactaccaaa gcagattccc gagtacctga 660 tttcgagcta atagaaactc atcagcttca gcttcaattt tgtccaatag tgcagcaaac 720 ttctttggat ccaaatccat ctctagtcta acagattcca acaaacaaat aggctcagaa 780 tcaaataaag cattattggt tgaaggggaa aaaggaaaag atttgataag tggtatagtc 840 aatcttccta atcctgtaca tctccatgtt aacctacatc ttcatgcaca aatcaaacca 900 caacgaagcc agaaaaaaag tgaagctatg gatactttgt ttgcttaagc tcaatttcaa 960 agactctaat tagtaccata agccactcca tgttttcgtt gtatgtatgt accaacatca 1020 tactaaacct ttcaccatcg tttcagcttc tattctatag agtgattatt gaattcccaa 1080 atttttcaat attgatacga ttaatgattt catccaattt cagagaaacg attatagaaa 1140 ttaacaattc tgataaaccc taaatggtta aaaccctgaa ttccgaaacc ctaaaaccta 1200 attcacaaaa tctctcgttc ctacgatgca ataacgaagg caaccacaac accaacaaaa 1260 gtgtaaaacg attctttaat tcatttttgc agcatcttaa caagagggcg agacgaagta 1320 actcagaaac aaaataataa cctttaaatg gatttacaga cgaagattta ataagcagag 1380 tccggtgaga atctcggtga cgcaatgaga gggacgaagt ttattaacaa tataaattac 1440 attaaacccc cgagttgagt tatgggctag gtatttgggc tataaaaggc ccggcccact 1500 aatcatacca ttgaaaggtg cgatcgttcc cgccgtcgta tcgtagcaca agaaaatgta 1560 aattttattt atttggtatt taaaaatata cctatttaat aatttgagat gataaagtca 1620 taaaacagag taactttttt gcatccaaaa acgagttcgt gatatcaact aagcttgtca 1680 atgtgttttt ttgtacacga gacattaaaa ttcacaatgt ttcgtagaga aggggacaag 1740 aaacacataa tataatcaac caattattat tactcgaatc cattataata ttttttcaaa 1800 aggataagat tttgttcatt gaaagacatc tgggtgggtc catttcactg ctaagcccct 1860 tgtggtggcg tctccaacgc atccacttgg cattgtttat ttgaccactt tttcgaatca 1920 tagattttga atattcaagc tcgatggctt atgttaactc ttatataaac atattatcaa 1980 cctccaaata ttttcatcat caatcttctc cacacttgaa tcacaaacaa caac 2034
<210> 4
<211> 2013
<212> DNA
<213> Arabidopsis thaliana
<400> 4 tattggaaac tgtgagtcaa gctgaataaa cttgatgcaa acgaatagat atgaaagaaa 60 ttgggttgat caacaaaaaa acaagccgac aaaaactgaa tctcatgata tgttttcgtc 120 cgtcagtttt aaccgttcgt ttgatcggat aatcaaaacc agtaaatgcc aataatacac 180 accagaaaag taagatgata atttttgttc gttagtagta ttttattatt atcatgctca 240 tcattttctt actttggcgg cccatctttt ctcttcccct ctttctgagt agtttagtaa 300 aacagaacct gacagaaaaa aatagaaaat gccaggaaac acacacttag caggaagact 360 atgggagtct ctaaccaatg cacatgcttt atttagttca actactctct agtggtctat 420 aaatatcgtt gagttcttta tttctattac tcatcatagt tctcaataga gagaatcaaa 480 aatcaaaatg tcttctccta ctgcgactaa gtacgacgtc tttctgagtt tcagaggact 540 cgacactcgt cgcaacttca tcagttttct ctacaaagaa ctcgttcgaa gaaaaattcg 600 aactttcaaa gacgacaagg agctcgagaa tggccggagg atttctccgg agctcaaacg 660 cgccatcgag gagtcgaaat tcgccgtcgt tgtggtctct gtgaactacg ctgcgtctcc 720 ttggtgtctc gatgagcttg tgaagattat ggatttcgaa aacaagggtt ccatcaccgt 780 gatgcctatc ttctacggcg tggatccttg tcatttgagg aggcagatcg gagatgtcgc 840 tgaacagttt aagaagcacg aggctagaga agaagatcat gagaaagtag cttcgtggag 900 acgagcattg accagtttag cgagtatctc cggcgaatgt tcattgaaat ggtgaaattt 960 cgtagcgttg gttagaaaga ttggattttt atttgatatg tctcgttact aaagtttcga 1020 tttttatttt gttttctctc tgtagggaag atgaagcgaa tctggtggac gaaattgctg 1080 acaagatatc aaaaaatctg atgactgtta caacaataag caatgggagg gacctagttg 1140 ggattgatac acacatgaag gctcttaaca aaaaattgga tttgaattcc aacaaaagtt 1200 tgagagtggt tgggatttgg gcaagaggat acaatggtag atcagcttta gctaaatatg 1260 tttatcaaga catctgtcat tggttgaagg ttggttggtt gaaggtttat caagatatca 1320 agacagcttc caaactttat gttttaactc gttggttgaa ggttgcacat ttgcttcata 1380 gatcacatca ctcttttaaa actgagtagc cagccaatca ataacccgaa tttcactgag 1440 aaatccccac atttgttatg tttccatact acaaaatcat ctattgacac cactagttag 1500 ctgtgatctt ataaagatga gtaagccaac caaaccaata acccaaattg gaaaatgtga 1560 gtcaagccga ataaacttga tgtaataacc tattcgttct aacttgaaat tgttattaaa 1620 atcatcctta tcaattagcc tttctcgtcc gtcaattgac ttagttctaa ccattcgttt 1680 gatcgataat caaacaaaca gttgaatctt cttcaaccaa ttaaattaga ctacgagtaa 1740 ctaaatgcca atacacgaga aaactagact aagaggagaa tttgtgttcg ttaatatgtt 1800 atccatactc attatcttat tttggcggcc catctattct cttccctctt tctgagtagt 1860 ttagtgaaac agaagctgac aagaaaaaat agaaaatgcc aggaaacaca cagttagcag 1920 taggaagact cttataggtc aacttctgta gtggtagtgc ctataaatat ccttgagttt 1980 tttatttcta tcactcattg agaatcaaaa ate 2013
<210> 5 <211> 1829 <212> DNA
<213> Arabidopsis thaliana
<400> 5 tgtcgaacct taacagtggc ggttctacgg gaacaactac tcaaatcaac gactttgggg 60 gtttccaggc ttcgaaatca aacacattct catcaggagg tagcttcaat gcttcaaacg 120 ttgattttgg tgtgcaacca agtggtccac aatcttcgag tgcaaacgat gatccattag 180 gaatgttctc taattccaaa ccctcagctg caccacctac gccacagacc gaagattggg 240 gatttgaaag ttttgacggt ggggctggtt ccaccaccga gcttgatggt ttgccaccgc 300 ctccaccggg tgtatcagct acatcagcta agaacaaggg cattgataac cagagacaag 360 gtcagtatgc tgatgccata aagtggctct cttgggctgt gattcttatg gatagagctg 420 gtgatgaagc tggatcagcc gaggttcttt caacaagggc ttcgtgttac aaagaagttg 480 gagagtataa gaaagctgta gccgactgca caaaggtatg ttaagaaagg cacatttttc 540 tttggatgca tcatggacca tggttcatat cattctcatc tcagatcctt agtttacgaa 600 gtctctagtt gtattggaat cagtttaagg actcttgatg aaattgatac tgataagagt 660 tttcaggtat atgaaacttg acaatatgag atttaggttc ttatggaatc ttaagataga 720 ttacgagtta gaaatactgt gcacgtatag aaagttttag tgtcttgctt tgtaggaaaa 780 tataccacaa ctaaacagtt cttgggatcg cgtcatcgta gctagttgat tggttgttat 840 gctccataga tggtttcttg tagactcaga acaaaaccaa catcttcgaa acttgttctg 900 attgtcagtt atgcttcaca gatgtttttt gttcactttg aaaacgtgat tgcaggtact 960 tgatcacgac aagaagaatg tgaccattct ggttcaacga gcactgttat acgagagcat 1020 ggagaaatac aaactcggag ctgaagacct aaggatggtt ctaaagattg atcccgggaa 1080 tagaatcgct agaagtactg ttcaccgcct aaccaaaatg gctggttgat ccttcatata 1140 tacggatact actcaaagaa aaaaaacaaa aaaccctcaa atatctataa tccttcacat 1200 tgctttgctc aataccgttt cctctctcat ccatggacca tgcgtgtgtg tatttttttt 1260 ctgagattta ttggttcgtc tataatgcgg cagcttcttg tagtaagtgg gtcatgagtg 1320 tatttttttg gatggtgcat ttatatgttg ttgattttct tacacactaa ttttttttga 1380 gtttatcatg aaatgtttga tcatcgatgt catatgaaat gccaaagatg accgtaaatt 1440 tcgcatcgta actcgcaaag atgaacctta ctttgagacg ctgcctgcgt gatggacgtg 1500 agacgcataa cgcatactat taaattacca gacgctcgtg gacacaacac ttcaataccg 1560 aaaactaaac taatcacatt gtgatacgtt ttgtattcag agagccaaaa aaaggcttac 1620 aattttgtcg ttatgtggat tagccttcaa tttgttgtaa ccgtcatgta tcaaggaaσa 1680 taaaactatt agaattatta ttctaatata aagtaattca tttcactcac actttattac 1740 cataaaacat ttattttgcg cctataaagg catttcagct ccaccgtagg aaactttctc 1800 ttgaaagaaa cccacagcaa caaacagag 1829
<210> 6 <211> 1767 <212> DNA
<213> Arabidopsis thaliana
<400> 6 ttgagacttg ggagacggtc aaaacgttac aataatattc ttagtttaaa gctcgatttg 60 gtagaaagtt tgactttaca tatttgtgta ttttttggat aggataggtc cgataggatt 120 tgggatatga tatcatgggt tcgtccaagt tatagtaatg ttgggattgg gggtaaattg 180 gtaaaactat aattaggcaa agcgtgaaga aaaaaaaaat tgacattatg atgagaggag 240 ttcgggttgt tgcatgggaa cattatttgt gctcccataa aggtaaataa aaaccaaatt 300 ataatcatgg gatatttaac taagaaaact tgatatcacg aggaaaatca ttataagttc 360 agatttaaag ccttttcaaa tatagcgtac ataactatat tctatatgta gtcgatgtct 420 cgatgaaatg cttgtttatt aaggagacga atccactagc aatttttata tccaactaat 480 ttatttattt tagtttcttt ctcaaaatat aaatttcgat tgtagtttaa ttgataagac 540 caaattcatt gagcatgcag taccctagca accaactaat cgtgagaaca gtttgaaaat 600 tctaattggg atagtcaagt caacgacctc aacgcgtatt ctttaagttc ttccgttttc 660 tttttttctt tctaagccgt gattaccgcc tcggaaaatc atggagtttt cacggaaaat 720 aatttttaga ctatagttat atactctaat aaatacaatt gcaatacccg ctaattcagc 780 aggaaactat gacaactggt ttccgtgtac ggaatgactc tcttgcccgc atcattcttg 840 atgatgtgtc atacactaca aggcaagaaa aaatcaataa ataaatcaaa gttaatagta 900 gtatagtata tagtataatc tgttttgctc agtcatattc actcaccgta aatgccaaag 960 tgaaagcatc agtttgaagc cagttagctt ctgttttttt attatgtcat attcaaaatc 1020 attcaattat tcatatgtca ataattactc ggttacaggg tgaaatttta taatctgatt 1080 cgtatgaacc aacgactcaa aagtttagct agctagtagc tactcaaaaa aaaaaaaaaa 1140 ctacttgtta aaactacaag tttagattta gacatatgtt cactagattt tgacagctac 1200 gcattcctta aaatgtctga tgtttaatta gcatgattta ctaaaataga cttgtattta 1260 ttatgttaaa acaaaatacg cagtttttgt tcgctgaaaa tgttccaaaa caataatatt 1320 ctttaagcga aaaatagaaa ttcatataat tatattacag ttaagggaaa caaaaagagg 1380 aagaattcgt tggcaacaat tgacaaatac ctaaagcgtg cgaggtcagt aaatcacacg 1440 cttgagcagt tagtggcggt tagaacggta aataaaggag acggaattaa caagtaatca 1500 cgttaaaaaa agctaaacga agaatcaaat tgtgtaggag acgacgtcgt atagatacat 1560 aacggccgtc tgtcttaacg gctctttttg gtgtcgtctt cttttatttt tattttggtc 1620 tcccaccaga ttctataaga ggaccaatat atctccaaac tcgtacattc actgtcttta 1680 actctcacta ctctctctct ctctctcctc tgttctaatt ctctaggaac agagtaaaag 1740 actttggctc agcttctgcc tctccaa 1767
<210> 7
<211> 1772
<212> DNA
<213> Arabidopsis thaliana
<400> 7 cacagaaagg gcatcaaaag catcaattta tttttaattg accaaatagc tttagtgatt 60 cttattatta gtaatttaat actagttggc gagaaaaaaa aatacaaaga agatgaacaa 120 gtgaaacaaa cttgagaaat atccgtggtc aaagaagata gacgagtggg ggtggcaata 180 gagtaaatag aaaggaacac gaagggtttg tttgtaagga atatgttaat aatggaaggg 240 cgaagaaaaa gatgagacag agagtgggac ccataaacgc gtagctgagg ttttggtaag 300 gatcgtcgtg ggttcctcct ctctcaccct ataaaccact tctttctcac gcgcctcttt 360 cacatttatt attacaccta aatttaaata aattattatt attattgtgg tcgaccactt 420 ctttcaaaag ttgttgtcat ttacctattc ccaacacata tattctactc caccttaaga 480 atatctattt ccttttgtca ttagaaacac ttatacgaac ctgttacata tttgcaaggt 540 cgtatcaata tgtttatcga cataatcgtt aggatctttg ggtttctcaa tgtttaatag 600 tcttttacct ctcgttaatt agatataaag attggattta tatacataaa tacaaattta 660 aaattagcgt ttcaatatca tattattatg gatgtacaat gtatgggaat aatgtaaagc 720 ttactcaaag ccaaagggcg agatatatta tgcctaatgg ggtattattt taatgtttaa 780 cgtatgtgat catgaaagcg acaaatgcgt aatggaggat tttgttgctt ggatactgtt 840 cggcaaagta gcttttttta attcaattat ttcgtattat tattgacaac ttggcttaat 900 tatttagtgt taatcatttt gtttgtcaac aattacaaac atctgcacaa taacctttga 960 tgtgacttca tttttgtgta gggggtgata taccatttct tattcaaaat attgaaatag 1020 tcctcagtgt aagaagtaga gttttgggat cagcagccat atgtcctctt caaattatat 1080 atgtttataa agttatctaa tgctttaaaa tatgcatagc tttttttcta ttttttataa 1140 agtaatttaa tgctttcaat tgtacttata gatgtaagat attaggttta aattttaatc 1200 tctggccttt aattttgaca aattaccttt gaacttttga atttggagat tgtaatcata 1260 catcacacag aatatcacat atttgaatac ttttaatttt ttgaaatggt ataggaaatg 1320 aaaaatgtaa agcttcttat tgaatgataa cacacatatg tgatgagaat aaaaaagaaa 1380 agaatacaga atttatgtga catatatctt attcacaacc atagtattga tccattgatt 1440 aacatatcaa ggaaagtaat tataaagtta aaggaaaaaa aaaaaaaaaa aaaagctaaa 1500 caaaaaacaa aaaaggaaga gacaactaag cgcgtgtagt tcacaaacca gaagccgaga 1560 gtcggttaag aaaccgtctt aagctgttct tggacacgct gaagcaaatt taatcgtgta 1620 taaaactatc cttcttccac cttctcatta tattcatttc catctttcta atttatcttt 1680 ccatttccga gccgttgaga attttttctg agagataatt taacaaattt cttcttcttc 1740 ttctgtttct gaaccaccaa atctgccttt ct 1772
<210> 8
<211> 2030
<212> DNA
<213> Arabidopsis thaliana
<400> 8 cggagataga tgaagattgt cttctaatga ttgccacttt ctgagaatag tatgttactg 60 ttgattgttg tacctagctc tagttgtaaa taataatatc aaactaagta acaagagcaa 120 cttgttgaca ctaaagggac cccaagttac cctcaatcaa aaattgtgga tcagttcatc 180 atcgaagtaa cgtgttaatt atcgtcatat gctccacaac atcgactccc caccataccc 240 actcaataat gtcaattcag ctctctcttt ttgtattttt ataaagaatg tatttaaatt 300 ttcaacccat aatttataga ttcatgatca tctattaata attcattaaa tacttttctg 360 catattgatt tatttaaaag ccgtacgcaa gctcaaatcg gataagacta aaataatatc 420 acccagttaa gttaactaac ttattttatt ttgaatagta gtattaatta cttttgttca 480 actactccaa tactgtttat atcctttcga cggtattagt tattttcatg tttcacgcat 540 ttttgtaatt gcaaatacag acacctttgt tgtaacacat gttgattaat tttccgaaac 600 tatatacttt tgaagacaaa aatttaaaac aaagaaacct cagatctaca gttccacctc 660 gtgtctgaca cgagtttcta tgcgataaga tcgaacatgt atatcagcag actttactat 720 agatgaactc atattagtaa taagatccaa atacggaaat aaacaacttg gaaagtgaat 780 ggaagtttta agagaaaaat catttctaga attatttttt agatcatttc tttttttgga 840 gcatccatta gagttttgtt tttagacttt atgatttatt tttcttttgt tatcagcaca 900 ataattgatt ttcaaaagat attgtatctt ttttttatct cttgagaagc acagcacaac 960 aacgagcaat gctgacttgt cggggtcagc gattattatt agtcaccgaa cgaatttttc 1020 ttgggttgga gccaaaacct agcaaaaaaa tatctaagtg ggggcactac attattgttt 1080 taacaccaag gaaaagtaaa caacaatggc ccatttttac tgtatcgaga aattgatctc 1140 gaaaggacaa atgtaccctt ccacttcggg aatccattat tgtgcataac acacgctgtt 1200 tcactatata tactttcaca taggatctca acactctcac taaaccaaaa tcctcaaaag 1260 cctatttggg ggatcatcaa ccttctatca tcaccatgga tccttacaag gtatcttcga 1320 tcatattctt cttacttttt ctttgttttt gtgtggtgta tgtgtatctt aattagaatt 1380 aggttcaact atatatgctc gttttctaaa ctatttttta attggattga tgttcttaaa 1440 tcttaagggt caaaatactt tttatgctca aaaacttact taaattctgt gatcgcttga 1500 acctaagttg atgatgttga tttctgtttt ggctggctat ctttaattaa aacgtttaac 1560 cactgcgtga gaaagacacg ccacatgtgg tttttgttgt ttttttcctt agattagaag 1620 ttattttgtt gttgtttttt tttttattat tattacacac atgtgttcta aaaatcggac 1680 gttcaaatga tataatcgat tgtttagacg tccgaccgta tattatttta gtgatatcag 1740 ccaaatcaga ttaagtaatc atcaacaaaa tgattgatca gatctactaa acacctgagt 1800 aatcaaatct acacgtttga ttaatcaaat taaataaccg atcagttaga tctatacata 1860 tgataaatct ttttttacaa cattcggtta atcaaaccat tgtcggtcga tctatcaata 1920 caagtgtatt tttttttcac atacaaaaaa attatctcac cgacgaaaaa aaaataaaaa 1980 attattatgt agatccatcg aacaaaaggc ttgaatatac ggaagtcact 2030
<210> 9 <211> 1591 <212> DNA
<213> Arabidopsis thaliana
<400> 9 aacagctaca ctcacgactc acgtcgccta aatcaccaca ttatcatttt tggtactagt 60 atccaaactg ttcggatatg tatttacagt ctttgtaaga gaatttcaat catttatagc 120 tgaaggtgta ccatatagat agatagggac accagcaatt tttacatata aaaaagaaaa 180 aaatatcaaa cgatttacaa tgtatcatgt cgcgattggg tcttgctgcc tcaagaatag 240 ggctgatatc tcgttttgtc caatcaaaga aaaaacacgt catattctta ttttgatgat 300 ttatatatta gcattatcca aagtccaaag attataaact gagattcatc ttctcgccct 360 tgcacattgt agcaacgtgt gaggcatgtg cgatgcctat acctacaatt cacatgtgat 420 ccggtatatt aaaatataca tgatttatgg atacactcga atacttctcc gatattcatt 480 tcgcggaaag ttgcatatga tccactatct cctatatatt tgttatataa cacatggcaa 540 atctctctat atatattttt taaatacaca tatatccaaa ataggcttaa ctataacaca 600 aagacttttg tttactgtat attagtaaga agtaaatgta ttttttaata ttatgataaa 660 gtttgtgaaa tcaccatttg caatagccat atagggtcgt gttttaattt tacagtttgt 720 attgttataa ttcgattcca aggttgagaa tatgtgtgta ctattagact atacaaataa 780 taattcgttg acgatattga atatttacta attataggaa gagaaaatta tttactaact 840 atagtacgat atatttcttc tatatgtgtt tttaacgttt tttttttttt aaatttaagt 900 cttaacttta cttctcattt ttaatcaaaa ggaaaaaaat accaatcaat ttttcctaac 960 acagtttact tatcattttc atttgaaatg tgttcacttt ctgataaaat gctaatccta 1020 caatcaaata caccattgtc gtgataacac gtgtacggct ctaaagcaat cagaacaatc 1080 attggacagt ttttacaccg tcagataagt acctatccac ttgctgactc agccggataa 1140 accctaaacc ggaagtttgc cccaccgtca aaattggaag aaaccggaca aaagagaatg 1200 taaagactaa gaagtaagaa cccatcggac gtcgtaagaa ggttaattaa cacgtggaaa 1260 cagctggtca gagttatccg gtaacttatc cggttacaag taaaaaaata atttgttccc 1320 atacacgact ccttcagaac caaacgcgac atcacggcgc cgtttagtgt ctataaatag 1380 agcaatcggt cgtagaaaac caagacatca aaaacacgaa tatcgatagt acacttctac 1440 gtgcaatttt ctcctttctc ttcctggaca tctgtctgtt tattacattt tcttgtaatc 1500 tctttttggg gttttacaat atctatcccc taaagtttcg gaaaattctg tttttctgtt 1560 ctcattcttc gtgatctttt tcactttctt 1591
<210> 10 <211> 24 <212> DNA
< 13> Artificial Sequence
<220>
<223> Description of Artificial Sequence: rimer sequence <400> 10 tagttcagtt cttttgccgc ttct 24
<210> 11
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:primer sequence
<400> 11 cacttcttct ttgaaacttc ttttag 26
<210> 12 <211> 24 <212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence : rimer sequence
<400> 12 aaactgaacc aaactgaacc gatg 24
<210> 13
<211> 26
<212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence :primer sequence
<400> 13 gttaatatgt agatgtatgt gttgtg 26
<210> 14 <211> 25 <212> DNA <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence :primer sequence <400> 14 ccctagttcc gatataaaca gagct 25
<210> 15 <211> 26 <212> DNA <213> Artificial Sequence
<220> <223> Description of Artificial Sequence :primer sequence
<400> 15 gttgttgttt gtgattcaag tgtgga 26 <210> 16
<211> 24
<212> DNA <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: rimer sequence <400> 16 tattggaaac tgtgagtcaa gctg 24
<210> 17 <211> 26 <212> DNA <213> Artificial Sequence
<220> <223> Description of Artificial Sequence -.primer sequence
<400> 17 gatttttgat tctcaatgag tgatag 26
<210> 18
<211> 23
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence :primer sequence
<400> 18 tgtcgaacct taacagtggc ggt 23
<210> 19
<211> 23 <212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence :primer sequence
<400> 19 ctctgtttgt tgctgtgggt ttc 23
<210> 20
<211> 19
<212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence :primer sequence
<400> 20 ttgagacttg ggagacggt 19
<210> 21 <211> 19 <212> DNA <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence : rimer sequence
<400> 21 ttggagaggc agaagctga 19
<210> 22
<211> 24
<212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence : primer sequence
<400> 22 cacagaaagg gcatcaaaag catc 24
<210> 23 <211> 24 <212> DNA <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:primer sequence <400> 23 agaaaggcag atttggtggt tcag 24
<210> 24 <211> 24 <212> DNA <213> Artificial Sequence
<220> <223> Description of Artificial Sequence:primer sequence
<400> 24 cggagataga tgaagattgt cttc 24
<210> 25
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:primer sequence
<400> 25 agtgacttcc gtatattcaa gcct 24
<210> 26 <211> 23 <212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence : primer sequence <400> 26 ttgtcgatgt gagtgctgag tgc 23
<210> 27
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:primer sequence
<400> 27 cttctttcac tttttctagt gcttc 25
<210> 28
<211> 23 <212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:primer sequence
<400> 28 aacatgtgga tctttcaagt gcc 23
<210> 29 <211> 19 <212> DNA <213> Artificial Sequence <220>
<223> Description of Artificial Sequence:primer sequence
<400> 29 gtcgttgctt tcctccatcg 20
<210> 30 <211> 23 <212> DNA <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:primer sequence <400> 30 gcatcttctg cttaccagat cga 23
<210> 31 <211> 18 <212> DNA <213> Artificial Sequence
<220> <223> Description of Artificial Sequence:primer sequence
<400> 31 cccaaatcgg ctccacct 18 <210> 32
<211> 20
<212> DNA <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:primer sequence <400> 32 gtttcgtctc gggtcatgga 20
<210> 33 <211> 23 <212> DNA <213> Artificial Sequence
<220> <223> Description of Artificial Sequence : primer sequence
<400> 33 gcagcaactt gttattcctt gga 23
<210> 34
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:primer sequence
<400> 34 aaagttctta aatggaggca agca 24
<210> 35 <211> 23 <212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence :primer sequence
<400> 35 agcttcgagt catcatcacc tga 23
<210> 36 <211> 15 <212> DNA <213> Artificial Sequence <220>
<223> Description of Artificial Sequence :primer sequence
<400> 36 cgccgccgac gactt 15
<210> 37 <211> 23 <212> DNA <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:primer sequence
<400> 37 gcctcctctt tcacgttaac ttg 23
<210> 38
<211> 22
<212 > DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence:primer sequence
<400> 38 catcccacca accaagttaa eg 22
<210> 39 <211> 17 <212> DNA <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:primer sequence <400> 39 ttccccggct ctgttgc 17
<210> 40 <211> 22 <212> DNA <213> Artificial Sequence
<220> <223> Description of Artificial Sequence:primer sequence
<400> 40 ccctttgcta aagaggtgac ga 22
<210> 41
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: rimer sequence
<400> 41 ctccttccga tctccaattc c 21
<210> 42 <211> 25 <212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:primer sequence <400> 42 acaccagaga gggaaacttt gatct 25
<210> 43
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:primer sequence
<400> 43 tcccatcacg gatgaagaac a 21
<210> 44 <211> 21 <212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:primer sequence
<400> 44 attgttgtca cggtgaacgg a 21
<210> 45 <211> 23 <212> DNA <213> Artificial Sequence <220>
<223> Description of Artificial Sequence -.primer sequence
<400> 45 cggaacttgt gattcttcag acg 23
<210> 46
<211> 23
<212> DNA <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence :primer sequence <400> 46 agtggtcgta caaccggtat tgt 23
<210> 47 <211> 24
<212> DNA
<213> Artificial Sequence
<220> <223> Description of Artificial Sequence:primer sequence
<400> 47 gatggcatgg aggaagagag aaac 24 <210> 48 <211> 22 <212> DNA <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:primer sequence <400> 48 gaggaagagc attcccctcg ta 22
<210> 49 <211> 24 <212> DNA <213> Artificial Sequence
<220> <223> Description of Artificial Sequence:primer sequence
<400> 49 gaggatagca tgtggaactg agaa 24
<210> 50
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:primer sequence
<400> 50 catggatcca tttttaattc agtcc 25
<210> 51 <211> 22 <212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:primer sequence
<400> 51 ctaatctttc accaagtccc ac 22
<210> 52
<211> 25
<212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: rimer sequence
<400> 52 gatggaggat cctgatatca agagg 25
<210> 53 <211> 24 <212> DNA <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence : rimer sequence
<400> 53 atcatattgg ctccttcagg actc 24
<210> 54 <211> 21 <212> DNA <213> Artificial Sequence <220>
<223> Description of Artificial Sequence :primer sequence
<400> 54 gatggacacc accattgatg g 21
<210> 55 <211> 20 <212> DNA <213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:primer sequence <400> 55 ctcaccgacg acgatgagag 20

Claims

1. A promoter operative in a plant cell, said promoter comprising: i) isolated DNA from Arabidopsis thaliana having a sequence as defined in any one of SEQ ID NO: 1 to SEQ ID NO: 9; ii) isolated DNA which is a plant genome-derived variant of the DNA of (i); iii) a promoter-active portion of the isolated DNA of (i) or (ii); iv) a regulatory cw-acting sequence element of the isolated DNA of (i) or (ii); v) isolated DNA which hybridizes under stringent conditions to the DNA of (i) or (ii); or vi) a promoter-active portion of the isolated DNA of (v) .
2. The promoter according to claim 1, wherein said DNA is single stranded or double stranded.
3. DNA corresponding to either strand of DNA comprising a promoter according to claim 1.
4. The promoter according to claim 1, comprising elements as illustrated in any one of Figures 6 to 14.
5. A DNA constract comprising at least one gene having at least one promoter according to claim 1 operatively linked to a coding sequence, which coding sequence encodes a desired product or products.
6. The constract according to claim 5, wherein said DNA is single stranded or double stranded.
7. DNA corresponding to either strand of a DNA constract according to claim 5.
8. A DNA construct comprising: i) a first gene having one or more promoters according to the first embodiment operatively linked to a coding sequence of interest; and ii) a second gene having a promoter operatively linked to a coding sequence, wherein the expression product of said coding sequence modulates activity of the expression product of said first gene coding sequence.
9. The construct according to claim 8, wherein said DNA is single stranded or double stranded.
10. DNA corresponding to either strand of a DNA constract according to claim 8.
11. A method of expressing a product in a plant cell, said method comprising introducing a DNA construct according to claim 5 or an RNA transcript of said constract into cells of a plant, wherein said DNA constract or RNA transcript coding sequence encodes said product.
12. A method of expressing a product in a plant cell, said method comprising introducing a DNA construct according to claim 8 or an RNA transcript of said constract into cells of a plant, wherein said DNA construct or RNA transcript coding sequence encodes said product.
13. A plant cell, wherein the genome of said plant cell includes a DNA constract according to claim 5.
14. A plant cell, wherein the genome of said plant cell includes a DNA constract according to claim 8.
15. A plant, plant tissue or reproductive material of a plant, wherein said plant, plant tissue or reproductive material comprises cells according to claim 13.
16. A plant, plant tissue or reproductive material of a plant, wherein said plant, plant tissue or reproductive material comprises cells according to claim 14.
17. A method of identifying a chemical or regulatory molecule which modulates the activity of a promoter according to claim 1, the method comprising the steps of: introducing a DNA construct according to the second embodiment into a plant cell; contacting said plant cell with said chemical or regulatory molecule; and determining the effect of said chemical or regulatory molecule on the activity of said promoter.
18. A chemical or regulatory molecule identified by the method of claim 17.
PCT/AU2001/000961 2000-08-04 2001-08-06 Promoters of plant defence-associated genes WO2002012483A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001277407A AU2001277407A1 (en) 2000-08-04 2001-08-06 Promoters of plant defence-associated genes

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPQ9206 2000-08-04
AUPQ9206A AUPQ920600A0 (en) 2000-08-04 2000-08-04 Promoters of plant defense-associated genes

Publications (2)

Publication Number Publication Date
WO2002012483A1 true WO2002012483A1 (en) 2002-02-14
WO2002012483B1 WO2002012483B1 (en) 2002-04-18

Family

ID=3823264

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2001/000961 WO2002012483A1 (en) 2000-08-04 2001-08-06 Promoters of plant defence-associated genes

Country Status (2)

Country Link
AU (1) AUPQ920600A0 (en)
WO (1) WO2002012483A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101255428B (en) * 2008-01-03 2010-07-21 广州大学 Plant salt resistance related gene and use thereof in plant breeding

Non-Patent Citations (17)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [online] 1 December 1999 (1999-12-01), KROCZYNSKA ET AL.: "The nucleotide sequence of a cDNA encoding the AtTIR, a TIR-like resistance protein in arabidopsis thaliana", Database accession no. AF188334 *
DATABASE GENBANK [online] 1 July 2000 (2000-07-01), ECKER ET AL.: "Genomic sequence of arabidopsis thaliana BAC F2D10 from chromosome 1", Database accession no. AC069251 *
DATABASE GENBANK [online] 16 March 2000 (2000-03-16), ROSE ET AL.: "Arabidopsis thaliana DNA chromosome 4, contig fragment no 84", accession no. EMBL Database accession no. AL161588 *
DATABASE GENBANK [online] 16 March 2000 (2000-03-16), TERRYN ET AL., accession no. EMBL Database accession no. AL161580 *
DATABASE GENBANK [online] 18 September 1999 (1999-09-18), ALTAFI ET AL., Database accession no. AC008017 *
DATABASE GENBANK [online] 19 July 1999 (1999-07-19), CHOISNE ET AL., accession no. EMBL Database accession no. AL096860 *
DATABASE GENBANK [online] 19 July 2000 (2000-07-19), KANEKO ET AL.: "Structural analysis of arabidopsis thaliana chromosome 3", Database accession no. AP000413 *
DATABASE GENBANK [online] 21 June 1999 (1999-06-21), WATERSTON R., Database accession no. AF149413 *
DATABASE GENBANK [online] 23 July 1997 (1997-07-23), THEOLOGIS A.: "The sequence of BAC T1G11 from arabidopsis thaliana chromosome 1", Database accession no. AC002376 *
DATABASE GENBANK [online] 27 December 2000 (2000-12-27), SATO ET AL.: "Structural analysis of arabidopsis thaliana chromosome 3", Database accession no. AB022217 *
DATABASE GENBANK [online] 27 July 1997 (1997-07-27), OKAMURO ET AL.: "The AP2 domain of APETALA2 defines a large new family of DNA binding protein in arabidopsis", Database accession no. AF003096 *
DATABASE GENBANK [online] OH ET AL.: "A senescence-associated gene of arabidopsis thaliana is distinctively regulated during natural and artificially induced leaf senescence", accession no. EMBL Database accession no. AAA80302 *
DNA RES., vol. 7, 2000, pages 131 - 135 *
DNA RES., vol. 7, 2000, pages 217 - 221 *
PLANT MOL. BIOL., vol. 30, 1996, pages 739 - 754 *
PLANT PHYSIOL., vol. 121, 1999, pages 1055 *
PROC. NATL. ACAD. SCI. USA, vol. 94, 1997, pages 7076 - 7081 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101255428B (en) * 2008-01-03 2010-07-21 广州大学 Plant salt resistance related gene and use thereof in plant breeding

Also Published As

Publication number Publication date
AUPQ920600A0 (en) 2000-08-31
WO2002012483B1 (en) 2002-04-18

Similar Documents

Publication Publication Date Title
Liu et al. Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought-and low-temperature-responsive gene expression, respectively, in Arabidopsis
Huh et al. ATAF2, a NAC transcription factor, binds to the promoter and regulates NIT2 gene expression involved in auxin biosynthesis
US6670528B1 (en) Environmental stress-tolerant plants
US7186563B2 (en) Methods and compositions for regulating plant stress tolerance
Liu et al. Identification of an Arabidopsis thaliana ribulose-1, 5-bisphosphate carboxylase/oxygenase activase (RCA) minimal promoter regulated by light and the circadian clock
JP2003144175A (en) Environmental stress-responding promoter
AU2010202610B2 (en) Plant Polynucleotide Promoter Sequences From Lolium Festuca And Arabidopsis
KR100578461B1 (en) Stress-induced promoter derived from rice
CN100359012C (en) Environmental stress-responsive promoters and genes encoding transcriptional factor
US7659448B2 (en) Plant regulatory sequences for selective control of gene expression
US20030217393A1 (en) Development of a stress-responsive promoter from maize
EP2247610A2 (en) Transcriptional activators involved in abiotic stress tolerance
Shen et al. AtbZIP16 and AtbZIP68, two new members of GBFs, can interact with other G group bZIPs in Arabidopsis thaliana
KR100703115B1 (en) Stress-induced promoter and method of using the same
AU780425B2 (en) Promoter expressed specifically in the cells of plant roots, recombinant vectors and host cells comprising same and transgenic plants obtained
CN106676132B (en) Efficient plant binary induction gene expression recombinant plasmid
CN111607598B (en) Application of soybean DDT structural domain gene GmDDT1
CA2313465A1 (en) Constitutive plant promoters
AU2002222940A1 (en) Lipoxygenase genes, promoters, transit peptides and proteins thereof
WO2002012483A1 (en) Promoters of plant defence-associated genes
WO2006006236A1 (en) Regulation of environmental stress-tolerance in plants using modified dreb2a gene
Roychoudhury et al. The promoter-elements of some abiotic stress-inducible genes from cereals interact with a nuclear protein from tobacco
Iqbal et al. Isolation and characterization of sucrose phosphate synthase promoter from cotton ('Gossypium hirsutum'L.)
KR102194283B1 (en) Recombinant vector for promoting plant length growth and volumetric growth and uses thereof
RU2697014C2 (en) Method for efficient biosynthesis of recombinant proteins in dicot plants using promoter of pro-smamp1 gene from stellaria media

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
AK Designated states

Kind code of ref document: B1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: B1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

B Later publication of amended claims
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP