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WO1997046692A1 - Promoteur de gene de plante inductible par nematodes - Google Patents

Promoteur de gene de plante inductible par nematodes Download PDF

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Publication number
WO1997046692A1
WO1997046692A1 PCT/EP1996/002437 EP9602437W WO9746692A1 WO 1997046692 A1 WO1997046692 A1 WO 1997046692A1 EP 9602437 W EP9602437 W EP 9602437W WO 9746692 A1 WO9746692 A1 WO 9746692A1
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WIPO (PCT)
Prior art keywords
plant
dna
dna sequence
sequence
nematode
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PCT/EP1996/002437
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English (en)
Inventor
Stephan Andreas Ohl
Peter Christiaan Sijmons
Frédérique Marianne VAN DER LEE
Oscar Johannes Maria Goddijn
Joke Klap
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Mogen International N.V.
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Priority to PCT/EP1996/002437 priority Critical patent/WO1997046692A1/fr
Priority to RU99100083/13A priority patent/RU2198219C2/ru
Priority to HU9903512A priority patent/HUP9903512A3/hu
Priority to JP10500116A priority patent/JP2000511427A/ja
Priority to US09/117,927 priority patent/US6262344B1/en
Priority to EP96920791A priority patent/EP0904387A1/fr
Priority to AU62222/96A priority patent/AU707563B2/en
Priority to CA002257149A priority patent/CA2257149A1/fr
Priority to BR9612635-3A priority patent/BR9612635A/pt
Publication of WO1997046692A1 publication Critical patent/WO1997046692A1/fr
Priority to BG103043A priority patent/BG103043A/xx

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8285—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for nematode resistance
    • 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
    • C12N15/8239—Externally regulated expression systems pathogen inducible
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the invention relates to regulatory DNA sequences which can be used for expressing DNA sequences in plant cells.
  • the invention further comprises chimeric DNA comprising said regulatory DNA sequences operably linked to DNA to be expressed in plant cells, as well as plants containing such chimeric DNA in their cells.
  • the invention further relates to methods for making plant3 that are resistant, or at least less susceptible to plant parasitic nematodes, or their effects, as well as to cells, plants and parts thereof.
  • WO 93/06710 Another regulatory sequence that is inducible by the root-knot nematode Meloidogyne incognita is disclosed in WO 93/06710.
  • a disadvantage of this regulatory sequence TobRb7 is that it is not activated by a number of cyst nematodes, among which the Heterodera and Giojbodera species. This makes the TobRB7 sequence unsuitable for use in chimeric constructs aiming at, for example, cyst nematode resistance in potato.
  • the invention provides a DNA fragment obtainable from Arabidopsis thaliana that is capable of promoting root knot and cyst nematode- inducible transcription of an associated DNA sequence when re-introduced into a plant.
  • Preferred according to the invention are sequences represented by nucleotides 1 to 2361 in SEQIDNO: 4.
  • portions or variants of a DNA fragment according to the invention capable of promoting root knot and cyst nematode-inducible transcription of an associated DNA sequence when re-introduced into a plant.
  • a still further preferred aspect of the invention comprises a regulatory DNA fragment that is substantially nematode feeding site-specific.
  • chimeric DNA sequences comprising in the direction of transcription a regulatory DNA fragment according to the invention and a DNA sequence to be expressed under the transcriptional control thereof and which is not naturally under transcriptional control of said DNA fragment.
  • Preferred among the chimeric DNA sequences according to the invention are those wherein the DNA sequence to be expressed causes the production of a plant cell- disruptive substance, such as barnase.
  • the cell-disruptive substance comprises RNA complementary to RNA essential to cell viability.
  • the DNA sequence to be expressed causes the production of a substance toxic to the inducing nematode.
  • the invention finds further use in a replicon comprising a DNA fragment or chimeric DNA sequence according to the invention, a microorganism containing such a replicon, as well as plant cells having incorporated into their genome a chimeric DNA sequence according to the invention.
  • a root system of a plant essentially consisting of cells according to the invention as well as full grown plants essentially consisting of cells according to the invention, preferably a dicotyledonous plant, more preferably a potato plant.
  • plants grafted on a root system according to the invention as well as plant parts selected from seeds, flowers, tubers, roots, leaves, fruits, pollen and wood and crops comprising such plants.
  • the invention also encompasses the use of a DNA fragment according to the invention for identifying subfragments capable of promoting transcription of an associated DNA sequence in a plant. Also envisaged is the use of a chimeric DNA sequence according to the invention for transforming plants. The invention further provides the use of a fragment, portion or variant of a regulatory DNA according to the invention for making hybrid regulatory DNA sequences.
  • FIGURES Figure 1. Schematic plasmid map of Binary vector pMOG23.
  • Figure 2. Schematic plasmid map of Binary vector pMOG ⁇ OO.
  • Figure 3. Schematic plasmid map of Binary vector pMOG553.
  • Figure 4. Schematic plasmid map of Binary vector pMOG819.
  • Figure 5. Schematic plasmid map of Binary vector pMOG849.
  • Figure 6. Expression patterns outside the NFS of several pMOG849 transformed Arabidopsis thaliana lines.
  • Figure . Schematic representation of a NFS disrupter gene and a neutraliser gene in a two component system for engineering of nematode resistanct plants
  • Figure 8. Schematic pla ⁇ mid map of Binary vector pMOG893.
  • the present invention provides regulatory DNA sequences obtainable from Arabidopsis thaliana, which are inducible by root knot and cyst nematodes and which show a high preference of expression of any associated DNA inside the special nematode feeding structures of the plant root.
  • a nematode feeding structure is used by an invading nematode as source of food, whereby the nematode induces a change in the plant tissue thereby forming either a giant cell (root-knot nematodes) or a syncytium (cyst nematodes) .
  • a method of isolating regulatory DNA sequences has been disclosed and claimed in a prior application, WO92/17054, which is incorporated herein by reference.
  • the regulatory DNA sequences according to the invention can be used to express any heterologous DNA in any plant of choice, by placing said DNA under the control of said regulatory DNA sequences and transforming plants with the resulting chimeric DNA sequence using known methods.
  • the heterologous DNA is expressed upon infection of the roots by various root knot nematodes, such as Meloidogyne incognita, and cyst nematodes, such as eterodera schachtii and Globodera pallida (a more comprehensive, but by no means limiting, list is presented in table 2) .
  • the heterologous DNA may consist of a gene coding for a substance that is toxic or inhibitive to a plant parasitic nematode in order to create plants with reduced susceptibility to plant parasitic nematodes.
  • toxic substances such as the endotoxins of Bacillus thuringiensis (e . g. EP 0 352 052), lectins, and the like.
  • a more preferred approach for making plants with reduced susceptibility to plant parasitic nematodes consists in the disruption of the specialised feeding structure of the plant roots by expressing a phytotoxic substance under the control of the regulatory DNA sequences according to the invention.
  • the general principles of this approach have been disclosed and claimed in International patent applications W092/21757, WO93/10251 and WO94/10320, which are hereby incorporated by reference.
  • the phytotoxic substance shall be referred to hereinafter as the nematode feedings site (NFS) disruptive substance .
  • the regulatory DNA sequences according to the invention are substantially specific for the nematode feeding structure, it may be that due to expression in non-target (i.e. non-NFS) tissue the NFS disruptive substances under the control thereof have adverse effects on plant viability and/or yields. Moreover, it was found that the regulatory DNA sequences according to the invention are active during the tissue culture phase in the transformation procedure, necessitating the use of a neutralising substance during this phase. In order to reduce or eliminate (potential) adverse effects, it is therefore strongly preferred to use a chimeric NFS-disruptive construct according to the invention in conjunction with a neutralising gene construct.
  • NFS- disrupter compound (coding sequence-A) is placed under the control of a promoter that is at least active in the NFS, and preferably not or hardly outside the NFS, whereas the unwanted phytotoxic effects outside the NFS are neutralised by a neutralising compound (coding sequence-B) that is expressed at least in those tissues wherein the disruptive substance is produced except for the NFS.
  • a suitable promoter-A is defined as a promoter that drives expression of a downstream coding sequence inside the NFS, at levels sufficient to be detrimental to the metabolism and/or functioning and/or viability of the NFS, while this promoter should preferably, but not necessarily, be inactive in tissues outside the NFS; it should at least never be active outside NFS at such levels that the activity of the disruptive substance, encoded by coding 3equence-A, can not be neutralized sufficiently by products from coding sequence-B.
  • the properties of the regulatory DNA sequences according to the invention make them highly useful in the two-component approach, as is illustrated by way of Examples herein.
  • numerous mutations such as deletions, additions and changes in nucleotide sequence and/or combinations of those are possible in the regulatory DNA sequences according to the invention which do not alter the properties of these sequences in a way crucial to their intended use. Such mutations do, therefore, not depart from the present invention.
  • regulatory regions of plant genes consist of disctinct subregions with interesting properties in terms of gene expression. Examples of subregions as meant here, are enhancers but also silencers of transcription. These elements may work in a general (constitutive) way, or in a tissue-specific manner. As is illustrated in the examples, several deletions may be made in the regulatory DNA sequences according to the invention, and the subfragments may be tested for expression patterns of the associated DNA. Various subfragments so obtained, or even combinations thereof, may be useful in methods of engineering nematode resistance, or other applications involving the expression of heterologous DNA in plants.
  • NFS disruptive substance and neutralizing substance embraces a series of selected compounds that are encoded by DNA whose gene products (either protein or RNA or antisense-RNA) are detrimental to the metabolism and/or functioning and/or viability of NFS or organelles therein and for which neutralizing substances are known that are able, when expressed simultaneously in the same cell as the disruptive substance, to repress the activity of the disrupting substance.
  • Preferred combinations of disrupting and neutralizing substances are e.g.
  • RNAses such as RNAse TI, ribonucleases or proteases and ribozymes against mRNA that code for phytotoxic proteins.
  • combinations of disrupting and neutralizing substances comprise respectively genes inhibitory to an endogenous gene that encodes a protein or polypeptide product that is essential for cell viability and, as a neutralizing gene, a gene that encodes a protein or polypeptide product capable of substituting the function of the endogenous protein or polypeptide product.
  • Such disruptive genes may be selected from the group consisting of (a) genes encoding ribozymes against an endogenous RNA transcript, (b) genes which when transcribed produce RNA transcripts that are complementary or at least partially complementary to RNA transcripts of endogenous genes that are essential for cell viability, a method known as antisense inhibition of gene expression (disclosed in EP-A 240 208), or (c) genes that when transcribed produce RNA transcripts that are identical or at least very similar to transcripts of endogenous genes that are essential for cell viability, an as yet unknown way of inhibition of gene expression referred to as co-suppression (disclosed by Napoli C. et ai., 1990, The Plant Cell 2, 279-289) .
  • antisense genes to inhibit expression of endogenous genes essential for cell viability, which genes are expressed in the nematode feeding structures by virtue of regulatory DNA sequences according to the invention fused upstream to the said antisense gene.
  • the disruptive effect brought about by the antisense gene inhibitory to the vital endogenous gene is neutralized by the expression of a neutralizing compound-B, which expression is under the control of a promoter-B as defined, said compound-B being a protein or polypeptide product which is identical or similar to the protein or polypeptide encoded by the endogenous vital gene and capable of substituting the function of the endogenous gene product in the host plant. It is preferred that the nucleotide sequence of the RNA transcript encoded by the neutralizing gene is divergent from the endogenous vital gene RNA transcript to avoid a possible co-suppressive effect.
  • the neutralizing gene encodes a protein or polypeptide with essentially the same function as the endogenous vital gene, but through an RNA transcript intermediate that is divergent; neutralizing genes which fit this description can be suitably obtained by screening a database for genes obtainable from a different plant species, or even a different non-plant species, such as yeasts, animal eukaryotes or prokaryotes.
  • the nucleotide sequence identity of the transcripts encoded by the disruptive antisense transgene and the neutralizing sense transgene is less than 90%, preferably less than 80%, yet more preferably said neutralizing sense transgene encodes a protein or polypeptide gene product that is not identical in amino acid sequence to the disrupted gene product and wherein the nucleotide sequence identity of the transcripts encoded by the neutralizing transgene is less than 75%.
  • Target genes for antisense disrupter genes are selected from those coding for enzymes that are essential for cell viability, also called housekeeping enzymes, and should be nuclear encoded, preferably as single copy genes, although a small size gene family would also be suitable for the purpose of the invention. Furthermore, the effect of antisense expression of said genes must not be nullified by diffusion or translocation from other cells or organelles of enzyme products normally synthesized by such enzymes. Preferably, genes coding for membrane-translocating enzymes are chosen as these are involved in establishing chemical gradients across organellar membranes. Inhibition of such proteins by antisense expression can not, by definition, be cancelled by diffusion of substrates across the membrane in which these proteins reside.
  • the translocated compound is not limited to organic molecules but can be of inorganic nature; e.g. P, H, OH or electrons.
  • the membrane-translocating enzymes should be present in organelles that increase in numbers during parasitism, thereby illustrating the essential role that such organelles have in cells
  • SUBSTITUTE SHEET tiiuLE 26 comprising the NFS.
  • organelles include mitochondria, endoplasmic reticulum and plasmodesmata (Hussey et al. 1992 Protoplasma 167; 55-65, Magnusson & Golinowski 1991 Can. J. Botany 6 ; 44-52) .
  • a list of target enzymes is given in Table 1 by way of example but the invention is not limited to the enzymes mentioned in this table. More detailed listings can be assembled from series as Biochemistry of Plants (Eds. Stu pf & Conn, 1988-1991, Vols. 1-16 Academic Press) or Encyclopedia of Plant Physiology (New Series, 1976, Springer-Verlag, Berlin) . Although only in some cases, the genes coding for these enzymes have been isolated and, therefore, the number of gene copies are not known, the criteria that have to be met are described in this invention.
  • NADPH-cytochrome P450 reductase lipid metabolism fatty acid synthase complex lipid metabolism glycerol-3P-acyltransferase lipid metabolism hydroxymethyl-glutaryl CoA reductase mevalonic acid pathway aminoacyl transferase nucleic acid metabolism transcription factors nucleic acid metabolism elongation factors nucleic acid metabolism
  • a suitable promoter-B is defined as a promoter that drives expression in substantially all cells wherein coding sequence-A is expressed, with the proviso that it does not drive expression inside a nematode feeding structure, or not effectively. (With 'substantially all cells' is meant at least those cells that should be viable in order to get normal plant growth and or development required for commercial exploitation of such plants) .
  • plants in which the disruptive effect is not neutralized in exactly all cells of the host plant and which are nevertheless viable and suitable for commercial exploitation are those which express a disrupter gene according to this invention in stamen cells; this may yield male-sterile plants, which is even regarded as a commercially attractive trait in some crops.
  • Suitable examples of the promoter-B type can be obtained from plants or plant viruses, or may be chemically synthesized.
  • the regulatory sequences may also include enhancer sequences, such as found in the 35S promoter of CaMV (Kay et al . , 1987, Science 236. 1299-1302), and mRNA stabilizing sequences such as the leader sequence of Alfalfa Mosaic Virus RNA4 (Brederode et al . , 1980, Nucl. Acids Res. £., 2213-2223) or any other sequences functioning in a like manner.
  • a promoter-B/coding-sequence-B can be complemented with a second promoter-B'/coding-sequence-B having an expression pattern which is partly overlapping or entirely complementary to promoter-B/coding- sequence-B, with the proviso that neither promoter-B nor promoter-B' drives expression in the NFS.
  • hybrid promoters comprising (parts of) different promoters combined as to provide for the required expression pattern as defined herein, fall within the scope of the present invention.
  • promoter-B is the Cauliflower Mosaic Virus 35S promoter or derivatives thereof, which is generally considered to be a 3trong constitutive promoter in plant tissues (Odell et al. 1985 Nature 313. 810-812) .
  • Another preferred example for promoter-B is the strong root promoter rolD (Leach & Aoyagi 1991 Plant Sci. 22; 69-76) from plasmid pRiA4 of Agrobacterium rhizogenes; the 5' flanking region of ORF15
  • Terminor sequences and polyadenylation signals include any such sequence functioning as ⁇ uch in plants, the choice of which is within the level of skill of the average skilled person in the art.
  • An example of such sequences is the 3* flanking region of the nopaline synthase (nos) gene of Agrobacterium tumefaciens (Bevan, 1984, Nucl. Acids Res. 12, 8711-8721) .
  • Plant genera which are damaged during agricultural practice by PPN and which can be made significantly less susceptible to PPN by ways of the present invention include but are not limited to the genera mentioned in Table 2.
  • Nematode species as defined in the context of the present invention include all plant-parasitic nematodes that modify host cells into specially adapted feeding structures which range from migratory ectoparasites (e . g. Xiphinema spp.) to the more evolved sedentary endoparasites (e . g. Heteroderidae, Meloidogynae or Rotylenchulinae) .
  • a plant is said to show reduced susceptibility to plant parasitic nematodes (PPN) if a statistically significant decrease in the number of mature females developing at the surface of plant roots can be observed as compared to control plants.
  • PPN plant parasitic nematodes
  • Susceptible / resistance classification according to the number of maturing females is standard practice both for cyst- and root-knot nematodes (e.g. LaMondia, 1991, Plant Disease 15., 453-454; O wega et al . , 1990, Phytopathol. Mi 745-748) .
  • a nematode feeding structure shall include an initial feeding cell, which shall mean the cell or a very limited number of cells destined to become a nematode feeding structure, upon induction of the invading nematode.
  • a NFS disruptive effect according to the invention is not limited to adverse effects on the NFS only; also disruptive effects are contemplated that, in addition, have an adverse effect on nematode development by way of direct interaction.
  • transformation systems involving vectors are widely available, such as viral vectors (e.g. from the Cauliflower Mosaic Virus (CaMV) and bacterial vectors (e.g. from the genus Agrobacterium) (Potrykus, 1990, Bio/Technol. £, 535-542).
  • viral vectors e.g. from the Cauliflower Mosaic Virus (CaMV)
  • bacterial vectors e.g. from the genus Agrobacterium
  • the protoplasts, cells or plant parts that have been transformed can be regenerated into whole plants, using methods known in the art (Horsch et al . , 1985, Science 225. 1229-1231) .
  • the choice of the transformation and/or regeneration techniques is not critical for this invention.
  • binary vector system (disclosed in EP-A 120 516) in which Agrobacterium strains are used which contain a helper plasmid with the virulence genes and a compatible plasmid, the binary vector, containing the gene construct to be transferred.
  • This vector can replicate in both E. coli and in Agroj acteriu/n; the one used here is derived from the binary vector Binl9 (Bevan, 1984, Nucl. Acids Res. 12, 8711-8721) .
  • the binary vectors as used in this example contain between the left- and right-border sequences of the T-DNA, an identical NPTII-gene coding for kanamycin resistance (Bevan, 1984, Nucl. Acids Res. 12, 8711-8721) and a multiple cloning site to clone in the required gene constructs.
  • kanamycin resistance Bevan, 1984, Nucl. Acids Res. 12, 8711-8721
  • a multiple cloning site to clone in the required gene constructs Recent scientific progress shows that in principle monocots are amenable to transformation and that fertile transgenic plants can be regenerated from transformed cells. The development of reproducible tissue culture systems for these crops, together with the powerful methods for introduction of genetic material into plant cells has facilitated transformation.
  • Transgenic maize plants have been obtained by introducing the Streptomyces hygroscopicus bar gene, which encodes phosphinothricin acetyltransferase (an enzyme which inactivates the herbicide phosphinothricin) , into embryogenic cells of a maize suspension culture by microparticle bombardment (Gordon-Kamm, 1990, Plant Cell, 2, 603-618) .
  • Transformation was carried out using co-cultivation of Arabidopsis thaliana (ecotype C24) root segments with AgroJ acteriu/n strain MOG101 containing a suitable binary vector as described by Valvekens et al . (1988, Proc. Nat. Acad. Sci. USA ££, 5536-5540) which is as follows:
  • Arabidopsis seeds were vernalized for 7 days at 4°C before germination. Seeds were surface-sterilized for 2 min in 70% EtOH, transferred to 5% NaOCl/0.5% NaDodS0 4 for 15 min rinsed five times with sterile distilled water, and placed on 150 x 25 mm Petri dishes containing germination medium (GM) (Table 3) to germinate. Petri dishes were sealed with gas-permeable medical tape (Urgopore, Chenove France) . Plants were grown at 22°C in a 16-hr light/8-hr dark cycle. The same growth-room conditions were used for tissue culture procedures.
  • GM germination medium
  • Roots were then cut into small pieces of about 0.5 cm (herein referred to as "root explants") and transferred to 10 ml of liquid 0.5/0.05 medium; 0.5-1.0 ml of an overnight Agrobacterium culture was added. The root explants and bacteria were mixed by gentle shaking for about 2 min.
  • the root explants were blotted on sterile filter paper to remove most of the liquid medium and cocultivated for 48 hr on 0.5/0.05 agar.
  • the explants were then rinsed in liquid 0.5/0.05 medium containing 1000 mg of vancomycin (Sigma) per liter.
  • the pieces were blotted and then incubated on 0.15/5 agar (Table 3) supplemented with 750 mg of vancomycin and 50 mg of Km per liter.
  • Three weeks after infection with agrobacteria containing a chimeric neo gene, green Km-resistant (Km R ) calli were formed in a background of yellowish root explants.
  • L liter
  • IAA indole-3-acetic acid
  • Kin kinetin
  • 2ipAde N 6 -(2- isopentenyl) adenine
  • CIM callus-inducing medium
  • SIM shoot-inducing medium
  • MS Murashige & Skoog medium
  • B5 Gamborg B5 medium
  • Peeled surface-sterilized potato tubers were cut in 2 mm thick slices. These were used to cut out disks of 1 cm in diameter around the periphery of the slice. The disks were collected in WM (Murashige & Skoog medium, containing 1 mg/1 thiamine HCl, 0.5 mg/1 pyridoxine Hcl, 0.5 mg/1 nicotinic acid, 100 mg/1 myo-inositol, 30 g/1 sucrose, 0.5 g/1 MES pH 5.8) . Inoculation with Agrobacterium tumefaciens strain EHA105 (Hood et al .
  • Arabidopsis seeds were surface sterilized and sown in petri dishes (0: 9 cm) on B5 medium containing 20 g/1 glucose and 20 mg/1 kanamycin. After 3 days at 4°C the plates were incubated for 2 weeks in a growth chamber at 22°C with 16-hr light/8 hr-dark cycle. Kanamycin-resistant plants were then transferred to soil-filled translucent plastic tubes (30x15x120 mm, Kelder plastibox b.v.. The Netherlands) . The tubes were placed tilted at an angle of 60 degrees to the vertical axis causing the roots to grow on the lower side of the tubes. This allows to monitor the infection process by eye and facilitates removal of the root system from the soil for GUS analysis.
  • Infection was done after two more weeks by injecting a suspension containing 500 second stage larvae of Heterodera schachtii (in 3 ml H 2 0) per root system or 300 second stage larvae of Meloidogyne incognita per root system into the soil.
  • GUS activity was determined at various times during the infection process by thoroughly washing the root systems to remove most of the adhering soil and incubating them in X-Gluc solution (1 mg/ml X-Gluc, 50mM NaP0 4 (pH7), ImM K 4 Fe(CN) 6 , ImM K ⁇ 3 Fe(CN) 6 , lOmM EDTA, 0.1% Triton XlOO) at 37°C over night. After removal of the chlorophyll from the tissue by incubation with 70% ethanol for several hours GUS staining was monitored under the microscope.
  • the binary vector pMOG ⁇ OO is a derivative of pMOG23 (Fig. 1, deposited at the Centraal Bureau voor schimmelcultures, Ooster ⁇ traat 1, Baarn, The Netherlands on January 29, 1990 under number CBS 102.90) in which an additional Kpnl restriction site was introduced into the polylinker between EcoRI and Smal.
  • This plasmid contains between the left and right borders of T-DNA a kanamycin resistance gene for selection of transgenic plant cells (Fig. 2) .
  • Example 2 Construction of promoterless GUS construct pMOG553 Construction of this vector is described in Goddijn et al . 1993 Plant J 4, 863-873. In this reference an error occurs; the construct contains a CaMV 35S RNA terminator behind the ⁇ -glucuronidase gene instead of the indicated nos terminator. The sequence between the T-DNA borders of this binary vector is available from the EMBL database under accession number: X84105.pMOG553 carries the HygR marker for plant transformation (Fig. 3) .
  • Example 3 Identification and Isolation of a trapped NFS-preferential promoter fragment in Arabidopsis thaliana
  • the binary vector pMOG553 was mobilized by triparental mating to Agrobacterium tumefaciens strain MOG101. The resulting strain was used for Arabidopsis root transformation. More than 1100 transgenic Arabidopsis plant lines were obtained in this way. Transgenic plants were grown to maturity, allowed to self-fertilize and the resulting seeds (SI) were harvested and vernalized. Subsequently SI seeds were germinated on nutrient solution (Goddijn et al .
  • Line pMOG553#1164 was identified as a line which showed rather strong GUS expression inside syncytia and giant cells induced by Heterodera schachtii and Meloidogyne incognita, respectively.
  • un-infected control plants (as well as in the infected plants) of this line very weak GUS expression was detected in a few cells at the base of young lateral roots and in some green parts of the plant.
  • Genomic DNA of this line was cleaved with the restriction enzyme MscI, which cleaves once in the GUS coding region, and religated.
  • MscI restriction enzyme
  • SnaBI a linear fragment was obtained with known GUS sequences at the ends and the flanking plant sequence in between.
  • This fragment was amplified using the primer set GUSinv5 (5' CTT TCC CAC CAA CGC TGA TC 3' SEQIDNO: 1) and GUS7 (5' GTA ATG CTC TAC ACC ACG CCG 3' SEQIDNO: 2), cloned in a multi-copy vector and sequenced (see below) .
  • GUSinv5 5' CTT TCC CAC CAA CGC TGA TC 3' SEQIDNO: 1
  • GUS7 5' GTA ATG CTC TAC ACC ACG CCG 3' SEQIDNO: 2
  • Primer 1164XBM introduces a BamHI site at the 5 end of the promoter, which allowed to clone the 1480 bp BamHI promoter fragment back in front of GUS in construct pMOG819 without changing the sequence between the GUS open reading frame and the plant promoter.
  • This vector was constructed by cloning the GUSintron coding region (Vancanneyt et al . 1990, Mol. Gen. Genet. 220; 245-250) of pMOG553 as a BamHI-EcoRI fragment in the polylinker of pMOG800.
  • the binary vector pMOG819 (Fig. 4) serves to introduce the cloned promoter fragments for further expression analysis after transformation of plants.
  • GUS-expression was also found in giant cells induced by infection with Meloidogyne incognita in the same lines which expressed GUS in syncytia induced by Heterodera schachtii .
  • the #1164 regulatory sequence was also active as a promoter, thus prompting the need to use a neutralizing gene if the #1164 promoter fragment is transferred to Arabidopsis with a plant cell disruptive gene under its control, such as barnase (see Example 8 and 9) .
  • the 553#1164-based PCR fragment was used as a probe to isolate the corresponding genomic clone.
  • a genomic fragment of 2.1 Kb (see SEQIDNO: 4) was then used in a similar approach as described above (pMOG889 contains genomic 55311164 fused to GUSintron) .
  • pMOG889 contains genomic 55311164 fused to GUSintron
  • Example 6 Sequence determination of promoter tag pMOG553#1164
  • the sequence of the genomic clone of #1164 was determined by the primer walking strategy on CsCl purified DNA, using the automatic sequencer ALF of Pharmacia. Fluor dATP was used in combination with the AutoRead sequencing kit. The procedure is described in " Voss et al. (1992) Mol Cell Biol 3, 153-155. The sequence is depicted in SEQIDNO: 4.
  • Example 7 Cloning of promoter subfrag ent (s) Five subfragments of promoter #1164 were made by PCR using the primers as shown in table 4. The primer numbering is the same as that used in the Sequence Listing. For all amplifications the proofreading DNA polymerase pfu was used and pMOG849 served as target DNA. All 5' end primers contain an Xhol site. Thus, all PCR generated deletion fragments of the 1164 promoter could be reintroduced in pMOG819 using this Xhol site and the BamHI site, which is located in the multiple cloning site of pMOG553 and was retained in the tagged linell64 between the GUS coding region and the tagged plant sequence. The numbers refer to the constructs resulting from the subfragments cloned in pMOG819; the primers 6044-1 to 6044-6 correspond with SEQIDNO' s 6 to 11, respectively.
  • timing and the like can be determined as described for the 1.5 Kb #1164 fragment in Example 3. Fragments found to have useful patterns and/or timing may subsequently be used to drive expression of other heterologous DNA sequences (both sense/coding and antisense) and/or used to make hybrid promoter constructs. Furthermore, further analysis yields insight in several regulatory elements such as silencers, enhancers and the like, and creates the possibility of willfully influencing expression patterns and/or timing.
  • Example 8 Cloning of #1164 in front of barnase A 2.1 Kb genomic DNA fragment containing the 5' tagged sequence from line 1164 was cloned in front of barnase, a Bacillus amyloliquefaciens derived RNase gene, to engineer plant ⁇ resistant to sedentary plant nematodes.
  • the genomic fragment was obtained by screening 400000 clones of a genomic library of Arabidopsis ecotype C24 with the #1164 iPCR product (see Example 3) . From one of the hybridizing clones a 4 kb EcoRI fragment was isolated and subcloned in the multicopy plasmid pKS (Stratagene) .
  • a fragment containing the barnase coding region was PCR amplified on pMT416 DNA (Hartley, sub) using primers 5' CGGACTCTGGATCCGGAAAGTG 3' (SEQIDNO: 12) and 5' CTGCTCGAGCCTAGGCACAGGTTATCAACACGTTTG 3' (SEQIDNO: 13) . These primers introduce flanking BamHI and Xhol restriction sites to facilitate cloning of the fragment. The fragment was cloned in the multiple cloning site in a vector containing the barstar gene under control of a Taq promoter (necessary to overcome toxicity of barnase in bacteria) .
  • the 5' untranslated sequence of barnase was further modified to resemble the corresponding sequence in the original line pMOG553#1164 by annealing the following oligonucleotides 5' GATCTAGACTCGAGAAGCTTGGATCCCCGGGTAGGTCAGTCCCC 3' (SEQIDNO: 16) and 5' CATGGGGGACTGACCTACCCGGGGATCCAAGCTTCTCGAGTCTA 3' (SEQIDNO: 17) and ligating the resulting adapter between the Bglll site and the Ncol site of pOGl ⁇ .l, resulting in clone pFL8.
  • the adapter introduced a Hindlll site 5' to the barnase coding region which was used to insert the 1164 promoter yielding pFL15.
  • a fragment containing the Tag promoter and the barstar gene were exchanged with this adapter.
  • Construct pFLll contains a chimeric barstar gene in a binary vector. This construct was cloned in the following way. The barstar coding region resides on a Hindlll/BamHI fragment in construct pMT316 (Hartley (1988) J Mol Biol 202, 913-915) . The Hindlll site was changed into a BamHI site by ligating in this site the self-annealing adapter 5' AGCTCGGATCCG '3 (SEQIDNO: 18) .
  • the resulting BamHI fragment was cloned between a double enhanced CaMV 35S promoter and a nos terminator in the expression cassette pMOGl ⁇ O, described in WO93/10251, resulting in p ⁇ G30.
  • the adapter 5' GGCTGCTCGAGC 3' SEQIDNO: 19
  • the Hindlll site at the 3' end of the nos terminator was changed into an Xhol site and the EcoRI site at the 5' end of the promoter was changed into a Hindlll site using the adapter 5' AATTGACGAAGCTTCGTC 3' (SEQIDNO: 20) .
  • the 35S promoter was replaced by the promoter from the Agrobacterium rhizogenes RolD gene.
  • This promoter was excised as a Hindlll/BamHI fragment from construct pD02, obtained from F. Leach (Leach and Aoyagi (1991) Plant Sci 23., 69-76) .
  • the barstar gene including promoter and terminator was excised by digestion with Hindlll and Xhol and inserted in the respective sites of the polylinker in pMOG800, resulting in pFLll.
  • chimeric #1164 promoter-barnase gene was cleaved out of pFL15 as an EcoRI fragment and inserted in the unique EcoRI site of pFLll between barstar and the Nptll marker gene in a tandem orientation, resulting in pMOG893.
  • the binary vector pMOG893 was mobilised to Agrobacterium tumefaciens and the resulting strain was used for transformation of tuber discs from the potato cultivar Kardal as described in the Experimental part. A total of 98 transgenic lines were obtained. These lines were propagated vegetatively by cutting shoots in segments containing at least one node and rooting them in vitro . Per line 15 plants are tested for increased resistance to Globodera pallida as described in the Experimental part. It is expected that potato plants transformed with the pMOG893 contained barnase/Barstar construct show reduced susceptibility to Globodera pallida due to the nematode-induced expression of Barnase inside the (developing) nematode feeding structure.
  • MOLECULE TYPE DNA (genomic)
  • CTTAACACTG CAGGTGAAGG CTGGCTCAAT CTTTGACAAT AT TTGATCT GCGATGACCC 300
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Arabidopsis thaliana
  • STRAIN C24
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL YES
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL YES
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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Abstract

La présente invention se rapporte à des fragments d'ADN pouvant être obtenus à partir de Arabidopsis thaliana, capables de stimuler la transcription inductible par nématodes à kyste et cécidogènes d'une séquence d'ADN associée lorsqu'ils sont réintroduits dans une plante, ainsi que leur utilisation.
PCT/EP1996/002437 1995-06-13 1996-06-04 Promoteur de gene de plante inductible par nematodes WO1997046692A1 (fr)

Priority Applications (10)

Application Number Priority Date Filing Date Title
PCT/EP1996/002437 WO1997046692A1 (fr) 1996-06-04 1996-06-04 Promoteur de gene de plante inductible par nematodes
RU99100083/13A RU2198219C2 (ru) 1996-06-04 1996-06-04 ФРАГМЕНТ ДНК, ПОЛУЧАЕМЫЙ ИЗ Arabidopsis thaliana, ЕГО СУБФРАГМЕНТ ИЛИ КОМБИНАЦИЯ СУБФРАГМЕНТОВ, ПОСЛЕДОВАТЕЛЬНОСТЬ ХИМЕРНОЙ ДНК И ЕЕ ИСПОЛЬЗОВАНИЕ, РЕПЛИКОН (ВАРИАНТЫ)
HU9903512A HUP9903512A3 (en) 1996-06-04 1996-06-04 Nematode-inducible plant gene promoter
JP10500116A JP2000511427A (ja) 1996-06-04 1996-06-04 線虫誘導性植物遺伝子プロモーター
US09/117,927 US6262344B1 (en) 1995-06-13 1996-06-04 Nematode-inducible plant gene promoter
EP96920791A EP0904387A1 (fr) 1996-06-04 1996-06-04 Promoteur de gene de plante inductible par nematodes
AU62222/96A AU707563B2 (en) 1996-06-04 1996-06-04 Nematode-inducible plant gene promoter
CA002257149A CA2257149A1 (fr) 1996-06-04 1996-06-04 Promoteur de gene de plante inductible par nematodes
BR9612635-3A BR9612635A (pt) 1996-06-04 1996-06-04 Promotor de gene de planta nematódio-indutìvel.
BG103043A BG103043A (en) 1995-06-13 1998-12-30 Nematode-induceable growth gene promotor

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998022599A1 (fr) * 1996-11-18 1998-05-28 Mogen International N.V. Sequences d'adn de regulation inductibles par les nematodes
WO2000001832A3 (fr) * 1998-07-02 2000-04-20 Plant Bioscience Ltd Promoteurs inductibles
US6271437B1 (en) 1998-05-18 2001-08-07 Pioneer Hi-Bred International, Inc. Soybean gene promoters
US6448471B1 (en) * 1998-01-22 2002-09-10 Piotr S. Puzio Nematode-feeding structure specific gene and its application to produce nematode resistant plants
US7572950B2 (en) 2002-07-04 2009-08-11 Sungene Gmbh & Co. Kgaa Methods for obtaining pathogen resistance in plants

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102011122267A1 (de) * 2011-12-23 2013-06-27 Kws Saat Ag Neue aus Pflanzen stammende cis-regulatorische Elemente für die Entwicklung Pathogen-responsiver chimärer Promotoren

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WO1992021757A1 (fr) * 1991-05-30 1992-12-10 Plant Genetic Systems, N.V. Promoteurs destines aux plantes et reagissant aux nematodes
WO1993010251A1 (fr) * 1991-11-20 1993-05-27 Mogen International N.V. Procede de production de plantes a sensibilite reduite aux nematodes parasites des plantes
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998022599A1 (fr) * 1996-11-18 1998-05-28 Mogen International N.V. Sequences d'adn de regulation inductibles par les nematodes
US6395963B1 (en) 1996-11-18 2002-05-28 Zeneca Limited Nematode-inducible regulatory DNA sequences
US6448471B1 (en) * 1998-01-22 2002-09-10 Piotr S. Puzio Nematode-feeding structure specific gene and its application to produce nematode resistant plants
US6271437B1 (en) 1998-05-18 2001-08-07 Pioneer Hi-Bred International, Inc. Soybean gene promoters
WO1999060109A3 (fr) * 1998-05-18 2002-09-19 Pioneer Hi Bred Int Promoteurs de gene de soja
WO2000001832A3 (fr) * 1998-07-02 2000-04-20 Plant Bioscience Ltd Promoteurs inductibles
US7572950B2 (en) 2002-07-04 2009-08-11 Sungene Gmbh & Co. Kgaa Methods for obtaining pathogen resistance in plants

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HUP9903512A3 (en) 2000-04-28
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