EP1034285A2 - Isolation und charakterisierung von regulatorischen sequenzen aus pfanzen - Google Patents

Isolation und charakterisierung von regulatorischen sequenzen aus pfanzen

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Publication number
EP1034285A2
EP1034285A2 EP98966826A EP98966826A EP1034285A2 EP 1034285 A2 EP1034285 A2 EP 1034285A2 EP 98966826 A EP98966826 A EP 98966826A EP 98966826 A EP98966826 A EP 98966826A EP 1034285 A2 EP1034285 A2 EP 1034285A2
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EP
European Patent Office
Prior art keywords
plant
nematode
dna
gus
promoter
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EP98966826A
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English (en)
French (fr)
Inventor
Nathalie Barthels
Mansour Karimi Dehkor Di
Godelieve Gheysen
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Vlaams Instituut voor Biotechnologie VIB
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Vlaams Instituut voor Biotechnologie VIB
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Priority to EP98966826A priority Critical patent/EP1034285A2/de
Publication of EP1034285A2 publication Critical patent/EP1034285A2/de
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems
    • C12N15/8239—Externally regulated expression systems pathogen inducible
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • 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 present invention relates to new pathogen-, in particular nematode-, induced promoters.
  • Said promoters induce an expression in very selective pathogen infection sites, in particular nematode infection sites of a plant.
  • Nematodes also called roundworms, have been enormously successful in colonizing nearly every habitat on earth. Although only about 100.000 species of nematodes have been named, there may be as many as 500.000. Nematodes are simple worms consisting of an elongate stomach and reproduction system inside a resistant outer cuticle (outer skin). Most nematodes are so small, between 200 micrometers to 5 mm long, that a microscope is needed to see them. Their small size, resistant cuticle, and ability to adapt to severe and changing environments have made nematodes one of the most abundant types of animals on earth.
  • nematodes feed on bacteria, fungi and other soil organisms. Others are parasitic, obtaining their food from animals , humans (such as the pinworm) and plants.
  • Agricultural cultivation encourages an increase in parasitic nematodes that feed on the crops being grown.
  • new kinds of plant parasitic nematodes may be introduced into a field by contaminated plant parts, soil on farm equipment and irrigation water.
  • Nematodes which parasitize plants may cause yield losses by themselves or they may join with other soilborne organisms such as viruses, fungi and bacteria to promote disease development in plants.
  • nematode feeding reduces the flow of water and nutrients into the plant, increasing the plant's susceptibility to other stress factors such as heat, water and nutritional deficiencies.
  • plant-parasitic nematodes move through the soil to find areas on plant roots to feed. Some nematodes stay outside the root and use long stylets to puncture cells inside the root. Nematodes which enter the root may move throughout the root (lesion nematodes) and feed at many sites (causing root lesions), or stay at one feeding site (cyst and root-knot nematodes). Nematodes which stay at one feeding site swell from eel-shaped to pear-shaped and stay at the same site until they die.
  • plant root-parasitic nematodes belong to two orders, Tylenchida and Dorylaimida.
  • the tylenchids show a broad diversity in parasitic adaptations and comprise both migratory and sedentary ectoparasites and endoparasites.
  • Sedentary endoparasitic nematode-plant interactions, involving species such as root knot and cyst nematodes, are intriguing to study because of the highly specialized feeding structures (giant cells and syncytia, respectively) that become established in the root during the prolonged and unique relationship between the nematode and host.
  • a variety of other feeding structures besides giant cells and syncytia is induced by other nematodes with different parasitic strategies such as the reniform nematode Rotylenchulus reniformis and the false root-knot nematode Nacobbus aberrans (Wyss,1997, in Cellular and Molecular Aspects of Plant-Nematode Interactions, 5-22, eds. C.Fenoll et al., Kluwer Academic Publ.). Feeding structure maintenance and nematode survival are preserved only when the mutual interaction remains established. These interactions often cause extensive crop damage and, hence, severe economic losses in infested fields.
  • Dorylaimida comprises migratory ectoparasites. Some of these parasites can feed at a particular site for long periods of time (e.g. Xiphinema spp.) rather than browsing along the roots. Similarly, these ectoparasitic species represent an economically important threat as vectors of soil-borne viruses and as cause of direct damage to the roots.
  • Infective second-stage (J2) juveniles of cyst and root knot nematodes migrate intracellularly or intercellularly, respectively, toward the vascular cylinder where they select an initial feeding cell.
  • Secretions are injected via the stylet, eliciting a series of cellular responses that result in the production of metabolically active, multinucleate feeding cells with elaborate cell wall ingrowths characteristic of transfer cells.
  • the infective juveniles become sedentary, after which they will mature by ingesting food from the feeding cell. This process is an absolute requirement for the nematode to complete its life cycle.
  • migratory dorylaimid ectoparasitic nematodes feed at multiple sites, although mainly at root tips, which then cease to grow and develop into terminal galls. With their long needle-like stylets these nematodes pierce a column of subepidermal cells, injecting secretions to predigest the cytoplasm of the recipient cell. A path of collapsed necrotic cells is left behind. They are surrounded by multinucleate and expanding meristematic cells that are responsible for gall formation.
  • nematode resistant plants For example expression of recombinant DNA encoding for a product which has a direct interaction with the pathogen, particularly peptides or proteins. Preferably said DNA is expressed at the site of nematode feeding area.
  • WO 93/10251 a method is disclosed for obtaining plants with reduced susceptibility to plant-parasitic nematodes by providing recombinant DNA that disrupts or at least delays the formation of a nematode feeding structure.
  • a number of strategies including the so-called bamase/barstar combination to inhibit nematode feeding cell development using the published TobRB7 promoter from Opperman et al (Science,263,221- 223,1994).
  • WO 95/32288 describes different genes (cDNAs) that are highly upregulated in nematode infection sites. These cDNAs can be used for the isolation of the corresponding promoters, but the specificity of those promoters is not known, since for most of the genes no detailed expression analysis (in situ hybridisation) has been performed. Because no promoter has been cloned, no promoter-reporter fusions have been studied and the specificity of promoter-activity in nematode infection sites is therefore not known.
  • nematode-induced promoters are searched for with for instance improved selectivity, strength and/or specific expression pattern for different types of nematodes.
  • a T-DNA system based on a randomly integrated promoter tag containing a promoteriess dominant screenable marker was used.
  • transgenic Arabidopsis lines harboring a promoteriess ⁇ -glucuronidase (gus; uidA gene from Esche chia coli) gene were generated and subsequently large-scale screening for activation of the reporter gene in the nematode feeding structures (NFSs) was performed.
  • the present invention thus relates to nematode-responsive promoters which are isolated from plants and which either induce, stimulate or repress the expression of genes or DNA fragments, under their control, at least substantially selectively in specific cells (e.g. fixed feeding site, pericycle, endodermis, cortex or vascular cells) of the plants' roots, preferably in cells of the plant's fixed feeding sites, in response to the nematode infection.
  • specific cells e.g. fixed feeding site, pericycle, endodermis, cortex or vascular cells
  • the nematode-inducible promoters according to the invention are especially useful in transgenic plants for controlling foreign DNAs that are to be expressed selectively in the specific root cells of plants, so as to render said plants resistant to nematodes.
  • an isolated DNA sequence comprising the nucleotide sequence of SEQ ID NO's 1 and/or 2. These nucleotide sequences are so-called nematode-responsive regulatory sequences.
  • SEQ ID NO's 1 and 2 as provided in the Sequence Listing , is indicated by arrows the location in the sequence where the plant sequence starts and ends respectively.
  • the remaining flanking sequences are vector/primer sequences.
  • Part of the invention is also a recombinant DNA comprising a plant-expressible promoter region having a sequence according to SEQ ID NO 1 and/or SEQ ID NO 2 or a fragment thereof. Furthermore said recombinant DNA may comprise a suitable foreign DNA under the expression of the promoter sequence according to the invention.
  • a plant cell comprising said recombinant DNA and a plant comprising the recombinant DNA integrated in its genome belong to the invention.
  • Another aspect of the invention is a method for obtaining said plant with reduced susceptibility to a plant nematode comprising the steps of
  • Nematode reproduction on a plant can be prevented by planting a plant obtained by the invention wherein the above-mentioned recombinant DNA is incorporated in the genome of said plant in an area susceptible to nematode- infection.
  • To the present invention also relates a method for suppressing plant pathogen activity comprising expression of a suitable foreign DNA in a plant under the control of a promoter region having the DNA sequence according to SEQ ID NO 1 and/or SEQ ID NO 2.
  • the promoter region comprising the DNA sequence according to the invention can be used to express a gene in specific root cells of a plant being infected with nematode(s).
  • the current promoters can be used for other purposes as well.
  • the nematode-inducible promoters are used to express a foreign gene predominantly, preferably selectively in fixed feeding cells, or specialized root cells of the plant.
  • the expressed foreign DNA encodes a polypeptide/protein which can kill or disable nematodes.
  • toxins for this purpose are toxins, collagenases, chitinases, lectins, antibacterial peptides or enzyme inhibitors like proteinase-inhibitors.
  • the polypeptide/protein be non-toxic to animals and certainly non- toxic to humans.
  • promoters according to the invention are used to express a foreign DNA sequence encoding an RNA, polypeptide or protein that when expressed, in for example a fixed feeding plant cell, will disable said plant cell by interfering in metabolic activities necessary for survival of the infecting nematodes.
  • foreign DNA sequences which can be expressed under the control of the promoters according to the invention in order to inhibit the development of fixed feeding cells are DNA sequences encoding antibodies immunoreactive with molecules (such as proteins, carbohydrates or compounds secreted through the nematode's infecting process) in the plant cells.
  • Said antibody can have a known variety of forms including Fv, Fab, or single chain antibodies and the like.
  • the foreign DNA adjacent to a nematode-inducible promoter can also encode an enzyme transforming an otherwise harmless substance into a cytotoxic product.
  • the isolated promoters according to the invention have specific advantages over the currently known nematode-inducible promoters in their enhanced specificity and/or time of induction and /or the fact that they are activated by different types of nematodes. Indeed, the Att0728 and Att1712 show very little expression in other tissues than the nematode infection sites itself.
  • the tobacco TobRB7 promoter (Opperman et al., Science, vol.263,p.221 , 1994) is also quite specific but is only activated by root knot nematodes and not by cyst nematodes.
  • sequences according to the invention will lead to the identification of more specific sequences (so- called fragments) needed for the nematode-induced promoter activity.
  • fragments sequences
  • structural features such as repeats and the like.
  • a preferred nematode-induced promoter comprises the nucleotide sequence of SEQ.ID.NO.2 from nucleotide position 371 to 1045 (both positions included).
  • variant promoter sequences obtained by modification including for instance exchange of a nucleotide for another nucleotide, inversions, deletions or insertions of a limited number of nucleotides remaining however the same specificity as the sequences described in SEQ ID NO's 1 and/or 2.
  • Promoters with essentially similar sequence as the nematode-inducible promoters or fragments thereof according to the current invention can be isolated from other plant species using the inventive promoter sequence(s) as, for instance, hybridization probe under conditions known to a skilled person.
  • inventive promoter sequence(s) can be identified, isolated and characterised via the cDNA coding sequence operably linked to the promoter sequence or fragment thereof according to the current invention.
  • nucleotide sequences or fragments thereof according to the invention can be used by skilled persons as so-called amplification primers in order to isolate promoter fragments with essentially similar sequences from other plant species by so- called amplification techniques like the PCR method.
  • the recombinant DNA of the current invention using the promoters or the fragments derived therefrom will be useful in other plant species to obtain resistance to nematode attack and/or infection.
  • Preferred host plants for said nematode-inducible recombinant DNA or fragments thereof are potato plants or oilseed rape plants, but also suitable plants are soybean, cereals such as corn, rice or barley and wheat, tomato, carrots or tobacco.
  • polynucleotide refers to a polymeric form of nucleotides of any length. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA. It also includes known types of modifications, for example, methylation, "caps" substitution of one or more of the naturally occuring nucleotides with analogs.
  • nucleic acid sequence/nucleotide sequence or polynucleotide sequence as mentioned above, “essentially similar” means that if two sequences are aligned, the percent sequence identity is higher than 80%, preferably higher than 85% and in particular higher than 95% especially regarding the promoter/regulatory regions.
  • sequence identity has to be understood the number of positions with identical nucleotides divided by the number of nucleotides in the shorter of the two sequences aligned. The alignment of the sequences is performed by the so-called Wilbur and Lipmann algorithm known to a skilled person using for instance a programs of Intelligenetics Inc. (USA)
  • Promoter fragment means a fragment of a promoter, in particular a nematode- induced promoter, that determines timing, selectivity or strength of the expression induced by said promoter.
  • Said fragment can comprise an autonomously functioning promoter or functions as a promoter, in particular a nematode-induced promoter when combined with other homologous or heterologous promoter fragments ( like for example a TATA box region).
  • the promoters according to the invention comprise at least one promoter fragment as described above.
  • fragment "of a sequence or "part” of a sequence means a truncated sequence of the original sequence referred to.
  • the truncated sequence can vary widely in length; the minimum size being a sequence of sufficient size to provide a sequence with at least a comparable function and/or activity of the original sequence referred to, while the maximum size is not critical. In some applications, the maximum size usually is not substantially greater than that required to provide the desired activity and/or function(s) of the original sequence.
  • Transformation refers to the insertion of an exogenous polynucleotide into a protoplast or a host cell, irrespective of the method used for the insertion, for example, direct uptake, transduction, Agrobacterium infection or eiectroporation.
  • the exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively may be integrated into the host genome. After selection and/or screening, the protoplasts, cells or plant parts that have been transformed can be regenerated into whole plants, using methods known in the art.
  • transformation and/or regeneration techniques are not critical for the present invention.
  • recombinant DNA is meant a hybrid DNA produced by joining pieces of DNA from different sources.
  • promoter is meant a recognition site on a DNA strand to which RNA polymerase binds, thereby initiating transcription.
  • foreign DNA, foreign sequence or foreign gene a DNA sequence which is not in the same genomic environment (e.g. not operably linked to the same promoter and/or 3' end) in a plant cell, transformed with said DNA according to said invention, as is such DNA when it naturally occurs in a plant cell or the organism (bacterium, fungi, virus or the like) from which the DNA originates.
  • Fixed feeding sites are specialized feeding sites such as giant cells, syncytia, nurse cells or galls, which are induced by (semi)-sedentary nematodes in susceptible plants. At such sites the plant cells serve as food transfer cells for the various developmental stages of the nematodes.
  • Giant cells and syncytia refer to multinucleate plant root cells induced by specific nematodes known to a skilled person while “nurse cells” refer to a group of six to ten uninucleated plant root cells induced by Tylenchulus spp.
  • Galls refer to a proliferation of cortical or pericycle plant cells / tissue induced by nematodes; typically giant cells reside within galls.
  • SEQ ID NO 1 nucleotide sequence of Att1712 left border T-DNA/plant flanking sequence (pZ1712LB1.7)
  • SEQ ID NO 2 nucleotide sequence of Att0728 left border T-DNA/plant flanking sequence (pZ728LB1.2)
  • nucleotide sequence region between arrows as indicated is plant sequence.
  • FIG. 1 depicts the T-DNA constructs. Transformation efficiencies, T-DNA integration events, and reporter gene expression patterns were compared for the two binary vectors.
  • Att lines for Arabidopsis thaliana tag
  • beet cyst nematode and root-knot nematode to identify promoter activities in NFSs.
  • 37 Att lines were found displaying various levels of GUS activity within the developing syncytia. Except for the GUS-positive NFSs, no differences in expression patterns were observed when screening was performed in the presence or absence of nematodes.
  • Att0728 (p ⁇ gusBin19 T-DNA) responded very early after infection with cyst nematodes. GUS histochemical staining could be observed within 6 hr after inoculation. Mechanical wounding experiments, however, did not result in the induction of reporter gene expression, indicating that the rapid activation of expression was independent of a wound response. Under in vitro conditions, GUS activity was found to be primarily located in the developing NFSs ( Figure 2H), but some activity was also detectable at sites of lateral root initiation. Soil-grown plants showed stronger activation in initiating lateral roots, together with some staining of the root vascular tissue. DNA gel blot hybridization analysis demonstrated the presence of a single T-DNA insertion.
  • AttOOOI and Att1712 had similar gus expression patterns after nematode inoculation. Strong GUS staining was observed in the syncytia 4 days post-inoculation (dpi) ( Figures 2A, 2B, 2D, and 2E). Sites of lateral root initiation were also clearly stained in both lines ( Figures 2C and 2F). Nevertheless, these tags were located at different chromosomal loci, as confirmed by DNA gel blotting. Some AttOOOI plants showed additional GUS activity at sites as indicated in Table 1 (in both soil- and in vitro-grown plants). This pattern of staining was never observed in Att1712 under in vitro conditions; however, soil-grown Att1712 plants occasionally displayed GUS activity in the leaf vascular tissue.
  • ARMIa was demonstrated to be the tagged sequence responsible for the observed nematode-induced gus expression. Sequence analysis indicated that the T- DNAs were inserted into a short putative ORF of 159 bp (EMBL accession number Y12834). However, no homology in the database was found.
  • iPCR was performed on Att0728 genomic DNA in order to isolate the T-DNA LB (see Methods).
  • a 1269 bp fragment was cloned and from this two different promoter-gus fusions were constructed containing either a 1012 bp (pTHW728PN) or a 675 bp fragment (pTHW728S) (see Methods).
  • the resulting binary vectors were mobilised to Agrobacterium tumefaciens for Arabidopsis transformation.
  • Feeding cell development by nematodes is a dynamic process and involves gene sets regulated by developmental stage-specific factors produced by nematode and host. Because such a temporal gene regulation might be reflected in the four selected lines, gus expression at different time points during syncytium development was monitored. Ten-day-old plants were inoculated, and the number of GUS-positive syncytia was determined at various intervals. Maximum GUS levels in syncytia were found at different times after inoculation of each of the lines.
  • Att1712 and AttOOOI showed maximum activities at 4 dpi, whereas Att0728 and Att1012 showed maximum GUS activities over a longer period during nematode infection rather than exhibiting transient patterns of activity.
  • AttOOOI -R/1 retained the timing of maximum response from the original line. However they displayed blue-stained syncytia with a lower frequency than did AttOOOI .
  • AttOOOI and Att1712 have a similar expression pattern ( Figures 3A,3B,3C and 3D), as previously mentioned for cyst nematode-infected lines, the different nature of the tag in these two lines became clearly apparent by infection with the root knot nematode: an average of 70% of the established Att1712 galls showed an unstained zone at 4 dpi ( Figure 3G). Subsequent cross-sectioning of these galls revealed strongly GUS-stained parenchymatous cells surrounding unstained giant cells ( Figure 3F). In contrast, AttOOOI galls displayed very strong and uniform staining ( Figures 3D and 3E) with GUS inside the giant cells.
  • Xiphinema nematodes feed for longer periods and can be regarded as being semi-sedentary. Their ability to transform root tips into galls made this nematode species interesting to add.
  • AttOOOI , Att0728, and Att1712 exhibited GUS-positive galls when infected with Xiphinema ( Figure 31).
  • a cross-section through a X/p ⁇ /nema-induced Att1712 gall indicated that reporter gene expression in this line occurred in the multinucleate cells induced at the nematode penetration site ( Figure 3H). The same pattern was observed in AttOOOI galls.
  • transgenic plants engineered with a nematode-inducible promoter/cytotoxin construct should be ensured when one seeks to develop nematode resistance in plants. This implies the absence of tagged promoter activity in the reproductive organs.
  • AttOOOI and Att1712 both expressed GUS in the root vascular tissue regions abutting the protruding calluses ( Figure 4A). In the case of Att1712, this GUS staining occasionally extended into the vascular cylinder of developing lateral roots. Att1712 also showed clear reporter gene activation in cells at the cut surfaces of the explants.
  • AttOOOI displayed GUS staining in the tips of main and lateral roots, a so-called "three-zone pattern" ( Figure 4B), which was also seen in auxin-treated Arabidopsis plants containing a cell cycle regulator (cdc2) promoter-gws construct (Hemerly et al., 1993, Plant Cell,5 ,p.1711-1723).
  • cdc2 cell cycle regulator
  • Agrobacterium infection was determined (see Methods) by mimicking the initial steps of the root explant transformation process. In general, Agrobacterium infection did not significantly alter the patterns seen after callus induction alone, except for the cut surfaces that showed weak GUS staining for all tags.
  • GUS activity in AttOOOI explants was no longer confined to vascular regions juxtaposed to the developing calluses, as described earlier, but extended throughout the entire vascular cylinder. Att0728 displayed weak GUS activity in a few calluses, which was not observed after hormone treatment alone.
  • pGV1047 has been reported by Kertbundit et al. (1991 ).
  • p ⁇ gusBin19 comprises the uidA-cod ' mg region at the left T-DNA border and has been described by Topping et al. (1991 ).
  • Mobilization of pGV1047 from Escherichia coli into Agrobacterium has been described by Kertbundit et al. (1991 ).
  • p ⁇ gusBin19 was transferred from the E.
  • Roots from 2-week-old plants were incubated on CIM (Valvekens et al., 1991). Wild-type C24 and transgenic 35S-gus Arabidopsis thaliana (L.) Heynh plants were used for control purposes. GUS histochemical assays were performed after 6 and 11 days of incubation.
  • Roots from 14-day-old promoter-tagged plants were incubated for 3 days on CIM (Valvekens et al., 1991 ). Subsequently, whole-root systems were cut into small explants and mixed with an C58C1Rif R (pGV2260) Agrobacterium solution (OD 600 of 0.1 ), after which the cocultivated material was further incubated on CIM for an additional 3 days. Root explants were washed several times to remove all overgrowing agrobacteria and were stained for GUS activity. Wild-type C24 and transgenic 35S-gus Arabidopsis plants were used for control purposes. Nematode cultures and hatching procedures
  • Root knot nematode (Meloidogyne incognita) cultures were maintained in vitro on tomato (Lycopersicon esculentum) hairy roots continuously subcultured on hormone-free Gamborg's B5 medium (Flow Laboratories, Bioggio, Switzerland; pH 6.2) supplemented with 2% sucrose and 1.5% Bacto agar (Difco, Detroit, Ml). Cyst nematodes (Heterodera schachtii) were grown in vitro on mustard (Sinapis alba) roots in Knop medium (Sijmons et al., 1991). Hatching was stimulated by putting cysts (H. schachtii) or galls (M.
  • Inoculations were performed after 2 more weeks of growth at 22°C and 16 hr of light by injecting a suspension containing 250 second-stage juveniles (5 to 7 days after hatching) of beet cyst or root knot nematodes in 1.5 mi H 2 O per root system. One to two weeks later, three to five plants were washed carefully and stained for GUS.
  • roots were inoculated with 5- to 7-day-old hatched beet cyst or root knot nematode second-stage juveniles at an average density of 20 juveniles per root system. The plants were then incubated again under the same tissue culture conditions. Five to ten plants were examined for the presence of GUS activity 4 to 6 days post-inoculation (dpi).
  • X-gluc Europa research products, Ely, U.K.
  • Jefferson (1987) 50 ⁇ L of X-gluc (20 mg in 1 mL of ⁇ /, ⁇ /-dimethylformamide) was diluted to a final concentration of 2 mM in 1 mL of 0.1 M NaPO4, pH 7.2.
  • Oxidative dimerization of the produced indoxyl derivative was enhanced by adding the oxidation catalyst K + ferricyanide/ferrocyanide to a final concentration of 0.5 mM.
  • Sectioned material was sometimes stained for examination using bright-field optics (Diaplan, Leitz): after removing the butyl-methylacrylate resin with acetone (15 min incubation), sections were immersed in a 0.1% ruthenium red (Sigma) solution during 7 to 20 min.
  • Bright-field optics Diaplan, Leitz: after removing the butyl-methylacrylate resin with acetone (15 min incubation), sections were immersed in a 0.1% ruthenium red (Sigma) solution during 7 to 20 min.
  • Nematodes inside root tissues can be visualized according to the McBryde method (described in Daykin and Hussey, 1985). Following GUS histochemistry, fixation in 2.5% glutaraldehyde and clearing in chlorallactophenol, root material was left in acid fuchsin dye for 16 hr and subsequently destained for 3 hr in a saturated chloral hydrate solution.
  • 0.2 to 2 g of plant material was used for the preparation of DNA as described by Dellaporta et al. (1983), with some modifications.
  • the DNA pellets were dissolved in 400 ⁇ l of Tris-EDTA to which 20 ⁇ g of RNase was added. After an incubation period of 20 min (37°C), 400 ⁇ l of 0.2 M Tris-HCI, pH 7.5, 2 M NaCI, 0.05 M EDTA, 2% (w/v) cetyltrimethylammonium bromide was added; the mixtures were incubated for an additional 15 min at 65°C.
  • the samples were extracted with 800 ⁇ l of chloroform-isoamylalcohol (24:1 ) and precipitated.
  • AttOOOI , Att0728, Att1012 and Att1712 plant DNA was digested with Hindlll and/or EcoRI in a double or single digest. Separation of the digested samples on a 1% agarose gel was followed by an overnight blotting to a Hybond-N membrane (Amersham, Aylesbury, UK). The DNA on the membrane was fixed through UV cross-linking (GS Gene Linker; Bio-Rad, Hercules, CA). The 1.7-kb Nrul gus-coding region of pGUS1 (Peleman et al., 1989) was used as a probe.
  • Radioactive labeling was performed using the Ready-To-Go DNA labeling kit (-dCTP) (Pharmacia, Uppsala, Sweden) according to the manufacturer's instructions.
  • the nylon membrane was incubated in a hybridization buffer (450 mM NaCI and 45 mM Na3-citrate, pH 7.0), 0.1% SDS, 0.25% milk powder (Gloria, Vevey, Switzerland), and 20 ⁇ g mL "1 herring sperm DNA (Promega, Madison, Wl)) for 3 hr at 65°C. Hybridization was performed overnight in fresh hybridization buffer to which the corresponding a 32 P-dCTP-labeled probe was added.
  • a hybridization buffer 450 mM NaCI and 45 mM Na3-citrate, pH 7.0
  • SDS 0.1% SDS
  • 0.25% milk powder 0.25% milk powder
  • 20 ⁇ g mL "1 herring sperm DNA Promega, Madison, Wl
  • suitable sized fragments are identified by analysis of restriction enzyme digests of the DNA from a particular interesting tag line through Southern hybridisation with a labelled nucleotide sequence that is homologous to the integrated T-DNA sequence.
  • AttOOOI DNA was digested with Sspl and EcoRI and circularized in conditions favoring self-ligation (Sambrook et al., 1989).
  • IPCR Inverse polymerase chain reaction (IPCR) was conducted using primer sets 1 and 2 and 1 and 3, respectively ( Figure 5A).
  • the PCR mix consisted of 50 ng self-ligated DNA, 200 ng of each primer, 1 mM MgCI 2 , 0.2 mM deoxynucleotide triphosphates, 2.5 ⁇ l 10x Taq buffer, 0.5 ⁇ l of Taq polymerase (5 units ⁇ l "1 ) (Beckman, Fullerton, CA), in a total volume of 25 ⁇ l, and 25 ⁇ l mineral oil. A total of 35 cycles were used. For both primer sets 1 and 2 and 1 and 3, the same temperature program was followed with an exception for the annealing temperature being 64°C and 60°C, respectively.
  • the cycle order was: cycle 1 , 4 min at 95°C, 2 min at 64°C/60°C, 10 min at 72°C; cycles 2 to 35, 1 min at 95°C, 2 min at 64°C/60°C, 3 min at 72°C; and finally, for 10 min at 72°C.
  • primer 1 5' CCAGCGTGGACCGCTTGCTGGAAC 3' primer 2: 5 * GTATTGCCAACGAACCGGATACCCG 3' primer 3: 5' CCCAGTCACGACGTTGTAAAAC 3'.
  • Primer 5 5' CCC CGA TCG TTC AAA CAT TT 3'; primer 6: 5' CGG GCT ATT CTT TTG ATT TAT 3'
  • Att1712 total DNA was digested with Accl ( Figure 5B).
  • the recircularisation reaction was performed as described by Topping et al. (1995). Following fenolisation and precipitation, the recircularised DNA was dissolved in 20 ⁇ l H 2 O from which 2 ⁇ l was added in a PCR mix as described for the Att0728 LB. Inverse PCR was conducted as described for the Att0728 LB. A fragment of 1642bp, containing 1126bp plant sequence, was recovered from a 1 % agarose gel in 10 ⁇ l H 2 O using the GENECLEAN II KIT.
  • the 3' protruding ends of the iPCR products were converted to blunt ends using T4 DNA polymerase in a reaction mix containing following components 10 ⁇ l GENECLEAN pure iPCR fragment, 3 ⁇ l ONE PHOR ALL buffer (10X Pharmacia), 1.5 ⁇ l 2mM dNTP's, 0.5 ⁇ l T4 DNA polymerase (7.3U/ ⁇ l Pharmacia), H 2 O up to a final volume of 30 ⁇ l. Following phenolisation and precipitation, iPCR fragments were dissolved in 15 ⁇ l H 2 O.
  • PZ728LB1.2 was digested with Sspl and Pvull/Nsil to generate Att0728 left border plant sequence fragments of respectively 675bp and 1016bp ( Figure A), the latter fragment consisting of 896bp plant derived sequence and 119bp T-DNA sequence.
  • PZ1712LB1.7 was digested with BamHI/Nsil, generating an Att1712 left border flanking T-DNA plant sequence fragment of 1443bp containing 1126bp plant derived sequence, 248 bp T- DNA sequence and 69bp pZErOTM-2 derived sequence as indicated in Figure B.
  • the recessed 3' ends generated by the restriction enzyme BamHI and the 3' protruding ends generated by the restriction enzyme Nsil were converted to blunt ends using respectively 1 U Klenow Fragment of DNA Polymerase I (Pharmacia) in the presence of 0.8mM dNTP's in 1X ONE PHOR ALL buffer and 3.65U T4 DNA Polymerase (Pharmacia) in the presence of 0.1 mM dNTP's in 1x ONE PHOR ALL . No treatment of the ends generated by Sspl and Pvull was required as these are blunt end generating restriction enzymes.
  • the 5'35S sequence in the binary vector pTHW136 was substituted with the 675bp Sspl and the 1016bp Pvull/Nsil fragments from pZ728LB1.2 and the 1443bp BamHI/Nsil fragment from pZ1712LB1.7 to make the respective constructs pTHW728S, pTHW728PN ( Figure A) and pTHW1712BN ( Figure B).
  • both LB-flanking regions were isolated by using IPCR.
  • Amplified fragments of »0.6 kb and »2.8 kb, designated ARMIa and ARM1b, respectively, were cloned in front of gus in pTHW136.
  • pthARM1-a600 corresponding to the cloned »0.6-kb ARMIa fragment, revealed a nematode response similar to that of the original tag.
  • the binary vectors pTHW728S, pTHW728PN and pTHW1712BN were mobilised to the Agrobacterium tumefaciens strain C ⁇ CI Ri ⁇ pMP ⁇ O) (Holsters er a/., 1980; Koncz and Schell, 1986). Introduction of the T-DNA's in the Arabidopsis genome was mediated according to the root explant transformation method of Clarke et al. (1992) with some modifications as described by Barthels et a/.(1994).
  • a fast method was optimised making use of the infecting nematodes that takes along agrobacteria into the infection site.
  • the wounding caused by the nematode triggers the Agrobacterium to transfer its T-DNA into the plant cells.
  • a mixture of second stage juveniles and C58C1 Rif R (pMP90)(pTHW728S), C58C1 Ri (pMP90)(pTHW728PN) or C58C1 Rif R (pMP90)(pTHW1712BN) was inoculated on roots of 3 to 4 weeks old A. thaliana plants.
  • the infected plant roots were analysed for gus expression in the feeding structures as seen in Figure C for pTHW728PN.
  • FIG. 1 T-DNA structures in the binary vectors pGV1047 and p ⁇ gusBin19.
  • gus gus gene encoding the ⁇ -glucuronidase (GUS) reporter
  • 3'nos nopaline synthetase 3' sequence
  • pnos nopaline synthetase promoter
  • nos nopaline synthetase gene
  • nptll neomycin phosphotransferase gene
  • 3'ocs octopine synthetase 3' sequence
  • P35S cauliflower mosaic virus 35S promoter
  • supF suppressor gene.
  • Figure 2 Reporter gene activation in syncytia after cyst nematode infection of Arabidopsis promoter tag lines.
  • FIG. 1 GUS patterns in roots of transgenic Arabidopsis lines after incubation on callus-inducing medium.
  • Att1712 displays strong GUS staining in the root vascular tissue at the base of protruding calluses.
  • AttOOOI root tips show a typical three-zone pattern 6 days after incubation on CIM medium.
  • primerset 5 and 6 were used to perform IPCR.
  • the DNA of these lines was digested respectively with Nsil and Accl prior to the IPCR.
  • Figure 6 Vector constructions used for reintroduction of promoter-containing plant sequences into Arabidopsis.
  • Figures A and B Isolation and cloning of the left border T-DNA / plant flanking sequences of Att0728 and Att1712 respectively
  • FIG. 1 GUS pattern in galls induced on Arabidopsis C24 roots 10 days after treatment with an Meloidogyne incognita/ Agrobacterium C ⁇ SCIRif (pMP90)(pTHW728PN) mixture.
  • Figure D GUS pattern in roots of Arabidopsis transformed with pTHW728PN as observed 3 days after inoculation with H.schachtii.
  • FIG. 1 Overview of the GUS patterns from five retained promoter trap lines after infection with cyst nematodes (Heterodera schachtii, Hs), root-knot nematodes (Meloidogyne incognita, Mi), and migratory ectoparasitic nematodes (Xiphinema diversicaudatum, Xd)

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