CA2257149A1 - Nematode-inducible plant gene promoter - Google Patents

Abstract

The invention provides DNA fragments obtainable from Arabidopsis thaliana that is capable of promoting root knot and cyst nematode-inducible transcription of an associated DNA sequence when re-introduced into a plant, and the use of said DNA fragments.

Description

CA 022~7l49 l998-l2-02 2 PCT~EP96tO2437 NEMATODE-rNDUClBLE PLANT GENE PROMOTER

The invention relates to regulatory DNA sequences which can be u~ed for expressing DNA sequences in plant cells. The invention further comprises chimeric DNA comprising said regulatory DNA ~equences operably linked to DNA to be expre~sed in plant cells, as well as plants cont~;n;~ 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 ~equences ~LEMMI's, for Lycopersicon esculentum - Meloidogyne incognita) are stimulated, whereas others appear to be repressed by the nematode. It i9 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 speci~ic way.
SU~ RY OF THE INVEN~ION
The invention provides a DNA fragment obtainable from Arabidopsis thaliana that is capable of promoting root knot and cyst nematode-SUBSTITUTE SHEET (RULE 26) . . ~.

CA 022~7149 1998-12-02 W097/46692 PCT~P96/02437 inducible transcription of an a~sociated 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 ~equence to be expre~sed causes the production of a plant cell-disruptive 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) . T

CA 022~7l49 l998-l2-02 WO 97/46692 PCT~EP96/02437 invention for making hybrid regulatory DNA sequences.
The following figures further illustrate the invention.

~S~TPTION OF T~E FIGURES
Figure 1. Schematic plasmid map of Binary vector pMOG23.
- Figure 2. Schematic plasmid map of Binary vector pMOG800.
Figure 3. Schematic plasmid map of Binary vector pMOG553.
Figure 4. Schematic plasmid map of Binary vector pMOG819.
Figure 5. Schematic plasmid map of Binary vector pMOG849.
Figure 6. Expression patterns outside the NFS of several pMOG849 transformed Arabidopsis thaliana line~.
Figure 7. Schematic representation of a NFS disrupter gene and a neutraliser gene in a two component system for engineering of nematode resistanct plants Figure 8. Schematic plasmid map of Binary vector pMOG893.

Some ways of practicing the invention a~ well as the -aning of variouR
phrases are explained in more detail below.

DETAILED DESCRIPTION OF T~E INV~.... ION
The present invention provides regulatory DNA sequences obtainable from Arabidopsis thaliana, which are inducible by root knot and cyst nematodes and which show a high preference of expression of any associated DNA inside the special nematode feeding structures of the plant root. Such a nematode feeding structure is used by an invading nematode as source of food, whereby the nematode induces a change in the plant tissue thereby forming either a giant cell (root-knot nematodes) or a ~yncytium ~cy~t nematodes). A method of isolating regulatory DNA
sequences has been disclosed and clai -.d in a prior application, WO92/17054, which is incorporated herein by reference.
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
~equences and transforming plants with the resulting chimeric DNA
sequence using known methods. The heterologous DNA is expressed upon infection of the roots by various root knot nematodes, such as ~eloidogyne incognita, and cyst nematodes, such as Heterodera schachtii and Globodera pallida ~a more comprehensive, but by no means limiting, SUBSTITUTE SHEET (RULE 26) CA 022~7l49 l998-l2-02 WO 97/46692 PCT~P96102437 list is presented in table 2). Advantageouqly, 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 (e.g. EP O 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, W093tlO251 and W094tlO320, 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 (i.e. non-NFS) tissue the NFS
disruptive substances under the control thereof have adverse effects on plant viability and/or yields. Moreover, it was found that the regulatory DNA sequences according to the invention are active during the tissue culture phase in the transformation procedure, necessitating the use of a neutralising substance during this phase. In order to reduce or eliminate (potential) adverse effects, it is therefore strongly preferred to use a chimeric NFS-disruptive construct according to the invention in conjunction with a neutralising gene construct. The details of such a so-called two-component approach for the engineering of nematode resistant plants are set out in W093/10251. According to this approach a NFS-disrupter compound (coding sequence-A) is placed under the control of a promoter that is at least active in the NFS, and preferably not or hardly outside the NFS, whereas the unwanted phytotoxic 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 SUBSTI~UTE SHEET (RULE 26~

CA 022~7l49 l998-l2-02 W 097/46692 PCT~EP96/02437 s 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 di~ruptive ~ubstance, encoded by coding sequence-A, can not be neutralized sufficiently by product~ from coding sequence-B.
The properties of the regulatory DNA ~equences 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. Variou~
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 encompa~sed 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 expres~ed 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) CA 022~7l49 l998-l2-02 Bacillus amyloliquefaciens (Hartley, 1988, J. Mol. Biol. 2Q2, gl3-915), restriction endonuclea~es / corresponding methyla~es ~uch a~ E~QRI from E.coli (Green et al., 1981, J. Biol. Chem. 2~, 2143-2153) and EcoRI
methylase or similar combination~ as described in the review for type II
restriction modification systems ~Wilson, 1991, Nucl. Acid Res. 12, 2539-2566), bacteriocins and corresponding immunity proteins, e.g.
colicin E3 / immunity protein from E. coli tLau et al. 1985, Nucl. Acid Res. 12, 8733-8745) or any disruptive substance coding gene which may be neutralized by simultaneou~ production of antisense RNA under control of promoter-B, such as DNA sequences encoding Diptheria Toxin Chain A (Czako & An, 1991, Plant Phy~iol. 95, 687-692), RNAses such as RNAse Tl, ribonucleaseq or proteases and ribozymes against mRNA that code for phytotoxic proteins.
According to another aspect of the invention combination~ of disrupting and neutralizing substances comprise respectively gene4 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 ~ubstituting the function of the endogenous protein or polypeptide product. Such disruptive genes may be selected from the group consisting of (a) genes encoding ribozymes against an endogenous RNA transcript, (b) gene~ which when transcribed produce RNA transcripts that are complementary or at least partially complementary to RNA transcripts of endogenous genes that are essential for cell viability, a method known as antisense inhibition of gene expression (disclosed in EP-A 240 208), or (c) genes that when transcribed produce RNA transcripts that are identical or at least very similar to transcripts of endogenous genes that are essential for cell viability, an as yet unknown way of inhibition of gene expression referred to as co-suppression (disclosed by Napoli C. et 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) CA 022~7149 1998-12-02 W 097l46692 PCTrEP96/02437 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-suppre~sive effect. Hence, it i~
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 les~ than 80%, yet more preferably said neutralizing sense transgene encodes a protein or polypeptide gene product that is not identical in amino acid sequence to the disrupted gene product and wherein the nucleotide sequence identity of the transcripts encoded by the neutralizing transgene is less than 75%.
Target genes for anti~ense 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 tranQlocation from other cells or organelles of enzyme products normally qynthesized by such enzymes. Preferably, genes coding for membrane-translocating enzymes are chosen as these are involved in establish;ng chemical gradients across organellar membranes. Inhibition of such proteins by antisense expres~ion 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 ~i~ULE 26) CA 022~7149 1998-12-02 WO 97/46692 PCT~EP96/02437 comprising the NFS. Specific examples for such organelles are mitochondria, endoplasmic reticulum and plasmodesmata (Hussey et ~1. 1992 Protoplasma 167; 55-65, Magnusson & Golinowski 1991 Can. J. Botany 5~;
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 Pre~s) or Encyclopedia of Plant Physiology (New Series, 1976, Springer-Verlag, Berlin).
Although only in some cases, the gene 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.

15 ~XAMPT.~.~ OF TAR~.FT FNZyMF~ FOR ANTISENSE ExPRF~SION IN NFS ~n SF.N.
EXPRF~SION ouTsIn~ NFS

~nzym~ pathway/or~anelle 20 ATP synthase mitochondrion adenine nucleotide translocator mitochondrion phosphate translocator mitochondrion tricarboxylate translocator mitochondrion dicarboxylate translocator mitochondrion 25 2-oxo-glutarate translocator mitochondrion cytochrome C mitochondrion pyruvate kinase glycolysis glyceraldehyde-3P-dehydrogena~e glycolysis NADPH-cytochrome P450 reductase lipid metabolism fatty acid synthase complex lipid metabolism glycerol-3P-acyltransferase lipid metabolism~5 hydroxymethyl-glutaryl CoA reductase mevalonic acid pathway aminoacyl transferase nucleic acid metabolism transcription factors nucleic acid metabolism elongation factors nucleic acid metabolism A suitable promoter-B is defined as a promoter that drives expression in substantially all cells wherein coding sequence-A is expressed, with the proviso that it does not drive expression inside a nematode feeding structure, or not effectively. (With 'substantially all SUBSTITUTE SHEET ~RliLE 261 CA 022~7l49 l998-l2-02 W 097/46692 PCT~EP96102437 cells' iq 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 i9 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 (Ray et al., 1987, Science 2~, 1299-1302), and mRNA stabilizing sequences quch 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 qecond promoter-B'/coding-sequence-B having an expression pattern which is partly overlapping or entirely complementary to promoter-B/coding-sequence-B, with the proviso that neither promoter-B nor promoter-B' drives expression in the NFS. 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 preqent 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 ~
810-812). Another preferred example for promoter-B is the strong root promoter ~QlD (Leach & Aoyagi 1991 Plant Sci. 79; 69-76) from plasmid pRiA4 of Agrobacterium rhizogenes; the 5' flanking region of ORF15 (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 fuqion to marker genes such as GUS (Jefferson, 1987, Plant Mol. Biol.
Reporter 5, 387-405), transfer of these constructs to plants and histochemical analysis of such transgenic plants after infection with PPN.

SUBSTITUTE SHEET (F~ULE 26) CA 022~7l49 l998-l2-02 W 097/46692 PCT~P96102437 Other regulatory sequences such as t~ ;n~tor 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 skille~ person in the art. An example of such sequences is the 3' fl~nk; ng region of the nopaline synthase ~nos) gene of Agrobacterium tumefaciens ~Bevan, 1984, Nucl. Acids Res. 12, 8711-~721).
Further details of the two component approach can be found in WO93~10251 (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 (e.g. Xiphlnema spp.) to the more evolved sedentary endoparasites (e.g. 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).

T~RL~ 2 F.~AMPT.F..S OF PLANT-PAR~SITIC NFM~TODES ANn THFIR
PRTNCIP~T HOST PL~NTS
Nematode Species Principal Host Plants MF~loi~ Jy~e M. hapla wide range M. incognita wide range M. exigua coffee, tea, Capsicum, Citrullus M. indica Citrus 35 M. javanica wide range M. africana coffee M. graminis cereals, grasses M. graminicola rice M. arenaria wide range Heterodera L Globo~ra H. mexicana Lycopersicon esculentum, Solanum spp.
H. punctata cereals, grasses G. rostochiensis Solanum tuberosum, Solanum spp, Lycopersicon esculentum SUBSTITUTE SHEET (~ULE 26) CA 022~7l49 l998-l2-02 W 097/46692 PCT~EP96/02437 G. pall~A Solanum tuberosum G. tabacum Nicotiana tabacum, Nicotiana 8pp.
H. cajani Cajanus cajan, Vigna sinenqis H. glycinea Glycine max, Glycine qpp.
5 H. oryzae Oryza sativa H. schachtii Beta qpp, Braaqica qpp, H. trifolii Trifolium spp.
H. avenae cerealq, grasses H. carotae Daucuq carota 10 H. cruciferae Cruciferae H. goettingiana Pisum sativum, Vicia 8pp.

Within the context of this invention, a plant i~ said to show reduced qusceptibility to plant paraqitic nematodes tPPN) if a statistically significant decrea~e in the number of mature females developing at the qurface of plant rootq can be observed as compared to control plants. Suqceptible / reaistance classification according to the number of maturing females is ctandard practice both for cyst- and root-knot nematodeq (e.g. T~Mon~;a, 1991, Plant Disease 75, 453-454;
20 Omwega et al., 1990, Phytopathol. 80, 745-748).
A nematode feeding qtructure according to the preqent invention qhall include an initial feeding cell, which shall mean the cell or a very limited number of cells destined to become a nematode feeding ~tructure, upon induction of the invading nematode.
A NFS disruptive effect according to the invention iq 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 rec~ ';nAnt DNA contsining the DNA sequences as described in the present invention into plant hosts. Such techniques include but are not limited to transformation of protoplastq using the calcium/polyethylene glycol method, electroporation and microinjection or (coated) particle bombardment tPotrykus, 1990, Bio/Technol. 8, 535-542).
In addition to these so-called direct DNA transformation methods, transformation systems involving vectors are widely available, such aq viral vectors ~e.g. from the Cauliflower Mosaic Virus tcaMv) and bacterial vectors te.g. from the genus Agrobacterium) tPotrykus, 1990, Bio/Technol. ~, 535-542). After qelection and/or screening, the protoplasts, cell~ or plant parts that have been transformed can be regenerated into whole plants, using methods known in the art ~Horsch et SUBST~lUTE SHEEI (~ULE ~6~

CA 022~7l49 l998-l2-02 W 097/46692 PCT~P96/02437 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 9y9tem (diqclosed in EP-A 120 516) in which Agrobacterium strains are used which contain a helper pla~mid with the virulence genes and a compatible pla~;A, the binary vector, conta;n;ng 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, 198g, Nucl. Acids Res. 12, 8711-8721). The binary vectors as used in thiq example contain between the left- and right-border sequences of the T-DNA, an identical NPTII-gene coding for k~ cin resistance (Bevan, 1984, Nucl. Acids Res.
12, 8711-8721) and a multiple cloning 9ite 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 tiqsue culture systems for these crops, together with the powerful methods for introduction of genetic material into plant cell~ has facilitated transformation. Presently, preferred methods for transformation of monocots are microprojectile bombardment of explants or suqpension cells, and direct DNA uptake or electroporation (Sh;m~m~to, 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 protopla~ts 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 95tl6031). 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) CA 022~7149 1998-12-02 The following examples are given only for purposeq 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 qtandard methods described in Maniatis ~Molecular Cloning, A laboratory Manual 2nd Edition, Cold Spring Harbor Laboratory, 1990).
Trans~ormat~on o~ Ar~b i dop~ ~ ~
Transformation wa~ carried out using co-cultivation of Arabidopsis thaliana (ecotype C24) root segments with Agrobacterium strain MOG101 containing a suitable binary vector as described by Valvekenq et al. (1988, Proc. Nat. Acad. Sci. USA ~, 5536-5540) which i8 as follows:
Arabidopsis seeds were vernalized for 7 days at 4~C before germination. Seeds were surface-~terilized for 2 min in 70% EtOH, transferred to 5% NaOCl/0.5~ NaDodSO4 for 15 min rinsed five times with sterile distilled water, and placed on 150 x 25 mm Petri disheA
cont~;n;ng germination medium ~GM) (Table 3) to germinate. Petri disheq were sealed with gas-permeable medical tape (Urgopore, Chenove France).
Plants were grown at 22~C in a 16-hr light/8-hr dark cycle. The same growth-room conditions were used for tissue culture procedures.
All plant media were buffered with 2-(N-morpholino)ethanesulfonic acid at 0.5g/liter (pH 5.7: adjusted with 1 M KOH), solidified with 0.8~ Difco Bacto agar, and autoclaved at 121~C for 15 min. Hormone9 and antibiotics were dissolved in dimethyl Aulfoxide and water, respectively, and were added to the medium after autoclaving and cooling to 65~C.
Intact roots were incubated for 3 days on solidified 0.5/0.05 medium (Table 3). 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 explanta were blotted on sterile filter paper to remove most of the liquid medium and cocultivated for 48 hr on 0.5/0.05 SUBSTITUTE S~EET (RiJLE 26) CA 022~7l49 l998-l2-02 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 Xm 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 0.15t5 agar containing only 500 mg of vanc: ~in 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 ~halian~ 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.

20 TART~ 3 PT,~T ~F'.nTA

CIM SIM
GM R3~ PG1* 0.5/0.05 0.05/7* 0.15/5*
Salts + ~itamins MS MS B5 B5 MS B5 Sucrose, g/L 10 30 -- -- 30 --Glucose, g/L -- -- 20 20 ~~ 20 30 IAA, mg/L -- 5 -- -- 0.05 0.15 2,4-D, mg/L -- 0.5 2 0.5 -- --2ipAde, mg/L -- -- -- -- 7 5 Xin, mg/L -- 0.3 0.05 0.05 -- --L, liter; IAA, indole-3-acetic acid; Kin, kinetin; 2ipAde, N6-(2-isopentenyl)adenine; CIM, callus-inducing medium; SIM, shoot-inducing medium; MS, Murashige & Skoog medium ; B5, Gam~org B5 medium Transformation o~ 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) . ~ . . ~

CA 022~7l49 l998-l2-02 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 ~hi; ;ne HCl, 0.5 mg/l pyridoxine Hcl, 0.5 mg~l ~ nicotinic acid, 100 mg/l myo-inositol, 30 g/l sucrose, 0.5 g/l MES pH
5.8). Inoculation with Agrobacterium tumefaciens 9train EHA105 ~Hood et al. 1993 TransgeniC Research 2, 20a-218) was done by replacing the WX
with 100 ml fresh WM containing the resuspended pellet of 10 ml Agrobacterium culture grown freshly in LB + appropriate antibiotic to an OD600 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/l indole acetic acid) at a density of 20 explants/petridish. After two days the disks were transferred to PM (CM supplemented with 200 mg/l cefotaxime, 100 mg/l 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 tran~fer 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.

~anAl ~n~ of nematode~, growth and infection of plant roots Arabidopsis seeds were surface sterilized and sown in petri dishes ~0: 9 cm) on B5 medium containing 20 g/l glucose and 20 mg~l kanamycin. After 3 days at 4~C the plates were incubated for 2 weeks in a growth chamber at 22~C with 16-hr light/8 hr-dark cycle. Kanamycin-resistant plants were then transferred to soil-filled translucent plastic tubes ~30x15x120 mm, Kelder plastibox b.v., The Netherlands). The tube9 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 SIJBSTITUTE SHEET (RULE 26) .. ... . ... . . ..

CA 022~7l49 l998-l2-02 W O 97/46692 PCT~P96/02437 auspension containing 500 second stage larvae of Heterodera schachtii ~in 3 ml H2O) per root system or 300 second stage larvae of Meloidogyne incorJn;ta per root system into the 30il.
S~milarly, potato shoots which had rooted on k~- yCin~COntaining RM
medium were transferred to qoil-filled tran~lucent plastic tubeq (30x15x120 mm, Kelder plactibox b.v., The Netherland~) and grown tilted for another 2 week~ at 22~C with 16 h light/8 h dark cycle. Infection was done by injecting a suspension conta;ning 500 ~econd stage larvae of Globodera pallida (in 3 ml H2O) per root sy~tem into the soil.

GUS aaaay GUS activity was determined at various times during the infection proce~
by thoroughly washing the root sy~tems to remove most of the adhering soil and incubating them in X-Gluc solution (1 mg/ml X-Gluc, 50mM NaPO4 (pH7), lmM K4Fe(CN)6, lmM K K3Fe(CN)6, 10mM EDTA, 0.1% Triton X100) at 37~C over night. After removal of the chlorophyll from the ti~que by incubation with 70% ethanol for aeveral hour~ GUS staining was monitored under the microscope.

E~ample ConJtruction of binary vector pMOG800 The binary vector pMOG800 i~ a derivative of pMOG23 (Fig. 1, depo~ited at the Centraal Bureau voor sch; -lcultures, Oosterstraat 1, Baarn, The Netherlands on January 29, 1990 under number CRS 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 cell~ (Fig. 2). A sample of E. Coli DH5 alpha, harbouring pMOG800, was deposited at the Centraal Bureau voor Schimmelcultures, Oosterstraat 1, Baarn, The Netherlands, on Augu~t 12, 1993 under number CBS 414.93.

E~ample 2 Conatruction of promoterleaa GUS con~truct pMOG553 Construction of this vector is described in Goddijn et al. 1993 Plant J 4, 863-873. In this reference an error occurs; the construct contains a CaMV 35S RNA terminator behind the ~-glucuronidase gene SUBSTITUTE SHEET (RULE 26) t CA 022~7l49 l998-l2-02 WO97/46692 PCT/~l~ f~2437 inqtead of the indicated nos terminator. The sequence between the T-DNA
borders of this binary vector is available from the EMBL databaqe under acceqqion number: X84105.pMOG553 carries the HygR marker for plant transformation (Fig. 3).

Esample 3 Identification and isolation of a trapped NFS-prefQrential promoter fragment in Arabidop~i~ thaliana The binary vector pMOG553 waq mobilized by triparental mating to Agro~acterium tumefaciens strain MOG101. The resulting strain was used for Arabidopsis root transformation. More than 1100 transgenic Arabidopsis plant lines were obtained in this way. Tranqgenic plants were grown to maturity, allowed to self-fertilize and the resulting qeeds (S1) were harvested and vernalized. Subqequently S1 qeedq were germinated on nutrient solution (Goddijn et al. 1993 Plant J 4, 863-873) solidified with 0.6% agar, 10 mg/l hyy-olllycin and stored at 4~C for a 4 day imbibition period. At day 5 the plateq were transferred to room temperature and moderate light (1000 lux, 16 h L / B h D) for germination. Fourteen days old see~l;ngs were transferred to potting soil in tilted translucent plastic tubes (30x15x120 mm) for further growth at 5000 lux (20~C). 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 80il and tube. After two weeks the roots were infected with nematodes as described in the Experimental part. At qeveral 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 qyncytia 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 analyqis.
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) CA 022~7149 1998-12-02 W097/46692 PCT~EP96/02437 GUS coding region, and religated. By subsequent digestion of the circular DNA with the enzyme SnaBI a linear fragment waq obtained with known GUS
sequences at the ends and the fl~nk;n~ plant sequence in between. This fragment was amplified using the primer set GUSinvS (5' CTT TCC CAC CAA
CGC TGA TC 3' SEQIDNO: 1) and GUS7 (5' GTA ATG CTC TAC ACC ACG CCG 3' SEQIDNO: 2), cloned in a multi-copy vector and sequenced (see below).
To clone this amplified fragment back in front of GUS the plant sequence was re-amplified from Arabidopsis genomic DNA using the primers GUSinv5 and 1164XBM (5' TCT AGA GGA TCC TGG CCA TAC AAA TCA ACG TTT AC 3' SEQIDNO: 3). A pfu DNA polymerase carrying a proofreading activity was used to reduce the error rate. Primer 1164XBM introduces a ~amHI 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.

Erample 4 Construction of promoterleQ3 GUS construct pMOG819 This vector was constructed by cloning the GUSintron coding region (V~nc~nneyt et al. 1990, Mol. Gen. Genet. 220; 245-250) of pMOG553 as a BamHI-Eco~I fragment in the polylinker of pMOG800. The binary vector pMOG819 (Fig. 4) qerves to introduce the cloned promoter fragments for further expression analysis after transformation of plantq.

25Esampl~ 5 Analysi~ of promoter fragments after re-introduction into Arabidop~is 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. 5). 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 waq mobilised to Agrobacterium tumefaciens and the corresponding strain was used to transform wildtype Arabidopsis thaliana plants. Per construct 24-30 transformants were produced. Seeds from the primary transformants were harvested and grown up for infection asqays with 8eterodera schachtii as deqcribed in the Experimental part. GUS analysis after nematode infection SUBSTITUTE SHEET (RULE 26) CA 022~7l49 l998-l2-02 W 097l46692 PCTAEP96/02437 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
expre~sion outside the syncytium showed strong variation from line to line (see Fig. 6). Presumably, this variation is a result of genome position effects on the introduced requlatory sequences. NevertheleRs, in most lines, an expression pattern was found that was very similar to the originally tagged line 553#1164.
~ven though the activity of the promoter fragment in the various pMOG849 lines waq 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 ~eloidogyne 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 Rb (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~

CA 022~7149 1998-12-02 W097/46692 PCT~EP96/02437 Mol Cell Biol 3, 153-155. The sequence is depicted in SEQIDNO: 4.

Example 7 Cloning of promoter ~ub~ragment (8) 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 10 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 15 6044-6 correspond with SEQIDNO's 6 to 11, respectively.

pMOG 5' end primer 3'end primer After reintroduction of these gene cassettès into plants expression patterns, timing and the like can be determined as described for the 1.5 Kb #1164 fragment in Example 3. Fragments found to have useful patterns and/or timing may subsequently be used to drive expression of other heterologous DNA sequences (both sense/coding and antisense) and/or used to make hybrid promoter constructs. Furthermore, further analysis yields insight in several regulatory elements such as silencers, enhancers and the like, and creates the possibility of willfully influencing expression patterns and/or timing. 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) t CA 022~7l49 l998-l2-02 W 097/46692 PCT~EP96/02437 cloned in front of Barnase, as an example of a NFS-disrupter gene.

EYample 8 Cloning of #1164 in front of barnase A 2.1 Kb genomic DNA fragment containing the S' tagged sequence from line 1164 was cloned in front of barna~e, a Bacillu~
amyloli4uefaciens 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 10 #1164 iPCR product (see Example 3). From one of the hybridizing cloneq a 4 kb EcoRI fragment was isolated and subcloned in the multicopy pla~mid 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 pMOG849 Apanning 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 pFL15.
A fragment containing the barnase coding region was PCR
amplified on pMT416 DNA (Hartley, sub) using primer~ 5' CGGACTCTGGATCCGGAAAGTG 3' (SEQIDNO: 12) and 5' C~GClCGAGCCTAGGCACAGGTTATCAACACGTTTG 3' (SEQIDNO: 13). These primers introduce flanking Bam~I 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-LSl intron was inserted in the StyI site of barnase. An NcoI site was created at the barnase translation initiation codon by recombinant PCR using the primers 5' CGGACTCTGGATCCGGAAAGTG 3' (SEQIDNO: 14) and 5' CTTACTCGAGCCATGGTAA~lllClGC 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 5' GATCTAGACTCr,A~.AA~CTTGGATCCCCGGGTAGGTCAGTCCCC 3' (SEQIDNO: 16) and 5' SUBSTITUTE SHEET (~ULE 26) .. .. . , . . ~,, .

CA 022~7l49 l998-l2-02 CATGGGGGACTGACCTACCCGGGGATCCAAGCTTCTCGAGTCTA 3' tSEQIDNO: 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 5' AGCTCGGATCCG '3 (SEQIDNO: 18). Subsequently, the resulting BamHI fragment was cloned between a double enhanced CaMV 35S promoter and a nos tenminator in the expres~ion cassette pMOG180, described in WO93/10251, re~ulting 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:
20). 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.

Exa~ple 10 ~ransformation of potato plants with pMOG893 and testing for increased resistance against Globodera pallida The binary vector pMOG~93 was mobilised to Agrobacterium tumefaciens and SUBSTITUTE Sl IEET (RULE 26) . . . , ~ . .

CA 022~7l49 l998-l2-02 W097/46692 PCT~EP96/02437 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 S 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 Barna~e/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 it~ limits. Numerous modifications will readily occur to the person skilled in the art which are within the scope of the invention.

SUBSTITUTE SHEET (RUEE 26) . .

CA 022~7149 1998-12-02 SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:
(A) NAME: MOGEN International N.V.
(B) STREET: Einsteinweg 97 (C) CITY: Leiden (D) STATE: Zuid-Holland (E) COUNTRY: The Netherlands (F) POSTAL CODE (ZIP): NL-2333 CB
(G) TELEPHONE: 31-71258282 (H) TELEFAX: 31-71-221471 (ii) TITLE OF INVENTION: REGULATORY DNA SEQUENCES
(iii) NUMBER OF SEQUENCES: 20 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPO) (2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs ~B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES
(ix) FEATURE:
(A) NAME/KEY: primer_bind (B) LOCATION: 1..20 (D) 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:

(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid SUBSTITUTE SHEET (RULE 26) CA 022~7l49 l998-l2-02 WO 97/46692 PCT~P96/02437 (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA ~genomic) (iii) HYPOTHETICAL: YES
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 1..12 (D) OTHER INFORMATION: /note= "5'overhang with a XbaI and a BamHI site"
(ix) FEATURE:
(A) NAME/KEY: primer_bind (B) LOCATION: 13..35 (D) 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:

(2) INFORMATION FOR SEQ ID NO: 4:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2163 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Arabidopsis thaliana SUBST~lUTE SHELI (RULE 26) CA 022~7l49 l998-l2-02 W 097/4~692 PCT/EP96/02437 (B) STRAIN: C24 (ix) FEATURE:
(A) NAME/KEY: CDS
~B) LOCATION: 2161... 2163 (D) OTHER INFORMATION: /codon_start= 2161 (ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 2128... 2163 (D) OTHER INFORMATION: /note= "Sequence of pMOG553 upstream (5') of the uid A translation initiation codon up to the RB/plant genome transition."
15 (ix) FEATURE:
(A) NAME/KEY: promoter (B) LOCATION: 1..2127 (ix) FEATURE:
(A) NAME/KEY: primer_bind (B) LOCATION: 787..804 (D) OTHER INFORMATION: /label= primer6044-1 /note= "annealing of primer 6044-1 (table 4) to amplify subfragment"
(ix) FEATURE:
(A) NAME/KEY: primer_bind (B) LOCATION: 1147..1169 (D) OTHER INFORMATION: /label= primer6044-2 /note= "annealing of primer 6044-2 (table 4) to amplify subfragments"
(ix) FEATURE:
(A) NAME/KEY: primer_bind 35 (B) LOCATION: 1853.. 1880 (D) OTHER INFORMATION: /label= primer6044-3 /note= "annealing of primer 6044-3 (table 4) to amplify subfragments"
40 (ix) FEATURE:
(A) NAME/KEY: primer_bind (B) LOCATION: 1918..1940 (D) OTHER INFORMATION: /label= primer6044-4 /note= "annealing of primer 6044-4 (table 4) to amplify subfragments"
(ix) FEATURE:
(A) NAME/KEY: primer_bind (B) LOCATION: 1897..1917 (D) 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 (i-lULE 26~
I

CA 022~7l49 l998-l2-02 GTCATCCAAA TTTTTAGGGT TCAAAACAAA ACrAAAAr~AA ACA~AAAAGA TCTGATAAAA 180 AGTCTTCATT TTAGAr~.A~.G GATCAAACTT ATCTAGTGCA TCTGAATGAA AAAAAATGAT 240 AG'lC~ 7lA TATC.l-l-G ~llCll-lIG GATAAAATCA AAGAAGTTTT TTGATCTTGC 420 G~AAG~A~AG AAArAAAGAA AAGCTAGAGA AGATGAGGTT TGTAGTTCAC AAAAAAGTTC 600 1.~-~ ~.~ TTCAAGTCTT CTCTGTATAT CCTAGTTAAC GAGCATGGCC ATACAAATCA 660 CrACCGGTAT Gr.A~ArAG~A GGAGGCGTGA CAAACGGGTA AGTACTTATT TGAGTCCAAA 780 AA-GC71llGG .~ L~ ~11~1 11 lGl 11 ~GC AGCCGAATCC ACGTGATTAT ATGGATGATT 900 ACCATGTAAG TC~lC~--~- ATCTCAACCA CTTTAAAAAG AATGGTTTAT GCATTTTAGT 960 ACTGAATCAT CTTAACTGTT CTAAAAATGT AA~7~ 7llA TGATTCTGAA ~llCGI~lAG 1020 ATTTTGCCTT TGAATGTGTA C~l~7lll~7lA ATTTCATAAT TTGTAACCTT TTGTATTCAT 1140 ATTCTTATAA TGTATTTTGG CATGAAAACT TGACTTGTTA illIlCCCTT CCAATACAAA 1200 GTGGTAATTT TAAllGl--'C ACr.ArAGAAA AIII~lClAT ATCCTGAAGA AGATAGCTGA 1320 45 GTTGAACTGA GAGGTTGGCG TTTCTTAGTG AAAATAr.AAA AAATAGAAAT CTTTAGCTAG 1380 TACTCCACCT AGCTAATATT lli~ii-AAC TAATGTTAGA AAGCCACCTA TTTGCATCCG 1560 TAATGATAAA AACTAAAAAA ATATTAGATT ATTAGAGTGA TACATTTTGT GTGAAAAcGT 1620 55 AAAC~AAAGT rAAAAGAAAG AAAAACGAAA GAAATTTAAA TGCGGTTTAT GGTGGGCACA 1680 SUBSTITUTE SHEET (RULE 26) ... .. . . . ...... . .

CA 022~7l49 l998-l2-02 W097/46692 PCT~EP96/02437 AAl~l.vLGA CCTGGTGTGT CCClllCCCA CTTAAATGTA CGGCTGATAA TCACATCAGT 1740 ACTAGACATT TTTGTTATCT ~lCCll,AGT G~l-C~1~lA ATCTGGAACG TCCTTATAAT 1860 ACTAATGGTA ATTACTAATT AATTGCGGAA AGCC~AGA~A GGTGATGGTG CACG~-lGCAT 1980 GT~.AA~AGCT TTTGATACGT AAGTGGAGCA CTCATGATAA GCGAAGTTGT CTATTTATAA 2040 Metl (2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1 am1no acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
Met (2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS
(A) LSNGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) ~iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO

~ix) FEATURE:
(A) NAME/KEY: misc_feature ~B) LOCATION: 1..12 (D) OTHER INFORMATION: /note= "5' overhang containing the XhoI and the EcoRI sites"

SIJBSTITUTE SHEET (RULE 26) CA 022~7l49 l998-l2-02 W 097/46692 PCT/EP96tO2437 (ix) FEATURE:
(A) NAME/REY: primer_bind ~B) LOCATION: 13..30 ~D) 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:

~2) INFORMATION FOR SEQ ID NO: 7:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 35 base pairs ~B) TYPE: nucleic acid ~C) STRANDEDNESS: single ~D) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA ~genomic) ~vi) ORIGINAL SOURCE:
~A) ORGANISM: Arabidopsis thaliana ~B) STRAIN: C24 ~ix) FEATURE:
~A) NAME/KEY: misc_feature ~B) LOCATION: 1..12 ~D) OTHER INFORMATION: /note= "5' overhang containing the XhoI and EcoRI site"
~ix) FEATURE:
~A) NAME/REY: primer_bind 35 ~B) LOCATION: 13.. 35 ~D) 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:
cTcGAr~AATT CTATAATGTA TTTTGGCATG AAAAC 35 ~2) INFORMATION FOR SEQ ID NO: 8:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 37 base pairs ~B) TYPE: nucleic acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA ~genomic) ~iii) HYPOTHETICAL: YES

SUBSTITUTE SHEET (RULE 26) CA 022~7149 1998-12-02 W 097l46692 PCT~P96/02437 ~ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 1..12 (D) OTHER INFORMATION: /note= "5' overhang containing a XhoI and a EcoRI site"
(ix) FEATURE:
(A) NAME/KEY: primer_bind (B) LOCATION: 13..37 (D) 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:

(2) INFORMATION FOR SEQ ID NO: 9:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 35 base pairs ~B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA ~genomic) ~iii) HYPOTHETICAL: YES
(ix) FEATURE:
(A) NAME/KEY: misc_feature (B) LOCATION: 1..12 (D) OTHER INFORMATION: /note= "5' overhang containing the XhoI and EcoRI site"
~ix) FEATURE:
(A) NAME/KEY: primer_bind (B) LOCATION: 13..35 (D) 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:
45 CTcGA~AATT CTATAACTAA TGGTAATTAC TAATT 35 ~2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA (genomic) SUBSTITUTE SHEET (RULE 26) CA 022~7149 1998-12-02 W 097/46692 PCT~EP96tO2437 ~iii) HYPOTHETICAL: YES
~ix) FEATURE:
IA) NAME/KEY: misc_feature ~B) LOCATION: 1..14 tD) OTHER INFORMATION: /note= "part of primer restores sequence of 6044-0 tSEQIDNO: 4) from position 2128 to 2142 while causing a deletion of 10 fragment 1909 to 2127 (ix) FEATURE:
(A) NAME/KEY: primer_bind ~B) LOCATION: 15..35 (D) OTHER INFORMATION: /note= "this part of the primer anneals to the sequence of 6044-0 (SEQIDNO: 4) at position 1897-1917"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

~2) INFORMATION FOR SEQ ID NO: 11:
~i) SEQUENCE CHARACTERISTICS:
tA) LENGTH: 20 base pair~
tB) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA tgenomic) tiii) HYPOTHETICAL: YES
~ix) FEATURE:
(A) NAME/KEY: primer_bind (B) LOCATION: 1..20 (D) OTHER INFORMATION: /notes "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:

~2) INFORMATION FOR SEQ ID NO: 12:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 22 base pairs (B) TYPE: nucleic acid tC) STRANDEDNESS: single (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: DNA (genomic) SUBSTITUTE SHEET (Rl ILE 26) .. .. . . . . ...

CA 022~7l49 l998-l2-02 W097/46692 PCT~P96/02437 ~iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 ba~e pairs ~B) TYPE: nucleic acid (C) STRANDEDNESS: ~ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
GC~CGAGC CTAGGCACAG GTTATCAACA CGTTTG 36 (2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
CTTACTCGAG CCATGGTAAG ~ GC 27 (2) INFORMATION FOR SEQ ID NO: 16:

SUBSTITUTE SHEET ~ULE 26~

CA 022~7l49 l998-l2-02 (i) SEQUSNCE CHARACTERISTICS:
~A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44 base pairs (B) TYPE: nucleic acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

(2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear SU~STITUTE SHEET (RULE 26) .. . , .. ~, ..... ~ .. ...... . . .. . .. .

CA 022~7l49 l998-l2-02 (ii) MOLECULE TYPE: DNA (genomic) ~iii) HYPOTHETICAL: YES
~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

(2) INFORMATION FOR SEQ ID NO: 20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA ~genomic) ~iii) HYPOTHETICAL: YES
~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

SUBSTITUTE SHEET (RULE 26) T- .

Claims (23)

1. A DNA fragment obtainable 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.

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.

6. A chimeric DNA sequence according to claim 5, wherein the DNA 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-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.

10. 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 ore 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.

15. A plant essentially consisting of cells according to claim 13.

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.

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 15 to 18 and comprising plant cells according to claim 13.

20. A crop consisting essentially of plants according to any one of claims 15 to 18.

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 DNA sequence 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.