AU707563B2 - Nematode-inducible plant gene promoter - Google Patents

Description

WO 97/46692 PCT/EP96/02437 1 NEMATODE-INDUCIBLE PLANT GENE PROMOTER 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 plants that are resistant, or at least less susceptible to plant parasitic nematodes, or their effects, as well as to cells, plants and parts thereof.

STATE OF THE ART In International patent application W092/17054, a method is disclosed for the identification and subsequent isolation of nematode responsive regulatory DNA sequences from Arabidopsis thaliana.

In WO 92/21757 several regulatory DNA sequences have been isolated from Lycopersicon esculentum, which are responsive to the root-knot nematode Meloidogyne incognita. Some of these regulatory sequences (LEMMI's, for Lycopersicon esculentum Meloidogyne incognita) are stimulated, whereas others appear to be repressed by the nematode. It is not known whether any of the inducible regulatory sequences are stimulated by a broader range of nematodes.

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 Globodera species. This makes the TobRB7 sequence unsuitable for use in chimeric constructs aiming at, for example, cyst nematode resistance in potato.

It is an object of the invention to provide regulatory DNA sequences which are inducible by both cyst and root knot nematodes and which can be used to express heterologous DNA sequences under their control inside the feeding structure of the nematode, preferably, but not necessarily in a substantially feeding site specific way.

SUMMARY OF THE INVENTION The invention provides a DNA fragment obtainable from Arabidopsis thaliana that is capable of promoting root knot and cyst nematode- SUBSTITUTE SHEET (RULE 26) WO 97/46692 PCT/EP96/02437 2 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. Also envisaged are 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.

Further embodiments of the invention comprise 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 celldisruptive substance, such as barnase. In a different embodiment the cell-disruptive substance comprises RNA complementary to RNA essential to cell viability. Yet in another embodiment 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. Further useful embodiments are 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. Also envisaged are 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 SUBSTITUTE SHEET (RULE 26) invention The Figure Figure Figure Figure Figure for making hybrid regulatory DNA sequencesfollowing figures further illustrate the invention.

DESCRIPTION OF THE FIGURES Schematic plasmid map of Binary vector pMOG23.

Schematic plasmid map of Binary vector pMOG800.

Schematic plasmid map of Binary vector pMOG553.

Schematic plasmid map of Binary vector pMOG819.

Schematic plasmid map of Binary vector pMOG849.

Expression patterns outside the NFS of several pMOG849 transformed Arabidopsis thaliana lines.

Schematic representation of a NFS disrupter gene and a neutraliser gene in a two component system for engineering of nematode resistanct plants Schematic plasmid map of Binary vector pMOG893.

Figure 7.

Figure 8.

D" Some ways of practicing the invention as well as the meaning of various phrases are explained in more detail below.

DETAILED DESCRIPTION OF THE INVENTION 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 Sassociated DNA inside the special nematode feeding structures of the plant root. Such a nematode feeding structure is used by an invading D! nematode as source of food, whereby the nematode induces a change in the S 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, W092/17054, which is incorporated herein by reference.

3a Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

In principle 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 sequences 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 Heterodera schachtii and Globodera pallida (a more comprehensive, but by no :means limiting,

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C:Vk\MNORD\STACVICDYF\SPECIJR561 549.DOC WO 97/46692 PCT/EP96/02437 4 list is presented in table Advantageously, 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. There exist numerous examples of such toxic substances, such as the endotoxins of Bacillus thuringiensis 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, W093/10251 and W094/10320, which are hereby incorporated by reference. For the sake of consistency, the phytotoxic substance shall be referred to hereinafter as the nematode feedings site (NFS) disruptive substance.

Although 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 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. The details of such a socalled two-component approach for the engineering of nematode resistant plants are set out in W093/10251. According to this approach a NFSdisrupter 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 efects 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.

According to the two-component approach 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 SUBSTITUTE SHEET (RULE 26) WO 97/46692 PCT/EP96/02437 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 sequence-A, can not be neutralized sufficiently by products from coding sequence-B.

The properties of the regulatory DNA sequences according to the invention, in particular the 4, 2.1 and 1.5 kBp fragments of #1164, make them highly useful in the two-component approach, as is illustrated by way of Examples herein. Obviously, 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.

Moreover, as is well known to those of skill in the art, 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. The use of DNA sequences according to the invention to identify functional subregions, and the subsequent use thereof to promote or suppress gene expression in plants is also encompassed by the present invention.

Within the context of this invention, the terms 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. barnase barstar from SUBSTITUTE SHEET (RULE 26) WO 97/46692 PCT/EP96/02437 6 Bacillus amyloliquefaciens (Hartley, 1988, J. Mol. Biol. 2.2, 913-915), restriction endonucleases corresponding methylases such as EcQRI from E.coli (Green et al., 1981, J. Biol. Chem. 256, 2143-2153) and EcoRI methylase or similar combinations as described in the review for type II restriction modification systems (Wilson, 1991, Nucl. Acid Res. 19, 2539-2566), bacteriocins and corresponding immunity proteins, e.g.

colicin E3 immunity protein from E. coli (Lau et al. 1985, Nucl. Acid Res. 12, 8733-8745) or any disruptive substance coding gene which may be neutralized by simultaneous production of antisense RNA under control of promoter-B, such as DNA sequences encoding Diptheria Toxin Chain A (Czako An, 1991, Plant Physiol. .95, 687-692), RNAses such as RNAse Tl, ribonucleases or proteases and ribozymes against mRNA that code for phytotoxic proteins.

According to another aspect of the invention 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 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 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 al., 1990, The Plant Cell 2, 279-289).

According to a preferred embodiment of the invention use is made of 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 SUBSTITUTE SHEET (RULE 26) WO 97/46692 PCT/EP96/02437 7 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. Hence, it is preferred that 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. Preferably, 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 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 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.

Preferably, 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 (RULE 26) WO 97/46692 PCT/EP96/02437 8 comprising the NFS. Specific examples for such organelles are mitochondria, endoplasmic reticulum and plasmodesmata (Hussey et al. 1992 Protoplasma 167; 55-65, Magnusson Golinowski 1991 Can. J. Botany 69; 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. Stumpf 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.

TABLE 1 EXAMPLES OF TARGET ENZYMES FOR ANTISENSE EXPRESSION IN NFS AND SENSE EXPRESSION OUTSIDE NFS enzyme ATP synthase adenine nucleotide translocator phosphate translocator tricarboxylate translocator dicarboxylate translocator 2-oxo-glutarate translocator cytochrome C pyruvate kinase glyceraldehyde-3P-dehydrogenase NADPH-cytochrome P450 reductase fatty acid synthase complex glycerol-3P-acyltransferase pathway/organelle mitochondrion mitochondrion mitochondrion mitochondrion mitochondrion mitochondrion mitochondrion glycolysis glycolysis lipid metabolism lipid metabolism lipid metabolism hydroxymethyl-glutaryl CoA reductase mevalonic acid pathway aminoacyl transferase transcription factors elongation factors nucleic acid metabolism nucleic acid metabolism 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 SUBSTITUTE SHEET (RULE 26) WO 97/46692 PCT/EP96/02437 9 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). As an illustration of 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.

Alternatively, to provide for expression in all or effectively all plant tissues, 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/codingsequence-B, with the proviso that neither promoter-B nor promoter-B' drives expression in the NFS. Also 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.

Preferebly, promoter-B is the Cauliflower Mosaic Virus 35S promoter or derivatives thereof, which is generally considered to be a strong constitutive promoter in plant tissues (Odell et al. 1985 Nature 313, 810-812). Another preferred example for promoter-B is the strong root promoter rJlD (Leach Aoyagi 1991 Plant Sci. 79; 69-76) from plasmid pRiA4 of Agrobacterium rhizogenes; the 5' flanking region of (Slightom et al. 1986, J. Biol. Chem. 261, 108-121). The suitability of other constitutive promoters such as the nopaline synthase promoter (Bevan, 1984, Nucl. Acids Res. 12, 8711-8721) or figwort mosaic virus promoter (EP-A 426 641) for use as promoter-B can be tested through fusion to marker genes such as GUS (Jefferson, 1987, Plant Mol. Biol.

Reporter 387-405), transfer of these constructs to plants and histochemical analysis of such transgenic plants after infection with

PPN.

SUBSTITUTE SHEET (RULE 26) WO 97/46692 PCT/EP96/02437 Other regulatory sequences such as terminator sequences and polyadenylation signals include any such sequence functioning as such 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).

Further details of the two component approach can be found in W093/10251 (herein incorporated by reference).

The choice of the plant species is primarily determined by the amount of damage through PPN infections estimated to occur in agriculture and the amenability of the plant species to transformation. 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 Xiphinema spp.) to the more evolved sedentary endoparasites Heteroderidae, Meloidogynae or Rotylenchulinae). A list of parasitic nematodes are given in Table 2, but the invention is not limited to the species mentioned in this table. More detailed listings are presented in Zuckerman et al. (eds., in: Plant Parasitic Nematodes, Vol. I 1971, New York, pp. 139-162).

TABLE 2 EXAMPLES OF PLANT-PARASITIC NEMATODES AND THEIR PRINCIPAL HOST PLANTS Nematode Species Principal Host Plants Meloidogyne M. hapla wide range M. incognita wide range M. exigua coffee, tea, Capsicum, Citrullus M. indica Citrus M. javanica wide range M. africana coffee M. graminis cereals, grasses M. graminicola rice M. arenaria wide range Heterodera Globodera H. mexicana Lycopersicon esculentum, Solanum spp.

H. punctata cereals, grasses G. rostochiensis Solanum tuberosum, Solanum spp, Lycopersicon esculentum SUBSTITUTE SHEET (RULE 26) WO 97/46692 PCT/EP96/02437 11 G. pallida Solanum tuberosum G. tabacum Nicotiana tabacum, Nicotiana spp.

H. cajani Cajanus cajan, Vigna sinensis H. glycines Glycine max, Glycine spp.

H. oryzae Oryza sativa H. schachtii Beta spp, Brassica spp, H. trifolii Trifolium spp.

H. avenae cereals, grasses H. carotae Daucus carota H. cruciferae Cruciferae H. goettingiana Pisum sativum, Vicia spp.

Within the context of this invention, 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. Susceptible resistance classification according to the number of maturing females is standard practice both for cyst- and root-knot nematodes LaMondia, 1991, Plant Disease 25, 453-454; Omwega et al., 1990, Phytopathol. IM, 745-748).

A nematode feeding structure according to the present invention 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.

Several techniques are available for the introduction of recombinant DNA containing the DNA sequences as described in the present invention into plant hosts. Such techniques include but are not limited to transformation of protoplasts using the calcium/polyethylene glycol method, electroporation and microinjection or (coated) particle bombardment (Potrykus, 1990, Bio/Technol. 535-542).

In addition to these so-called direct DNA transformation methods, transformation systems involving vectors are widely available, such as viral vectors from the Cauliflower Mosaic Virus (CaMV) and bacterial vectors from the genus Agrobacterium) (Potrykus, 1990, Bio/Technol. E, 535-542). 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 (Horsch et SUBSTITUTE SHEET (RULE 26) WO 97/46692 PCT/EP96/02437 12 al., 1985, Science 225, 1229-1231). The choice of the transformation and/or regeneration techniques is not critical for this invention.

According to a preferred embodiment of the present invention use is made of so-called 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 Agrobacterium; 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.

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. Presently, preferred methods for transformation of monocots are microprojectile bombardment of explants or suspension cells, and direct DNA uptake or electroporation (Shimamoto, et al., 1989, Nature 338, 274-276). 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). The introduction of genetic material into aleurone protoplasts of other monocot crops such as wheat and barley has been reported (Lee, 1989, Plant Mol. Biol. 13, 21-30). Wheat plants have been regenerated from embryogenic suspension culture by selection only the aged compact and nodular embryogenic callus tissues for the establishment of the embryogenic suspension cultures (Vasil, 1990 Bio/Technol. 429-434). Also an Agrobacterium-using method for the transformation of rice has been disclosed recently (WO 95/16031). The combination with transformation systems for these crops enables the application of the present invention to monocots. These methods may also be applied for the transformation and regeneration of dicots.

SUBSTITUTE SHEET (RULE 26) WO 97/46692 PCT/EP96/02437 13 The following examples are given only for purposes of illustration and do not intend to limit the scope of the invention.

EXPERIMENTAL PART DNA procedures All DNA procedures were carried out according to standard methods described in Maniatis (Molecular Cloning, A laboratory Manual 2nd Edition, Cold Spring Harbor Laboratory, 1990).

Transformation of Arabidopsis Transformation was carried out using co-cultivation of Arabidopsis thaliana (ecotype C24) root segments with Agrobacterium strain MOG101 containing a suitable binary vector as described by Valvekens et al. (1988, Proc. Nat. Acad. Sci. USA 85, 5536-5540) which is as follows: Arabidopsis seeds were vernalized for 7 days at 4 0 C before germination. Seeds were surface-sterilized for 2 min in 70% EtOH, transferred to 5% NaOCl/0.5% NaDodSO 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 0 C in a 16-hr light/8-hr dark cycle. The same growth-room conditions were used for tissue culture procedures.

All plant media were buffered with 2-(N-morpholino)ethanesulfonic acid at (pH 5.7: adjusted with 1 M KOH), solidified with 0.8% Difco Bacto agar, and autoclaved at 121 0 C for 15 min. Hormones and antibiotics were dissolved in dimethyl sulfoxide and water, respectively, and were added to the medium after autoclaving and cooling to 65 0

C.

Intact roots were incubated for 3 days on solidified 0.5/0.05 medium (Table 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.

Subsequently, 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 SUBSTITUTE SHEET (RULE 26) WO 97/46692 PCT/EP96/02437 14 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 (KmR) calli were formed in a background of yellowish root explants. At this point the root explants were transferred to fresh 0.15/5 agar containing only 500 mg of vancomycin and 50 mg of Km per liter. Three weeks later most green call had formed shoots. Transformed shoots were transferred to 150 x 25 mm Petri dishes containing GM to form roots or seeds or both. In these Petri dishes, many regenerants formed seeds without rooting. Rooted plants could also be transferred to soil to set seed. The following modification was made to obtain the initial root material 6 sterilized Arabidopsis thaliana C24 seeds were germinated in 50 ml GM (250 ml Erlenmeyer) on a rotary shaker (100 rpm) in a growth room for 9 days under low light conditions. Transgenic plants were regenerated from shoots grown on selection medium (50 mg/l kanamycin), rooted and transferred to germination medium or soil.

TABLE 3 PLANT MEDIA CIM SIM GM R3* PG1* 0.5/0.05 0.05/7* 0.15/5* Salts vitamins MS MS B5 B5 MS Sucrose, g/L 10 30 Glucose, g/L 20 20 IAA, mg/L 5 0.05 0.15 2,4-D, mg/L 0.5 2 2ipAde, mg/L 7 Kin, mg/L 0.3 0.05 0.05 L, liter; IAA, indole-3-acetic acid; Kin, kinetin; 2ipAde, N 6 isopentenyl)adenine; CIM, callus-inducing medium; SIM, shoot-inducing medium; MS, Murashige Skoog medium B5, Gamborg B5 medium Transformation of potato For the transformation of Solanum tuberosum var. Kardal a protocol as described in Hoekema et al. 1989 Bio/Technology 7, 273-278 was used with several modifications.

Peeled surface-sterilized potato tubers were cut in 2 mm thick slices.

SUBSTITUTE SHEET (RULE 26) WO 97/46692 PCT/EP96/02437 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/l thiamine HC1, 0.5 mg/l pyridoxine Hcl, 0.5 mg/l nicotinic acid, 100 mg/1 myo-inositol, 30 g/l sucrose, 0.5 g/l MES pH Inoculation with Agrobacterium tumefaciens strain EHA105 (Hood et al. 1993 Transgenic Research 2, 208-218) was done by replacing the WM with 100 ml fresh WM containing the resuspended pellet of 10 ml Agrobacterium culture grown freshly in LB appropriate antibiotic to an

OD

600 of 0.5-0.7. After incubating the tuber disks for 20 min in the bacterium suspension they were transferred to solidified CM (WM supplemented with 8 g/l agar, 3.5 mg/l zeatin riboside, 0.03 mg/1 indole acetic acid) at a density of 20 explants/petridish. After two days the disks were transferred to PM (CM supplemented with 200 mg/1 cefotaxime, 100 mg/1 vancomycin) to select against the Agrobacteria. Three days later the disks were transferred to SIM plates (CM supplemented with 250 mg/l carbenicillin, 100 mg/l kanamycin) at a density of 10 explants/petridish to select for the regeneration of transformed shoots. After 2 weeks the tissue disks were transferred to fresh SIM, and after another 3 weeks they were transferred to SEM (SIM with 10 x lower concentration of hormones). About 8-9 weeks after co-cultivation the shoots were large enough to cut them from the callus tissue and transfer them to glass tubes (Sigma, Cat.nr. C5916) containing 10 ml of RM (WM containing 0.5 x MS salts, 0.5 x vitamins, 10 g/l sucrose, 100 mg/l cefotaxime, 50 mg/l vancomycin and 50 mg/l kanamycin) for rooting maintenance in vitro and vegetative propagation.

Handling of nematodes, growth and infection of plant roots Arabidopsis seeds were surface sterilized and sown in petri dishes 9 cm) on B5 medium containing 20 g/1 glucose and 20 mg/l kanamycin. After 3 days at 4 0 C the plates were incubated for 2 weeks in a growth chamber at 22 0 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 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 SUBSTITUTE SHEET (RULE 26) WO 97/46692 PCT/EP96/02437 16 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.

Similarly, potato shoots which had rooted on kanamycin-containing RM medium were transferred to soil-filled translucent plastic tubes (30x15x120 mm, Kelder plastibox The Netherlands) and grown tilted for another 2 weeks at 22°C with 16 h light/8 h dark cycle. Infection was done by injecting a suspension containing 500 second stage larvae of Globodera pallida (in 3 ml H20) per root system into the soil.

GUS assay 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 NaPO 4 (pH7), imM K 4 Fe(CN) 6 ImM K K 3 Fe(CN) 6 10mM EDTA, 0.1% Triton X100) 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.

Example 1 Construction of binary vector pMOG800 The binary vector pMOG800 is a derivative of pMOG23 (Fig. 1, deposited at the Centraal Bureau voor schimmelcultures, Oosterstraat 1, Baarn, The Netherlands on January 29, 1990 under number CBS 102.90) in which an additional KpnI restriction site was introduced into the polylinker between EcoRI and SmaI. This plasmid contains between the left and right borders of T-DNA a kanamycin resistance gene for selection of transgenic plant cells (Fig. A sample of E. Coli DH5 alpha, harbouring pMOG800, was deposited at the Centraal Bureau voor Schimmelcultures, Oosterstraat 1, Baarn, The Netherlands, on August 12, 1993 under number CBS 414.93.

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 8-glucuronidase gene SUBSTITUTE SHEET (RULE 26) WO 97/46692 PCT/EP96/02437 17 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 (Sl) were harvested and vernalized. Subsequently Sl seeds were germinated on nutrient solution (Goddijn et al. 1993 Plant J 4, 863-873) solidified with 0.6% agar, 10 mg/1 hygromycin and stored at 40C for a 4 day imbibition period. At day 5 the plates were transferred to room temperature and moderate light (1000 lux, 16 h L 8 h D) for germination. Fourteen days old seedlings were transferred to potting soil in tilted translucent plastic tubes (30x15x120 mm) for further growth at 5000 lux (20 0 Growing the plants in this way causes most of the root system to grow on the lower side of the tubes in the interphase between soil and tube. After two weeks the roots were infected with nematodes as described in the Experimental part. At several time points after inoculation (ranging from 2 -14 days), the root systems were analyzed for GUS activity as described in the Experimental part. 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. In 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.

In line 1164 this phenotype was found to segregate at a 1:3 ratio, indicating that the GUS construct is present at one locus per genome. The presence of only one T-DNA copy was confirmed by Southern analysis.

A 1.5 kb fragment of the trapped promoter sequence adjacent to the GUS open reading frame was isolated by inverted PCR. Genomic DNA of this line was cleaved with the restriction enzyme MscI, which cleaves once in the SUBSTITUTE SHEET (RULE 26) WO 97/46692 PCT/EP96/02437 18 GUS coding region, and religated. By subsequent digestion of the circular DNA with the 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 CTT TCC CAC CAA CGC TGA TC 3' SEQIDNO: 1) and GUS7 GTA ATG CTC TAC ACC ACG CCG 3' SEQIDNO: cloned in a multi-copy vector and sequenced (see below).

To clone this amplified fragment back in front of GUS the plant sequence was re-amplified from Arabidopsis genomic DNA using the primers and 1164XBM TCT AGA GGA TCC TGG CCA TAC AAA TCA ACG TTT AC 3' SEQIDNO: A pfu DNA polymerase carrying a proofreading activity was used to reduce the error rate. 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.

Example 4 Construction of promoterless GUS construct pMOG819 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.

Example Analysis of promoter fragments after re-introduction into Arabidopsis The PCR product from tag 553#1164 was cloned back in front of a GUS gene on the binary vector pMOG819 to make pMOG849 (Fig. A sample of E. coli DH5a harbouring pMOG849 has been deposited at the Centraal Bureau voor schimmelcultures, Oosterstraat 1, Baarn, The Netherlands, on May 4, 1995 under number CBS 308.95. To determine the tissue-specific activity of the cloned promoter fragment the resulting clone pMOG849 was mobilised to Agrobacterium tumefaciens and the corresponding strain was used to transform wildtype Arabidopsis thaliana plants. Per construct 24transformants were produced. Seeds from the primary transformants were harvested and grown up for infection assays with Heterodera schachtii as described in the Experimental part. GUS analysis after nematode infection SUBSTITUTE SHEET (RULE 26) WO 97/46692 PCT/EP96/02437 19 showed that 79% of the lines transformed with pMOG849 expressed the reporter gene in syncytia. Some weak expression was also found in the area of lateral root branching, in the vascular tissue of roots and leaves, in the centre of the rozette and in some flower tissues. GUS expression outside the syncytium showed strong variation from line to line (see Fig. Presumably, this variation is a result of genome position effects on the introduced regulatory sequences. Nevertheless, in most lines, an expression pattern was found that was very similar to the originally tagged line 553#1164.

Even though the activity of the promoter fragment in the various pMOG849 lines was generally much weaker than the GUS-activity inside syncytia, none of the syncytium-positive lines was entirely specific for the feeding sites.

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. This shows that the #1164 fragment can be used as a nearly feeding site specific promoter to engineer plants having reduced susceptibility to Meloidogyne incognita and Heterodera schachtii.

During the tissue culture phase, it was observed that 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 553#1164 fused to GUSintron). Again, nematode-induced GUS expression could be observed in syncytia and giant cells after nematode infection of Arabidopsis roots with H. Schachtii and M.

incognita respectively.

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) SUBSTITUTE SHEET (RULE 26) WO 97/46692 PCT/EP96/02437 Mol Cell Biol 3, 153-155. The sequence is depicted in SEQIDNO: 4.

Example 7 Cloning of promoter subfragment(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 XhoI site. Thus, all PCR generated deletion fragments of the 1164 promoter could be reintroduced in pMOG819 using this XhoI 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.

TABLE 4 pMOG 5' end primer 3'end primer 958 6044-1 6044-6 959 6044-2 6044-6 960 6044-3 6044-6 961 6044-4 6044-6 962 6044-1 6044-5 After reintroduction of these gene cassettes into plants expression patterns, timing and the like can be determined as described for the 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. To illustrate how the promoter fragments according to invention can be used to impart reduced susceptibility to nematodes this is now illustrated for the genomic 2.1 Kb #1164 fragment, SUBSTITUTE SHEET (RULE 26) WO 97/46692 PCT/EP96/02437 21 cloned in front of Barnase, as an example of a NFS-disrupter gene.

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 plants 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 From one of the hybridizing clones a 4 kb EcoRI fragment was isolated and subcloned in the multicopy plasmid pKS (Stratagene). Sequence analysis revealed that this clone contained 2.1 kb of sequence 5' to the T-DNA insertion in line 1164 and 1.9 kb of 3' sequence.

To restore the exact sequence context in front of the GUS coding region a 546 bp SnaBI fragment from pMOGB49 spanning the promoter- GUS fusion was inserted at the SnaBI site of the genomic clone. A 2325 bp HindIII fragment was isolated from the resulting clone, containing the entire 5' tagged sequence from the genomic EcoRI subclone. This fragment was cloned in front of the barnase gene in construct pFL8 (described below), resulting in clone A fragment containing the barnase coding region was PCR amplified on pMT416 DNA (Hartley, sub) using primers CGGACTCTGGATCCGGAAAGTG 3' (SEQIDNO: 12) and CTGCTCGAGCCTAGGCACAGGTTATCAACACGTTTG 3' (SEQIDNO: 13). These primers introduce flanking BamHI and XhoI 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). To eliminate toxicity of barnase expression in subsequent cloning steps a ST-LS1 intron was inserted in the Styl site of barnase. An Ncol site was created at the barnase translation initiation codon by recombinant PCR using the primers 5' CGGACTCTGGATCCGGAAAGTG 3' (SEQIDNO: 14) and CTTACTCGAGCCATGGTAAGTTTCTGC 3' (SEQIDNO: 15), resulting in pOG16.1. 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 GATCTAGACTCGAGAAGCTTGGATCCCCGGGTAGGTCAGTCCCC 3' (SEQIDNO: 16) and SUBSTITUTE SHEET (RULE 26) WO 97/46692 PCT/EP96/02437 22 CATGGGGGACTGACCTACCCGGGGATCCAAGCTTCTCGAGTCTA 3' (SEQIDNO: 17) and ligating the resulting adapter between the BglII site and the NcoI site of pOG16.1, resulting in clone pFL8. The adapter introduced a HindIII site 5' to the barnase coding region which was used to insert the 1164 promoter yielding pFL15. In addition, by this procedure a fragment containing the Tag promoter and the barstar gene were exchanged with this adapter.

Example 9 Construction rolD-B* Construct pFL11 contains a chimeric barstar gene in a binary vector. This construct was cloned in the following way. The barstar coding region resides on a HindIII/BamHI fragment in construct pMT316 (Hartley (1988) J Mol Biol 202, 913-915). The HindIII site was changed into a BamHI site by ligating in this site the self-annealing adapter AGCTCGGATCCG '3 (SEQIDNO: 18). Subsequently, the resulting BamHI fragment was cloned between a double enhanced CaMV 35S promoter and a nos terminator in the expression cassette pMOG180, described in W093/10251, resulting in pOG30. Using the adapter 5' GGCTGCTCGAGC 3' (SEQIDNO: 19) the HindIII site at the 3' end of the nos terminator was changed into an XhoI site and the EcoRI site at the 5' end of the promoter was changed into a HindIII site using the adapter 5' AATTGACGAAGCTTCGTC 3' (SEQIDNO: Then the 35S promoter was replaced by the promoter from the Agrobacterium rhizogenes RolD gene. This promoter was excised as a HindIII/BamHI fragment from construct pDO2, obtained from F. Leach (Leach and Aoyagi (1991) Plant Sci 79, 69-76). From the resulting clone, pOG38, the barstar gene including promoter and terminator was excised by digestion with HindIII and XhoI and inserted in the respective sites of the polylinker in pMOG800, resulting in pFL11.

Finally, the chimeric #1164 promoter-barnase gene was cleaved out of pFL15 as an EcoRI fragment and inserted in the unique EcoRI site of pFL11 between barstar and the NptII marker gene in a tandem orientation, resulting in pMOG893.

Example Transformation of potato plants with pMOG893 and testing for increased resistance against Globodera pallida The binary vector pMOG893 was mobilised to Agrobacterium tumefaciens and SUBSTITUTE SHEET (RULE 26) WO 97/46692 PCT/EP96/02437 23 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.

The above examples merely serve to illustrate the invention and are not meant to indicate its limits. Numerous modifications will readily occur to the person skilled in the art which are within the scope of the invention.

SUBSTITUTE SHEET (RULE 26) WO 97/46692 PCT/EP96/02437 24 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT:

NAME: MOGEN International N.V.

STREET: Einsteinweg 97 CITY: Leiden STATE: Zuid-Holland COUNTRY: The Netherlands POSTAL CODE (ZIP): NL-2333 CB TELEPHONE: 31-71258282 TELEFAX: 31-71-221471 (ii) TITLE OF INVENTION: REGULATORY DNA SEQUENCES (iii) NUMBER OF SEQUENCES: (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (EPO) INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (ix) FEATURE: NAME/KEY: primer bind LOCATION: 1..20 OTHER INFORMATION: /note= "primer that anneals to uidA gene (Beta-glucuronidase) at position 224-205 from the tagging construct pMOG553.(X83420)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: CTTTCCCACC AACGCTGATC INFORMATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 21 base pairs TYPE: nucleic acid SUBSTITUTE SHEET (RULE 26) WO 97/46692 PCT/EP9602437 STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: GTAATGCTCT ACACCACGCC G 21 INFORMATION FOR SEQ ID NO: 3: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (ix) FEATURE: NAME/KEY: miscfeature LOCATION: 1..12 OTHER INFORMATION: /note= "5'overhang with a XbaI and a BamHI site" (ix) FEATURE: NAME/KEY: primerbind LOCATION: 13..35 OTHER INFORMATION: /note= "this part of the primer anneals to sequence 6044-0 at position 646 to 668" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: TCTAGAGGAT CCTGGCCATA CAAATCAACG TTTAC INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 2163 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (vi) ORIGINAL SOURCE: ORGANISM: Arabidopsis thaliana SUBSTITUTE SHEET (RULE 26) WO 97/46692 PCT/EP96/02437 26 STRAIN: C24 (ix) FEATURE: NAME/KEY: CDS LOCATION: 2161..2163 OTHER INFORMATION: /codon_start= 2161 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 2128..2163 OTHER INFORMATION: /note= "Sequence of pMOG553 upstream of the uid A translation initiation codon up to the RB/plant genome transition." (ix) FEATURE: NAME/KEY: promoter LOCATION: 1..2127 (ix) FEATURE: NAME/KEY: primer_bind LOCATION: 787..804 OTHER INFORMATION: /label= primer6044-1 /note= "annealing of primer 6044-1 (table 4) to amplify subfragment" (ix) FEATURE: NAME/KEY: primer_bind LOCATION: 1147..1169 OTHER INFORMATION: /label= primer6044-2 /note= "annealing of primer 6044-2 (table 4) to amplify subfragments" (ix) FEATURE: NAME/KEY: primer_bind LOCATION: 1853..1880 OTHER INFORMATION: /label= primer6044-3 /note= "annealing of primer 6044-3 (table 4) to amplify subfragments" (ix) FEATURE: NAME/KEY: primerbind LOCATION: 1918..1940 OTHER INFORMATION: /label= primer6044-4 /note= "annealing of primer 6044-4 (table 4) to amplify subfragments" (ix) FEATURE: NAME/KEY: primer_bind LOCATION: 1897..1917 OTHER INFORMATION: /label= primer6044-5 /note= "annealing of primer 6044-5 (table 4) to amplify subfragments (opposite strand)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: SUBSTITUTE SHEET (RULE 26) WO 97/46692 WO 9746692PCT/EP96/02437

GAATTCCATC

TTATATAATA

GTCATCCAAA

AGTCTTCATT

CTTAACACTG

GGCATATGCA

AGTCTTTGTA

AAGTGTGTAG

AGTGAATTTG

GGAAGCAGAG

TTGTTCTCTT

ACGTTTACAG

CCACCGGTAT

TGAATTATAA

AATGGTTTGG

ACCATGTAAG

ACTGAATCAT

GACGAGCTAT

ATTTTGCCTT

ATTCTTATAA

AATTCTAAAA

GTGGTAATTT

GTTGAACTGA

AAAGTGTGGT

ATTAGCTAAA

TACTCCACCT

TAATGATAAA

AAACGAAAGT

AAATATTAAC

TTCACTGCCA

TTTTTAGGGT

TTAGACGAGG

CAGGTGAAGG

AGAAGCATCG

TATCTTTTTG

TAATTGCAAA

ATATGCTATA

AAAGAAAGAA

TTCAAGTCTT

GAAGCTCGGA

GGAGACAGGA

CCTTCTCAAC

TTTGTTTTGT

TGTTCCTTCT

CTTAACTGTT

GAGGCGCAGA

TGAATGTGTA

TGTATTTTGG

TTGGCAAGAA

TAATTGTTTC

GAGGTTGGCG

GTGGACCCGA

AGCATATTGT

AGCTAATATT

AACTAAAAAA

CAAAAGAAAG

TTTTAATATC

ATTAGTTCTT

TCAAAACAAA

GATCAAACTT

CTGGCTCAAT

TGGATGACTA

CTTCTTTTTG

TGGATTTTCT

TACTTGGCCA

AAGCTAGAGA

CTCTGTATAT

TAGCACGGGA

GGAGGCGTGA

TCTGTTTTAT

TTTGTTTTGC

ATCTCAACCA

CTAAAAATGT

GTGGTGGGCA

CCTGTTTGTA

CATGAAAACT

CGACTTACTA

ACCACAGAAA

TTTCTTAGTG

CTGATGGTAA

TGAGTGTTGA

TTTTTTTAAC

ATATTAGATT

ACTACATCAT

TTAACATATA

ACCAAAAGAA

ATCTAGTGCA

CTTTGACAAT

TTTTGCCCAA

GATAAAATCA

GCATGCTATT

TATGCACCAG

AGATGAGGTT

CCTAGTTAAC

AGAAGGTGAA

CAAACGGGTA

CTGGAAACCA

AGCCGAATCC

CTTTAAAAAG

AAGTTTGTTA

TTTGGCAAAG

ATTTCATAAT

TGACTTGTTA

CCATGCAGTG

ATTTCTCTAT

AAAATACAAA

CCATGTTCAT

CAAAATGACA

TAATGTTAGA

ATTAGAGTGA

CACCAGATAT

TATGCTGCTT

ACAGAAAAGA

TCTGAATGAA

ATATTGATCT

CACAGGGAGG

AAGAAGTTTT

ATATACGAAA

TCTGAGZLAGG

TGTAGTTCAC

GAGCATGGCC

CGCAGGCGGA

AGTACTTATT

AGTGAGTGAA

ACGTGATTAT

AATGGTTTAT

TGATTCTGAA

CATTGGGGGA

TTGTAACCTT

TTTTTCCCTT

ATTTGTGAAG

ATCCTGAAGA

AAATAGAAAT

TTGGAGGAAC

ACAGATAAAT

AAGCCACCTA

TACATTTTGT

TGCGGTTTAT

GGATGAATAT

GTACATATAG

TCTGATAAAA

AAAAAZATGAT

GCGATGACCC

TAGATTTCCA

TTGATCTTGC

ATGTCTTATT

AGTTATTTGC

AAAAAAGTTC

ATACAAATCA

AAGAAAGGGA

TGAGTCCAAA

TATTGTTGGA

ATGGATGATT

GCATTTTAGT

TTTCGTGTAG

AATTATCTAT

TTGTATTCAT

CCAATACAAA

TTTGATAGTG

AGATAGCTGA

CTTTAGCTAG

TAATGTGAAT

CGTCAAATAC

TTTGCATCCG

GTGAAAACGT

GGTGGGCACA

120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 AAAAACGAAA GAAATTTAAA SUBSTITUTE SHEET (RULE 26) WO 97/46692 WO 9746692PCT/EP96/02437

AATGTTGTGA

GGCGACTTTA

ACTAGACATT

AACATAAGAT

ACTAATGGTA

GTGAAGAGCT

AGTTTAATTT

TTTATTTATA

ATG

Met

CCTGGTGTGT

GGAAATAGAA

TTTGTTATCT

AAATATTTAC

ATTACTAATT

TTTGATACGT

ACTGTGCTTT

TGTAAAAAAA

CCCTTTCCCA CTTAAATGTA AATTCGCACA ATTGACTCGA GTCCTTTAGT GGTTCGTTTA TTAATTAGCT ACGGAACTAC AATTGCGGAA AGCCGAGAGA AAGTGGAGCA. CTCATGATAA TTATAATGTG ACACACTATT AAAGTCTCAA AGCTTGGATC

CGGCTGATAA

TACGCATTAA

ATCTGGAACG

ATTAGTATTC

GGTGATGGTG

GCGAAGTTGT

GGAATCCAAT

CCCGGGTAGG

TCACATCAGT

AGTCGTAATC

TCCTTATAAT

AATTGATATA

CACGGTGCAT

CTATTTATAA

GACTGCATTA

TCAGTCCCTT

1740 1800 1860 1920 1980 2040 2100 2160 2163 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 1 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Met INFORMATION FOR SEQ ID NO: 6: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1. .12 OTHER INFORMATION: /note= "15' overhang containing the XhoI and the EcoRI sites" SUBSTITUTE SHEET (RULE 26) WO 97/46692 PCT/EP96/02437 29 (ix) FEATURE: NAME/KEY: primer bind LOCATION: 13..30 OTHER INFORMATION: /note= "this part of the primer anneals the sequence of 6044-0 (SEQIDNO: 4) at position 787-804" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CTCGAGAATT CTATAACCTT CTCAACTCTG INFORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (vi) ORIGINAL SOURCE: ORGANISM: Arabidopsis thaliana STRAIN: C24 (ix) FEATURE: NAME/KEY: miscfeature LOCATION: 1..12 OTHER INFORMATION: /note= overhang containing the XhoI and EcoRI site" (ix) FEATURE: NAME/KEY: primer bind LOCATION: 13..35 OTHER INFORMATION: /note= "this part of the primer anneals to the sequence of 6044-0 (SEQIDNO: 4) at position 1147-1169" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CTCGAGAATT CTATAATGTA TTTTGGCATG AAAAC INFORMATION FOR SEQ ID NO: 8: SEQUENCE CHARACTERISTICS: LENGTH: 37 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES SUBSTITUTE SHEET (RULE 26) WO 97/46692 PCT/EP96/02437 (ix) FEATURE: NAME/KEY: miscfeature LOCATION: 1..12 OTHER INFORMATION: /note= overhang containing a XhoI and a EcoRI site" (ix) FEATURE: NAME/KEY: primer_bind LOCATION: 13..37 OTHER INFORMATION: /note= "this part of the primer anneals to the sequence of 6044-0 (SEQIDNO: 4) at position 1853-1880" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: CTCGAGAATT CTATAATAAC ATAAGATAAA TATTTAC 37 INFORMATION FOR SEQ ID NO: 9: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (ix) FEATURE: NAME/KEY: miscfeature LOCATION: 1..12 OTHER INFORMATION: /note= overhang containing the XhoI and EcoRI site" (ix) FEATURE: NAME/KEY: primerbind LOCATION: 13..35 OTHER INFORMATION: /note= "part of primer annealing to the sequence of 6044-0 (SEQIDNO: 4) at position 1918-1940" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: CTCGAGAATT CTATAACTAA TGGTAATTAC TAATT INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) SUBSTITUTE SHEET (RULE 26) WO 97/46692 PCT/EP96/02437 (iii) HYPOTHETICAL: YES (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1..14 OTHER INFORMATION: /note= "part of primer restores sequence of 6044-0 (SEQIDNO: 4) from position 2128 to 2142 while causing a deletion of fragment 1909 to 2127 (ix) FEATURE: NAME/KEY: primer_bind LOCATION: 15..35 OTHER INFORMATION: /note= "this part anneals to the sequence of 6044-0 position 1897-1917" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: GGATCCAAGC TTTGATCAAT TGAATACTAA TGTAG of the primer (SEQIDNO: 4) at INFORMATION FOR SEQ ID NO: 11: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (ix) FEATURE: NAME/KEY: primer bind LOCATION: 1..20 OTHER INFORMATION: /note= "primer that anneals to uidA gene (Beta-glucuronidase) at position 224-205 from the tagging construct pMOG553.(X83420)" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: CTTTCCCACC AACGCTGATC INFORMATION FOR SEQ ID NO: 12: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) SUBSTITUTE SHEET (RULE 26) WO 97/46692 PCT/EP96/02437 32 (iii) HYPOTHETICAL: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: CGGACTCTGG ATCCGGAAAG TG 22 INFORMATION FOR SEQ ID NO: 13: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: CTGCTCGAGC CTAGGCACAG GTTATCAACA CGTTTG 36 INFORMATION FOR SEQ ID NO: 14: SEQUENCE CHARACTERISTICS: LENGTH: 22 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: CGGACTCTGG ATCCGGAAAG TG 22 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 27 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: CTTACTCGAG CCATGGTAAG TTTCTGC 27 INFORMATION FOR SEQ ID NO: 16: SUBSTITUTE SHEET (RULE 26) WO 97/46692 PCT/EP96/02437 33 SEQUENCE CHARACTERISTICS: LENGTH: 44 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: GATCTAGACT CGAGAAGCTT GGATCCCCGG GTAGGTCAGT CCCC 44 INFORMATION FOR SEQ ID NO: 17: SEQUENCE CHARACTERISTICS: LENGTH: 44 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: CATGGGGGAC TGACCTACCC GGGGATCCAA GCTTCTCGAG TCTA 44 INFORMATION FOR SEQ ID NO: 18: SEQUENCE CHARACTERISTICS: LENGTH: 12 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: AGCTCGGATC CG 12 INFORMATION FOR SEQ ID NO: 19: SEQUENCE CHARACTERISTICS: LENGTH: 12 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear SUBSTITUTE SHEET (RULE 26) WO 97/46692 PCT/EP96/02437 34 (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: GGCTGCTCGA GC 12 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 18 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: AATTGACGAA GCTTCGTC 18 SUBSTITUTE SHEET (RULE 26)

Claims (20)

1. An isolated and purified DNA fragment obtained from Arabidopsis thaliana, capable of promoting root knot and cyst nematode-inducible transcription of an associated DNA sequence when re-introduced into a plant comprising the nucleotide sequence represented by nucleotides 646 to 2141 in SEQIDNO: 4.

2. A DNA fragment according to claim 1 comprising the nucleotide sequence represented by nucleotides 1 to 2141 in SEQIDNO: 4.

3. A portion or subfragment or combination of subfragments of a DNA fragment according to any one of claims 1 or 2, capable of promoting root knot and cyst nematode-inducible transcription of an associated DNA sequence when re-introduced into a plant.

4. A DNA fragment according to any one of claims 1 to 3, which is substantially nematode feeding site-specific. *i 20 5. A chimeric DNA sequence comprising in the direction of transcription a DNA fragment according to any one of claims 1 to 4 and a DNA sequence to be expressed under the transcriptional control thereof and which is not naturally under transcriptional control of said DNA fragment. 25 6. A chimeric DNA sequence according to claim 5, wherein the DNA S*sequence to be expressed causes the production of a plant cell-disruptive substance.

7. A chimeric DNA sequence according to claim 6, wherein said cell- 30 disruptive substance is barnase.

8. A chimeric DNA sequence according to claim 6, wherein said cell-disruptive substance comprises RNA complementary to RNA essential to cell viability.

9. A chimeric DNA sequence according to claim 5, wherein the DNA sequence to be expressed causes the production of a substance toxic to the inducing nematode. A replicon comprising a chimeric DNA sequence according to any one of claims 5 to 9.

11. A replicon comprising in the direction of transcription a DNA fragment according to any one of claims 1 to 4 and at least one recognition site for a restriction endonuclease for insertion of a DNA sequence to be expressed under the control of said DNA fragment.

12. A microorganism containing a replicon according to any one of claims 10 or 11.

13. A plant cell having incorporated into its genome a chimeric DNA sequence according to any one of claims 5 to 9.

14. A root system of a plant essentially consisting of cells according to claim 13. A plant essentially consisting of cells according to claim

16. A plant according to claim 15 which is a dicotyledonous plant.

17. A plant according to claim 16 which is a potato plant.

18. A plant grafted on a root system according to claim 14. SH

19. A part of a plant selected from seeds, flowers, tubers, roots, leaves, fruits, pollen and wood, obtained from a plant according to any one of claims to 18 and comprising plant cells according to claim 13.

20. to 18. A crop consisting essentially of plants according to any one of claims SS S S. 6@ S. S Se S. S 0 S. OS S 0 @0 .6 U. 0 60 S S S OSSS @0 6@ 0 0 0060 6 6005

21. Use of a DNA fragment according to any one of claims 1 to 4 for identifying subfragments capable of promoting transcription of an associated 10 DNA sequences in a plant.

22. Use of a chimeric DNA sequence according to any one of claims 6 to 9 for transforming plants.

23. Use of a portion or subfragment or combination of subfragments according to claim 3 for making hybrid regulatory DNA sequences.

24. A DNA fragment according to claim 1 substantially as hereinbefore described with reference to the examples. DATED: 30 November, 1998 PHILLIPS ORMONDE FITZPATRICK Attorneys for: MOGEN INTERNATIONAL NV C:\WINWORD\STACVIC\DYF\SPECN\R561 54gDOC