AU2513199A - Isolation and characterization of plant regulatory sequences - Google Patents

Description

WO 99/28483 PCT/EP98/07761 Isolation and characterization of plant regulatory sequences The present invention relates to new pathogen-, in particular nematode-, induced promoters. Said promoters induce an expression in very selective pathogen infection sites, in particular nematode infection sites of a plant. Background of the invention Nematodes, also called roundworms, have been enormously successful in colonizing nearly every habitat on earth. Although only about 100.000 species of nematodes have been named, there may be as many as 500.000. Nematodes are simple worms consisting of an elongate stomach and reproduction system inside a resistant outer cuticle (outer skin). Most nematodes are so small, between 200 micrometers to 5 mm long, that a microscope is needed to see them. Their small size, resistant cuticle, and ability to adapt to severe and changing environments have made nematodes one of the most abundant types of animals on earth. Most nematodes feed on bacteria, fungi and other soil organisms. Others are parasitic, obtaining their food from animals , humans (such as the pinworm) and plants. Agricultural cultivation encourages an increase in parasitic nematodes that feed on the crops being grown. Occasionally, new kinds of plant parasitic nematodes may be introduced into a field by contaminated plant parts, soil on farm equipment and irrigation water. Nematodes which parasitize plants may cause yield losses by themselves or they may join with other soilborne organisms such as viruses, fungi and bacteria to promote disease development in plants. Most often, nematode feeding reduces the flow of water and nutrients into the plant, increasing the plant's susceptibility to other stress factors such as heat, water and nutritional deficiencies. After hatching, plant-parasitic nematodes move through the soil to find areas on plant roots to feed. Some nematodes stay outside the root and use long stylets WO 99/28483 PCT/EP98/07761 to puncture cells inside the root. Nematodes which enter the root may move throughout the root (lesion nematodes) and feed at many sites (causing root lesions), or stay at one feeding site (cyst and root-knot nematodes). Nematodes which stay at one feeding site swell from eel-shaped to pear-shaped and stay at the same site until they die. In general, plant root-parasitic nematodes belong to two orders, Tylenchida and Dorylaimida. The tylenchids show a broad diversity in parasitic adaptations and comprise both migratory and sedentary ectoparasites and endoparasites. Sedentary endoparasitic nematode-plant interactions, involving species such as root knot and cyst nematodes, are intriguing to study because of the highly specialized feeding structures (giant cells and syncytia, respectively) that become established in the root during the prolonged and unique relationship between the nematode and host. A variety of other feeding structures besides giant cells and syncytia is induced by other nematodes with different parasitic strategies such as the reniform nematode Rotylenchulus reniformis and the false root-knot nematode Nacobbus aberrans (Wyss,1997, in Cellular and Molecular Aspects of Plant-Nematode Interactions, 5-22, eds. C.Fenoll et al., Kluwer Academic Publ.). Feeding structure maintenance and nematode survival are preserved only when the mutual interaction remains established. These interactions often cause extensive crop damage and, hence, severe economic losses in infested fields. The order Dorylaimida comprises migratory ectoparasites. Some of these parasites can feed at a particular site for long periods of time (e.g. Xiphinema spp.) rather than browsing along the roots. Similarly, these ectoparasitic species represent an economically important threat as vectors of soil-borne viruses and as cause of direct damage to the roots. Infective second-stage (J2) juveniles of cyst and root knot nematodes migrate intracellularly or intercellularly, respectively, toward the vascular cylinder where they select an initial feeding cell. Secretions are injected via the stylet, eliciting a series of cellular responses that result in the production of metabolically active, multinucleate feeding cells with elaborate cell wall ingrowths characteristic of transfer cells. Once feeding begins, the infective 2 WO 99/28483 PCT/EP98/07761 juveniles become sedentary, after which they will mature by ingesting food from the feeding cell. This process is an absolute requirement for the nematode to complete its life cycle. In contrast, migratory dorylaimid ectoparasitic nematodes feed at multiple sites, although mainly at root tips, which then cease to grow and develop into terminal galls. With their long needle-like stylets these nematodes pierce a column of subepidermal cells, injecting secretions to predigest the cytoplasm of the recipient cell. A path of collapsed necrotic cells is left behind. They are surrounded by multinucleate and expanding meristematic cells that are responsible for gall formation. For many years, the ultrastructure of such nematode-plant interactions has been comprehensively investigated (Endo, B.Y. ,1991, Ultrastructure of nematode-plant interactions; in: Electron Microscopy of Plant Pathogens, K. Mendgen and D.E. Lesemann, eds (Berlin: Springer-Verlag), pp. 291-305). Anatomical and cytological observations using advanced microscopical techniques have broadened the knowledge of the nematode feeding apparatus and the secreted glandular granules as well as of plant tissue responses to nematode attack (Wyss,1997, in Cellular and Molecular Aspects of Plant Nematode Interactions, 5-22, eds. C.Fenoll et al., Kluwer Academic Publ.). High-resolution video-enhanced contrast microscopy has also allowed the direct observation of feeding nematodes parasitizing living plant cells. Underlying mechanisms are now being explored by using molecular genetic techniques to further elucidate the redifferentiation processes involved and to develop genetic engineering strategies for effective nematode control in crops (Gheysen et al., 1996, Pestic.Sci,47, p.95-101). It is known that plant-parasitic nematodes worldwide cause diseases of nearly all crop plants of economic importance with estimated losses of about USD 6 billion annually in the USA alone and exceeding USD 100 billion annually worldwide. Without any doubt there is definitely an enormous need for plants with reduced or no susceptibility to plant nematodes. Several methods have been designed to obtain nematode resistant plants. For example expression of recombinant 3 WO 99/28483 PCT/EP98/07761 DNA encoding for a product which has a direct interaction with the pathogen, particularly peptides or proteins. Preferably said DNA is expressed at the site of nematode feeding area. In WO 93/10251 a method is disclosed for obtaining plants with reduced susceptibility to plant-parasitic nematodes by providing recombinant DNA that disrupts or at least delays the formation of a nematode feeding structure. In this application is disclosed a number of strategies including the so-called barnase/barstar combination to inhibit nematode feeding cell development using the published TobRB7 promoter from Opperman et al (Science,263,221 223,1994). In Goddijn et al.,1993,(Plant Journal, 4, p.863-873) the analysis of promoter gus fusions in plants in response to nematode infection is disclosed. Most of the promoters analysed are from characterized genes and are not specific for the infection site, others are from newly identified genes. However the promoter isolation, characterisation and sequence are not described in this paper. The disclosed line 553-2 is not interesting for a potential application either, since the promoter is not activated by nematodes in soil grown plants. Furthermore the so-called 553-25 line is not available from the seed stock center and can therefore not further be tested for useful applications. The line 553-35 shows expression in tissues outside of the nematode infection site but the study was not continued. WO 95/32288 describes different genes (cDNAs) that are highly upregulated in nematode infection sites. These cDNAs can be used for the isolation of the corresponding promoters, but the specificity of those promoters is not known, since for most of the genes no detailed expression analysis (in situ hybridisation) has been performed. Because no promoter has been cloned, no promoter-reporter fusions have been studied and the specificity of promoter-activity in nematode infection sites is therefore not known. To broaden the applicability of above-captioned strategy new nematode induced promoters are searched for with for instance improved selectivity, strength and/or specific expression pattern for different types of nematodes. 4 WO 99/28483 PCT/EP98/07761 To pursue the identification of nematode-responsive plant promoters, in the current invention a T-DNA system based on a randomly integrated promoter tag containing a promoterless dominant screenable marker was used. Therefor large collections of transgenic Arabidopsis lines, harboring a promoterless p-glucuronidase (gus; uidA gene from Escherichia coli) gene were generated and subsequently large-scale screening for activation of the reporter gene in the nematode feeding structures (NFSs) was performed. The strength of this promoter-tagging strategy, compared to other molecular approaches (Topping and Lindsey, 1995, Transgenic Research,4, p.291-305), rests on the immediate visual detection of tagged promoter activity and assessment of its spatial specificity. According to the current invention it appears that there are several expression patterns at specific tissues in a plant resulting from the induction of the gus reporter gene by a number of tagged nematode-responsive Arabidopsis regulatory sequences. Detailed description of the invention The present invention thus relates to nematode-responsive promoters which are isolated from plants and which either induce, stimulate or repress the expression of genes or DNA fragments, under their control, at least substantially selectively in specific cells (e.g. fixed feeding site, pericycle, endodermis, cortex or vascular cells) of the plants' roots, preferably in cells of the plant's fixed feeding sites, in response to the nematode infection. The nematode-inducible promoters according to the invention are especially useful in transgenic plants for controlling foreign DNAs that are to be expressed selectively in the specific root cells of plants, so as to render said plants resistant to nematodes. In accordance with the current invention an isolated DNA sequence is provided comprising the nucleotide sequence of SEQ ID NO's 1 and/or 2. These nucleotide sequences are so-called nematode-responsive regulatory sequences. 5 WO 99/28483 PCT/EP98/07761 In the SEQ ID NO's 1 and 2, as provided in the Sequence Listing , is indicated by arrows the location in the sequence where the plant sequence starts and ends respectively. The remaining flanking sequences are vector/primer sequences. Part of the invention is also a recombinant DNA comprising a plant-expressible promoter region having a sequence according to SEQ ID NO 1 and/or SEQ ID NO 2 or a fragment thereof. Furthermore said recombinant DNA may comprise a suitable foreign DNA under the expression of the promoter sequence according to the invention. In addition a plant cell comprising said recombinant DNA and a plant comprising the recombinant DNA integrated in its genome belong to the invention. Another aspect of the invention is a method for obtaining said plant with reduced susceptibility to a plant nematode comprising the steps of 1) transforming a recipient plant cell with a recombinant DNA according to the invention, 2) generating a whole plant from a transformed cell and 3) identifying a transformed plant with said reduced susceptibility. Furthermore to the invention belongs a transformation vector containing recombinant DNA according to the invention and an Agrobacterium strain containing this transformation vector as well. Nematode reproduction on a plant can be prevented by planting a plant obtained by the invention wherein the above-mentioned recombinant DNA is incorporated in the genome of said plant in an area susceptible to nematode infection. To the present invention also relates a method for suppressing plant pathogen activity comprising expression of a suitable foreign DNA in a plant under the control of a promoter region having the DNA sequence according to SEQ ID NO 1 and/or SEQ ID NO 2. 6 WO 99/28483 PCT/EP98/07761 In addition the promoter region comprising the DNA sequence according to the invention can be used to express a gene in specific root cells of a plant being infected with nematode(s). It is known to a skilled person that expression driven by pathogen-responsive regions can often be influenced by more than one pathogen. Therefore the current promoters can be used for other purposes as well. In an embodiment of the invention the nematode-inducible promoters are used to express a foreign gene predominantly, preferably selectively in fixed feeding cells, or specialized root cells of the plant. In a preferred embodiment the expressed foreign DNA encodes a polypeptide/protein which can kill or disable nematodes. To mention some examples, without being limited thereto, for this purpose are toxins, collagenases, chitinases, lectins, antibacterial peptides or enzyme inhibitors like proteinase-inhibitors. In case the plant is a food plant it is obvious that the polypeptide/protein be non-toxic to animals and certainly non toxic to humans. Another embodiment is that the promoters according to the invention are used to express a foreign DNA sequence encoding an RNA, polypeptide or protein that when expressed, in for example a fixed feeding plant cell, will disable said plant cell by interfering in metabolic activities necessary for survival of the infecting nematodes. Other examples of foreign DNA sequences which can be expressed under the control of the promoters according to the invention in order to inhibit the development of fixed feeding cells are DNA sequences encoding antibodies immunoreactive with molecules (such as proteins, carbohydrates or compounds secreted through the nematode's infecting process) in the plant cells. Said antibody can have a known variety of forms including Fv, Fab, or single chain antibodies and the like. The foreign DNA adjacent to a nematode-inducible promoter can also encode an enzyme transforming an otherwise harmless substance into a cytotoxic product. In this embodiment it is preferred to have another foreign DNA 7 WO 99/28483 PCT/EP98/07761 expressed which product thereof inhibits or reverses the cytotoxic effect of the first mentioned foreign DNA gene product in order to prevent any detrimental effects on yield or performance of the plant in the field under several conditions. The isolated promoters according to the invention have specific advantages over the currently known nematode-inducible promoters in their enhanced specificity and/or time of induction and /or the fact that they are activated by different types of nematodes. Indeed, the Att0728 and Att1712 show very little expression in other tissues than the nematode infection sites itself. The tobacco TobRB7 promoter (Opperman et al., Science, vol.263,p.221, 1994) is also quite specific but is only activated by root knot nematodes and not by cyst nematodes. In addition it is expected that a further refinement of the sequences according to the invention will lead to the identification of more specific sequences (so called fragments) needed for the nematode-induced promoter activity. In particular are interesting those sequences exhibiting structural features such as repeats and the like. Particularly interesting for this purpose is the isolated DNA sequence from position 371 to 1045 (both nucleotides included) of SEQ.ID.NO.2. So a preferred nematode-induced promoter comprises the nucleotide sequence of SEQ.ID.NO.2 from nucleotide position 371 to 1045 (both positions included). To the scope of the invention also belongs those variant promoter sequences obtained by modification including for instance exchange of a nucleotide for another nucleotide, inversions, deletions or insertions of a limited number of nucleotides remaining however the same specificity as the sequences described in SEQ ID NO's 1 and/or 2. Promoters with essentially similar sequence as the nematode-inducible promoters or fragments thereof according to the current invention, which have comparable or identical characteristics, can be isolated from other plant species using the inventive promoter sequence(s) as, for instance, hybridization probe under conditions known to a skilled person. Alternatively other promoter 8 WO 99/28483 PCT/EP98/07761 sequences can be identified, isolated and characterised via the cDNA coding sequence operably linked to the promoter sequence or fragment thereof according to the current invention. In another approach the nucleotide sequences or fragments thereof according to the invention can be used by skilled persons as so-called amplification primers in order to isolate promoter fragments with essentially similar sequences from other plant species by so called amplification techniques like the PCR method. The recombinant DNA of the current invention using the promoters or the fragments derived therefrom will be useful in other plant species to obtain resistance to nematode attack and/or infection. Preferred host plants for said nematode-inducible recombinant DNA or fragments thereof are potato plants or oilseed rape plants, but also suitable plants are soybean, cereals such as corn, rice or barley and wheat, tomato, carrots or tobacco. In order to clarify what is meant in this description by some terms a further explanation is hereunder given. The terms "polynucleotide", "nucleic acid sequence" or "nucleotide sequence" as used herein refers to a polymeric form of nucleotides of any length. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA. It also includes known types of modifications, for example, methylation, "caps" substitution of one or more of the naturally occuring nucleotides with analogs. With regard to nucleic acid sequence/nucleotide sequence or polynucleotide sequence as mentioned above, "essentially similar" means that if two sequences are aligned, the percent sequence identity is higher than 80%, preferably higher than 85% and in particular higher than 95% especially regarding the promoter/regulatory regions. As "sequence identity" has to be understood the number of positions with identical nucleotides divided by the number of nucleotides in the shorter of the two sequences aligned. The 9 WO 99/28483 PCT/EP98/07761 alignment of the sequences is performed by the so-called Wilbur and Lipmann algorithm known to a skilled person using for instance a programs of Intelligenetics Inc. (USA) "Promoter fragment" means a fragment of a promoter, in particular a nematode induced promoter, that determines timing, selectivity or strength of the expression induced by said promoter. Said fragment can comprise an autonomously functioning promoter or functions as a promoter, in particular a nematode-induced promoter when combined with other homologous or heterologous promoter fragments ( like for example a TATA box region). The promoters according to the invention comprise at least one promoter fragment as described above. The term "fragment "of a sequence or "part" of a sequence means a truncated sequence of the original sequence referred to. The truncated sequence (nucleic acid or protein sequence) can vary widely in length; the minimum size being a sequence of sufficient size to provide a sequence with at least a comparable function and/or activity of the original sequence referred to, while the maximum size is not critical. In some applications, the maximum size usually is not substantially greater than that required to provide the desired activity and/or function(s) of the original sequence. "Transformation" as used herein, refers to the insertion of an exogenous polynucleotide into a protoplast or a host cell, irrespective of the method used for the insertion, for example, direct uptake, transduction, Agrobacterium infection or electroporation. The exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively may be integrated into the host genome. After selection and/or screening, the protoplasts, cells or plant parts that have been transformed can be regenerated into whole plants, using methods known in the art. The choice of the transformation and/or regeneration techniques is not critical for the present invention. 10 WO 99/28483 PCT/EP98/07761 With "recombinant DNA" is meant a hybrid DNA produced by joining pieces of DNA from different sources. With "promoter" is meant a recognition site on a DNA strand to which RNA polymerase binds, thereby initiating transcription. In the current invention is meant by "foreign DNA, foreign sequence or foreign gene" , a DNA sequence which is not in the same genomic environment (e.g. not operably linked to the same promoter and/or 3' end) in a plant cell, transformed with said DNA according to said invention, as is such DNA when it naturally occurs in a plant cell or the organism (bacterium, fungi, virus or the like) from which the DNA originates. "Fixed feeding sites" are specialized feeding sites such as giant cells, syncytia, nurse cells or galls, which are induced by (semi)-sedentary nematodes in susceptible plants. At such sites the plant cells serve as food transfer cells for the various developmental stages of the nematodes. "Giant cells and syncytia" refer to multinucleate plant root cells induced by specific nematodes known to a skilled person while "nurse cells" refer to a group of six to ten uninucleated plant root cells induced by Tylenchulus spp. "Galls" refer to a proliferation of cortical or pericycle plant cells / tissue induced by nematodes; typically giant cells reside within galls. In the Examples and in the description , reference is made to the following sequences of the Sequence Listing: SEQ ID NO 1: nucleotide sequence of Att1712 left border T-DNA/plant flanking sequence (pZ1712LB1.7) The nucleotide sequence region between arrows as indicated is plant sequence. 11 WO 99/28483 PCT/EP98/07761 SEQ ID NO 2: nucleotide sequence of Att0728 left border T-DNA/plant flanking sequence (pZ728LB1.2) The nucleotide sequence region between arrows as indicated is plant sequence. Examples and Methods Example 1. Taqqing novel promoter activities in NFSs Using the above-mentioned promoter-tagging technique and described in detail in the Methods section hereafter, Arabidopsis promoter tag lines using binary T-DNA vectors pGV1 047 and pAgusBin19 were generated. Figure 1 depicts the T-DNA constructs. Transformation efficiencies, T-DNA integration events, and reporter gene expression patterns were compared for the two binary vectors. A total of 284 independent Att lines (for Arabidopsis thaliana tag) were screened with the beet cyst nematode and root-knot nematode to identify promoter activities in NFSs. Within this group, 37 Att lines were found displaying various levels of GUS activity within the developing syncytia. Except for the GUS-positive NFSs, no differences in expression patterns were observed when screening was performed in the presence or absence of nematodes. Four lines were examined in more detail. Their selection was based on the significant levels and/or specificity of GUS activity in the NFSs. The gus expression characteristics of these four original tags (AttOO01, Att0728, Att1012 and Att1712) and one reintroduced promoter-gus fusion (AttOO01-R/1) are summarized in Table 1 and Figures 2 and 3. The lines studied contain the pAgusBin19 T-DNA (AttOO01 also called "ARM-1", Att0728, Att1712 and Att1012); no line transformed with pGV1047 was promising enough for further characterization. To avoid potential artifacts in inducible GUS activity, as 12 WO 99/28483 PCT/EP98/07761 described by Goddijn et al. (1993, Plant Journal,4, p. 863-873), inoculations were repeated both under in vitro and soil conditions. Att0728 (pAgusBin19 T-DNA) responded very early after infection with cyst nematodes. GUS histochemical staining could be observed within 6 hr after inoculation. Mechanical wounding experiments, however, did not result in the induction of reporter gene expression, indicating that the rapid activation of expression was independent of a wound response. Under in vitro conditions, GUS activity was found to be primarily located in the developing NFSs (Figure 2H), but some activity was also detectable at sites of lateral root initiation. Soil-grown plants showed stronger activation in initiating lateral roots, together with some staining of the root vascular tissue. DNA gel blot hybridization analysis demonstrated the presence of a single T-DNA insertion. AttOO01 and Att1712 had similar gus expression patterns after nematode inoculation. Strong GUS staining was observed in the syncytia 4 days post-inoculation (dpi) (Figures 2A, 2B, 2D, and 2E). Sites of lateral root initiation were also clearly stained in both lines (Figures 2C and 2F). Nevertheless, these tags were located at different chromosomal loci, as confirmed by DNA gel blotting. Some AttOO01 plants showed additional GUS activity at sites as indicated in Table 1 (in both soil- and in vitro-grown plants). This pattern of staining was never observed in Att1712 under in vitro conditions; however, soil-grown Att1712 plants occasionally displayed GUS activity in the leaf vascular tissue. DNA gel blot analysis of S, segregants (originating from seeds of the primary transformant) from AttOO01 revealed the presence of three T-DNAs. Two of these T-DNAs were arranged in an inverted repeat over the RB. After segregation, the locus containing the inverted T-DNA arrangement appeared to be responsible for NFS-directed expression and was analyzed further. Att1712 contained two T-DNAs in the S, generation, but analysis of S2 progeny showed only one insert for progenitor Att171 2 A, indicating independent segregation of the two T-DNAs in the original line. This segregant showed a GUS activity pattern identical to that of the original Attl712. 13 WO 99/28483 PCT/EP98/07761 On genomic Att0001 plant DNA, iPCR was carried out to isolate the plant DNA regions flanking both T-DNA LB. Both clones, designated ARM1a and ARM1b, were reintroduced into Arabidopsis (Methods). ARM1a was demonstrated to be the tagged sequence responsible for the observed nematode-induced gus expression. Sequence analysis indicated that the T DNAs were inserted into a short putative ORF of 159 bp (EMBL accession number Y12834). However, no homology in the database was found. A GUS histochemical assay at 4 dpi on root knot nematode-infected Att0001-R/1 S, plants (Table 1) confirmed the regulatory character and revealed a gus expression pattern in the NFSs with a timing similar to that of the original AttOO01 line. Example 1a Nematode-responsive promoter activity in a 675 bp fragment from Att0728 iPCR was performed on Att0728 genomic DNA in order to isolate the T-DNA LB (see Methods). A 1269 bp fragment was cloned and from this two different promoter-gus fusions were constructed containing either a 1012 bp (pTHW728PN) or a 675 bp fragment (pTHW728S) (see Methods). The resulting binary vectors were mobilised to Agrobacterium tumefaciens for Arabidopsis transformation. The transformation procedure resulted in the harvesting of 16 pTHW728S and 15 pTHW728PN independent transformed lines of which respectively 11 and 10 lines were inoculated with H. schachti. A GUS assay was performed 3 days post inoculation. Gus expression levels in syncytia in pTHW728PN lines were slightly stronger when compared with the pTHW728S lines. Also more gus expression beyond the feeding structures was observed in the pTHW728PN plants. Compared with the original Att0728 line, GUS levels in syncytia of reintroduced lines were in general similar (see Figure C). So far, one pTHW728PN reintroduction line was inoculated with M.incognita and showed clear staining of the galls (see Figures D and E). 14 WO 99/28483 PCT/EP98/07761 Example 2 gus expression patterns are temporally regulated Feeding cell development by nematodes is a dynamic process and involves gene sets regulated by developmental stage-specific factors produced by nematode and host. Because such a temporal gene regulation might be reflected in the four selected lines, gus expression at different time points during syncytium development was monitored. Ten-day-old plants were inoculated, and the number of GUS-positive syncytia was determined at various intervals. Maximum GUS levels in syncytia were found at different times after inoculation of each of the lines. Lines Att1712 and AttOO01 showed maximum activities at 4 dpi, whereas Att0728 and Att1012 showed maximum GUS activities over a longer period during nematode infection rather than exhibiting transient patterns of activity. AttOO01-R/1 retained the timing of maximum response from the original line. However they displayed blue-stained syncytia with a lower frequency than did AttOO01. It should be emphasized that these data correlate with the number of stained syncytia irrespective of the GUS level. Therefore, the time point corresponding to the maximum GUS activity in a given syncytium does not necessarily coincide with the time point corresponding to the maximum number of GUS-positive syncytia during the infection period. The fact that not all syncytia are stained at a given time point might be the consequence of the transient expression and because infections are not completely synchronous. Example 3 Infections with qall-forminq nematodes All lines with induced GUS activity in galls after root knot and ectoparasitic nematode infections are depicted in Figure 3. GUS staining levels in galls induced by root knot nematodes were similar to those observed in syncytia for line Att0728 (Figures 3M). Different levels of GUS staining in galls and syncytia 15 WO 99/28483 PCT/EP98/07761 appeared in lines Att1712 and Att1012, suggesting lower promoter activity in galls (Attl012) or syncytia (Attl712) Although AttOO01 and Att1712 have a similar expression pattern (Figures 3A,3B,3C and 3D), as previously mentioned for cyst nematode-infected lines, the different nature of the tag in these two lines became clearly apparent by infection with the root knot nematode: an average of 70% of the established Attl712 galls showed an unstained zone at 4 dpi (Figure 3G). Subsequent cross-sectioning of these galls revealed strongly GUS-stained parenchymatous cells surrounding unstained giant cells (Figure 3F). In contrast, AttOO01 galls displayed very strong and uniform staining (Figures 3D and 3E) with GUS inside the giant cells. Despite their ectoparasitic nature, Xiphinema nematodes feed for longer periods and can be regarded as being semi-sedentary. Their ability to transform root tips into galls made this nematode species interesting to add. AttOO01, Att0728, and Att1712 exhibited GUS-positive galls when infected with Xiphinema (Figure 31). A cross-section through a Xiphinema-induced Att1712 gall indicated that reporter gene expression in this line occurred in the multinucleate cells induced at the nematode penetration site (Figure 3H). The same pattern was observed in AttOO01 galls. Example 4 Screening for GUS activity in callus tissue, flowers, and pods The reproduction capacity of transgenic plants, engineered with a nematode-inducible promoter/cytotoxin construct should be ensured when one seeks to develop nematode resistance in plants. This implies the absence of tagged promoter activity in the reproductive organs. From the four selected lines (infected and noninfected), flowers were carefully dissected to screen for GUS activity. Pollen was GUS negative for all four lines. No GUS staining was observed in either flowers or seeds. As demonstrated in Figure 4, monitoring GUS activity after 6 days of incubation on callus-inducing medium (see Methods) three of the four 16 WO 99/28483 PCT/EP98/07761 uninfected lines revealed a variety of responses. AttOO01 and Att1712 both expressed GUS in the root vascular tissue regions abutting the protruding calluses (Figure 4A). In the case of Att1712, this GUS staining occasionally extended into the vascular cylinder of developing lateral roots. Att1712 also showed clear reporter gene activation in cells at the cut surfaces of the explants. Intriguingly different from Att1712 was the observation that AttOO01 displayed GUS staining in the tips of main and lateral roots, a so-called "three-zone pattern" (Figure 4B), which was also seen in auxin-treated Arabidopsis plants containing a cell cycle regulator (cdc2) promoter-gus construct (Hemerly et al., 1993, Plant Cell,5 ,p.1711-1723). Explants of Att0728 showed no significant effect when an external hormone was applied. Extension of the incubation period for 5 more days did not alter the observed patterns. A peculiar response was seen in the cauliflower mosaic virus 35S-gus control root explants. Except for root tips and calluses, GUS activity in the remaining root parts was seen to decline after 6 days of incubation on callus-inducing medium (Figure 4E). Newly formed lateral roots did not show any GUS staining except in the root tips (Figure 4D). Extension of the incubation time up to 12 days led to a 35S promoter activity confined solely to root tips and callus tissue (Figure 4F). The influence of Agrobacterium infection was determined (see Methods) by mimicking the initial steps of the root explant transformation process. In general, Agrobacterium infection did not significantly alter the patterns seen after callus induction alone, except for the cut surfaces that showed weak GUS staining for all tags. By adding an Agrobacterium cocultivation step, GUS activity in AttOO01 explants was no longer confined to vascular regions juxtaposed to the developing calluses, as described earlier, but extended throughout the entire vascular cylinder. Att0728 displayed weak GUS activity in a few calluses, which was not observed after hormone treatment alone. 17 WO 99/28483 PCT/EP98/07761 METHODS Binary T-DNA vectors and Agrobacterium tumefaciens strains Construction of pGV1047 has been reported by Kertbundit et al. (1991). pAgusBin19 comprises the uidA-coding region at the left T-DNA border and has been described by Topping et al. (1991). Mobilization of pGV1047 from Escherichia coli into Agrobacterium has been described by Kertbundit et al. (1991). pAgusBin19 was transferred from the E. coli strain MC1022 into the Agrobacterium strain C58C1RifR (Holsters et al., 1980) harboring either the octopine vir plasmid pGV2260 (Deblaere et al., 1985) or the nopaline vir plasmid pMP90 (Koncz and Schell, 1986). Plant transformation The recovery of Att0001, Att0728, Att1012 and Att1712 was accomplished according to the transformation procedure described by Clarke et al. (1992) with some modifications (Barthels et al., 1994; Karimi et al., 1994). Callus induction Roots from 2-week-old plants were incubated on CIM (Valvekens et al., 1991). Wild-type C24 and transgenic 35S-gus Arabidopsis thaliana (L.) Heynh plants were used for control purposes. GUS histochemical assays were performed after 6 and 11 days of incubation. Agrobacterium infections Roots from 14-day-old promoter-tagged plants were incubated for 3 days on CIM (Valvekens et al., 1991). Subsequently, whole-root systems were cut into small explants and mixed with an C58C1 Rif(pGV2260) Agrobacterium solution

(OD

60 0 of 0.1), after which the cocultivated material was further incubated on CIM for an additional 3 days. Root explants were washed several times to remove all overgrowing agrobacteria and were stained for GUS activity. Wild-type C24 and transgenic 35S-gus Arabidopsis plants were used for control purposes. 18 WO 99/28483 PCT/EP98/07761 Nematode cultures and hatching procedures Root knot nematode (Meloidogyne incognita) cultures were maintained in vitro on tomato (Lycopersicon esculentum) hairy roots continuously subcultured on hormone-free Gamborg's B5 medium (Flow Laboratories, Bioggio, Switzerland; pH 6.2) supplemented with 2% sucrose and 1.5% Bacto agar (Difco, Detroit, MI). Cyst nematodes (Heterodera schachtii) were grown in vitro on mustard (Sinapis alba) roots in Knop medium (Sijmons et al., 1991). Hatching was stimulated by putting cysts (H. schachti) or galls (M. incognita) on 70-pm nylon sieves (Falcon 2350 Cell Strainer; Becton Dickinson, Bedford, MA) submerged in filter-sterile root exudate extracted from rapeseed (Brassica napus) and sterile deionized water, respectively. The migratory ectoparasitic nematodes (Xiphinema diversicaudatum) were cultured on raspberry, Rubus ideas CV Gpen Moy in soil. Its size of 6 mm allowed us to isolate nematodes quite easily from the sand by using a sieve. Nematode inoculation of tagged Arabidopsis lines For the X diversicaudatum inoculations, seven to 10 Arabidopsis seeds were sown in a 1:1 sand/compost mixture in 30-ml plastic pots. These pots were then arranged in small propagation trays with a clear plastic cover. The trays were placed in a greenhouse at 18 0 C with 16 hr of light. The Arabidopsis plants were grown for 14 days prior to infection. Pots were inoculated twice - one week apart - with five to 10 nematodes. After one more week, the roots were washed and stained for GUS activity. Plants were scored immediately using reflected light to clearly detect the GUS activity signal in the optically dense galls. For cyst and root knot nematode soil inoculations, 2-week-old Arabidopsis seedlings were transferred to a 1:2 mixture of cutting soil (M. Snebbout s.a., Kaprijke, Belgium) and potting soil (M. Snebbout s.a.) in open translucent plastic tubes. By placing these tubes slanting in rectangular flower boxes, the roots were forced to grow along one side of the tube, allowing more controlled inoculations and reproducible infections. Inoculations were performed after 2 more weeks of growth at 22'C and 16 hr of light by injecting a suspension containing 250 second-stage juveniles (5 to 7 days after hatching) of beet cyst 19 WO 99/28483 PCT/EP98/07761 or root knot nematodes in 1.5 ml H 2 0 per root system. One to two weeks later, three to five plants were washed carefully and stained for GUS. In vitro inoculations with cyst and root knot nematodes

S

2 seeds can be sown directly on selective Knop medium (Sijmons et al., 1991). On the other hand, S, plants frequently showed abnormal growth when cultured for 2 weeks or longer on Knop medium, impeding sound analysis of inoculation and staining results after this time. Surface-sterilized seeds (2 min in 70% EtOH and 15 min in 5% sodium hypochlorite) were germinated on germination medium (Valvekens et al., 1988) supplemented with either 50 mg L 1 kanamycin monosulfate (Sigma) or 20 mg L 1 hygromycin B (Calbiochem, La Jolla, CA). Two-week-old seedlings were subsequently transferred to and lined up on a thin layer of Knop medium. Petri dishes were placed slightly tilted to promote unidirectional root growth. After 2 more days of growth at 220C (16-hr-light/8-hr-dark cycle), roots were inoculated with 5- to 7-day-old hatched beet cyst or root knot nematode second-stage juveniles at an average density of 20 juveniles per root system. The plants were then incubated again under the same tissue culture conditions. Five to ten plants were examined for the presence of GUS activity 4 to 6 days post-inoculation (dpi). GUS histochemical assay Histochemical localization of GUS activity was performed using the substrate 5-bromo-4-chloro-3-indolyl glucuronide (X-gluc: Europa research products, Ely, U.K.) according to Jefferson (1987) with minor modifications: 50 pL of X-gluc (20 mg in 1 mL of NN-dimethylformamide) was diluted to a final concentration of 2 mM in 1 mL of 0.1 M NaPO4, pH 7.2. Oxidative dimerization of the produced indoxyl derivative was enhanced by adding the oxidation catalyst K* ferricyanide/ferrocyanide to a final concentration of 0.5 mM. Incubation of whole plantlets in phosphate buffer was preceded by a short treatment (15 to 30 min) with 90% ice-cold acetone followed by several washes with 0.1 M sodium phosphate, pH 7.2. The GUS reaction was incubated overnight at 370C. 20 WO 99/28483 PCT/EP98/07761 Stained tissues were subsequently fixed for a few hours to overnight in 2.5% glutaraldehyde (Agar Scientific Ltd., Stansted, U.K.) at 40C to prevent diffusion of the GUS product during the subsequent incubation in chlorallactophenol (2:1:1 mixture of choral hydrate, lactic acid, and phenol). Incubation of the material in chlorallactophenol removes all pigments and brown phenolics producing transparent tissues which were further monitored for GUS activity using a dissecting light microscope (Jenalumar; Zeiss, Oberkochen, Germany). Sectioning and microscopic analysis Blue stained syncytia and galls were fixed in 4% paraformaldehyde/1% glutaraldehyde; samples were vacuum infiltrated for 20 min and further incubated overnight at 40C in fresh fixing solution. After several dehydration steps, material was embedded in LR white medium grade resin (The London Resin Co., Basingstoke, U.K.) or butyl-methylacrylate resin (Merck-Schuchardt, Hohenbrunn bei Minchen, Germany and BDH Laboratory Supplies, Poole, U.K.). Sections (2- to 2.5-pm-thick) were examined using dark-field optics (Diaplan; Leitz, Wetzlar, Germany). Sectioned material was sometimes stained for examination using bright-field optics (Diaplan, Leitz): after removing the butyl-methylacrylate resin with acetone (15 min incubation), sections were immersed in a 0.1% ruthenium red (Sigma) solution during 7 to 20 min. Nematode staining Nematodes inside root tissues can be visualized according to the McBryde method (described in Daykin and Hussey, 1985). Following GUS histochemistry, fixation in 2.5% glutaraldehyde and clearing in chlorallactophenol, root material was left in acid fuchsin dye for 16 hr and subsequently destained for 3 hr in a saturated chloral hydrate solution. Inoculation time course experiments All time course experiments were performed in vitro (Knop medium containing 1% sucrose) with cyst nematodes. For each line, several 9-cm Petri dishes were prepared with 20 seeds lined up in two rows. The seeds had been 21 WO 99/28483 PCT/EP98/07761 vernalized at 40C for 3 days to break dormancy. Ten days after germination, each plant was inoculated with 30 second-stage juveniles. Plants were monitored for GUS activity at 2, 4, 7, 12, and 30 dpi. The GUS assays with the infected plants were performed immediately. In a Petri dish, 4 mL of X-gluc solution, including Fe-cyanide, was poured on top of the agar, and the plates were incubated at 37*C for 24 hr. Subsequently, plants were examined for GUS activity in the nematode feeding structures (NFSs). DNA extraction 0.2 to 2 g of plant material was used for the preparation of DNA as described by Dellaporta et al. (1983), with some modifications. The DNA pellets were dissolved in 400 d of Tris-EDTA to which 20 tg of RNase was added. After an incubation period of 20 min (370C), 400 d of 0.2 M Tris-HCI, pH 7.5, 2 M NaCl, 0.05 M EDTA, 2% (w/v) cetyltrimethylammonium bromide was added; the mixtures were incubated for an additional 15 min at 650C. The samples were extracted with 800 ptl of chloroform-isoamylalcohol (24:1) and precipitated. T-DNA number determination by DNA gel blot analysis AttOO01, Att0728, Att1012 and Att1712 plant DNA was digested with HindIll and/or EcoRI in a double or single digest. Separation of the digested samples on a 1% agarose gel was followed by an overnight blotting to a Hybond-N membrane (Amersham, Aylesbury, UK). The DNA on the membrane was fixed through UV cross-linking (GS Gene Linker; Bio-Rad, Hercules, CA). The 1.7-kb Nrul gus-coding region of pGUS1 (Peleman et al., 1989) was used as a probe. Radioactive labeling was performed using the Ready-To-Go DNA labeling kit (-dCTP) (Pharmacia, Uppsala, Sweden) according to the manufacturer's instructions. The nylon membrane was incubated in a hybridization buffer (450 mM NaCl and 45 mM Na3-citrate, pH 7.0), 0.1% SDS, 0.25% milk powder (Gloria, Vevey, Switzerland), and 20 ptg mL herring sperm DNA (Promega, Madison, WI)) for 3 hr at 650C. Hybridization was performed overnight in fresh hybridization buffer to which the corresponding a 32 P-dCTP-labeled probe was added. 22 WO 99/28483 PCT/EP98/07761 Inverse polymerase chain reaction In a first step, suitable sized fragments are identified by analysis of restriction enzyme digests of the DNA from a particular interesting tag line through Southern hybridisation with a labelled nucleotide sequence that is homologous to the integrated T-DNA sequence. Target DNAs with the corresponding primer annealing sites are shown in Figure 5. AttOO01 DNA was digested with Sspl and EcoRI and circularized in conditions favoring self-ligation (Sambrook et al., 1989). Inverse polymerase chain reaction (IPCR) was conducted using primer sets 1 and 2 and 1 and 3, respectively (Figure 5A). The PCR mix consisted of 50 ng self-ligated DNA, 200 ng of each primer, 1 mM MgCl 2 , 0.2 mM deoxynucleotide triphosphates, 2.5 pl 10x Taq buffer, 0.5 pl of Taq polymerase (5 units pl -1) (Beckman, Fullerton, CA), in a total volume of 25 pl, and 25 pl mineral oil. A total of 35 cycles were used. For both primer sets 1 and 2 and 1 and 3, the same temperature program was followed with an exception for the annealing temperature being 64 0 C and 600C, respectively. The cycle order was: cycle 1, 4 min at 950C, 2 min at 640C/600C, 10 min at 720C; cycles 2 to 35, 1 min at 95 0 C, 2 min at 640C/600C, 3 min at 720C; and finally, for 10 min at 720C. Following primers were used: primer 1: 5' CCAGCGTGGACCGCTTGCTGGAAC 3' primer 2: 5' GTATTGCCAACGAACCGGATACCCG 3' primer 3: 5' CCCAGTCACGACGTTGTAAAAC 3'. Att0728 and Att1712 left border (LB) flanking plant sequences (promoter regions): Att0728 total DNA was digested with Nsil (Figure 5B). The restricted DNA was ligated under circumstances that allow preferentially circularisation and this self-ligated DNA was subsequently used as a template in a standard PCR reaction. The recircularisation reaction was performed as described by Topping et al. (1995). Following phenolisation and precipitation, the recircularised DNA was dissolved in 20 ptl H 2 0 from which 2 l1 was mixed with 23 WO 99/28483 PCT/EP98/07761 5 pl PCR buffer (1OX; Perkin Elmer Cetus, Branchburg, NJ), 5 ld 2mM dNTP's (100mM; Pharmacia, Uppsala, Sweden), 1 pl primer 5 (150 ng/pl), 1 ptl primer 6 (100 ng/pl), 2 pl 25mM MgCl 2 , 0.5 pl Ampli Taq@ DNA Polymerase (5U/ I; Perkin Elmer Cetus) in a final volume of 50 pl. Inverse PCR was carried out with following cycle order: cycle 1, 4min 950C, 2min 50*C, 10min 720C; cycles 2 to 31, 1min 950C, 1min 30s 500C, 2min 30s 720C; cycle 32, 1min 950C, 1min 30s 50*C, 10min 72C. Primer 5: 5' CCC CGA TCG TTC AAA CAT TT 3'; primer 6: 5' CGG GCT ATT CTT TTG ATT TAT 3' A fragment of 1269bp, containing 892bp plant sequence, was recovered from a 1% agarose gel in 10 pl H 2 0 using GENECLEAN 11 KIT (BO1101, Vista CA). Att1712 total DNA was digested with Acci (Figure 5B). The recircularisation reaction was performed as described by Topping et al. (1995). Following fenolisation and precipitation, the recircularised DNA was dissolved in 20 pl

H

2 0 from which 2 pl was added in a PCR mix as described for the Att0728 LB. Inverse PCR was conducted as described for the Att0728 LB. A fragment of 1642bp, containing 1126bp plant sequence, was recovered from a 1% agarose gel in 10 pl H 2 0 using the GENECLEAN II KIT. Cloning of the iPCR fragments from Att0728 and Att1712 in pZErO

TM

-2 The 3' protruding ends of the iPCR products were converted to blunt ends using T4 DNA polymerase in a reaction mix containing following components: 10 p GENECLEAN pure iPCR fragment, 3 p1 ONE PHOR ALL buffer (10X; Pharmacia), 1.5 p 2mM dNTP's, 0.5 pl T4 DNA polymerase (7.3U/pl; Pharmacia), H 2 0 up to a final volume of 30 pl. Following phenolisation and precipitation, iPCR fragments were dissolved in 15 pl H 2 0. Blunt ended iPCR fragments were cloned in pZErOTM-2 (Invitrogen, Leek, Nederland) which was digested with the enzyme EcoRV: 1 pg pZErOTM-2 was digested with 1 pl EcoRV (15U/pl; Pharmacia) in 1x ONE PHOR ALL (Pharmacia); following fenolisation and precipitation, linearised vector fragments were dissolved in 15 p H 2 0. Approximately 50 ng linearised pZErO 24 WO 99/28483 PCT/EP98/07761 was ligated with either of both isolated flanking plant regions corresponding to the Att0728-1269bp-iPCR-LB or the Att1712-1642bp-iPCR-LB fragments in a 1:1 and/or 1:3 molar ratio resulting in the respective constructs pZ728LB1.2 and pZ1712LBI.7.(Figure A and B respectively) Vector constructions for reintroduction of the Att0728 and Att1712 promoter containing sequences. Vectors used for reintroduction in Arabidopsis of promoter-containing T-DNA-flanking regions are presented in Figure 6. The binary T-DNA vector pTHW136 (Figure 8A) contains the P35S-gus-intron-3'35S cassette from P35SGUSINT (Vancanneyt et al., 1990). pTHW136 was kindly provided by Plant Genetic Systems nv (Gent, Belgium). Isolated sequences from the iPCR fragments were subsequently cloned in front of a promoterless reporter gene to confirm the nematode-inducible promoter activity. PZ728LB1.2 was digested with SspI and Pvull/Nsil to generate Att0728 left border plant sequence fragments of respectively 675bp and 1016bp (Figure A), the latter fragment consisting of 896bp plant derived sequence and 119bp T-DNA sequence. PZ1712LB1.7 was digested with BamHI/Nsil, generating an Attl712 left border flanking T-DNA/plant sequence fragment of 1443bp containing 1126bp plant derived sequence, 248 bp T DNA sequence and 69bp pZErO

TM

-2 derived sequence as indicated in Figure B. The recessed 3' ends generated by the restriction enzyme BamHI and the 3' protruding ends generated by the restriction enzyme Nsil were converted to blunt ends using respectively 1U Klenow Fragment of DNA Polymerase I (Pharmacia) in the presence of 0.8mM dNTP's in 1X ONE PHOR ALL buffer and 3.65U T4 DNA Polymerase (Pharmacia) in the presence of 0.1mM dNTP's in 1x ONE PHOR ALL . No treatment of the ends generated by Sspl and Pvull was required as these are blunt end generating restriction enzymes. In the next step, the blunt ended left border flanking plant sequences obtained from pZ728LB1.2 and pZ1712LB1.7 as described above, were cloned upstream of the gus-intron reporter gene in a blunt end linearised 25 WO 99/28483 PCT/EP98/07761 binary vector pTHW1 36. This was accomplished through a substitution of the 5'35S region controlling the gus-intron in pTHW136: cleavage of pTHW136 with the restriction enzyme Xbal was followed by a treatment with 1 U Klenow Fragment of DNA Polymerase I to blunt end the linearised binary vector fragments that were subsequently subjected to a Calf Intestinal Phosphatase (Promega) treatment to prevent self ligation later on. The 5'35S sequence in the binary vector pTHW136 was substituted with the 675bp Sspl and the 1016bp Pvull/Nsil fragments from pZ728LB1.2 and the 1443bp BamHI/Nsil fragment from pZ1712LB1.7 to make the respective constructs pTHW728S, pTHW728PN (Figure A) and pTHW1712BN (Figure B). Reintroduction of regulatory regions From AttOO01, both LB-flanking regions were isolated by using IPCR. Amplified fragments of ))0.6 kb and )2.8 kb, designated ARMIa and ARM1b, respectively, were cloned in front of gus in pTHW136. After introduction into Arabidopsis, pthARM1-a600, corresponding to the cloned )0.6-kb ARM1a fragment, revealed a nematode response similar to that of the original tag. The binary vectors pTHW728S, pTHW728PN and pTHW1712BN were mobilised to the Agrobacterium tumefaciens strain C58C1Rif"(pMP90) (Holsters et al., 1980; Koncz and Schell, 1986). Introduction of the T-DNA's in the Arabidopsis genome was mediated according to the root explant transformation method of Clarke et al. (1992) with some modifications as described by Barthels et al.(1994). To obtain results in a very short time, a fast method was optimised making use of the infecting nematodes that takes along agrobacteria into the infection site. The wounding caused by the nematode triggers the Agrobacterium to transfer its T-DNA into the plant cells. A mixture of second stage juveniles and C58C1RifR(pMP90)(pTHW728S), C58C1Rif"(pMP90)(pTHW728PN) or C58C1Rif(pMP90)(pTHW1712BN) was inoculated on roots of 3 to 4 weeks old A. thaliana plants. In a next step, the infected plant roots were analysed 26 WO 99/28483 PCT/EP98/07761 for gus expression in the feeding structures as seen in Figure C for pTHW728PN. REFERENCES Barthels, N., Karimi, M., Van Montagu, M., and Gheysen, G. (1994). Isolation and analysis of nematode-induced genes in Arabidopsis thaliana through in vivo b-glucuronidase fusions. Med. Fac. Landbouww. Univ. Gent 59/2b, 757-762. Clarke, M.C., Weli, W., and Lindsey, K. (1992). High-frequency transformation of Arabidopsis thaliana by Agrobacterium tumefaciens. Plant Mol. Biol. Rep. 10, 178-189. Daykin, M.E., and Hussey, R.S. (1985). Staining and histopathological techniques in nematology. In An Advanced Treatise on Meloidogyne. Volume 11. Methodology, K.R. Barker, C.C. Carter, and J.N. Sasser, eds (Raleigh, NC: Department of Plant Pathology and US Agency for International Development), pp. 39-48. Deblaere, R., Bytebier, B., De Greve, H., Deboeck, F., Schell, J., Van Montagu, M., and Leemans, J. (1985). Efficient octopine Ti plasmid-derived vectors for Agrobacterium-mediated gene transfer to plants. Nucleic Acids Res. 13, 4777-4788. Dellaporta, S.L., Wood, J., and Hicks, J.B. (1983). A plant DNA minipreparation: Version 11. Plant Mol. Biol. Rep. 1, 19-21. Does, M.P., Dekker, B.M.M., de Groot, M.J.A., and Offringa, R. (1991). A quick method to estimate the T-DNA copy number in transgenic plants at an early stage after transformation, using inverse PCR. Plant Mol. Biol. 17,151-153. Gheysen, G., Villarroel, R., and Van Montagu, M. (1991). Illegitimate recombination in plants: A model for T-DNA integration. Genes Dev. 5, 287-297. Goddijn, O.J.M., Lindsey, K., van der Lee, F.M., Klap, J.C., and Sijmons, P.C. (1993). Differential gene expression in nematode-induced feeding structures of transgenic plants harbouring promoter-gusA fusion constructs. Plant J. 4, 863-873. Holsters, M., Silva, B., Van Viliet, F., Genetello, C., De Block, M., Dhaese, P., Depicker, A., Inz6, D., Engler, G., Villarroel, R., Van Montagu, M., and Schell, J. (1980). The functional organization of the nopaline A. tumefaciens plasmid pTiC58. Plasmid 3, 212-230. Jefferson, R.A. (1987). Assaying chimeric genes in plants: The GUS gene fusion system. Plant Mol. Biol. Rep. 5, 387-405. Karimi, M., Barthels, N., Van Montagu, M., and Gheysen, G. (1994). Identification of root knot nematode-induced genes in Arabidopsis thaliana. Med. Fac. Landbouww. Univ. Gent 59/2b, 751-756. Kertbundit, S., De Greve, H., Deboeck, F., Van Montagu, M., and Hernalsteens, J.-P. (1991). In vivo random b-glucuronidase gene fusions in Arabidopsis thaliana. Proc. NatI. Acad. Sci. USA 88, 5212-5216. 27 WO 99/28483 PCT/EP98/07761 Koncz, C., and Schell, J. (1986). The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Mol. Gen. Genet. 204, 383-396. Krebbers, E., Herdies, L., De Clercq, A., Seurinck, J., Leemans, J., Van Damme, J., Segura, M., Gheysen, G., Van Montagu, M., and Vandekerckhove, J. (1988). Determination of the processing sites of an Arabidopsis 2S albumin and characterization of the complete gene family. Plant Physiol. 87, 859-866. Mettler, I.J. (1987). A simple and rapid method for minipreparation of DNA from tissue cultured plant cells. Plant Mol. Biol. Rep. 5, 346-349. Offringa, R. and van der Lee, F. (1995). Isolation and characterization of plant genomic DNA sequences via (inverse) PCR amplification. Methods in Molecular Biology, Vol 49, 181-95. Edited by H. Jones. Humana Press Inc., Totowa, NJ. Peleman, J., Boerjan, W., Engler, G., Seurinck, J., Botterman, J., Alliotte, T., Van Montagu, M., and lnze, D. (1989). Strong cellular preference in the expression of a housekeeping gene of Arabidopsis thaliana encoding S-adenosylmethionine synthetase. Plant Cell 1, 81-93. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press). Sijmons, P.C., Dekker, B.M.M., Schrammeijer, B., Verwoerd, T.C., van den Elzen, P.J.M., and Hoekema, A. (1990). Production of correctly processed human serum albumin in transgenic plants. Bio/technology 8, 217-221. Sijmons, P.C., Grundler, F.M.W., von Mende, N., Burrows, P.R., and Wyss, U. (1991). Arabidopsis thaliana as a new model host for plant-parasitic nematodes. Plant J. 1, 245-254. Topping, J.F., Wei, W., and Lindsey, K. (1991). Functional tagging of regulatory elements in the plant genome. Development 112, 1009-1019. Topping, J.F., Wei, W., Clarke, M.C., Muskett, P., and Lindsey, K. (1995). Agrobacterium-mediated transformation of Arabidopsis thaliana: application in T-DNA tagging. Methods in Molecular Biology, Vol 49: Plant Gene Transfer and Expression Protocols, 63-76. Edited by H. Jones. Humana Press Inc., Totowa, NJ. Valvekens, D., Van Montagu, M., and Van Lijsebettens, M. (1988). Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection. Proc. Nat. Acad. Sci. USA 85, 5536-5540. Valvekens, D., Van Lijsebettens, M., and Van Montagu, M. (1991). Arabidopsis regeneration and transformation (root explant system). In Plant Tissue Culture Manual: Fundamentals and Applications, Supplement 1, K. Lindsey, ed (Dordrecht, The Netherlands: Kluwer Academic Publishers), pp. A8/1-17. Vancanneyt, G., Schmidt, R., O'Connor-Sanchez, A., Willmitzer, L., and Rocha-Sosa, M. (1990). Construction of an intron-containing marker gene: Splicing of the intron in transgenic plants and its use in monitoring 28 WO 99/28483 PCT/EP98/07761 early events in Agrobacterium-mediated plant transformation. Mol. Gen. Genet. 220, 245-250. Vanderhaeghen, R., and Van Lijsebettens, M. (1992). The inverse polymerase chain reaction. In Plant Molecular Biology (EMBO Practical Course Manual), D. Inze, D. Van Der Straeten, and M. Van Montagu, eds (Gent: Laboratorium voor Genetica), pp. 73-80. Yenofsky, R.L., Fine, M., and Pellow, J.W. (1990). A mutant neomycin phosphotransferase 11 gene reduces the resistance of transformants to antibiotic selection pressure. Proc. Natl. Acad. Sci. USA 87, 3435-3439. 29 WO 99/28483 PCT/EP98/07761 LEGEND TO FIGURES Figure 1. T-DNA structures in the binary vectors pGV1 047 and pAgusBinl9. gus, gus gene encoding the B-glucuronidase (GUS) reporter; 3'nos, nopaline synthetase 3' sequence; pnos, nopaline synthetase promoter; nos, nopaline synthetase gene; nptll, neomycin phosphotransferase gene; 3'ocs, octopine synthetase 3' sequence; P35S, cauliflower mosaic virus 35S promoter; supF, suppressor gene. Figure 2. Reporter gene activation in syncytia after cyst nematode infection of Arabidopsis promoter tag lines. (A) Att1712 syncytium 4 dpi. (B) Cross-section through an Att1712 syncytium 4 dpi. Cell wall dissolution at this stage is still in progress. (C) GUS-stained lateral root initiation site in Att1 712. (D) AttOO01 syncytium 4 dpi. (E) Cross-section through an AttOO01 syncytium 4 dpi. (F) GUS-stained lateral root initiation site in AttOO01. (H) Longitudinal section through an Att0728 syncytium 3 dpi. (B) and (H) were taken using dark-field optics. GUS, p-glucuronidase; N, nematode; S, syncytium. Bars in (A) to (H) = 50 Im. Figure 3. Reporter gene activation in NFSs induced by gall-forming nematodes. Inoculations were performed with root knot nematodes, except when stated otherwise. (A) Att1712 gall 4 dpi. 30 WO 99/28483 PCT/EP98/07761 (B) Total Attl712 plant 4 dpi. (C) Total AttOO01 plant 4 dpi. (D) Close-up of AttOO01 gall (4 dpi) boxed in (C). (E) AttOO01 gall 4 dpi. The plant was inoculated in the soil. (F) Cross-section through an Att1712 gall 4 dpi. (G) Attl712 gall 4 dpi. A nonstained zone is frequently observed. (H) Cross-section through an Att1712 Xiphinema diversicaudatum-induced gall 4 dpi. (I) Att0001 X. diversicaudatum-induced gall 4 dpi. (M) Att0728 gall 2 dpi. (F) and (H) were taken using dark-field optics. G, gall; GC, giant cell; MC, multinucleate cells; N, nematode; Nps, nematode penetration site; VP, vascular parenchyma. Bars in (A) and (D) to (M) = 50 pm; bars in (B) and (C) = 1 mm. Figure 4. GUS patterns in roots of transgenic Arabidopsis lines after incubation on callus-inducing medium. (A) Att1712 displays strong GUS staining in the root vascular tissue at the base of protruding calluses. (B) AttOO01 root tips show a typical three-zone pattern 6 days after incubation on CIM medium. (D) Shown are roots of P35S-gus-transformed plants after 6 days of growth on CIM medium. Newly formed lateral roots are not stained for GUS except for the root tips. (E) Shown are P35S-gus roots after 6 days of growth on CIM medium. Strong promoter activity in callus is evident. GUS activity starts to diminish (arrows) in root parts juxtaposed to the callus. (F) Shown are roots of P35S-gus-transformed plants after 11 days of incubation on CIM medium. GUS staining is confined solely to root tips and callus tissue. C, callus; LR, lateral root; RT, root tip; RVT, root vascular tissue. Bars in (A) to (F) = 50 ptm. 31 WO 99/28483 PCT/EP98/07761 Figure 5. Schematic presentation of the performed IPCRs on Att0001, Att0728 and Att1 712. (A) pAgusBin19 inverted T-DNA repeat structure in Att0001. Primer sets 1 and 2 and 1 and 3 were used to isolate both LB-flanking sequences. Abbreviations are as given in Figure 1. Numbered arrows indicate the different primer annealing positions. (B) One single T-DNA copy is integrated in lines Att0728 and Att171 2 A For both lines, primerset 5 and 6 were used to perform IPCR. The DNA of these lines was digested respectively with Nsil and AccI prior to the IPCR. Figure 6. Vector constructions used for reintroduction of promoter-containing plant sequences into Arabidopsis. Both Att0001 LB-flanking regions and the LB-flanking regions of Att0728 and Att1712 were cloned in pTHW136 in front of the gus intron by replacing the P35S. Abbreviations are as given in Figure 1. Figures A and B. Isolation and cloning of the left border T-DNA / plant flanking sequences of Att0728 and Attl712 respectively Figure C. GUS pattern in galls induced on Arabidopsis C24 roots 10 days after treatment with an Meloidogyne incognita/Agrobacterium C58C1 Rif" (pMP90)(pTHW728PN) mixture. Figure D. GUS pattern in roots of Arabidopsis transformed with pTHW728PN as observed 3 days after inoculation with H.schachtii. Figure E. GUS pattern in roots of Arabidopsis transformed with pTHW728PN as observed 4 days after inoculation with Meloidogyne incognita. 32 WO 99/28483 PCT/EP98/07761 Table 1. Overview of the GUS patterns from five retained promoter trap lines after infection with cyst nematodes (Heterodera schachtii, Hs), root-knot nematodes (Meloidogyne incognita, Mi), and migratory ectoparasitic nematodes (Xiphinema diversicaudatum, Xd). Expression' Cultureb 0728' 1712 0001 0001-R/1 1012 Hs in vitro +++ +++ ++++ ND ++ in soil +++ +++ ++++ ND ND Mi in vitro +++ ++++ ++++ ++ ++ in soil +++ ++++ ++++ ND ND Xd in vitro ND ND ND ND ND in soil +++ ++++ ++++ ND ND Other tissues rvt +/- - rt ---- + (in vitro)* Iri +- +++ +++ +/- vs - - + - sti - - lvt - - ++ + hd - - f&p a Hs, Heterodera schachtii; Mi, Meloidogyne incognita; Xd, Xiphinema diversicaudatum. The asterisk indicates that expression patterns from in vitro-grown plants as described here occasionally slightly differ from those of soil-grown plants (see text for details). b f&p, flowers and pods; hd, hydathodes; Iri, lateral root initiation; lvt, leaf vascular tissue; rt, root tip; rvt, root vascular tissue; sti, stipules; vs, vegetative shoot. ' (++++) indicates high reporter gene expression; (+++), strong expression; (++) moderate expression; (+), weak expression. The (+/-) indicates weak staining for GUS activity; the (-) indicates that no GUS activity could be seen; ND, not determined. 33

Claims (12)

1. An isolated DNA sequence comprising the nucleotide sequence of SEQ ID NO 1 or fragments thereof.

2. An isolated DNA sequence comprising the nucleotide sequence of SEQ ID NO 2 or fragments thereof.

3. An isolated DNA sequence according to claim 2 characterized in that the fragment comprises the nucleotide sequence of SEQ.lD.NO.2 from position nucleotide 371 to 1045, both positions included.

4. A recombinant DNA comprising a plant-expressible promoter region having a sequence according to SEQ ID NO 1 and/or SEQ ID NO 2 or a fragment thereof.

5. A plant cell comprising a recombinant DNA according to claim 4.

6. A plant comprising the recombinant DNA according to claim 4 integrated in its genome.

7. A method for obtaining a plant with reduced susceptibility to a plant nematode comprising the steps of 1) transforming a recipient plant cell with a recombinant DNA according to claim 4, 2) generating a whole plant from a transformed cell and 3) identifying a transformed plant with said reduced susceptibility.

8. A transformation vector containing recombinant DNA according to claim 4.

9. An Agrobacterium strain containing a transformation vector according to claim 8.

10. A method for preventing nematode reproduction in a plant comprising planting a plant according to claim 6 in an area susceptible to nematode infection.

11. A method for suppressing plant pathogen activity comprising expression of a suitable foreign DNA in a plant under the control of a promoter region having the DNA sequence according to SEQ ID NO I and/or SEQ ID NO 2.

12. Use of a promoter region comprising the DNA sequence of claim 1 or 2 to express a gene in specific root cells of a plant being infected with nematode(s). 34