ZA200005115B - Salicylic acid pathway genes and their use for the induction of resistance in plants. - Google Patents

Description

SALICYLIC ACID PATHWAY GENES AND THEIR USE FOR THE INDUCTION OF

- RESISTANCE IN PLANTS.

FIELD OF THE INVENTION

This invention is related to genes in the salicylic acid biosynthesis pathway, more specifically in the salicylic acid pathway through isochorismic acid, and their use for the induction of * resistance through salicylic acid in plants. More specifically the invention is related to the use of isochorismate synthase (ICS) genes . 10 for the production of salicylic acid, specifically by a new plant isochorismate synthase gene, more specifically to the use of an isochorismate pyruvate lyase in addition to the isochorismate synthase. Further the invention is related to the use of the promoter of the new plant isochorismate synthase gene as a pathogen-inducible promoter.

BACKGROUND ART

Upon pathogen challenge, plants can react by raising a defense mechanism that acts both locally and systemically. In the hypersensitive response (HR) the local response consists of amongst others rapid cell necrosis/apoptosis, seen as the formation of lesions, and the accumulation of growth-inhibiting phenolic subslances (phytoalexins) and pathogenesis-related (PR) proteins. Also cell wall reinforcement and cell wall thickening are observed. This combined, multifactorial defense response leads to the restriction of pathogen growth and spread.

Systemically, induction of PR-proteins, which have strong anti- pathogen effects, occurs after this hypersensitive response, which is the likely reason for the state of Systemic Acquired Resistance (SAR) that plants exhibit after the HR. This state of SAR may last for several weeks and protects plants from pathogens to which it otherwise is susceptible.

The signalling pathways involved in both the development of the HR and

SAR are poorly understood. Early studies have shown that salicylic acid (SA) accumulates to substantial levels both locally and systemically. Also, treatment of plants with SA increases the level of expression of PR-genes, and increases the resistance of plants to pathogens suggesting that SA plays a crucial role in the establishment of SAR (see e.g. Ryals, Plant Cell §, 1809-1819, 1996).

Even more firm evidence for the crucial role of SA was obtained by ) making transgenic plants carrying the nahG gene from Pseudomonas putida. The geneproduct of the nahG gene hydroxylates SA, and renders it inactive. NahG-transgenic tobacco and Arabidopsis thaliana plants are compromised in their ability to raise an effective HR, since the pathogen grows and spreads from the initial infection site (Gaffney et al., Science 261, 754-756, 1993; Delaney et al., Science 266, 1247- 1250, 1994). The nahG-transgenic plants are also defective in raising a SAR response. ’

It is, however, clear that =.ternat.ve cathwzys fcr induction SI toe hypersensitive response are present, and overexpression of nahG in tomato does not compromise the hypersensitive response occurring after challenge of CfS8 or Cf2 plants with Cladosporium fulvum races containing AvrS and Avrz, respectively. (Hammond-Kosack & Jones, Plant

Cell 8, 1773-1791, 1596).

Overviews of the role of SA in plant disease signalling can be found in Durner et al., Trends. Plant. Sci. 2, 266-274, 1997; Chasan, Plant

Cell 7, 1519-1521, 1995; Klessig & Malamy, Plant Mol. Biol. 26, 1439- 1458, 1994)

SUMMARY OF THE INVENTION

This invention describes a method to induce pathogen resistance in plants, characterized in that plants are transformed with an expression cassette harboring a gene coding for an isochorismate synthase. More specifically, this method is characterized in that the gene coding for isochorismate synthase is selected from a group consisting of entC, orfA, pchA and ICS, this last gene preferably the

ICS gene from Catharantus roseus. Genes which can be used for this method are depicted in SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 17.

Another embodiment of the invention is a method according to the method described above, characterized in that plants are additionally transformed with a vector carrying an expression cassette harboring a gene coding for an isochorismate pyruvate lyase, preferably on the same vector as the expression cassette comprising the gene coding for

“ isochorismate synthase. The gene coding for isochorismate pyruvate . lyase is preferably selected from the group consisting of orfD and pchB.

BA specific embodiment of the invention is a method as described above, characterized in that the gene coding for isochorismate synthase is entC and the gene coding for isochorismate pyruvate lyase is orfD. - A further aspect of the invention is a protein having isochorismate synthase activity which is isolated from Catharantus roseus. This . 10 protein has a Molecular Weight of 67 kD. This protein preferably comprises the amino acid sequence of SEQ ID NO: 19.

Also part of the invention is a nucleotide sequence encoding this protein, which is preferably a nucleotide sequence comprising the nucieotide sequence of SEQ ID NC: 18

A further aspect of the invention is the nucleotide sequence comprising the 5' regulatory region which is naturally found to regulate the expression of the ICS gene in Catharantus roseus. This regulatory region can be used as a pathogen-inducible promoter, which can drive expression of a protein that has antifungal, antibacterizl or antiviral properties. Examples of such proteins are chitinases, glucanases, osmotins, defensins, magainins, cecropins, ribozymes.

Alternatively, such a pathogen-inducible promoter can be used to express elicitor proteins or resistance genes for use in a strategy aimed at the induction of a hypersensitive response (see WO 91/15585).

Vectors, Agrobacteria, plant cells and plants which comprise or are transformed with the above-mentioned genes form further part of the invention.

DESCRIPTION OF THE FIGURES

Figure 1: Schematic respresentation of the vector pM0G22 GUS ICS

Figure 2: Northern blot of RNA isolated from transgenic plants with the indicated constructs (3 transgenic lines per construct) and control plants hybridized with a probe for PR-la.

Figure 3: Schematic map with restriction sites of the regulatory sequence of the Catharantus roseus isochorismate synthase gene.

DETAILED DESCRIPTION OF THE INVENTION

Biosynthesis of salicylic acid in plants is normally thought to ) proceed via synthesis of transcinnamic acid and conversion to benzoic acid fcllowed by Z-hydroxylatiorn. In infected plants the synthesis pathway may be altered slightly, using ~rans-cinnamic acid to convert it into ortho-coumaric acid which is then converted into salicylic acid. In micro-organisms the biosynthesis of salicyl:c acid is known to proceed from chorismate to isochorismate (catalyzed by the enzyme ) isochorismete synthase, ICS). Genes required for this conversion have beer. cloned from Pseudomonas aeruginosa (the pchA gene encoding ICS-~ : a-tivity, Serine, L. et &l., Mci. Gen. Genet. 24%, 217-223, 12330 =ng from Escherischia coli (the entC gene encoding ICS activity,

Ozenberger, B.A. et &l., J. Bacteriol. 171, 775-783, 1989).

We have now surprisingly found that use of the chorismate pathway to produce salicylic acid can be introduced in plants by transformation of said plants with an expression cassette harboring a gene coding for isochorismate synthase. This gene can either be derived from bacteria such as the PchA and the entC genes identified above, or the orfA gene from Pseudomonas fluorescens, or from plants where the gene coding for isochorismate synthase has been found present, such as the ICS gene from Catharanthus roseus, as provided in this application. Also other genes, not yet identified, but having isochorismate synthase activity, are envisaged to be used in this invention. Such genes can be isolated from bacteria or from plants by probing them with a degenerated probe derived from the sequences present in this invention.

Although it is known (and it is also observed in this invention) that salicylic acid, probably because of its relative toxicity is rapidly inactivated in plants (either by degradation or by glucosidation) we have still been able to find an increased concentration of salicylic acid after transformation of the plants with genes coding for isochorismate synthase. Moreover, we also observed an induction of pathogenesis-related proteins, most probably caused by the overproduction of salicylic acid, showing that the endogenously produced salicylic acid can give lead to induction of the plants to impart pathogen resistance.

The genes which can be used in this invention are depicted in . SEQ ID NC's: 13, 15 and 18. It must be understood that the nucleotide sequences coding for the enzymes may be changed freely as long as the resulting gene product still has isochorismate synthase activity.

Changes which are apparent are changes to the codon usage to adapt it to the codon usage which is most similar to the plant to which the genes will be transformed. Also the pelynucleotide used for - transfcrmation may be modified in that mRNA instability encoding motifs and/or fortuitous splice regions may be removed so that expression of the thus modified polynucleotides yields substantially similar enzyme.

The genes of the invention encode enzymatically active proteins. The word protein means a sequence of amino acids connected trough peptide bonds. Polypeptides or peptides are also considered to be proteins. Muteins of the protein of the invention are proteins that are obtained from the proteins depicted in the sequence listing by replacing, adding and/or deleting one or more amino acids, while still retaining their enzymatic activity. Such muteins can readily be made by protein engineering in vivo, e.g. by changing the open reading frame capable of encoding the enzyme such that the amino acid sequence is thereby affected. As long as the changes in the amino acid sequences do not altogether abolish the enzymatical activity such muteins are embraced in the present invention. Further, it should be understood that mutations should be derivable from the proteins or the DNA sequences encoding these proteins depicted in the sequence listing while retaining biological activity, i.e. all, or sa great part of the intermediates between the mutated protein and the protein depicted in the sequence listing should have enzymatical activity. A great part would mean 30% or more of the intermediates, preferably 40% of more, more preferably 50% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferab:y 99% or more.

The present invention provides a chimeric DNA sequence which comprises an expression cassette according to the invention. The term chimeric DNA sequence shall mean to comprise any DNA sequence which comprises DNA sequences not naturally found in nature. For instance, 40 chimeric DNA shall mean to comprise DNA comprising the open reading frame coding for the enzyme in a non-natural location of the plant genome, notwithstanding the fact that said plant genome normally contains a copy of the said open reading frame in its natural chromosomal location. Similarly, the said open reading frame may be ’ incorporated in the plant genome wherein it is not naturally found, or in a replicon or vector where it is not naturally found, such as a bacterial plasmid or a viral vector. Chimeric DNA shall not be limited to DNA molecules which are replicable in a host, but shall also mean to comprise DNA capable of being ligated into a replicon, for instance by virtue of specific adaptor sequences, physically linked to the open reading frame according to the invention. The open reading frame may or may not be linked toc its natural upstream and downstream regulatory : elements.

The open reading frame may be derived from a genomic library. In this latter it may contain one or more introns separating the exons making up the open reading frame that encodes a protein according to the invention. The open reading frame may also be encoded by one uninterrupted exon, or by a cDNA to the mRNA encoding a protein according to the invention. Open reading frames according to the invention also comprise those in which one or mere introns have been artificially removed or added. Each of these variants is embraced by the present invention.

In order to be capable of being expressed in a host cell a chimeric DNA according to the invention will usually be provided with regulatory elements enabling it to be recognised by the biochemical machinery of the host and allowing for the open reading frame to be transcribed and/or translated in the host. It will usually comprise a transcriptional initiation region which may be suitably derived from any gene capable of being expressed in the host cell of choice, as well as a translational initiation region for ribosome recognition and attachment. In eukaryotic cells, an expression cassette usually comprises in addition a transcriptional termination region located downstream of said open reading frame, allowing transcription to terminate and polyadenylation of the primary transcript to occur. In addition, the codon usage may be adapted to accepted codon usage of the host of choice. Further, often a signal sequence may be encoded, which is responsible for the targeting of the gene expression product to subcellular compartments. The principles governing the expression of a chimeric DNA construct in a chosen host cell are commonly understood by those of ordinary skill in the art and the construction ) of expressible chimeric DNA constructs is now routine for any sort of host cell, be it prokaryotic or eukaryotic.

In order for the open reading frame to be maintained in a host cell it will usually be provided in the form of a replicon comprising said open reading frame according to the invention iinked to LFA wnici is recognised and replicated by the chosen host cell. Accordingly, the . selection of the replicon is determined largely by the host cell of choice. Such principles as govern the selection of suitable replicons for a particular chosen host are well within the realm of the ordinary ] skilled person in the art.

A special type of replicon is one capable of transferring itself, or a part thereof, to another host cell, such as a plant cell, thereby co-transferring the open reading frame according to the invention to said plant cell. Replicons with such capability are herein referred to as vectors. An example of such vector is a Ti- plasmid vector which, when present in a suitable host, such as

Agrobacterium tumefaciens, is capable of transferring part of itself, the so-called T-region, to a plant cell. Different types of Ti-plasmid vectors (vide: EP 0 116 718 Bl) are now routinely being used to transfer chimeric DNA sequences into plant cells, or protoplasts, from which new plants may be generated which stably incorporate said chimeric DNA in their genomes. A particularly preferred form of Ti- plasmid vectors are the so-called binary vectors as claimed in (EP 0 120 516 Bl and US 4,940,838). Other suitable vectors, which may be used to introduce DNA according to the invention into a plant host, may be selected from the viral vectors, e.g. non-integrative plant viral vectors, such as derivable from the double stranded plant viruses (e.g. CaMV) and single stranded viruses, gemini viruses and the like. The use of such vectors may be advantageous, particularly when it is difficult to stably transform the plant host. Such may be the case with woody species, especially trees and vines.

The expression "host cells incorporating a chimeric DNA sequence according to the invention in their genome" shall mean to comprise cells, as well as multicellular organisms comprising such cells, or essentially consisting of such cells, which stably incorporate said chimeric DNA into their genome thereby maintaining the chimeric DNA, and preferably transmitting a copy of such chimeric DNA to progeny cells, be it through mitosis or meiosis. According to a preferred embodiment of the invention plants are provided, which essentially consist of cells which incorporate one or more copies of said chimeric )

DNA into their genome, and which are capable of transmitting a copy or copies to their progeny, preferably in a Mendelian fashion. By virtue of the transcription and translation of the chimeric DNA according to the invention in some or all of the plant’s cells, those cells that produce the enzyme will show enhanced resistance to pathogen infections. Although the principles as indicated above govern ) transcription of DNA in plant cells are not always understood, the creation of chimeric DNA capable of being expressed in substantially a : cons—itutive fashicm, that is, in substantially most cell types oI the plant and substantially without serious temporal and/or developmental restrictions, is now routine. Transcription initiation regions routinely in use for that purpose are promoters obtainable from the cauliflower mosaic virus, notably the 355 RNA and 19S RNA transcript promoters and the so-called T-DNA promoters of Agrobacterium tumefaciens, in particular to be menticned are the nopaline synthase promoter, octopine synthase promoter (as disclosed in EP 0 122 791 Bl) and the mannopine synthase promoter. In addition plant promoters may be used, which may be substantially constitutive, such as the rice actin gene promoter, or e.g. organ-specific, such as the root-specific promoter RolD, or the potato tuber specific patatin promoter.

Alternatively, inducible promoters may be used which enable induction of pathogen resistance by an external factor, which can be applied at a time point which is most suitable. Thus it prevents unwanted effects, such as for instance can occur due to the relative toxicity of the salicylic acid. Inducible promoters include any promoter capable of increasing the amount of gene product produced by a given gene, in response to exposure to an inducer. In the absence of an inducer the DNA sequence will not be transcribed. Typically, the factor that binds specifically to an inducible promoter to activate transcription is present in an inactive form which is then directly or indirectly converted to the active form by the inducer. The inducer may be a chemical agent such as a protein, metabolite (sugar, alcohol, etc.), a growth regulator, herbicide, or a phenolic compound or a physiological stress imposed directly by heat, salt, wounding, toxic elements etc., or indirectly through the action of a pathogen or disease agent such as a virus. A plant cell containing an inducible ] promoter may be exposed to an inducer by externally applying the inducer to the cell such as by spraying, watering, heating, or similar methods. Inducible promoters are known to those familiar with the art and several exist that could conceivably be used to drive expression of the genes of the invention. Inducible promoters suitabie for use in accordance with the present invention include, but are not limited to, . the heat shock promoter, the mammalian steroid receptor system and any chemically inducible promoter. Examples of inducible promoters include the inducible 70 kD heat shock promoter of Drosophila melanogaster (Freeling, M. et al., Ann. Rev. Genet. 13, 297-323) and the alcohol dehydrogenase promoter which is induced by ethanol (Nagao, R.T. et al., in: Miflin, B.J. (ed.) Oxford Surveys of Plant Molecular and Cell

Biology, Vol. 3., pp. 384-438, oxford Univ. Press, 1986). A promoter that is inducible by a simple chemical is particularly useful.

Examples for the last category are the promoters described in WO 90/08826, WO 93/21334, WO 93/031294 and WO 96/37609. As examples of a pathogen-inducible promoter the PRP1 promoter (also named gstl promoter) obtainable from potato (Martini N. et al. (1993), Mol. Gen.

Genet. 263, 179-186), the Fisl promoter (Wo 96/34949), the Bet v 1 : promoter (Swoboda, I., et al., Plant, Cell and Env. 18, B65-874, 1995), the Vstl promoter (Fischer, R., Dissertation, Univ. of

Hohenheim, 1994; Schubert, R., et al. Plant Mol. Biol. 34, 417-426, 1997), the sesquiterpene cyclase promoter (yin, s., et al., Plant physiol. 115, 437-451, 1997) and the gstAl promoter (Mauch, F. and

Dudler, R., Plant Physiol. 102, 1133-1201, 1993) may be mentioned. Of course also the regulatory region of the ICS gene from Catharanthus rogeus, which forms part of this invention, may be used in this respect.

The choice of the promoter is not essential, although it must be gaid that constitutive high-level promoters and/or inducible promoters are slightly preferred. It is further known that duplication of certain elements, so-called enhancers, may considerably enhance the expression level of the DNA under its regime (vide for instance: Kay

R. et al., Science 236, 1299-1302, 1987: the duplication of the sequence between -343 and -90 of the CaMV 35S promoter increases the activity of that promoter). In addition to the 35S promoter, singly or doubly enhanced, examples of high-level promoters are the light-

inducible ribulose bisphosphate carboxylase small subunit (zrbcSSU) promoter and the chlorophyll a/b binding protein (Cab) promoter. Also envisaged by the present invention are hybrid promoters, which ’ comprise elements of different promoter regions physically linked. A well known example thereof is the so-called CaMV enhanced mannopine synthase promoter (US Patent 5,106,739), which comprises elements of the mannopine synthase promoter linked to the CaMV enhancer.

As is demonstrated in the Examples illustrating this invention, targeting of the enzymes to organelles in the plant cell can enhance the production of salicylic acid. This can be explained by the fact that the substrate for the enzymes of the invention is abundant in special organelles. Especially tarceting to the chloroplast, using a signal peptide derived from tobacco, yields good results. Of course, signal peptides obtained from other sources can be used.

As regards the necessity of a transcriptional terminator region, it is generally believed that such a region enhances the reliability as well as the efficiency of transcription in plant cells. Use thereof is therefore strongly preferred in the context of the present invention.

As regards the applicability of the invention in different plant species, it has to be mentioned that one particular embodiment of the invention is merely illustrated with transgenic tobacco and

Arabidopsis plants as an example, the actual applicability being in fact not limited to these plant species. Any plant species that is subject to some form of pathogen attack, may be transformed with genes according to the invention, allowing the enzyme(s) to be produced in some or all of the plant’s cells.

Although some of the embodiments of the invention may not be practicable at present, e.g. because some plant species are as yet recalcitrant to genetic transformation, the practicing of the invention in such plant species is merely a matter of time and not a matter of principle, because the amenability to genetic transformation as such is of no relevance to the underlying embodiment of the invention.

Transformation of plant species is now routine for an impressive number of plant species, including both the Dicotyledoneae as well as the Monocotyledoneae. In principle any transformation method may be used to introduce chimeric DNA according to the invention into a suitable ancestor cell, as long as the cells are capable of being ] regenerated into whole plants. Methods may suitably be selected from the calcium/polyethylene glycol method for protoplasts (Krens, F.A. et al., Nature 296, 72-74, 1982; Negrutiu I. et al,, Plant Mol. Biol. 8, 363-373, 1987), electroporation of protoplasts (shillito R.D. et al.,

Bio/Technol. 3, 1099-1102, 1885}, microinjection into pliant material (Crossway A. et al., Mol. Gen. Genet. 202, 179-185, 1986), DNA (or . RNA-coated) particle bombardment of various plant material (Klein T.M. et al., Nature 327, 70, 1987), infection with (non-integrative) viruses and the like. A preferred method according to the invention ) comprises Agrobacterium-mediated DNA transfer. Especially preferred is the use of the so-called binary vector technology as disclosed in EP A 120 516 and U.S. Patent 4,540,838.

Tomato transformation is preferably done essentially as described by van Roekel et al. (Plant Cell Rep. 12, 644-647, 1993). Potato transformation is preferably done essentially as described by Hoekema et al. (Hoekema, A. et al., Bio/Technology 7, 273-278, 1989).

Generally, after transformation plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant expressible genes co-transferred with the nucleic acid sequence : encoding the protein according to the invention, whereafter the transformed material is regenerated into a whole plant.

Although considered somewhat more recalcitrant towards genetic transformation, monocotyledonous plants are amenable to transformation and fertile transgenic plants can be regenerated from transformed cells or embryos, or other plant material. Presently, preferred methods for transformation of monocots are microprojectile bombardment of embryos, explants or suspension cells, and direct DNA uptake or electroporation (Shimamoto, et al, Nature 338, 274-276, 1989) .

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 microprojectile bombardment (Gordon-Kamm, , Plant Cell, 2, 603-618, 1990). The introduction of genetic material into aleurone protoplasts of other monocot crops such as wheat and barley has been reported (Lee, Plant Mol. Biol. 13, 21-30, 1989). Wheat plants have been regenerated from embryogenic suspension culture by selecting only the aged compact and nodular embryodgenic callus tissues for the establishment of the embryogenic suspension cultures (Vasil,

Bio/Technol. 8, 429-434, 1990). The combination with transformation ’ systems for these crops enables the application of the present invention to monocots.

Monocotyledonous plants, including commercially important crops such as rice and corn are also amenable to DNA transfer by

Agrobacterium strains (vide WO 94/00977; EP 0 159 418 Bl; Gould J, et al., Plant. Physiol. 95, 426-434, 1991). )

Following DNA transfer and regeneration, putatively transformed plants may be evaluated, for instance using Southern analysis, for the - presence of the chimeric DNA according to the invention. copy umber and/or genomic organization. Im addition, or alternatively, expression levels of the newly introduced DNA may be undertaken, using Northern and/or Western analysis, techniques well known to persons having ordinary skill in the art. After the initial analysis, which is optional, transformed plants showing the desired copy number and expression level of the newly introduced chimeric DNA according to the invention may be tested for resistance levels against pathogens..

Alternatively, the selected plants may be subjected to another round of transformation, for instance to introduce further genes, in order to enhance resistance levels, or broaden the resistance.

Other evaluations may include the testing of pathogen resistance under field conditions, checking fertility, yield, and other characteristics. Such testing is now routinely performed by persons having ordinary skill in the art.

Following such evaluations, the transformed plants may be grown directly, but usually they may be used as parental lines in the breeding of new varieties or in the creation of hybrids and the like.

To obtain transgenic plants capable of constitutively expressing more than one chimeric gene, a number of alternatives are available including the following:

A. The use of DNA, e.g a T-DNA on a binary plasmid, with a number of modified genes physically coupled to a selectable marker gene. The advantage of this method is that the chimeric genes are physically coupled and therefore migrate as a single Mendelian locus.

B. Cross-pollination of transgenic plants each already capable of ; expressing one or more chimeric genes, preferably coupled to a selectable marker gene, with pollen from a transgenic plant which contains one or more chimeric genes coupled to another selectable marker. Afterwards the seed, which is obtained by this crossing, maybe selected on the basis of the presence of the two selectable markers, or on the basis of the presence of the chimeric genes themselves. The . plants obtained from the selected seeds can afterwards be used for further crossing. In principle the chimeric genes are not on a single ] locus and the genes may therefore segregate as independent loci.

C. The use of a number of a plurality chimeric DNA molecules, e.g. plasmids, each having one or more chimeric genes and a selectable marker. If the frequency of co-transformation is high, then selection on the basis of only one marker is sufficient. In other cases, the selection on the basis of more than one marker is preferred.

D. Consecutive transformation of transgenic plants already containing a first, second, (etc), chimeric gene with new chimeric DNA, optionally comprising a selectable marker gene. As in method B, the chimeric genes are in principle not on a single locus and the chimeric genes may therefore segregate as independent loci.

E. Combinations of the above mentioned strategies.

The actual strategy may depend on several considerations as maybe easily determined such as the purpose of the parental lines (direct growing, use in a breeding programme, use to produce hybrids) but is not critical with respect to the described invention.

In this context it should be emphasised that plants already containing chimeric DNA capable of encoding an enzyme of the isochorismatic pathway may form a suitable genetic background for introducing chimeric DNA according tc the invention, for instance in order to enhance the production of salicylic acid, thereby enhancing the induction capability and thereby enhancing resistance levels. The cloning of other genes corresponding to proteins that can suitably be used in combination with DNA, and the obtention of transgenic plants, capable of relatively over-expressing same, as well as the assessment of their effect on pathogen resistance in planta, is now within the scope of the ordinary skilled person in the art.

plants, or parts thereof, which relatively over-express salicylic acid according to the invention, including plant varieties, with improved resistance against pathogens may be grown in the field, in the greenhouse, or at home or elsewhere. Plants or edible parts thereof may be used for animal feed or human consumption, or may be processed for food, feed or other purposes in any form of agriculture or industry. Agriculture shall mean to include horticulture, arboriculture, flower culture, and the like. Industries which may benefit from plant material according to the invention include but are not limited to the pharmaceutical industry, the paper and pulp manufacturing industry, sugar manufacturing industry, feed and food ’ industry, enzyme manufacturers and the like.

The advantages of the plants, or parts thereof, according to the invention are the decreased need for biocide treatment, thus lowering costs of material, labour, and environmental pollution, or prolonging shelf-life of products (e.g. fruit, seed, and the like) of such plants. Plants for the purpose of this invention shall mean multicellular organisms capable of photosynthesis, and subject to some form of pathogen attack. They ghall at least include angiosperms as well as gymnosperms, monocotyledonous as well as dicotyledonous plants.

The phrase "plants which relatively over-express an enzyme" shall mean plants which contain cells expressing a transgene-encoded enzyme which is either not naturally present in said plant, or if it is present by virtue of an endogenous gene encoding an identical enzyme, not in the same guantity, or not in the same cells, compartments of cells, tissues or organs of the plant.

A further aspect of the invention is the regulatory sequence naturally occurring in the 5’ untranslated region of the ICS-gene from

Catharanthus roseus. It has been found that upon pathogen infection the ICS gene is highly expressed, indicating pathogen inducibility.

Pathogen inducible promoters (such as the prpl-promoter described above) are of great value in biotechnological resistance engineering.

Examples of proteins that may be used in combination with the

ICS regulatory region according to the invention include, but are not limited to, $-1,3-glucanases and chitinases which are obtainable from barley (Swegle M. et al., Plant Mol. Biol. 12, 403-412, 1989; Balance

G.M. et al., Can. J. Plant Sci. 56, 459-466, 1976 ; Hoj P.B. et al.,

FEBS Lett. 230, 67-71, 1988; Hoj P.B. et al., Plant Mol. Biol. 13, 31- 42, 1989), bean (Boller T. et al., Planta 157, 22-31, 1983; Broglie

K.E. et al., Proc. Natl. Acad. Sci. USA 83, 6820-6824, 1986; Vdgeli U. et al., Planta 174, 364-372, 1988); Mauch F. & Staehelin L.A., Plant

Cell 1, 447-457, 1989); cucumber (Metraux J.P. & Boiler T., roysicli.

Mol. Plant Pathol. 28, 161-169, 1986); leek {spanu P. et al., Planta . 177, 447-455, 1989); maize (Nasser W. et al., Plant Mol. Biol. 11, 529-538, 1988), oat (Fink W. et al., Plant Physiol. 88, 270-275, 1988), pea (Mauch F. et al., Plant Physiol. 76, 607-611, 13984; Mauch ’ F. et al., Plant Physiol. 87, 325-333, 1988), poplar (Parsons, T.J. et al., Proc. Natl. Acad. Sci. USA 86, 7895-7899, 1989), potato (Gaynor

J.J., Nucl. Acids Res. 16, 5210, 1988; Kombrink E. et al., Proc. Natl.

Acad. Sci. USA 85, 782-786, 1988; Laflamme D. and Roxby R., Plant Mol.

Biol. 13, 249-250, 1989), tobacco (e.g. Legrand M. et al., Proc. Natl.

Acad. Sci. USA 84, 6750-6754, 1987; Shinshi H. et al. Proc. Natl.

Acad. Sci. USA 84, 89-93, 1987), tomato (Joosten M.H.A. & De Wit

P.J.G.M., Plant Physiol. 89, 945-931, 1989), wheat (Molano J. et al.,

J. Biol. Chem. 254, 4901-4907, 1979), magainins, lectins, toxins igolated from Bacillus thuringiensis, antifungal proteins isolated : from Mirabilis jalapa (EP 0 576 483) and Amaranthus (EP 0 593 501 and us 5,514,779), albumin-type proteins (such as thionine, napin, barley trypsin inhibitor, cereal gliadin and wheat-alpha-amylase, EP 0 602 098), proteins isolated from Raphanus, Brassica, Sinapis, Arabidopsis,

Dahlia, Cnicus, Lathyrus and Clitoria (EP 0 603 216), oxalate oxidase (EP 0 636 181 and EP 0 673 416), saccharide oxidase (PCT/EP 97/04923), antimicrobial proteins isolated from Allium seeds and proteins from

Aralia and Impatiens (WO 95/24485) and the like. another use of the inducible promoter is to drive proteins which play a role in the gene-for-gene resistance interaction (e.g. as described in WO 91/15585). Such proteins are, for example, plant proteins such as disclosed in Karrer, E.E. et al. (Plant Mcl. Biol. 36, 681-690, 1998), ndrl and edsl, Cf-proteins and Pto proteins from tomato, the avr-elicitor proteins from Cladosporium fulvum, and the avrpPto protein from Pseudomonas.

A clone harboring plasmid pMOG 1431 containing a 3kb insert which contains the ICS regulatory region according to the inventions was deposited under number 101670 with the Centraal Bureau voor

Schimmelcultures at Baarn, the Netherlands on March 19, 1999.

From the Examples it can be seen that an approximately two Kb } fragment of the promoter as listed in SEQ ID NO: 25 already shows the inducible properties. This 2 kb fragment can be obtained by splicing the sequence of SEQ ID NO:25 at the XhoI and NcoI sites, thereby forming the part of nucleotide number 1118 to 3275 of SEQ ID NO:25.

It is envisaged that this fragment can be truncated further while still maintaining the inducibility.

The following state of the art may be taken into consideration, : especially as illustrating the general level of skill in tre art tc which this invention pertains.

EP-A 392 225 A2; EP-A 440 304 Al; EP-A 460 753 A2; WOS0/07001 Al; US

Patent 4,940,840.

Evaluation of transgenic plants

Subsequently transformed plants are evaluated for the presence of the desired properties and/or the extent to which the desired properties are expressed. A first evaluation may include the level of expression of the newly introduced genes, the level of salicylic acid expressed, the level of induction of pathogen-related proteins, the pathogen resistance of the transformed plants, stable heritability of the desired properties, field trials and the like.

Secondly, if desirable, the transformed plants can be crossbred with other varieties, for instance varieties of higher commercial value or varieties in which other desired characteristics have already been introduced, or used for the creation of hybrid seeds, or be subject to another round of transformation and the like.

EXPERIMENTAL PART gtandard methods for the isolation, manipulation and amplification of DNA, as well as suitable vectors for replication of recombinant DNA, suitable bacterium strains, selection markers, media and the like are described for instance in Maniatis et al., molecular cloning: A Laboratory Manual 2nd. edition (1989) Cold Spring Harbor

Laboratory Press; DNA Cloning: Volumes I and II (D.N. Glover ed. 1985); and in: From Genes To Clones (E.-L. Winnacker ed. 1987).

Assay of isochorismate synthase activity

The incubation mixture (total volume 250 ui) contained U.i is 1iris~-aCi pH 7,5, 2mM chorismate, 10 mM MgCl,, and enzyme extract (crude . extracts 125 pl, column fractions 10-100 pl). The incubation was started by addition of chorismate. After incubation for 60 min at 30°C the reaction was stopped by the addition of 62.5 pl methanol- ] isobutanol (1:1 v/v). The samples were centrifuged and analyzed by

HPLC according to Poulsen, C. et al. Phytochem. 30, 2873-2878, 1991}.

EXAMPLE 1

Purification of ICS from Cathara tus roseus.

Catharanthus roseus (L.)G.Don cell cultures were grown in MS medium {Murashige and Skoog, 1962) supplemented with 30 g/1 sucrese as described previously (Moreno, P. et al. Plant Cell Rep. 12, 702-705, 1993). Cell cultures were elicited with Pythium aphanisermatum (CBS,

Baarn, The Netherlands) filtrate as described by Moreno et al. (1893).

Cells were harvested by suction 24 hours after elicitation, washed once with water, immediately frozen in liquid nitrogen and stored at - 80°C. Six hundred grams of frozen cells were homogenized in a Waring

Blender equipped with a stainless steel bucket. One ml of extraction buffer (0.1M Tris-HCl pH 7.5, 10% glycerol (v/v), 1 mM DIT, 0.2 mM

PMSF, 10 uM leupeptin and 1 mM EDTA) and 50 mg polyvinylpyrrolidone were added per gram fresh weight. After thawing, the homogenate was centrifuged at 10,000g for 30 min to remove cell debris. The supernatant is referred to as crude extract. The following operations were performed at 4°C. The crude extract was cleared by filtration through a 200 pum glassfiber filter. The filtrate was concentrated and desalted using a tangential flow ultrafiltration unit (Provario, PAL-

Filtron, Breda, The Netherlands) equipped with a 30 kD cut-off membrane. To the desalted extract solid ammonium sulfate was added to 30% saturation. After stirring for 20 min the precipitated protein was removed by centrifugation at 10, 000g for 30 min. Additional ammonium sulfate was added to the supernatant up to 60% saturation. The precipitated protein was collected by centrifugation at 10,000g for 30 min. The pellet was dissolved in 50 ml buffer A {2CnmM triethanolamine-

HCl pH 7.5, 10% (v/v) glycerol, 1 mM DTT and 0.2 mM PMSF], and solid

KCl was added to a final concentration of 2M. Ammonium sulfate precipitation yielded good and reproducible fractionation without ’ substantial loss of ICS activity. After centrifugation at 13,000g for 15 min, the supernatant was applied to a Phenyl Sepharose CL-4B column (72 ml, 2.6 x 13.5 cm) equilibrated in buffer B (A buffer + 2M KCl).

After washing the column with 300 ml buffer B, ICS activity was eluted with a 700 ml linear gradient from buffer B to A, followed by 150 ml buffer A, with a flow of 1 ml/min. Fractions of 10 ml were collected.

Fractions containing ICS activity were pooled and concentrated using the ultrafiltration unit. The concentrate was desalted by gel : filrration over Sephadex G-25 columns (PD-.C cc_umns, Fharmacia,

Uppsala) equilibrated in buffer A and applied to a 20 ml BlueA column.

After application the flow was stopped for one half-hour to allow binding. The column was washed with 40 ml buffer A, and ICS was eluted with a 160 ml gradient from buffer A to 50% buffer B. Dye affinity chromatography on a Blue A column proved to be a crucial purification step, which resulted in a 15-fold increase in specific activity.

Fractions containing ICS activity were pooled, concentrated and desalted on PD-10 columns equilibrated with buffer C (20 mM triethanolamine-HCl pH 8.0, 5% (v/v) glycerol and 1 mM DTT) . The deslated sample was applied to a MonoQ HR 5/5 column equilibrated in puffer C. The column was washed with 16 ml buffer C and ICS was eluted with a 80-ml linear gradient from buffer C to D (buffer C + 0.5 M

KCl). The flow was 0.5 ml/min and fractions of 0.5 ml were collected.

On this column, ICS activity was eparated into two peaks (ICS I and

ICS II). The specific activities were increased 532- and 754-fold relative to the crude extract for ICS I and II, respectively. ICS I and II had an activity ratio of 1 to 2, a number that was found in several independent purifications. Re-injection of either ICS resulted in the occurrence of only the injected ICS in the chromatogram.

Native PAGE of the MonoQ fractions showed that ICS I still contained some impurity whereas ICS II was obtained in a pure form. SDS-PAGE of 1Ccs II revealed that this protein is about 67 kD.

Biochemical characterization

Both isoforms showed an identical pH dependency with a broad pR optimum between 7.0 and 9.0, and 50% of the maximal activity at pH 6.5 and 10. The presence of Mg? was essential for product formation.

Separate incubations with divalent ions Mg?* other than Mg?* in a concentration of 10 mM did not sustain enzyme activity of either isoform. ICS activity of both isoforms was not inhibited by the presence of tyrosine, phenylalanine or tryptophan in the assay mixture. . Both isoforms showed Michaelis-Menten kinetics for chorismate. The Km values for chorismate were 558+5 pM and 31941 pM for ICS I and II, respectively. Typical saturation curves were obtained for the enzyme ) activity of both isoforms as a function of Mg®? concentration. The saturation curves for Mg?" followed Michaelis-Menten kinetics with Km values of 1.27+0.36 mM (ICS I) and 1.63%#0.12mM (ICS ITI).

EXAMPLE 2

Cloning of the ICS gene from Cathara thus roseus

The protein band containing ICS II was isolated from a native PAGE gel and digested with trypsin, which yielded about 50 peptides. Five peptides were isolated and sequenced. One of these peptides displayed high homology to bacterial isochorismate synthase sequences. . Therefore, a degenerate primer was developed against this peptide. PCR on a cDNA library of elicited cell cultures of C. roseus using this primer and the T7 primer of pBluescript yielded a fragment of 520 bp.

This fragment was cloned and sequenced. B 440 bp fragment of the amplified DNA was used to screen the cDNA library of elicited C. roseus cell cultures. Screening of 450,000 plaques identified 52 independent positive plaques. Twelve of these were isolated and subjected to a second screening using the same 440 bp probe. This resulted in the identification of 7 independent positive plaques.

These were in vivo excised and partially sequenced. The longest clone had inserts of 2.1 kb and contained the ATG initiation codon. The region around the first ATG (TCCAATGGC) closely resembles the consensus translation initiation sequence in plants (Litcke et al.

EMBO J., 6, 43-48, 1987). The cDNA with a complete length of 2081 bp contained an open reading frame of 1743 nucleotides encoding a protein of 581 amino acids. The calculated molecular mass was 64 kD and the isoelectric point 7.88. The protein is roughly 30% identical (40%

homologous) with isochorismate synthases from bacteria with most homology in the C-terminal region.

Construction of a plasmid containing ICS under control of a heterologous promoter.

The ICS cDNA was cloned between the EcoRI and Xhol sites in pBluescript II SK(Stratagene, CA USA). A 2 kbp BamHI-Xhol fragment containing the entire cDNA was ligated into the vector pIC-20H, digested with BglII and Sall. A further partial HindIII digestion released the 2 kbp fragment, and this was cloned into vector pMOGB43 : digested with HindIII. This places the ICS coding ssguaences COWLLZTISER from the 35S CaMV promoter and preceding the potato PI-TI terminator

Is sequences. The plasmid is named pMOG843-ICS.

Construction of a binary vector containing the ICS exprassion cassette.

First, a 355 CaMV promoter-GUS-nos terminator cassette was introduced into binary vector pMOG22. This was done by digestion of pMOG101 with ¥bal and EcoRI which releases the 2.6 kbp fragment containing the expression cassette, and ligation of this fragment into pMO0G22 digested with Xbal and EcoRI. The resulting vector is pMOG22-GUS.

Subsequently the ICS expression cassette was cloned into pMOG22-GUS.

This was done by partially digestion of pMOGB43-ICS with Xbal and ligating the 3.2 kbp fragment into pMOG22-GUS digested with Xbal. The resulting plasmid is pMOG22-GUS-ICS.

The binary vector pMOG22-GUS-ICS was mobilized into Agrobacterium tumefaciens strain LBA4404 using tri-parental mating. Tobacco transformation was performed essentially as described (Horsch et al.,

Science 227, 1229-1231, 1985) using hygromycin as a selectable marker.

EXAMPLE 3

EntC/orfD constructs

The entC coding sequence (Ozenberger et al., J. Bacteriol. 171, 775- 783, 1989) was isolated using a PCR strategy on E. coli genomic DNA.

For this purpose primers 1 (SEQ ID NO: 1) and 2 (SEQ ID NO: 2) were . used. These amplify the entire coding region of entC, and add an extra

BamHI site to both ends. This fragment was cloned into vector pMOG843 in which a BamHI site was introduced via an adapter sequence into the the HindIII site. The resulting pMOG834B-entC contains the entC coding sequences coupled to the 35S CaMV promoter and followed by a potato

PI-II 3’ untranslated sequence. The 35S-entC-PI cassette was then . mobilized into pIC20H by Xbal digestion and cloning into the Xbal site . of pIC20H. A partial Xbal digest of pIC20H was used. Therefore, the cassette is in the Xbal site flanked by the EcoRV site. The resultant vector is denoted pIC20H~entC

A chloroplast transit peptide (denoted ss) (Mazur and Chui, Nucl.

Acids Res. 13, 2373-2386, 1985) was isolated from tobacco genomic DNA using primers 3 (SEQ ID NO: 3) and 4 (SEQ ID NO: 4).

Primer 3 contains a KpnI site that was used to introduce the transit peptide in front of the entC gene. Vector pIC20H-entC was digested with NcoI, followed by blunting of the sticky sites, and then digestion with KpnI. The PCR product was digested with KpnI and cloned into this vector. The resulting vector pIC20H-entC+ss contains the transit peptide in frame with the entC coding sequences, lacking the first 36 nucleotides of the coding sequence of entC. The encoded truncated entC is still fully active.

The orfD sequence from Pseudomonas fluorescens was amplified using primers 5 (SEQ ID NO: 3) and 7 (SEQ ID NO: 7). In order to make a fusion of orfD coding sequences with the transit peptide coding sequences primers 6 (SEQ ID NO: 6) and 7 were used. The Rubisco chloroplast targeting signal was amplified from tobacco genomic DNA using primers 3 and 8 (SEQ ID NO: 8).

The amplified fragments obtained with primersets 6/7 and 3/8, respectively, were digested with NdeI, ligated, and re-amplified using primersets 3 and 7. The resulting PCR fragment has the orfD sequences fused in-frame to the transit peptide. This is denoted as orfD+ss.

Both the orfD and the orfD+ss PCR products were digested with Kpnl and

BamHI and ligated into pMOG843B digested with KpnI and BamHI. The resulting expression cassettes were mobilized into binary vector pMOG800 using the EcoRI and Xbal sites. This resulted in two binary vectors denoted pMOG800-orfD and pMOG800-orfD+ss.

Finally, entC sequences were added. Vector pIC20H-entC+ss was digested with XbaI and Scal and the Xbal fragment containing the entC+ss ) expression cassette was ligated into Xbal digested pMOG800, pMOG8Q0- orfD and pMOG800-orfD+ss to make the following constructs: pMOGBQ0-entC+ss pMOG800-entC+ss + orfD pMOG800-entC+ss + orfD+ss.

All five binary vectors were transformed into Agrobacterium rumefaciens strain LBA 4404 by electrotransformation. Transformation : ir tobacco (Samsun NN) was pesrformec as described using kanamycin selection.

Example 4

Analysis of transformants, enzyme activities

Transgenic Samsun NN tobacco plants were grown under 16 h light regimes at 23-25°C. Leaf samples of these primary transformants were harvested and kept at -80°C until further use.

For determination of enzyme activities of isochorismate synthase and isochorismate pyruvate lyase extract from plants carrying the entC + orfD construct and the entC + orfD+ss construct. Protein extracts were made essentially as decsribed (Moreno et al., Plant Cell Rep. 14, 188- 191, 1994), using 2.5 g of leaf material and 2.5 ml extraction buffer.

Deslating was done using a buffer containing 100 mM Tris-Cl (pH=7.5) supplemented with 1 mM DTT.

Isochorismate synthase activity was measured as described (Poulsen et al., Phytochem. 30, 2873-2876, 1991). A fluorescence detector and integrator were linked to the HPLC to allow quantification of SR (see below). The emission wavelength detector was set at 407 nm, the excitation wavelength is 305 nm.

Incubation of both extracts with Ba-chorismate leads to formation of isochorismate and SA, indicating that the enzymes are produced in an active form, irrespective of the presence of the transit peptide.

Analysis of transformants, salicylic acid accumulation.

Three primary transformants made with each of the constructs were . analysed for the accumulation of both bound and free SA. As positive and negative controls, respectively TMV-infected (2 days after infection) and untreated Samsun NN tobacco plants were included.

A modified version cf the protocol described by Meuwly and Metraux (Anal. Biochem. 214, 500-505, 1993) was used. Approximately 0.5 g of leaf tissue was ground in liquid nitrogen, and extracted using 1 ml . 90% methanol by incubation in a sonicator bath for 5 minutes. Then the mixture was centrifuged for 5 minutes in a table centrifuge at 13,000 rpm. The supernatant was removed and the pellet re-extracted with 0.5 ) ml 100% methanol using the procedure as outlined above. The supernatant fractions were then combined and dried down. The residue was resuspended in 250 pl 5% TCA in water, spun down, and the supernatant was collected and extracted twice with 800 nl ethylacetate: cyclohexane (1:1, v:v), after which the organic phase was dried down. The residue was then dissolved in 400 pl 0.1 M Na-acetate puffer (pH=5.0), containing 10% methanol. Before injection onto the

HPLC column, the sample was centrifuged briefly and the supernatant transferred to a new tube. To determine the amount of SA-glucoside the aqueous phase from the ethylacetate:cyclohexane extraction was acidified by adding an equal volume of BM Hcl. Then the mixture was incubated at 80°C for 1 hour. After this acid hydrolysis, the SA was extracted using ethylacetate:cyclohexane processed for HPLC analysis as described above.

Twenty ul of sample was injected onto the column. A reverse-phase

Lichrospher 60 RP-Select B (5pm) 125 mm x 4 mm column was used (Merck,

Darmstadt, Germany). In a first test run a Shimadzu fluorescence HPLC monitor RF-530 and Chrompack K-001 integrator were used to quantify SA levels. In a second test run a Shimadzu fluorescence HPLC monitor RF- 10Ax1 and Chrompack K-001 integrator were used.

The HPLC eluens is 0.1 M acetate buffer {(pH=5.0), 10% methanol. The flow rate employed was 0.9 ml/ minute.

The results are listed in Table 1. Substantial accumulation of bound salicylic acid (SA) is observed in plants containing entcggtorfd and entcggtorfdgs . In plants containing the latter constructs even free SA was detected, albeit at low levels. Some increase in bound SA was seeil in plants transformed with only the enfcs;. No free SA was observed when orfd alone was transformed.

TABLE 1:

SA accumulation in 1 gram of leaf material of primary transformants, free SA and after acid hydrolysis. o_o plants that have been ug free SA/g ug freeSA/g pg bound SA/ pghound SA / analysed leaf material leaf material g leaf material g leaf material

RF-10Ax1 RF-530 RF-10AxI RF-530 entc, 15 - - 0.09 - entc 5 - = - 0.14 entc,, 14 - - - - orfd 18 - - 0.87 - ’ orfd 4 - - 0.20 B orfd 9 - - - _ orfd, 10 - - - . orfd,, 22 - - - _ orfd,, 1 - - 1.01 . entc + orfd 4 - - - + 0.01 entc, + orfd 11 - - 0.80 0.84 entc+ orfd 13 - - 0.08 0.43 entc + orfd 4 0.25 - 0.41 0.18 entc + orfd, 16 0.93 = 0.01 6.46 7.36 entc + orfd, 20 0.37 - 4.39 6.10

TMY infected 1 1.01 042 6.51 8.32

TMV infected 2 0.75 6.22 control tobacco 1 - - 0.48 - control tobacco 2 - - -

P12 - nt 0.97 nt

Example 5

Infection assay of transgenic tobacco plants with Tobacco Mosaic Virus (TMV)

Transgenic tobacco plants transformed with the bacterial entC and/or orfD constructs (described in example 3 and 4) were tested for their . ability to inhibit spread of a plant pathogenic virus. Three plants per construct, 8 plants per line and 3 leaves per plant were jnoculated with a suspension containing 1 ug/ml TMV. As a control, tobacco transgenic P12 plants were included in this assay. Inoculation was done by rubbing the plants with carborundum powder and the virus suspension. After inoculation the leaves were rinsed with water to remove the carborundum powder again. Lesion size (8 lesions per leaf) was measured at 2, 4 and 7 days after inoculation.

The data were analysed and processed using a one way ANOVA test (a = 0.05, SPSS). The lesion size in the plants is expressed as the percentage of the lesion size determined in the tobacco P12 control plants.

Table 2. Representation of the lesion diameter measured in the transgenic tobacco plants relative to the lesion diameter measured on the P12 control plants.

Plant line T=2 T=4 T=7

EE

P12 100 100 100 entc+orfd, 4 57.3 44.6 45.7 entc+orfd, 16 53.3 36.4 37.7 entc+orfd,20 56.7 40.8 51.8 entc+orfds 94.3 94.3 90.1 entc+orfdll 84.0 85.7 89.4 entc+orfdl3 90.7 87.6 88.5 entch 97.6 101.4 82.7 entcs 121.8 91.7 85.6 entcl3 110.1 96.2 93.3 orfds 107.9 99.5 93.4 orfdli8 105.2 99.9 95.5 orfdl 135.3 93.9 86.7 orfd, 16 96.6 $0.7 96.7 orfd, 22 95.9 84.4 78.8 orfd, 10 96.8 84.7 B6.5 oe —————————————

Infection assay of transgenic tobacco plants with powdery mildew (0idium lycopersicon).

Based on the SA levels measured, tobacco primary transformants were selected for analysis of increased resistance to fungal infection. The following lines were selected: entc+orfd, 4, entc+orfd,16 and entc+orfd, 20. Next to these lines also non-transgenic control lines . {wt /Nt/ssnn-1 and -2) were included in the assay. Plants of 6 weeks old, 7 or 8 plants per line were taken. Plants originating from primary transformant entc+orfdsslé were smaller in size compared to the non-transgenic control plants and the other transgenic lines.

The plants were inoculated with the tomato fungal pathogen Oidium lycopersicon by spraying a spore suspension of 3.5x104 sp/ml (total volume of 400 ml). The plants were tested at a temperature of 20°C, a relative humidity (RH) of 80% and a 16h light/8h dark regime.

Disease severity was determined by measuring the percentage of leaf area covered by powdery mildew. Disease severity was scored at 13 days, 18 days and 24 days after inoculation.

Table3. Disease severity represented in the percentage of infected leaf area of the transgenic tobacco lines with entc+orfdss measured at 13, 18 and 24 days after inoculation (dai) . plant line disease disease disease severity (%) severity (%) severity (%) entc+orfd;4 o* o* ox 5 40 60 ox o* o* 5 10 20 <5 0 0 5 10 40 5 20 20 0 0 <5 entc+orfd,,16 5 30 50 5 30 50 10 50 40 5 35 50 10 50 40

’ 5 20 40 40 40 5 40 40 entc+orfd, 20 <5 20 60 <5 40 60 <5 40 60 <5 - - 3 50 50 5 <5 50 40 50 . wt/Nt/ssnn-1 5 30 40 50 40 5 20 30 5 20 50 : 5 40 30 5 30 50 5 40 50 10 25 40 wt/Nt/ssnn-2 10 40 60 10 40 70 10 25 60 5S 60 50 10 50 40 <5 - - 5 50 40 10 40 70 note: - = plants died during the test. * = plants have necrotic spots on leaves 5 The T1 progeny of the transgenic lines were not selected for the presence of the genes of interest. So the population tested may have segregating T-DNA loci and therefore also segregation of resistance can be observed (as in line entctorfdgs4). 10 EXAMPLE 6

Induction of PR gene expression

RNA was isolated from 0.5 gram of leaf material. The RNA was extracted by grinding the leaf material in liquid nitrogen and 15 extraction with 0.5 ml of a buffer containing 0.35M glycin, 0.048 M

NaOH, 0.34 M NaCl, 0.04 M EDTA and 4% SDS. The preparation was extracted subsequently with water saturated solutions of phenol/chloroform (1:1, v:v), phenol and phenol/chloroform. To the aqueous phase half a volume of 8M LiCl was added and the sample was stored overnight at 4°C. After centrifugation, the pellet was washed with 70% ethanol and dissolved in water.

Ten pg of RNA of each sample was glyoxylated for 1 hour at 50°C and run on a 15 mM Na-phosphate 1.5% agarose gel in 15 mM Na-phosphate buffer (pH=6.5) at 7 V/cm. Anode and cathode buffers were mixed regularly.

The gel was blotted upon Eybond-N+ nylon transfer membrane, cross- linked and baked for 2 hours at 80°C (see fig. 2).

A 450 bp PstI fragment was used as a PRla probe.It was labeled by random-prime labeling using 32P-dCTP. The blot was hybridized : overnight and suosequently washed with 2 x SSC, 01.% 3D3 az ci~C.

Exposure was for 3 days at -80°C, using an intensifier screen.

Procedures are as described in Feinberg and Vogelstein, Anal. Biochem 137, 266-267, 1984; Cornelissen, B. et al., Nucl. Acids Res. 17, 67993- 6811, 1987; Payne et al., Plant Mol. Biol. 11, 89-94, 1988; Pfitzner et al., Mol. Gen. Genet. 211, 290-295, 1988.

Table 4. Qualitative synthesis of SA and expression of PR-la in vitro and in transformed and control tobacco Samsun NN plants. i vitro Sa glucoside | expression

PET REE

PS EU NE EE

Pr EE RE I I cores comer |e | - | 0. | - (contest septs | wa | - | = | -

Results are shown in Table 4. In TMV-induced plants and in transformed plants containing entCtss + orfD+ss accumulation of PR-1la transcript is apparent.

Example 7

Infection assays in Cathara thus roseus with Phytophthora cactorum.

- C. roseus plants of about 50 cm in heigth were inoculated by laying a small droplet (15-20 pl) of a P. cactorum hyphal suspension on a small cutting of 0.5 cm made in the leaf to enable the fungus to § penetrate.

Fungal infection was allowed to proceed at 18°C and a high relative humidity (+ 90%). Leaf disks (diameter = 13 mm) containing the site of : infection were harvested at 48 hours after inoculation and 6 days after inoculation. Control leaf disks were harvested in non-infected i 10 leaf tissue 48 hours after inoculation and in the non-infected area of inoculated leaves.

RNA extraction from infected leaf tissue and cDNA synthesis.

Poly-A+ RNA was harvested from 100 mg of leaf tissue using the

Quickprep Micro mRNA purification Kit (Amersham Pharmacia Biotech,

Uppsala, Sweden). The relative amount of MRNA was determined using visualisation of nucleic acids by spotting 10 ul of the samples with 4 pl 1 pg/ml ethidium bromide on a UV illuminator.

Equal amounts of Poly-A+ RNA (+ 100 ng) were used to synthesize cDNA using 200 units of Superscript II RT RNAse H- reverse transcriptase (Gibco BRL) and 1 nl oligo(dT) 12-18 primers (500 ng/ml, Gibco BRL) as described by ihe manufacturer.

Construction of PCR MIMIC and analysis of samples by competitive RT-

PCR.

For the construction of the PCR MIMIC which served as a competitor in the cRT-PCR experiments the following primers were developed; FR-pUC- 257 (SEQ ID NO: 8) 5’ ATA GAA ACG AGG ACA CTT CCA CGT TAA GGG ATT TTG

G 3’, FR-pUC- 258 (SEQ ID NO: 10) 5' ATA AGC ACG GAT TAA TGG GCC GGA

GCT GAA TGA AGC C 3', FR-ICS-255 (SEQ ID NO: 11) 5’ ATA GAA ACG AGG

ACA CTT CC 3’ and FR-ICS-256 (SEQ ID NO: 12) 5’ ATA AGC ACG GAT TAA

TGG GC 3’. Primers FR-pUC-257 and FR-pUC-258 were used to amplify a fragment of 527 bp from the plasmid pUC18 (Yanisch-Perron, C. et al.,

Gene 33, 103-119, 1985) by PCR. From this PCR product 1 nl was amplified using primers FR-ICS-255 and FR-ICS-256 by PCR to produce a large amount of PCR MIMIC.

PCT/EP99/02176

Primers FR-ICS-255 and FR-ICS-256 will amplify a band of 443 bp from the ICS cDNA so it can be distinguished easily from the 527 bp MIMIC band when seperated on a 1.5% agarose gel.

PCR MIMIC dilutions were made in a range of 10 ng/pl to 0.1 ag/ul in

H20 containing 0.2 pg/nl glycogen as a carrier.

The cDNA samples were analysed in a competitive PCR. Therefore 2 nl of the cDNA samples was combined in a 0.5 ml tube with 1 pl diluted MIMIC (amounts: 0.1 pg, 10 fg, 1 fg and 0.1 fg) or no MIMIC. Amplification of cDNA and MIMIC was performed using 10 uM of the primers FR-ICS-255 and FR-ICS-256, 0.5 pl of 20 mM dNTP’s, 1x PCR buffer, MgCl2 and 2.5 } 4anits Recombinant Tag DONA polymerase (Gibco BRL. ant was a2lioMel Ts proceed for 35 cycles, 1° g5°c, 1’ 55°C, 2' 72°C.

Table 5: Induction levels of the ICS messenger after infection of

C. roseus leaves with P. cactorum relative tc the control. ee ——————————————————————————————

Sample fold induction ee ——

Control 1 48 hrs after inoculation >1002 6 days after inoculation 10%

Uninfected leaf area” 1 ee notes: 2: Fold induction compared to control. ». yninfected leaf area is the area of the inoculated leaf not infected by the fungus.

Example 8

Isolation of the isochorismate synthase promoter from Catharanthus roseus by iPCR

PCR primers were developed based on the sequence of the ICS cDNA (SEQ

ID NO: 18). Primers FR-ICS-259 57766 TGA TCC ARG AGC TCC GG3’ (SEQ ID

NO: 20) and FR-ICS-260 5’CCT GGT TGA AAG GTC TGT G3’ (SEQ ID NO: 21) for iPCR and primer FR-ICS~-261 5'GCA ACA CAA TGC CCT GTG3’ (SEQ ID NO: 22) for nested PCR.

C. roseus genomic DNA was isolated using a CTAB DNA extraction procedure. The gencmic DNA was subjected to restriction enzyme digestion with five different enzymes, Dde I, Kpn I, Msc I, Nco I and ; Nla IV. After restriction enzyme digestion the DNA was extracted with phenol /chloroform/isocamylalcohol and precipitated with ethanol. The

DNA pellet was dissolved in 50 pl water and 25 nl was used for furer iPCR. For this purpose the volume of the digested DNA mixture was increased to 300 nl in 1x ligase buffer (Gibco BRL) with 5 units of T4

DNA ligase (Gibco BRL). This mixture was incubated at 16°C for 16 - hours. After ligation the DNA was again extracted with phenol/chloroform/isoamylalcohol and precipitated with ethanol and dissolved in 50 pl water. 2 pl of this mixture was used as a template in a PCR reaction with 150 ng primers FR-ICS-259 and FR-ICS-269, 1x Klentaqg PCR buffer, 10 uM dNTP’s and 1.0 pl 50x Advantage cDNA polymerase mix (Clontech, Palo

Blto, CA, USA}. The complete reaction mixture was subjected to 1' at 94°C and 30 cycles of 30” 94°C, 1’ 55°C, 3’ 68°C. Then 1 pl of the reaction was used for nested PCR. Therefore a similar procedure was followed but primer FR-ICS-260 was replaced by primer FR~ICS-261. The results of the iPCR are listed in Table 6.

Table 6. Bands obtained after iPCR with five different enzymes ee ——————————————

Restriction enzyme iPCR band size (bp) ee —

Dde I 100

Kpn T 900

Msc I -

Nco I 3.000

Nla IV S00 ere etree

The resulting PCR bands from the Kpn I, Nco I and Nla IV digestions were cloned into the T/A cloning vector pGEM-T (Promega). The DNA sequences of the inserts were determined.

Example 9

Isolation of the ICS promoter by direct PCR

New PCR primers were developed based on the DNA sequence of the cloned

PCR fragments. These primers were located at the far upstream part of the promoter and at the ATG translational startcodon of the ICS open reading frame. Primer FR-ICS-295 5’/GCA AGC TTC ATG TAC CTT ATC TTG

GCC3’ (SEQ ID NO: 23) is located at the upstream end of the promoter and introduces a Hind III restriction site and primer FR- ICS-296 ’ 5’ TAG ATG CCA TGG GAT GGG AG3’ (SEQ ID NO: 24) is located at the startcodon of the ICS ORF introducing a Ncec I restriction site overlapping the ATG translational start.

These primers (150 ng) were used in a PCR reaction on C. roseus genomic DNA in 1x Klentag PCR buffer, 10 pM dNTP's and 2.0 pl 50x

Advantage cDNA polymerase mix (Clontech, Palo Alto, CA, USA). The complete reaction mixture (100 ul) was subjected to 1’ at 94°C and 30 cycles of 30" 94°C, 1’ 55°C, 4’ 68°C. A band of the correct size (3.0

Kb) was isolated from arn agarose gel, purified and cloned using restriction enzymes Hind III and Neco I inte a high copy cloning vector based on puUCl8 (Yanisch-Perron, C., Vieira, J. and Messing, J. (1985)

Gene 33, 103-119) forming plasmid pMOG 1431. The DNA sequence of the complete promoter fragment was determined using automated DNA sequencing (SEQ ID NO: 25 ). The promoter was cut out using Hind III and Neco T and ligated inte a Hind III, Nco I digested cloning vector containing GUSintron (Jefferson et al., (1987) EMBO J 6: 3901-3907) followed by the 3' untranslated region of the potato proteinase inhibitor II gene (Thornburg et al., 1987, Proc. Natl. Acad. Sci. UsA 84, 744-748) which contains sequences needed for polyadenylation (An et al., 1989, Plant cell 1, 115-122). The expression unit was then transferred to binary vector pMOG800 (deposited at the Centraal Bureau voor Schimmelcultures, Baarn, The Netherlands, under CBS 414.93, on august 12, 1993) using restriction enzyme Xho I. Using Xho I 2.0 kb of actual the 3.0 kb promoter was transferred to the binary vector. A clone was selected with the entire expression unit in the correct orientation e.g. the promoter on the T-DNA next to the right border repeat sequence. The resulting plasmid was designated pMOG1433.

Example 10

Transformation of the ICS promoter-GUS binary vector to potato pMOG 1433 was transformed to potato essentially as described by

Hoekema et al. (Hoekema, A. et al., Bio/Technology 7, 273-278, 1989).

In short, potatoes (Solanum tuberosum cv. Kardal) were transformed with the Agrobacterium strain EHA 105 pMOG 1433. The basic culture medium was MS30R3 medium consisting of MS salts (Murashige and Skoog

(1962) Physiol. Plant. 14, 473), R3 vitamins (Ooms et al. (1987) ] Theor. Appl. Genet. 73, 744), 30 g/l sucrose, 0.5 g/l MES with final pH 5.8 (adjusted with KOH) solidified when necessary with 8 g/l

Daichin agar. Tubers of Solanum tuberosum cv. Kardal were peeled and surface sterilized by burning them in 96% ethanol for 5 seconds. The flames were extinguished in sterile water and cut slices of approximately 2 mm thickness. Disks were cut with a bore from the . vascular tissue and incubated for 20 minutes in MS30R3 medium containing 1-5 x10° bacteria/ml of Agrobacterium EHA 105 containing the binary vector. The tuber discs were washed with MS30R3 medium and transferred to solidified postculture medium (PM). PM consisted of

M30R3 medium supplemented with 3.5 mg/l zeatin riboside and 0.03 mg/l indole acetic acid (IAA). After two days, discs were transferred to fresh PM medium with 200 mg/l cefotaxim and 100 mg/l vancomycin. Three days later, the tuber discs were transferred to shoot induction medium (SIM) which consisted of PM medium with 250 mg/l carbenicillin and 100 mg/l kanamycin. After 4-8 weeks, shoots emerging from the discs were excised and placed on rooting medium (MS30R3-medium with 100 mg/l cefotaxim, 50 mg/l vancomycin and 50 mg/l kanamycin). The shoots were propagated axenically by meristem cuttings.

Example 11

Testing of promoter function in transgenic potato plants

Transgenic potato plants harbouring the pMOG1433 ICS promoter-GUS construct were grown in tubes in vitro and assayed for expression of the GUS gene. For this purpose leaf, stem and root samples were taken and stained (results in table 7). GUS expression levels were determined visually, on a scale of 0 to 5, where 0 is no detectable expression and 5 is the highest level of GUS we have observed in leaves of a transgenic plant, of a rare tobacco 355~-GUS—-transgenic (line 96306). Samples from leaves of this plant were included in all experiments for internal reference.

Table 7: Expression of the GUS gene driven by the ICS promoter in leaves, stems and roots of small in vitro plantlets.

Plant number Leaf Stem Root ee ———————————————————— eee

PCT/EP9Y99/02176 1433-1 0 [0] 0 1433-2 [0] 6] 0] 1433-3 [0] [9] 0 1433-4 lo] 0 0 : 1433-5 0 0 0 1433-6 0 0 0 1433-7 0 0 0 1433-8 1 1 1 1433-9 [o] 0 0 1433-10 [0] 0} 0 1433-11 0 0 0 1433-12 0 0 0 1433-13 0 0 0 ’ 1433-14 0 0 9] 1433-15 1 0 0 1433-16 [0] 0 1 1433-17 0 0 0 1433-18 [0] 0 C 1433-18 0 0 0 1433-20 [4] 0 0 1433-21 0 0 0 1433-22 0 0 0 1433-23 [o] 0 0 1433-24 1 2 1 1433-25 1 2 1

In vitro plantlets of the same age were infected with the potato late blight causing fungus Phytophthora infestans. Small droplets of water : containing a high concentration of fungal spores were applied on the leaf surface. The infection was left to proceed at room temperature for 96 hours. Leaves which showed disease symptoms were removed from the plantlets and stained for expression of the GUS gene by histochemical GUS analysis (Goddijn et al., The Plant Journal (1993) 4(5): 863-873). Results are listed in table 8.. Expression was monitored in the lesion resulting from the fungal infection, in the primary zone (the area just around the site of infection) and in the uninfected part of the leaf (background) .

Table 8: Expression of the GUS gene driven by the ICS promoter in leaves of potato in vitro plantlets infected by P. infestans

Plant number before lesion primary background infection zone 1433-1 0 0 1 0 1433-2 0 0 1 0 1433-3 0 0 0 0 1433-4 0 0 0 0 1433-5 0 0 1 [o} 1433-6 0 0 1 0

1433-7 0] 0 0 0 1433-8 1 0 1 0 . 1433-9 0 0 0 0 1433-10 0 0* 0% ox 1433-11 0 0 0 0 1433-12 0 [+] 1 0 1433-13 0 0 0 0 1422-14 ¢} 0 0 0 1433-15 1 0 1 0 1433-16 0 0 2 0 1433-17 0 0 1 0 . 1433-18 0 0 1 0 1433-19 0 0 1 0 1433-20 0 ] 1 0 1433-21 0 0 1 0 1433-22 0 0 1 0 1433-23 0 0 1 0 1433-24 1 ox ox O* 1433-25 1 0 o} 0

Note: * = plants not infected/no disease symptoms visible promoter performance was also tested in the leaves of full grown potato plants before and after infection with P. infestans. Before

S inoculation leaves were detached and stained for expression of GUS.

The plants were then sprayed with a spore suspension of 5x105 spores/ml and the infection was allowed to develop for 4 days (96 hours). Again leaves were detached and stained for the expression of

GUS. GUS expression levels were scored in the lesion, primary zone and in the uninfected part of the leaf (background). The results are listed in table 9.

Table 9: Expression of the GUS gene driven by the ICS promoter in leaves of transgenic potato plants before and after infection with P. infestans. ee

Plant number before lesion primary background 1433-1 1 4 3 1 1433-2 1 0 2 0 1433-3 1 4 2 Ls 1433-4 1 0 0 0 1433-5 1 2 2 0 1433-6 1 0 2 1 1433-7 1 0 2 0 1433-8 3 0 2 2 1433-10 1 4 3 1 1433-11 D C 0 = 1433-12 1 4 4 1 1433-13 1 0 0 0 1433-14 1 0 0 0 1433-15 1 0 2 1 1433-16 1 4 4 1 1433-17 1 0 4 1 1433-18 2 4 4 0 1433-19 2 0 4 t 1433-20 1 2 3 1 1433-21 1 4 4 0 1433-22 1 2 2 1 1433-23 1 0 4 1 1433-24 3 2 3 :

JE SE SE SU —

PCT/EP99/02176

BUDAPEST TREATY ON THE INTERNATIONAL

RECOGNITION OF THE DEPOSIT OF MICROORGANISMS

FOR THE PURPOSES OF PATENT PROCEDURE

TNTERNATICNAD IoRM

Mogen international N.V. RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT

N Einsteinweg 97 issued pursuant to Rule 7.1 by the 2333 CB LEIDEN INTERNATIONAL DEPOSITARY AUTHORITY

Nedertand identified at the bottom of this page name and address of depositor

I. IDENTIFICATION OF THE MICROORGANISM

Identification reference given by the Accession number given by the

DEPOSITOR: INTERNATIONAL DEPOSITARY AUTHORITY:

E. coli strain DHSalpha pMOG1431 CBS 101670

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The microorganism identified under I above was accompanied by: [x] a scientific description : [] a proposed taxonomic designation (mark with a cross where applicable)

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Mark with a cross the applicable box.

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Authority or of authorized offici ): _— a ~~

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Claims (1)

1. J a PCT/EP99/02176 CLAIMS

   1. Method to induce pathogen resistance in plants, characterized in that plants are transformed with an expression cassette harboring a gene coding for an iscchorismets syniness.

   2. Method according to claim 1, characterized in that the gene coding for isochorismate synthase is selected from a group consisting of entC, orfA, pchA and /CS.

   3. Method according to claim 2, characterized in that the gene coding for isochorismate synthase is the /CS gene from Catharantus roseus.

   4. Method according to claim 2 or 3, characterized in that the gene coding for isochorismate synthase comprises a nucleotide sequence encoding the protein of SEQ ID NO: 14, SEQ ID NO: 16 or SEQ ID NO: 19.

   5. Method according to claim 5, characterized in that the gene coding for isochorismate synthase comprises the nucleotide sequence of the open reading frame of SEQ ID NO: 13, SEQ ID NO: 15 or SEQ ID NO: 18.

   6. Method according to any of claims 1-5, characterized in that plants are additionally transformed with an expression cassette harboring a gene coding for an isochorismate pyruvate lyase.

   7. Method according to claim 6, characterized in that the gene coding for isochorismate synthase and the gene coding for isochorismate pyruvate lyase both are present on the same vector.

   8. Method according to claim 6 or 7, characterized in that the gene coding for isochorismate pyruvate lyase is selected from the group consisting of orfD and pchB. AMENDED SHEET rs 4 t hd 1 - ry PCT/EP99/02176

   9. Method according to claim 8, characterized in that the gene coding for isochorismate synthase is entC and the gene coding for isochorismate syruvets yess is sil.

   10. A protein having isochorismate synthase activity which is isolated from Catharantus roseus (and has an MW of about 67 kD).

   11. A protein according to claim 10, characterized in that it comprises the amino acid sequence of SEQ ID NO: 19.

   12. A nucleotide sequence encoding the protein of claim 10 or 11.

   13. A nucleotide sequence, characterized in that it comprises the nucleotide sequence of SEQ ID NO: 18.

   14. The nucleotide sequence according to claim 12 or 13, characterized in that it also comprises the 5’ regulatory region which is naturally found to regulate the expression of the /CS gene in Catharantus roseus.

   15. A nucleotide sequence comprising the 5’ regulatory region which is naturally found to regulate the expression of the /CS gene in Catharantus roseus.

   16. A pathogen-inducible promoter, characterized in that it comprises the 5’ regulatory region which is naturally found to regulate the expression of the /CS gene in Catharantus roseus.

   17. A pathogen-inducible promoter according to claim 16, characterized in that it comprises a nucleotide sequence from nucleotide 1118 to nucleotide 3275 as depicted in SEQ ID NO: 25. REPLACEMENT SHEET c . Jit 1 PCT/EP99/02176

   18. A pathogen-inducible promoter according to claim 17, characterized in that it comprises a nucleotide sequence from nucleotide 1 to nucleotide 3275 as depicted in SEQ ID NO: 25.

   19. A pathogen-inducible promoter according to claim 16, characterized in that it comprises a nucleotide sequence from plasmid pM0OG1431 (deposited under no. 101670 at the Centraal Bureau voor Schimmelcultures, Baarn, The Netherlands) located between the restriction sites Hindlll and Ncol as shown in fig. 3.

   20. Use of the pathogen-inducible promoter according to any of claims 16- 19 to drive expression of a heterologous protein.

   21. Use according to claim 20, characterized in that the heterologous protein is an antipathogenic protein selected from the group consisting of chitinase, glucanase, osmotin, magainins, lectins, saccharide oxidase, oxalate oxidase, toxins from Bacillus thuringiensis, antifungal proteins isolated from Mirabilis jalapa, Amaranthus, Raphanus, Brassica, Sinapis, Arabidopsis, Dahlia, Cnicus, Lathyrus, Clitoria, Allium seeds, Aralia and Impatiens and albumin-type proteins, such as thionine, napin, barley trypsin inhibitor, cereal gliadin and wheat-alpha-amylase.

   22. Use according to claim 20, characterized in that the heterologous protein is a protein that can induce a hypersensitive response, preferably selected from the group consisting of Cf, Bs3 and Pto proteins from tomato, Rpm1 and Rps2 from Arabidopsis thaliana, N-protein from tobacco, avr proteins from Cladosporium fulvum, harpins from Erwinia and elicitor proteins (avrBs3, avrRpm1, avrRpt2) from Pseudomonas or Xanthomonas.

   23. Vector comprising a nucleotide sequence according to one of claims 11-

   15. AMENDED SHEET

   , * t - PCT/EP99/02176 24, Agrobacterium strain comprising a vector according to claim 23.

   22. Plznt calls registant to 2zthogens, characterized in that they arg transformed with a gene coding for isochorismate synthase.

   26. Plant cells according to claim 25, characterized in that the gene coding for isochorismate synthase is selected from the group consisting of entC, orfA, pchA and /CS.

   27. Plant cells according to claim 26, characterized in that the gene coding for isochorismate synthase is the /CS gene from Catharantus roseus.

   28. Piant cells according to any of claims 25-27, characterized in that they additionally comprise a gene coding for isochorismate pyruvate lyase.

   29. Plant cells according to claim 28, characterized in that the gene for isochorismate pyruvate lyase is selected from the group consisting of orfD and pchB.

   30. Plant cells according to claim 28, characterized in that the gene coding for isochorismate synthase is entC and the gene coding for isochorismate pyruvate lyase is orfD.

   31. Plants comprising plant cells according to any of claims 25-30.

   32. A method according to claim 1, substantially as herein described and illustrated.

   33. A protein according to claim 10, substantially as herein described and illustrated. AMENDED SHEET

   . ; . PCT/EP99/02176

   34. A nucleotide sequence according to claim 12, or claim 13, or claim 15, substantially as herein described and illustrated.

   2c. A cromctsr according to claim 18, eubetantiallyv 2g bargin daecrihed and illustrated.

   36. Use according to claim 20, substantially as herein described and illustrated.

   37. Vector according to claim 23, substantially as herein described and illustrated.

   38. A strain according to claim 24, substantially as herein described and itlustrated.

   39. Plant cells according to claim 25, substantially as herein described and illustrated. 40, A plant according to claim 31, substantially as herein described and illustrated. 41, A new method to induce pathogen resistance, a new protein, a new nucleotide sequence, a new promoter, a new use of a promoter according to any one of claims 16-19, a new vector, a new strain, new plant cells, or a new plant, substantially as herein described. AMENDED SHEET