AU746787B2 - Salicylic acid pathway genes and their use for the induction of resistance in plants - Google Patents

Description

WO 99/50423 PCT/EP99/02176 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 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 substances (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 antipathogen 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 1 pathogens suggesting that SA plays a crucial role in the establishment of SAR (see e.g. Ryals, Plant Cell 8, 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 alternative pathways for induction of the hypersensitive response are present, and overexpression of nahG in tomato does. not compromise the hypersensitive response occurring after challenge of Cf9 or Cf2 plants with Cladosporium fulvum races containing Avr9 and Avr2, respectively. (Hammond-Kosack Jones, Plant Cell 8, 1773-1791, 1996).

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- S1458, 1994) 2a SUMMARY OF THE INVENTION The present invention provides a method to induce pathogen resistance in plants, wherein the plants are transformed with an expression cassette harboring a gene coding for an isochorismate synthase, wherein the gene coding for isochorismate synthase is the ICS gene from Catharantus roseus.

Another aspect of the invention is a pathogen-inducible promoter, wherein it comprises the 5' regulatory region which is naturally found to regulate the expression of the ICS gene in Catharantus roseus.

The invention further provides a vector comprising a nucleotide sequence, wherein the nucleotide sequence is capable of encoding a protein having isochorismate synthase activity which is isolated from Catharantus roseus (and has an MW of about 67 kD).

Another aspect of the present invention is plant cells capable of overexpression of isochorismate synthase, wherein the cells have been transformed with a gene coding for isochorismate synthase, wherein the gene 20 coding for isochorismate synthase is the ICS gene from Catharantus roseus.

S* This invention describes a method to induce pathogen resistance in plants, wherein the plants are transformed with an expression cassette I:2i 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.

W:\llonaShamn\SJJspeci\sp36025 Another embodiment of the invention is a method according to the method described above, wherein the 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 e* *9 0 W:\llona\Sharon\SJJspecisp36025 isochorismate synthase. The gene coding for isochorismate pyruvate lyase is preferably selected from the group consisting of orfD and pchB.

A specific embodiment of the invention is a method as described above, wherein 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 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 nucleotide sequence of SEQ ID NO: 18 oo 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, antibacterial 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.

3* DESCRIPTION OF THE FIGURES Figure 1: Schematic respresentation of the vector pMOG22 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.

WO 99/50423 PCTIEP99/02176 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 2-hydroxylation. In infected plants the synthesis pathway may be altered slightly, using trans-cinnamic acid to convert it into ortho-coumaric acid which is then converted into salicylic acid. In micro-organisms the biosynthesis of salicylic acid is known to proceed from chorismate to isochorismate (catalyzed by the enzyme isochorismate synthase, ICS). Genes required for this conversion have been cloned from Pseudomonas aeruginosa (the pchA gene encoding ICSactivity, Serino, L. et al., Mol. Gen. Genet. 249, 217-228, 1995) and from Escherischia coil (the entC gene encoding ICS activity, Ozenberoer, B.A. et al., 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.

WO 99/50423 PCT/EP99/02176 The genes which can be used in this invention are depicted in SEQ ID NO'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 polynucleotide used for transformation 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 tc 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 a 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 or more, more preferably 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 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, chimeric DNA shall mean to comprise DNA comprising the open reading frame coding for the enzyme in a non-natural location of the plant WO 99/50423 PCT/EP99/02176 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 to 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 more 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 WO 99/50423 PCT/EP99/02176 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 linked to DNA which 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 Tiplasmid 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 B1) 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 Tiplasmid vectors are the so-called binary vectors as claimed in (EP 0 120 516 B1 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 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 7 WO 99/50423 PCT/EP99/02 176 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 constitutive fashion, that is, in substantially most cell types of 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 35S RNA and 19S RNA transcript promoters and the so-called T-DNA promoters of Agrobacterium tumefaciens, in particular to be mentioned are the nopaline synthase promoter, octopine synthase promoter (as disclosed in EP 0 122 791 B1) 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 WO 99/50423 PCT/EP99/02176 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 suitable 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. 19, 297-323) and the alcohol dehydrogenase promoter which is induced by ethanol (Nagao, R.T. et al., in: Miflin, B.J. Oxford Surveys of Plant Molecular and Cell Biology, Vol. 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, et al., Plant, Cell and Env. 18, 865-874, 1995), the Vstl promoter (Fischer, Dissertation, Univ. of Hohenheim, 1994; Schubert, et al. Plant Mol. Biol. 34, 417-426, 1997), the sesquiterpene cyclase promoter (Yin, et al., Plant Physiol. 115, 437-451, 1997) and the gstAl promoter (Mauch, F. and Dudler, Plant Physiol. 102, 1193-1201, 1993) may be mentioned. Of course also the regulatory region of the ICS gene from Catharanthus roseus, which forms part of this invention, may be used in this respect.

The choice of the promoter is not essential, although it must be said 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- WO 99/50423 PCT/EP99/02176 inducible ribulose bisphosphate carboxylase small subunit (rbcSSU) 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 targeting 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 WO 99/50423 PCT/EP99/02176 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, 1985), microinjection into plant 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 4,940,838.

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 WO 99/50423 PCT/EP99/02176 the aged compact and nodular embryogenic 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 B1; 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 number and/or genomic organization. In 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.

WO 99/50423 PCT/EP99/02176 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 to 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.

WO 99/50423 PCT/EP99/02 176 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 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 shall 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 quantity, 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, 9-l,3-glucanases and chitinases which are obtainable from barley (Swegle M. et al., Plant Mol. Biol. 12, 403-412, 1989; Balance WO 99/50423 PCT/EP99/02176 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; V6geli U.

et al., Planta 174, 364-372, 1988); Mauch F. Staehelin Plant Cell 1, 447-457, 1989); cucumber (Metraux J.P. Boiler Physiol.

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, 1984; 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 Nucl. Acids Res. 16, 5210, 1988; Kombrink E. et al., Proc. Natl.

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

Biol. 13, 249-250, 1989), tobacco 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 Plant Physiol. 89, 945-951, 1989), wheat (Molano J. et al., J. Biol. Chem. 254, 4901-4907, 1979), magainins, lectins, toxins isolated 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 as described in WO 91/15585). Such proteins are, for example, plant proteins such as disclosed in Karrer, E.E. et al. (Plant Mol. Biol.

36, 681-690, 1998), ndrl and edsl, Cf-proteins and Pto proteins from tomato, the avr-elicitor proteins from Cladosporium fulvum, and the avrPto protein from Pseudomonas.

A clone harboring plasmid pMOG 1431 containing a 3kb insert which contains the ICS regulatory region according to the inventions WO 99/50423 PCT/EP99/02176 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 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 the art to which this invention pertains.

EP-A 392 225 A2; EP-A 440 304 Al; EP-A 460 753 A2; W90/07001 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 Standard 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 WO 99/50423 PCT/EP99/02176 Laboratory Press; DNA Cloning: Volumes I and II Glover ed.

1985); and in: From Genes To Clones Winnacker ed. 1987).

Assay of isochorismate synthase activity The incubation mixture (total volume 250 pl) contained 0.1 M Tris-HC1 pH 7,5, 2mM chorismate, 10 mM MgCl 2 and enzyme extract (crude extracts 125 pl, column fractions 10-100 pi). The incubation was started by addition of chorismate. After incubation for 60 min at 30 0

C

the reaction was stopped by the addition of 62.5 pl methanolisobutanol (1:1 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/l sucrose 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. (1993) Cells were harvested by suction 24 hours after elicitation, washed once with water, immediately frozen in liquid nitrogen and stored at 0 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 1 mM DTT, 0.2 mM PMSF, 10 pM 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 0 C. The crude extract was cleared by filtration through a 200 pm 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 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 min. The pellet was dissolved in 50 ml buffer A [20mM triethanolamine- 17 WO 99/50423 PCT/EP99/02176 HC1 pH 7.5, 10% glycerol, 1 mM DTT and 0.2 mM PMSF], and solid KC1 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 KC1).

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 filtration over Sephadex G-25 columns (PD-10 columns, Pharmacia, 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% glycerol and 1 mM DTT). The deslated sample was applied to a MonoQ HR 5/5 column equilibrated in buffer 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 KC1). 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 ICS II revealed that this protein is about 67 kD.

Biochemical characterization Both isoforms showed an identical pH dependency with a broad pH optimum between 7.0 and 9.0, and 50% of the maximal activity at pH WO 99/50423 PCT/EP99/02176 and 10. The presence of Mg 2 was essential for product formation.

Separate incubations with divalent ions Mg 2 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 319±41 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 2 followed Michaelis-Menten kinetics with Km values of 1.27±0.36 mM (ICS I) and 1.63±0.12mM (ICS II).

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. A 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 (LUtcke et al.

EMBO 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 WO 99/50423 PCT/EP99/02176 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 XhoI sites in pBluescript II SK(Stratagene, CA USA). A 2 kbp BamHI-XhoI fragment containing the entire cDNA was ligated into the vector digested with BglII and SalI. A further partial HindIII digestion released the 2 kbp fragment, and this was cloned into vector pMOG843 digested with HindIII. This places the ICS coding sequences downstream from the 35S CaMV promoter and preceding the potato PI-II terminator sequences. The plasmid is named pMOG843-ICS.

Construction of a binary vector containing the ICS expression cassette.

First, a 35S CaMV promoter-GUS-nos terminator cassette was introduced into binary vector pMOG22. This was done by digestion of pMOG101 with XbaI and EcoRI which releases the 2.6 kbp fragment containing the expression cassette, and ligation of this fragment into pMOG22 digested with XbaI 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 pMOG843-ICS with XbaI and ligating the 3.2 kbp fragment into pMOG22-GUS digested with XbaI. 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.

WO 99/50423 PCT/EP99/02176 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 XbaI digestion and cloning into the XbaI site of pIC20H. A partial XbaI digest of pIC20H was used. Therefore, the cassette is in the XbaI site flanked by the EcoRV site. The resultant vector is denoted 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: 5) and 7 (SEQ ID NO: 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 KpnI 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 XbaI sites. This resulted in two binary vectors denoted pMOG800-orfD and pMOG800-orfD+ss.

WO 99/50423 PCT/EP99/02176 Finally, entC sequences were added. Vector pIC20H-entC+ss was digested with XbaI and Scal and the XbaI fragment containing the entC+ss expression cassette was ligated into XbaI digested pMOG800, pMOG800orfD and pMOG800-orfD+ss to make the following constructs: pMOG800-entC+ss pMOG800-entC+ss orfD pMOG800-entC+ss orfD+ss.

All five binary vectors were transformed into Agrobacterium tumefaciens strain LBA 4404 by electrotransformation. Transformation in tobacco (Samsun NN) was performed 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 0 C. Leaf samples of these primary transformants were harvested and kept at -80 0 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 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 SA (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.

WO 99/50423 PCT/EP99/02176 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 of 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 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 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 pl ethylacetate:cyclohexane after which the organic phase was dried down. The residue was then dissolved in 400.pl 0.1 M Na-acetate buffer 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 8M Hcl. Then the mixture was incubated at 80 0 C for 1 hour. After this acid hydrolysis, the SA was extracted using ethylacetate:cyclohexane processed for HPLC analysis as described above.

Twenty pl 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 RFand Chrompack K-001 integrator were used.

The HPLC eluens is 0.1 M acetate buffer 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 entcss+ofdand entcss+orfdss.In plants containing the latter constructs even free SA was detected, albeit at low levels. Some increase in bound SA was seen in WO 99/50423 WO 9950423PCT/EP99/02176 plants transformed with only the entess. No free SA was observed when oifd alone was transformed.

TABLE 1: SA accumulation in 1 gram of leaf material of primary transformants, free SA and after acid hydrolysis.

plants that have been pg free SA g pg free SA g ptg bound SA I pg bound SAI analysed leaf material leaf material g leaf material g leaf material RF-lOAxl RF-530 RF-lOAxI RF-530 entc. 15 0.09 entc,, 5 0.14 entc,, 14 orfd 18 0.87orfd 4 0.20orfd 9 orfd,, orfd,, 22 orfd 3 I 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,,+ orfdl, 20 0.37 4.39 6.10 TMV infected 1 1.01 0.42 6.51 8.32 TMV infected 2 0.75 6.22 control tobacco 1 0.48 control tobacco 2- P12 nt 0.97 nt WO 99/50423 PCT/EP99/02176 Example 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 inoculated with a suspension containing 1 pg/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 P12 100 100 100 entc+orfd,4 57.3 44.6 45.7 entc+orfdl6 53.3 36.4 37.7 56.7 40.8 51.8 entc+orfd4 94.3 94.3 90.1 entc+orfdll 84.0 85.7 89.4 entc+orfdl3 90.7 87.6 88.5 97.6 101.4 92.7 entc8 121.8 91.7 85.6 entcl3 110.1 96.2 93.3 107.9 99.5 93.4 orfdl8 105.2 99.9 95.5 orfdl 135.3 93.9 86.7 WO 99/50423 PCT/EP99/02176 orfd,16 96.6 90.7 96.7 orfd,22 95.9 84.4 78.8 orfdl0 96.8 84.7 86.5 Infection assay of transgenic tobacco plants with powdery mildew (Oidium 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+orfd1sl6 and Next to these lines also non-transgenic control lines (wt/Nt/ssnn-1 and were included in the assay. Plants of 6 weeks old, 7 or 8 plants per line were taken. Plants originating from primary transformant entc+orfdssl6 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 0 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 13 dai 18 dai 24 dai entc+orfd,,4 0* 0* 0* 40 0* 0* 0* 10 0 0 10 20 0 0 entc+orfd 6 1l6 5 30 30 50 35 50 WO 99/50423 PCT/EP99/02176 wt/Nt/ssnn-1 wt/Nt/ssnn-2 10 during the test.

necrotic spots on leaves note: plants died plants have 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 entc+orfds4).

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 extraction with 0.5 ml of a buffer containing 0.35M glycin, 0.048 M NaOH, 0.34 M NaC1, 0.04 M EDTA and 4% SDS. The preparation was extracted subsequently with water saturated solutions of phenol/chloroform phenol and phenol/chloroform. To the WO 99/50423 PCT/EP99/02176 aqueous phase half a volume of 8M LiCI was added and the sample was stored overnight at 4 0 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 0 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 Hybond-N+ nylon transfer membrane, crosslinked and baked for 2 hours at 80 0 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 subsequently washed with 2 x SSC, 01.% SDS at 65 0

C.

Exposure was for 3 days at -80 0 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, 6799- 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.

Plants SA synthesis Free SA PR-la i vitro SA glucoside expression e tC+ss nd orfD nd orfD+ss nd e tC+ss orfD e tC+ss orfD+ss control tobacco nd TMV infected nd Results are shown in Table 4. In TMV-induced plants and in transformed plants containing entC+ss orfD+ss accumulation of PR-la transcript is apparent.

Example 7 Infection assays in Cathara thus roseus with Phytophthora cactorum.

28 WO 99/50423 PCT/EP99/02176 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 0 C and a high relative humidity 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 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 pl of the samples with 4 p1 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 p1 oligo(dT)12-18 primers (500 pg/ml, Gibco BRL) as described by the 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: 9) 5' ATA GAA ACG AGG ACA CTT CCA CGT TAA GGG ATT TTG G FR-pUC- 258 (SEQ ID NO: 10) 5' ATA AGC ACG GAT TAA TGG GCC GGA GCT GAA TGA AGC C 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 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 pl was amplified using primers FR-ICS-255 and FR-ICS-256 by PCR to produce a large amount of PCR MIMIC.

WO 99/50423 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/pl in containing 0.2 pg/pl glycogen as a carrier.

The cDNA samples were analysed in a competitive PCR. Therefore 2 p1 of the cDNA samples was combined in a 0.5 ml tube with 1 p1 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 pM of the primers FR-ICS-255 and FR-ICS-256, 0.5 pl of 20 mM dNTP's, Ix PCR buffer, MgC12 and units Recombinant Taq DNA polymerase (Gibco BRL) and was allowed to proceed for 35 cycles, 1' 95 0 C, 1' 55 0 C, 2' 72°C.

Table 5: Induction levels of the ICS messenger after infection of C. roseus leaves with P. cactorum relative to the control.

Sample fold induction Control 1 48 hrs after inoculation >100a 6 days after inoculation Uninfected leaf areab 1 notes: Fold induction compared to control.

b: Uninfected leaf area is the area of the inoculated leaf not infected by the fungus.

Example 8 Isolation of the isochorismate synthase promoter from Catharan roseus by iPCR PCR primers were developed based on the sequence of the ICS cD ID NO: 18). Primers FR-ICS-259 5'TGG TGA TCC AAG AGC TCC GG3' NO: 20) and FR-ICS-260 5'CCT GGT TGA AAG GTC TGT G3' (SEQ ID N( for iPCR and primer FR-ICS-261 5'GCA ACA CAA TGC CCT GTG3' (SE( 22) for nested PCR.

C. roseus genomic DNA was isolated using a CTAB DNA extraction procedure. The genomic DNA was subjected to restriction enzyme thus NA (SEQ (SEQ ID 3: 21) Q ID NO: WO 99/50423 PCT/EP99/02176 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/isoamylalcohol and precipitated with ethanol. The DNA pellet was dissolved in 50 pi water and 25 p1 was used for furer iPCR. For this purpose the volume of the digested DNA mixture was increased to 300 pl in Ix ligase buffer (Gibco BRL) with 5 units of T4 DNA ligase (Gibco BRL). This mixture was incubated at 16 0 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, Ix Klentaq PCR buffer, 10 pM dNTP's and 1.0 pi 50x Advantage cDNA polymerase mix (Clontech, Palo Alto, CA, USA). The complete reaction mixture was subjected to 1' at 94 0 C and 30 cycles of 30" 94 0 C, 1' 55 0 C, 3' 68 0 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 Restriction enzyme iPCR band size (bp) Dde I 100 Kpn I 900 Msc I Nco I 3.000 Nla IV 900 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 WO 99/50423 PCT/EP99/02176 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 ATG CCA TGG GAT GGG AG3' (SEQ ID NO: 24) is located at the startcodon of the ICS ORF introducing a Nco I restriction site overlapping the ATG translational start.

These primers (150 ng) were used in a PCR reaction on C. roseus genomic DNA in Ix Klentaq PCR buffer, 10 pM dNTP's and 2.0 pl Advantage cDNA polymerase mix (Clontech, Palo Alto, CA, USA). The complete reaction mixture (100 pl) was subjected to 1' at 94 0 C and cycles of 30" 94°C, 1' 55 0 C, 4' 68 0 C. A band of the correct size Kb) was isolated from an agarose gel, purified and cloned using restriction enzymes Hind III and Nco I into a high copy cloning vector based on pUC18 (Yanisch-Perron, 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 Nco I and ligated into 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 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 WO 99/50423 PCT/EP99/02176 (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 xl10 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 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 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 (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 WO 99/50423 PCT/EP99/02176 1433-1 0 0 0 1433-2 0 0 0 1433-3 0 0 0 1433-4 0 0 0 1433-5 0 0 0 1433-6 0 0 0 1433-7 0 0 0 1433-8 1 1 1 1433-9 0 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 0 1433-15 1 0 0 1433-16 0 0 1 1433-17 0 0 0 1433-18 0 0 0 1433-19 0 0 0 1433-20 0 0 0 1433-21 0 0 0 1433-22 0 0 0 1433-23 0 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) 863-873). Results are listed in table 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 0 1433-6 0 0 1 0 WO 99/50423 PCT/EP99/02176 1433-7 1433-8 1433-9 1433-10 1433-11 1433-12 1433-13 1433-14 1433-15 1433-16 1433-17 1433-18 1433-19 1433-20 1433-21 1433-22 1433-23 1433-24 1433-25 Note: plants 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1 1 not infected/no 0 0 0 0* 0 0 0 0 0 0 0 0 0 0 0 0 0 0* 0 disease symptoms 0 1 0 0* 0 1 0 0 1 2 1 1 1 1 1 1 1 0* 0 visible Promoter performance was also tested in the leaves of full grown potato plants before and after infection with P. infestans. Before 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.

@0

S

0@e 0g

S

*000 *0

F

0@ 0 Plant number 1433-1 1433-2 1433-3 1433-4 1433-5 1433-6 1433-7 1433-8 1433-10 1433-11 1433-12 1433-13 1433-14 1433-15 1433- 6 1433-17 1433-18 1433-19 1433-20 1433-21 1433-22 1433-23 1433-24 1433-25 before infection 1 1 1 1 1 1 1 3 1 0 1 1 1 1 1 1 1 2 1 1 1 1 3 3 lesion primary zone 4 3 0 2 4 2 0 0 2 2 0 2 0 2 0 2 4 3 0 0 4 4 0 0 0 0 0 2 4 4 0 4 4 4 0 4 2 3 4 4 2 2 0 4 2 3 0 4 background 00 0* S *0 @0 0 F @0

OS

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

The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.

WO 99/50423 PCTIEP99/021 76 BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE INTERNATIONAL FORM4 Mogen International N.V. RECEIPT IN THE CASE OF AN ORIGINAL DEPOSIT 97 issued pursuant to Rule 7.1 by the Einsternweg 97INTERNATIONAL DEPOSITARY AUTHORITY 2333 CB LEIDEN identified at the bottom of this page Nederland name and address of depositor I. IDENTIFICATION OF THE MICROORGANISM II. SCIENTIFIC DESCRIPTION AND/OR PROPOSED TAXONM41C DESIGNATION The microorganis identified under I above was ecv ned by: nentonlDpstr A =hoit oa scint ti apcrpli on dt dimy fth rgnldpst n res to cronve te noigia desitatonadpstudrteBdps raywsrcie by ar iton t appcablhe (pli ae d-my frcito eus o ovrin II. RITAINACCDEPOTACAE

EOT

Nameie byeitaabeu ono S19mme3u99e Sigatur of eorsoinasl havoigthe poe The icrorgaism denifie undr Iabov t repesent thes International Depositary Addresst to tnertath 1 rgnldpstt eoi nerteBdps raywsrcie The ion ntheppliads Datee (dd- -yy) 24f3 reep/frqet o ovrin 1 VereRul 6.(d)appieS suh dte s te dteon whprseh the snteato a inena taly A dre ps i tr ta tI t o authority wa ac ui ed Form BP/4 (sole page) CBS/91O7 WO 99/50423 PCTIEP99/02176 BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE INTERNATIONAL FORM VIABILITY STATEMENT issued pursuant to Rule 10.2 by the INTERNATIONAL DEPOSITARY AUTHORITY identified on the following page Mogen Internaional N.V.

Ensteinweg 97 2333 CB LEIDEN Nederland name and address of the party to whom the viability statement is issued I. DEPOSITOR II. IDENTIFICATION OF THE MXCROORGAIISM Name: Mogen International N.V. Accession number given by the INTERNATIONAL DEPOSITARY AUTHORITY: CBS 101670 Address: Einsteinweg 97 2333 CB LEIDEN Date (dd-m-yy) of the deposit or of the Nederland transfer: 1 19-03-99 1iI. VIAB3ILITY STATEMENT The viability of the microorganism identified under II above was tested on 23-03-99 2 On that date (dd-mm-yy), the said microorganism was 3i viable 3 no longer viable IIndicate the date of the original deposit or, where a new deposit or a transfer has been made, the most recent relevant date (date of the new deposit or date of the transfer).

2 In the cases referred to in Rule 10.2(a) (ii) and (iii), refer to the most recent viability test.

3 Mark with a cross the applicable box.

Form BP/9 (first page) WO 99/50423 WO 9950423PCT/EP99/Oz 176 4 IV. CONDITIONS UNDER WHICH TEE VIA13XLXTY HAS EN PERYOOND V. INTERNATIONAL DEPOSITARY AUTHORITY Name: Centraalbureau voor Schimmelcultures Signature of person(s) having the power to rep resent the International Depositary Authority or of authorized of fici*r- Address: Oosterstraat I P.O. Box 273 3740 AG BAARN Mrs F.B. Snippe-Claus

SP

The Netherlands Date (dd-m-yy): 24-03-99 4Fill in if the information has been requested and if the results of the test reT negative.

EDITORIAL NOTE NO. 36025/99 This specification contains a sequence listing following the description and is numbered as follows: Sequence listing pages 1 Claim pages 40 43 SEQUENCE LISTING <110> MOGEN International nv <120> Salicylic acid pathway genes and their use for the induction of resistance in plants.

<130> 46049 PCT <140> <141> <150> US 60/080,625 <151> 1998-04-03 <150> US 60/080,203 <151> 1998-03-31 <160> <170> Patentln Ver. 2.1 <210> 1 <211> <212> DNA <213> Artificial Sequence <220> S<223> Description of Artificial Sequence:primer <400> 1 gtcgaggatc catggatacg tcactggctg <210> 2 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> 2 gaatggaatg cggatcctcg ctccttaatg c 31 <210> 3 <211> 33 <212> DNA S<213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> 3 gcgggtacca caatggcttc ctcagttctt tcc 33 <210> 4 <211>18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> 4 cattgcactc ttccgccg 18 <210> <211>30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> Sgcgggtacca tgctgccgct aaaaccgcca a a <210>6 <211>27 <212> DNA <213> Artificial Sequence <220> S<223> Description of Artificial Sequence:primer <400> 6 •gcgcatatgc tgccgctaaa accgcca 27 9a* <210> 7 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> 7 cgcggatcct catgacttgg cctgcgccga gta 33 <210> 8 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> 8 cgccatatgg cattgcactc ttccgccgtt gct 33 <210> 9 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer C<400> 9 atagaaacga ggacacttcc acgttaaggg attttgg 37 <210> <211> 37 <212>

DNA

<213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> ataagcacgg attaatgggc cggagctgaa tgaagcc 37 0 <210> 11 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> 11 atagaaacga ggacacttcc <210> 12 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> 12 ataagcacgg attaatgggc <210O> 13 <211> 1659 <212> DNA <213> Escherichia coli <220> <221> ODS <222> 552) /function= "isochorismate synthase" entC protein <400> 13 gagtctcaca aatcagcttc ctgttattaa taaggttaag ggcgtaatga caaattcgac aaagcgcaca atccgtcccc tcgcctttgg gagagggtta gggtgagggg aacagccagc 120 actggtgcga acattaaccc tcaccccagc cctcaccctg gaaggagagg gggcagaacg 180 gcgcaggaca tcacattgcg cttatgcgaa tccatcaata atgcttctca ttttcattgt 240 aaccacaacc agatgcaacc ccgagttgca gattgcgtta cctcaagagt tgacatagtg 300 cgcgtttgct tttaggttag cgaccgaaaa tataaatgat aatcattatt aaagccttta 360 tcattttgtg gaggatgat atg gat acg tca ctg gct gag gaa gta cag cag .412 Met Asp Thr Ser Leu Ala Glu Glu Val Gin Gin 1 5 acc atg gca aca ctt gcg ccc aat cgc ttt ttc ttt atg tcg ccg tac 460 Thr Met Ala Thr Leu Ala Pro Asn Arg Phe Phe Phe Met Ser Pro Tyr 20 cgc agt ttt acg acg tca gga tgt ttc gcc cgc ttc gat gaa ccg gct 508 Arg Ser Phe Thr Thr Ser Gly Cys Phe Ala Arg Phe Asp Glu Pro Ala 35 gtg aac ggg gat tcg ccc gac agt ccc ttc cag caa aaa ctc gcc gcg 556 Val Asn Gly Asp Ser Pro Asp Ser Pro Phe Gin Gin Lys Leu Ala Ala 50 ctg ttt gcc gat gcc aaa gcg cag ggc atc aaa aat ccg gtg atg gtc 604 Leu Phe Ala Asp Ala Lys Ala Gin Gly lie Lys Asn Pro Val Met Val 65 70 ggg gcg att ccc ttc gat cca cgt cag cct tcg tcg ctg tat att cct 652 Gly Ala lie Pro Phe Asp Pro Arg Gin Pro Ser Ser Leu Tyr lie Pro 85 gaa tcc tgg cag tcg ttc tcc cgt cag gaa aaa caa gct tcc gca cgc 700 Glu Ser Trp Gin Ser Phe Ser Arg Gin Glu Lys Gin Ala Ser Ala Arg 100 105 cgt ttc acc cgc agc cag tcg ctg aat gtg gtg gaa cgc cag gca att 748 Arg Phe Thr Arg Ser Gin Ser Leu Asn Val Val Glu Arg Gin Ala lie 110 115 120 ccg gag caa acc acg ttt gaa cag atg gtt gcc cgc gcc gcc gca ctt 796 Pro Glu Gin Thr Thr Phe Glu Gin Met Val Ala Arg Ala Ala Ala Leu 125 130 135 acc gcc acg ccg cag gtc gac aaa gtg gtg ttg tca cgg ttg att gat 844 Thr Ala Thr Pro Gin Val Asp Lys Val Val Leu Ser Arg Leu lie Asp :..140 145 150 155 S S atc acc act gac gcc gcc att gat agt ggc gta ttg ctg gaa cgg ttg 892 Slie Thr Thr Asp Ala Ala lie Asp Ser Gly Val Leu Leu Glu Arg Leu 160 165 170 att gcg caa aac ccg gtt agt tac aac ttc cat gtt ccg ctg gct gat 940 lie Ala Gin Asn Pro Val Ser Tyr Asn Phe His Val Pro Leu Ala Asp 175 180 185 ggt ggc gtc ctg ctg ggg gcc ago ccg gaa ctg ctg cta cgt aaa gac 988 Gly Gly Val Leu Leu Gly Ala Ser Pro Glu Leu Leu Leu Arg Lys Asp 190 195 200 ggc gag cgt ttt agc tcc att ccg tta gcc ggt tcc gcg cgt cgt cag 1036 Gly Glu Arg Phe Ser Ser lie Pro Leu Ala Gly Ser Ala Arg Arg Gin 205 210 215 ccg gat gaa gtg ctc gat cgc gaa gca ggt aat cgt ctg ctg gcg tca 1084 Pro Asp Glu Val Leu Asp Arg Glu Ala Gly Asn Arg Leu Leu Ala Ser 220 225 230 235 gaa aaa gat cgc cat gaa cat gaa ctg gtg act cag gcg atg aaa gag 1132 Glu Lys Asp Arg His Glu His Giu Leu Val Thr Gin Ala Met Lys Glu 240 245 250 gta ctg cgc gaa cgc agt agt gag tta cac gtt cct tct tct cca cag 1180 Val Leu Arg Giu Arg Ser Ser Giu Leu His Val Pro Ser Ser Pro Gin 255 260 265 ctg atc acc acg ccg acg ctg tgg cat ctc gca act ccc ttt gaa ggt 1228 Leu lie Thr Thr Pro Thr Leu Trp His Leu Ala Thr Pro Phe Glu Gly 270 275 280 aaa gcg aat tcg caa gaa aac gca ctg act ctg gcc tgt ctg ctg cat 1276 Lys Ala Asn Ser Gin Giu Asn Ala Leu Thr Leu Ala Cys Leu Leu His 285 290 295 ccg acc ccc gcg ctg agc ggt ttc ccg cat cag gcc gcg acc cag gtt 1324 Pro Thr Pro Ala Leu Ser Gly Phe Pro His Gin Ala Ala Thr Gin Val 300 305 310 315 aft gct gaa ctg gaa ccg ttc gac cgc gaa ctg tttggc ggc att gtg 1372 lie Ala Giu Leu Glu Pro Phe Asp Arg Giu Leu Phe Gly Gly lie Val 320 325 330 ggt tgg tgt gac agc gaa ggt aac ggc gaa tgg gtg gtg acc atc cgc 1420 Gly Trp Cys Asp Ser Giu Gly Asn Gly Giu Trp Val Val Thr lie Arg t 33 5 340 345 tgc gcg aag ctg cgg gaa aat cag gtg cgt ctg ttt gcc gga gcg ggg 1468 Cys Ala Lys Leu Arg Giu Asn Gin Val Arg Leu Phe Ala Gly Ala Gly 350 355 360 att gtg cct gcg tcg tca ccg ttg ggt gag tgg cgc gaa aca ggc gtc 1516 lie Val Pro Ala Ser Ser Pro Leu Gly Giu Trp Arg Giu Ihr Gly Val 365 370 375 ;aaa ctt tct acc atg ttg aac gtt ttt gga ttg cat taaggagcgagga 1565 Lys Leu Ser Thr Met Leu Asn Val Phe Gly Leu His 380 385 390 tgagcattcc attcacccgc tggccgaaga gtttgcccgt cgctatcgga aaaaggatac 1625 tgcagatttc gctgaccgac attctgacgc aact 1659 <210> 14 <211> 391 <212> PRT <213> Escherichia coli <400> 14 Met Asp Thr Ser Leu Ala Glu Glu Val Gin Gin Thr Met Ala Thr Leu 1 5 10 Ala Pro Asn Arg Phe Phe Phe Met Ser Pro Tyr Arg Ser Phe Thr Thr 25 Ser Gly Cys Phe Ala Arg Phe Asp Glu Pro Ala Val Asn Gly Asp Ser 40 Pro Asp Ser Pro Phe Gin Gin Lys Leu Ala Ala Leu Phe Ala Asp Ala 55 Lys Ala Gin Gly lie Lys Asn Pro Val Met Val Gly Ala lie Pro Phe 70 75 Asp Pro Arg Gin Pro Ser Ser Leu Tyr lie Pro Glu Ser Trp Gin Ser 90 Phe Ser Arg Gin Glu Lys Gin Ala Ser Ala Arg Arg Phe Thr Arg Ser 100 105 110 Gin Ser Leu Asn Val Val Glu Arg Gin Ala lie Pro Glu Gin Thr Thr 115 120 125 Phe Glu Gin Met Val Ala Arg Ala Ala Ala Leu Thr Ala Thr Pro Gin 130 135 140 Val Asp Lys Val Val Leu Ser Arg Leu lie Asp lie Thr Thr Asp Ala 145 150 155 160 Ala lie Asp Ser Gly Val Leu Leu Glu Arg Leu lie Ala Gin Asn Pro 165 170 175 Val Ser Tyr Asn Phe His Val Pro Leu Ala Asp Gly Gly Val Leu Leu 180 185 190 Gly Ala Ser Pro Glu Leu Leu Leu Arg Lys Asp Gly Glu Arg Phe Ser 195 200 205 Ser lie Pro Leu Ala Gly Ser Ala Arg Arg Gin Pro Asp Glu Val Leu 210 215 220 Asp Arg Glu Ala Gly Asn Arg Leu Leu Ala Ser Glu Lys Asp Arg His 225 230 235 240 Glu His Glu Leu Val Thr Gin Ala Met Lys Glu Val Leu Arg Glu Arg 245 250 255 Ser Ser Glu Leu His Val Pro Ser Ser Pro Gin Leu Ilie Thr Thr Pro 260 265 270 Thr Leu Trp His Leu Ala Thr Pro Phe Glu Giy Lys Ala Asn Ser Gin 275 280 285 Glu Asn Ala Leu Thr Leu Ala Cys Leu Leu His Pro Thr Pro Aia Leu 290 295 300 Ser Gly Phe Pro His GIn Ala Ala Thr GIn Val Ilie Ala Glu Leu Glu 305 310 315 320 Pro Phe Asp Arg Glu Leu Phe Gly Gly Ilie Val Gly Trp Cys Asp Ser 325 330 335 Glu Gly Asn Gly Giu Trp Val Val Thr Ilie Arg Cys Ala Lys Leu Arg 340 345 350 Glu Asn GIn Val Arg Leu Phe Ala Gly Ala Gly Ilie Val Pro Ala Ser 355 360 365 Ser Pro Leu Gly Glu Trp Arg Glu Thr Gly Val Lys Leu Ser Thr Met 370 375 380 .00 Leu Asn Val Phe Gly Leu His :385 390 <210> o o:<211> 5057 <212> DNA <213> Pseudomonas fluorescens <220> <221> CDS <222> (207)..(1379) <223> /function= "isochorismate synthase" orfA protein <220> <221> CDS <222> (4516)..(4848) <223> /function= "isochorismate pyruvate lyase" orfD protein <400> gaattcggca tacagatgag ctgaaacttt ccctaaaaac gcatgagcag tccgcccgat ccaacaggcg gcttgcattg aagttaactc tctcatggct cactcatgtg attgacaaat 120 cgtaatgatt agcatcacat attagaaacg ataatgattc tatttoaagt ttctttttat 180 ccctaggcca oggaggotto ccccaa atg aga acg ggc acc cta aga acg acc 233 Met Arg Thr Gly Thr Leu Arg Thr Thr 1 gag atg gaa gag gtg caa ctc gca gag gta caa cgt agt ttt tcg ttc 281 Glu Met Glu Glu Vat Gin Leu Ala Giu Val Gin Arg Ser Phe Ser Phe 15 20 aca tcc ggc gat cgc gag tta gcg gtc aco ggg atg ctg cag aga att 329 Thr Ser Gly Asp Arg Glu Leu Ala Val Thr Gly Met Leu Gin Arg lie 35 gaa aca cct gc att ggc ggc gat gac gcc aat ago ctg ttc cag cag 377 Glu Thr Pro Ala lie Gly Gly Asp Asp Ala Asn Ser Leu Phe Gin Gin 50 aca att gcg caa gcg ctt gat cgg gcg cgc gaa gct ggc cag agc aac 425 Thr lie Ala Gin Ala Leu Asp Arg Ala Arg Giu Ala Gly Gin Ser Asn 65 cca atc atc gtt ggc gcc atc cot ttc gac cot got gaa oct too tgo 473 Pro lie lie Vai Gly Ala lie Pro Phe Asp Pro Ala Giu Pro Ser Cys 75 80 otc tao att ccc gaa cac gog caa tgg cgg aco oga gao ato cac gcg 521 Leu Tyr lie Pro Glu His Aia Gin Trp Arg Thr Arg Asp lie His Ala 95 100 105 aaa acg ggt atg tog acg otg cot gag ttg ato gaa cag aaa aac att 569 Lys Thr Gly Met Ser Thr Leu Pro Glu Leu lie Glu Gin Lys Asn lie 110 115 120 cog gao gaa cag gc tto aaa cgg gog gtg gaa cac gc gto gtc aac 617 Pro Asp Giu Gin Aia Phe Lys Arg Ala Vai Glu His Aia Vai Vai Asn 125 130 135 ttc cgo cac ago gao gta cgo aag got gtg otc tog gtt caa cgo gag 665 Phe Arg His Ser Asp Vai Arg Lys Ala Vai Leu Ser Val Gin Arg Glu 140 145 150 otg ata ttt gca aao gat gtg gat gtg agt gc ctg cag cac aac otg 713 Leu lie Phe Ala Asn Asp Vai Asp Vat Ser Aia Leu Gin His Asn Leu 155 160 165 aaa gcc cag aac cog ago ggo tao cac tto cgt gtg oca atg cot gat 761 Lys Ala Gin Asn Pro Ser Gly Tyr His Phe Arg Val Pro Met Pro Asp 170 175 180 185 ggc acc acg ctg atc ggt gtc agt coo gaa ctt otg gtc cgc aag gaa 809 Gly Thr Thr Leu lie Giy Vat Ser Pro Giu Leu Leu Vat Arg Lys Glu 190 195 200 ggc ctg agc tct otg tcc aac ccg ctg gca ggg tca gcc aag cgc atg 857 Gly Leu Ser Ser Leu Ser Asn Pro Leu Ala Gly Ser Ala Lys Arg Met 205 210 215 gcc gat oca gaa gc gac cgg cg aat gca gao tgg ttg otg aca tog 905 Ala Asp Pro Glu Aia Asp Arg Arg Asn Aia Asp Trp Leu Leu Thr Ser 220 225 230 gaa aaa gat cac tac gaa cac ggg tto gtg acc cag gac ato gtc agc 953 Glu Lys Asp His Tyr Giu His Gly Phe Vai Thr Gin Asp ie Vat Ser 235 240 245 caa otg ggg aaa otg tgc acg cag otg aat gtg ocg caa cgc coo tcc 1001 Gin Leu Gly Lys Leu Cys Thr Gin Leu Asn Vai Pro Gin Arg Pro Ser 250 255 260 265 otc ato ago aog cc gog otc tgg cac otc tcg aoo cgo ato gaa ggt 1049 Leu ie Ser Thr Pro Ala Leu Trp His Leu Ser Thr Arg ie Glu Gly 270 275 280 acg otg goa gac ccg got gta tog goo ttg cag ott goo tgo cgo ttg 1097 Thr Leu Ala Asp Pro Aia Vai Ser Aia Leu Gin Leu Ala Cys Arg Leu 285 290 295 cac cc aoa cog got gtg tgo ggo tt cc aco gag ogo gc cgg cgo 1145 His Pro Thr Pro Aia Vat Cys Gly Phe Pro Thr Giu Arg Ala Arg Arg 300 305 310 otg att cgo tto gto gaa cc ifo gag cgo ggo ctg tto aco ggo atg 1193 Leu ie Arg Phe Vai Glu Pro Phe Glu Arg Giy Leu Phe Thr Gly Met 315 320 325 gtg ggt tgg tgo gat gc cag ggo aat ggc gaa tgg gto gta aog att 1241 Vai Gly Trp Cys Asp Aia Gin Gly Asn Giy Giu Trp Va Vat Thr Ile.

330 335 340 345 ogt tgo ggo aog gto agg oga aao aag gto cgo ctg tto gc ggc goa 1289 Arg Cys Gly Thr Vai Arg Arg Asn Lys Vat Arg Leu Phe Aia Gly Ala 350 355 360 ggo ato gtt gaa gc toa ago cc gao too gaa tgg gca gaa gtc cag 1337 Gly Ile Vai Giu Ala Ser Ser Pro Asp Ser Glu Trp Aia Glu Va GIn 365 370 375 acc aaa ctt ggc acc atc gtg cgc gcc tgc gga ttg gcc cac taa 1382 Thr Lys Leu Gly Thr lie Val Arg Ala Cys Gly Leu Ala His 380 385 390 ctcgaatttt ttcacacgtg aatactatga ctattgaatt taaccactgg cctctggaaa 1442 gcgcacagcg ttatcgggac aaaggctatt ggctcgacaa accgctcacc cacctccttc 1502 aggaacgcag ccagtcg caa cccgacgccc ccgcgattat ttg cgg cg at cgccacttca 1562 gctatgccga gttggaccaa ttgtcttcca acctggcctc gcgactggcc gccagcgggc 1622 ttggcaacgg tgacactgca ctggtgcagt tgcccaatat cgcggagttt tacattgtcc 1682 tttttgccct gctcaagtca ggaatcgcac ccctcaacgc gctctacagc catcgcaaac 1742 ttgaactcaa gagttacgcc aaacaaatcg cgccaacgtt gttgattgcc tcccgcgaac 1802 atgaagtctt ccgtgacgac agctatatcg ccgacttcaa ggaggtgggt tcaagtccag 1862 acatcatctt gctgttgggc gagcaacgtc acgaaaacaa cctcgccgac tggatcaata 1922 cgccgagcga gagcaacgtg aacgtctccc cctcggggcc cggcgaggtc gcattgttcc 1982 aactgtcagg cggcagcacg ggcaccccca aactcatccc ccgcactcac aacgactatt 2042 actacaacgc cagggcaagc gcgcaagtat gcgaacttac gccacgcacg cgctttctat 2102 gcgcgctacc tgccgcccat aacttcttgc tcagctcccc cggcgccctc ggtgttctac 2162 ~atg ctg gcgg ctg catcatc atggcg ccca g cccgg ag cc cttg acctgt ttttcg atca 2222 tccagcgcca agaagtcaat actgtggcct tggtgccaag tgcagtcgcc ttgtggctgc 2282 9 aggcagcgcc ggagcataaa gaacaactgc aatcgcttga gttcctccag gtcggtgg cg 2342 cctgttttgc cgactcgctg gcacgccagg tgcccggcgt gctcggttgt aagctgcaac 2402 aggtattcgg gatggccgaa ggcctgatca actacacccg gctaaatgac tccgacgaac 2462 agatttttac tacccagggc cgtccgatca gccccgacga tgaaatcaaa atcgttg acg 2522 aacaaggcct ccccgtcccg gacggagaac ctggcatgct cgccacacgt ggcccttaca 2582 ctttttgcgg gtactaccaa agccccgaac aaaatgccca ggcgttcgat aacgaggggt 2642 actactactc cggcgacctc gtccaactca tgcccagcgg cgatttgcgc gtggtcggca 2702 gggtcaagga ccagatcaac cgtggcggtg aaaaagtcgc ctcggaggaa atcgaaaacc 2762 tcatcgtcct ccatcctgat gtgactcacg cgggcttggt ggccatgccc gatgacaggc 2822 tgggagaaaa aagctgcgcg ttcgtcgtct cacgcaaccc gagcctgaag ccgcccgcgc 2882 tcagacgtca cctgatggaa ctcggcatcg ccgaatacaa actgcccgac cgcatccggt 2942 taatcgaaac catgccgctg acccccgtgg gcaagattga caagaagcat ctgcgtcagc 3002 ttctggcagc ggaaaccaca cgcgcctggt tgcagactcg cgtgcggcaa ctcgtcgagg 3062 actgtgaaga cctggacccc gaggaaaacc tgattttcta tggcctcgac tccttgcaag 3122 tgatgagact cgctgccgaa ctcaaggagc gtggcattgc cgtcagcttc gaagaactgg 3182 cggattcgcc cacgctcagc agctggtggt cattggtaga cgcgaggcag atagccgcct 3242 gaccgggcgg cgtcacccag tcgttttaaa aggagttaga catgacttta tcccctgccg 3302 accaaagcaa gctgaaggc ttctggcagc actgcgtgac acatcagtat ttcaacattg 3362 g gtatcccga atcagccg at tttg attact cccagctg ca ccgtttcttg cagttttcaa 3422 ttaacaactt gctggggact gggaatgagt acagcaacta cctgttgaac tcgttcgact 3482 ttgaaaaaga cgtcatgacg tatttcgccg agctgttcaa cattgccctt gaagacagtt 3542 ggggttacgt caccaatggc gggacggaag gcaatatgtt tggctgctac ctgggacgcg 3602 aactgtttcc ggacggcacc ctgtactact cgaaagacac ccactactcc gtggcaaaga 3662 tcgtcaaatt attgcggatc aaatgccgtg cggtcgaatc gctgcccaat ggcgaaatcg 3722 actacgacga cctgatggca aaaataaccg ccgaccagga gcgtcacccc atcatcttcg 3782 ccaacatcgg caccacgatg cgtggagccc tggataatat cgtgaccatc cagcaacgcc 3842 tgcaacaggc aggcattgcc cgocacgact actacctgca cgctgatgcg gccttgagcg 3902 ggatgatoot gooottcgto gatoaccoac aacocttctc gtttgoogac ggoatogaot 3962 cgatctgcgt otcoggcoao aagatgatcg gotogoocat tccttgcgga attgtcgtgg 4022 ooaaaogcaa caaogtogog cgcatttoag tggaagtgga ctatatccgc goooatgaoa 4082 agacoatoag cggotogogc aacggccaca oaooootgat gatgtgggcg goaotgogca 4142 gctactcatg ggotgaatgg ogocatogaa toaaacacag ootggaoaog gcaoagtaog 4202 oogtogaoog ctoaggcc tcgggcattg atgcctggog caacgaaaac tooatcacog 4262 togtgttcoo ttgccatoa gaaagaattg ogaogaaata otgcctggoo aootcoggta 4322 attcggcaca ootg atcacc acacotcatc atoacgactg cag oatg atc gacgccttg a 4382 togaogaagt ggttgccgaa gotoaaotga atacootg atooaagcga goattcactg 4442 aaoaaaoggt ogtogagoga ttgcccgcgg ogtcattcaa ottgogtaoo cattattgaa 4502 agagacoago oto atg otg cog cta aaa cog cca caa gcc tgo gag aac 4551 Met Leu Pro Leu Lys Pro Pro Gin Ala Cys Glu Asn *395 400 oto aat gac aft oga gog ggo ato gac ttt ttt gao cgc cag ato ott 4599 Leu Asn Asp Ilie Arg Aia Gly Ilie Asp Phe Phe Asp Arg Gin Ilie Leu 405 410 -415 420 gao tog ota oaa aaa ogo otg ogt tao gta aag got gog gog cag tto 4647 Asp Ser Leu Gin Lys Arg Leu Arg Tyr Val Lys Ala Ala Aia Gin Phe 425 .430 435 aaa goo aao gag oag gao att ooa goa oot gaa ogo gto gog goc atg 4695 Lys Ala Asn Glu Gin Asp Iie Pro Ala Pro Giu Arg Val Ala Aia Met :440 445 450 ott gag gag ogg oga ota tgg gog gta gaa goc gaa ott gat gto got 4743 Leu Giu Giu Arg Arg Leu Trp Ala Val Glu Ala Glu Leu Asp Val Ala 455 460 465 tto gto gag aag oto tao gag oag att aft oao tgg aat att caa cag 4791 Phe Vai Giu Lys Leu Tyr Giu Gin Ilie Ilie His Trp Asn Ilie Gin Gin 470 475 480 caa atc ctg cat tgg cgg gcc acc cga caa cca acg tac tcg gcg cag 4839 Gin lie Leu His Trp Arg Ala Thr Arg Gin Pro Thr Tyr Ser Ala Gin 485 490 495 500 gcc aag tca tga cgatggcggg tggatgcctg acgacttgag gtctgcgatt 4891 Ala Lys Ser cacgcgggct gtccttgttc gagtggtgtg tgttcacaac tatcaccgta gaccaagggc 4951 ggccttttct tcattttttt gtaatcagat cagcctgtta gctgttaatt aagtgccatt 5011 caaatctgtc ccctttttt ttcgccataa tgctgatatc gaattc 5057 <210> 16 <211> 391 <212> PRT <213> Pseudomonas fluorescens <400> 16 Met Arg Thr Gly Thr Leu Arg Thr Thr Glu Met Glu Glu Val Gin Leu 1 5 10 Ala Glu Val Gin Arg Ser Phe Ser Phe Thr Ser Gly Asp Arg Glu Leu 20 25 Ala Val Thr Gly Met Leu Gin Arg lie Glu Thr Pro Ala lie Gly Gly 40 Asp Asp Ala Asn Ser Leu Phe Gin Gin Thr lie Ala Gin Ala Leu Asp 55 Arg Ala Arg Glu Ala Gly Gin Ser Asn Pro lie lie Val Gly Ala lie 65 70 75 Pro Phe Asp Pro Ala Glu Pro Ser Cys Leu Tyr lie Pro Glu His Ala 85 90 Gin Trp Arg Thr Arg Asp lie His Ala Lys Thr Gly Met Ser Thr Leu 100 105 110 Pro Glu Leu lie Glu Gin Lys Asn lie Pro Asp Glu Gin Ala Phe Lys 115 120 125 Arg Ala Val Glu His Ala Val Val Asn Phe Arg His Ser Asp Val Arg 130 135 140 Lys Ala Val Leu Ser Val Gin Arg Glu Leu lie Phe Ala Asn Asp Val 145 150 155 160 Asp Val Ser Ala Leu Gin His Asn Leu Lys Ala Gin Asn Pro Ser Gly 165 170 175 Tyr His Phe Arg Val Pro Met Pro Asp Gly Thr Thr Leu lie Gly Val 180 185 190 Ser Pro Glu Leu Leu Val Arg Lys Glu Gly Leu Ser Ser Leu Ser Asn 195 200 205 Pro Leu Ala Gly Ser Ala Lys Arg Met Ala Asp Pro Glu Ala Asp Arg 210 215 220 Arg Asn Ala Asp Trp Leu Leu Thr Ser Glu Lys Asp His Tyr Glu His 225 230 235 240 Gly Phe Val Thr Gin Asp lie Val Ser Gin Leu Gly Lys Leu Cys Thr 245 250 255 Gin Leu Asn Val Pro Gin Arg Pro Ser Leu lie Ser Thr Pro Ala Leu 260 265 270 Trp His Leu Ser Thr Arg lie Glu Gly Thr Leu Ala Asp Pro Ala Val 275 280 285 Ser Ala Leu Gin Leu Ala Cys Arg Leu His Pro Thr Pro Ala Val Cys 290 295 300 Gly Phe Pro Thr Glu Arg Ala Arg Arg Leu lie Arg Phe Val Glu Pro 305 310 315 320 o oo Phe Glu Arg Gly Leu Phe Thr Gly Met Val Gly Trp Cys Asp Ala Gin 325 330 335 Gly Asn Gly Glu Trp Val Val Thr lie Arg Cys Gly Thr Val Arg Arg 340 345 350 Asn Lys Val Arg Leu Phe Ala Gly Ala Gly lie Val Glu Ala Ser Ser 355 360 365 Pro Asp Ser Glu Trp Ala Glu Val Gin Thr Lys Leu Gly Thr lie Val 370 375 380 Arg Ala Cys Gly Leu Ala His 385 390 <210> 17 <211>111 <212> PRT <213> Pseudomonas fluorescens <400> 17 Met Leu Pro Leu Lys Pro Pro Gin Ala Cys Giu Asn Leu Asn Asp Ilie 1 5 10 Arg Ala Gly Ilie Asp Phe Phe Asp Arg Gin Ilie Leu Asp Ser Leu Gin 25 Lys Arg Leu Arg Tyr Val Lys Ala Ala Ala Gin Phe Lys Ala Asn Giu 40 Gin Asp Ilie Pro Ala Pro Giu Arg Val Ala Ala Met Leu Giu Giu Arg 55 Arg Leu Trp Ala Val Giu Ala Giu Leu Asp Val Ala Phe Val Giu Lys 70 75 Leu Tyr Glu Gin Ilie Ilie His Trp Asn Ilie Gin Gin Gin Ilie Leu His 90 Trp Arg Ala Thr Arg Gin Pro Thr Tyr Ser Ala Gin Ala Lys Ser 100 105 110 18* <222 (3).(70 1 *t gc a5t c a t c c g a c c t t c 0 Va l i PeTrApLe e h rgLsSrSr h h e 15 aat tct aat aat aac tct tcc ctt ttt aga aga aag tct aca aat ata 150 Asn Ser Asn Asn Asn Ser Ser Leu Phe Arg Arg Lys Ser Thr Asn lie 30 35 gtc acc aga aaa aaa tat ata Utt tgt tct aca tca ttg tcc atg aat 198 Val Thr Arg Lys Lys Tyr lie Phe Cys Ser Thr Ser Leu Ser Met Asn 50 ggt tgc aat ggt gat cca aga gct ccg gtt gga act ata gaa acg agg 246 Gly Cys Asn Gly Asp Pro Arg Ala Pro Val Gly Thr lie Glu Thr Arg 65 aca cUt ccg gcg gtt tcg acg ccg gca Utg gcc atg gaa cgt cUt agc 294 Thr Leu Pro Ala Val Ser Thr Pro Ala Leu Ala Met Giu Arg Leu Ser 80 tcc gcc gtg gct aac ttg aaa tca act cta cct tct gct caa tca ggg 342 Ser Ala Val Ala Asn Leu Lys Ser Thr Leu Pro Ser Ala GIn Ser Gly 95 100 atc ato cgt ctt gag gta cca att gaa gaa cat ata gaa gca cta gac 390 lie lie Arg Leu Glu Val Pro lie Glu Giu His lie Glu Ala Leu Asp 105 110 115 120 tgg ctt cat tcg caa gac caa aaa aac ctt ctt ccc cgt tgc tat ttc 438 Trp Leu His Ser GIn Asp GIn Lys Asn Leu Leu Pro Arg Cys Tyr Phe 125 130 135 S. *S= tct ggt aga agt caa gtt acc ttc tct gat ttc aca tct aac gac ctt 486 Ser Gly Arg Ser Gin Val Thr Phe Ser Asp Phe Thr Ser Asn Asp Leu 140 145 150 aca aat aga aat ggg agt gcc gcc aat gga cat ctt caa cga att tct 534 Thr Asn Arg Asn Gly Ser Aia Ala Asn Gly His Leu Gin Arg lie Ser 155 160 165 act tca tct gat gat aag aat ctg gtc agt gtt gct ggt gtc ggt tct 582 Thr Ser Ser Asp Asp Lys Asn Leu Val Ser Val Ala Gly Vai Gly Ser 170 175 180 gca gtc ctc ttc cgg agc cca aat cca ttt tct ttt gat gat tgg ctc 630 Ala Val Leu Phe Arg Ser Pro Asn Pro Phe Ser Phe Asp Asp Trp Leu 185 190 195 200 tca att aag agg ttt ttg tcc aag aac tgc cca tta atc cgt gct tat 678 Ser lie Lys Arg Phe Leu Ser Lys Asn Cys Pro Leu lie Arg Ala Tyr 205 210 215 gga gca att cgc ttt gat gca agg cct cat ata gca cca gag tgg aag 726 Gly Ala lie Arg Phe Asp Ala Arg Pro His lie Ala Pro Glu Trp Lys 220 225 230 gct ttt ggc tca ttt tac ttc atg gtt cct cag gtt gag ttt gat gag 774 Ala Phe Gly Ser Phe Tyr Phe Met Val Pro Gin Val Glu Phe Asp Glu 235 240 245 cta cat gga agt tcc atg att gct gca aca gtt gca tgg gat aat gct 822 Leu His Gly Ser Ser Met lie Ala Ala Thr Val Ala Trp Asp Asn Ala 250 255 260 ctc tct ttg aca tat caa caa gca ata gtt cga ctt caa aca aca atg 870 Leu Ser Leu Thr Tyr Gin Gin Ala lie Val Arg Leu Gin Thr Thr Met 265 270 275 280 gag cag gtt tcc tct acc gtc tcc aaa cta aga caa gat gtc tct cat 918 Glu Gin Val Ser Ser Thr Val Ser Lys Leu Arg Gin Asp Val Ser His 285 290 295 act tct ttg gtg agc aag gct aat att cct gat aga aca tcc tgg gat 966 Thr Ser Leu Val Ser Lys Ala Asn lie Pro Asp Arg Thr Ser Trp Asp 300 305 310 ctt act ctt aac cga gtt ttg gaa gaa ata ggc aac aaa tat tcg cca 1014 Leu Thr Leu Asn Arg Val Leu Glu Glu lie Gly Asn Lys Tyr Ser Pro 315 320 325 ttg aca aag gtt gta ctt gca cgt cgt agt caa gtt atc aca aca tca 1062 Leu Thr Lys Val Val Leu Ala Arg Arg Ser Gin Val lie Thr Thr Ser 330 335 340 gat att gat cct ttg gct tgg ctg agt agt ttc aag gct gat ggg aaa 1110 Asp lie Asp Pro Leu Ala Trp Leu Ser Ser Phe Lys Ala Asp Gly Lys 345 350 355 360 gat gct tac caa ttt tgc ctt cag cct cat gaa gca cca gca ttc att 1158 Asp Ala Tyr Gin Phe Cys Leu Gin Pro His Glu Ala Pro Ala Phe lie 365 370 375 gga aac act cca gag caa cta ttt ggc cgg gac cag cta acc gtt ttt 1206 Gly Asn Thr Pro Glu Gin Leu Phe Gly Arg Asp Gin Leu Thr Val Phe 380 385 390 agt gag gct ttg gct gca acc cga gcc agg ggt gaa tca gat tcg tta 1254 Ser Glu Ala Leu Ala Ala Thr Arg Ala Arg Gly Glu Ser Asp Ser Leu 395 400 405 gat ctt cag atg gca cat gat ctc ttt tcc agt ccc aag gat aac cac 1302 Asp Leu Gin Met Ala His Asp Leu Phe Ser Ser Pro Lys Asp Asn His 410 415 420 gag ttt gcc ata gta cga gag aac atc aga cag aaa cta gat 9CC att 1350 Glu Phe Ala lie Val Arg Glu Asn lie Arg Gin Lys Leu Asp Ala lie 425 430 435 440 tgt act agt gta gaa act gaa cca atg aag tca gta aga aag ctt aag 1398 Cys Thr Ser Val Glu Thr Glu Pro Met Lys Ser Val Arg Lys Leu Lys 445 450 455 aga att caa cat ctt tat gct cga ttt gca ggc aga tta cgc tct gaa 1446 Arg lie GIn His Leu Tyr Ala Arg Phe Ala Gly Arg Leu Arg Ser Glu 460 465 470 gat gat gag ftc aag att ttg tct tcc ctt cat cct act cca gct gtt 1494 Asp Asp Glu Phe Lys lie Leu Ser Ser Leu His Pro Thr Pro Ala Val 475 480 485 tgt ggg ftt cct atg gaa gat gca cgg aaa ttt att gcg gaa aat gaa 1542 Cys Gly Phe Pro Met Giu Asp Ala Arg Lys Phe lie Ala Glu Asn Glu 490 495 500 atg ttt gac cga gga tta tac gct ggc cct gtt ggt ttc ttt gga gga 1590 Met Phe Asp Arg Gly Leu Tyr Ala Gly Pro Val Gly Phe Phe Gly Gly 505 510 515 520 gct cag agt gat ttt tct gtt gga ata aga tct gcc ttg att gga aag 1638 Ala GIn Ser Asp Phe Ser Val Gly lie Arg Ser Ala Leu lie Gly Lys 525 530 535 gat gcc ggt gca tta ata tat gcg ggg ctt ggg gtt gta gaa gga agt 1686 Asp Ala Gly Ala Leu lie Tyr Ala Gly Leu Gly Val Val Glu Gly Ser 540 545 550 gat cca gct cta gaa tgg cag gaa cta gag ctc aag gca tcg cag ttt 1734 Asp Pro Ala Leu Glu Trp Gin Glu Leu Glu Leu Lys Ala Ser Gin Phe 555 560 565 atg aag ttg atg aaa tta gag gca cct gct ttg aag tga aaattaggac 1783 Met Lys Leu Met Lys Leu Glu Ala Pro Ala Leu Lys 570 575 580 tgaaaaatca ataaaaagat tgcgatagaa atttcagata attcgttagc cagaagatct 1843 tgttgagccg ttattaaatg tgtcctctac agtttaactg ataaccagat gaagaaaacc 1903 tatatctagt atatatatat ctaccatata taaatatatt gtacattttt gttttttctc 1963 ccacaaattt tatttgtatc tttttgaaca ttgtgccagc tggtttattg tattccatta 2023 tcttaattca ttattcaata agatgtgtca attcattcaa aaaaaaaaaa aaaaa 2078 <210> 19 <211> 580 <212> PRT <213> Catharanthus roseus <400> 19 Met Ala Ser lie Thr Gly His Cys Val Ala His Phe Thr Asp Leu Ser 1 5 10 Thr Arg Lys Ser Ser Phe Phe Ser Asn Ser Asn Asn Asn Ser Ser Leu 25 Phe Arg Arg Lys Ser Thr Asn lie Val Thr Arg Lys Lys Tyr lie Phe 40 Cys Ser Thr Ser Leu Ser Met Asn Gly Cys Asn Gly Asp Pro Arg Ala 55 Pro Val Gly Thr lie Glu Thr Arg Thr Leu Pro Ala Val Ser Thr Pro 70 75 Ala Leu Ala Met Glu Arg Leu Ser Ser Ala Val Ala Asn Leu Lys Ser 90 Thr Leu Pro Ser Ala Gin Ser Gly lie lie Arg Leu Glu Val Pro lie 100 105 110 Glu Glu His lie Glu Ala Leu Asp Trp Leu His Ser Gin Asp Gin Lys 115 120 125 Asn Leu Leu Pro Arg Cys Tyr Phe Ser Gly Arg Ser Gin Val Thr Phe 130 135 140 Ser Asp Phe Thr Ser Asn Asp Leu Thr Asn Arg Asn Gly Ser Ala Ala 145 150 155 160 Asn Gly His Leu Gin Arg lie Ser Thr Ser Ser Asp Asp Lys Asn Leu 165 170 175 Val Ser Val Ala Gly Val Gly Ser Ala Val Leu Phe Arg Ser Pro Asn 180 185 190 Pro Phe Ser Phe Asp Asp Trp Leu Ser lie Lys Arg Phe Leu Ser Lys 195 200 205 Asn Cys Pro Leu lie Arg Ala Tyr Gly Ala lie Arg Phe Asp Ala Arg 210 215 220 Pro His lie Ala Pro Glu Trp Lys Ala Phe Gly Ser Phe Tyr Phe Met 225 230 235 240 Val Pro Gin Val Glu Phe Asp Glu Leu His Gly Ser Ser Met lie Ala 245 250 255 Ala Thr Val Ala Trp Asp Asn Ala Leu Ser Leu Thr Tyr Gin Gin Ala 260 265 270 lie Val Arg Leu Gin Thr Thr Met Glu Gin Val Ser Ser Thr Val Ser 275 280 285 Lys Leu Arg Gin Asp Val Ser His Thr Ser Leu Val Ser Lys Ala Asn 290 295 300 lie Pro Asp Arg Thr Ser Trp Asp Leu Thr Leu Asn Arg Val Leu Glu 305 310 315 320 Glu lie Gly Asn Lys Tyr Ser Pro Leu Thr Lys Val Val Leu Ala Arg 325 330 335 Arg Ser Gin Val lie Thr Thr Ser Asp lie Asp Pro Leu Ala Trp Leu 340 345 350 Ser Ser Phe Lys Ala Asp Gly Lys Asp Ala Tyr Gin Phe Cys Leu Gin 355 360 365 Pro His Glu Ala Pro Ala Phe lie Gly Asn Thr Pro Glu Gin Leu Phe 370 375 380 :*Gly Arg Asp Gin Leu Thr Val Phe Ser Glu Ala Leu Ala Ala Thr Arg 385 390 395 400 Ala Arg Gly Glu Ser Asp Ser Leu Asp Leu Gin Met Ala His Asp Leu 405 410 415 Phe Ser Ser Pro Lys Asp Asn His Glu Phe Ala lie Val Arg Glu Asn 420 425 430 lie Arg Gin Lys Leu Asp Ala lie Cys Thr Ser Val Glu Thr Glu Pro 435 440 445 "Met Lys Ser Val Arg Lys Leu Lys Arg lie Gin His Leu Tyr Ala Arg 450 455 460 Phe Ala Gly Arg Leu Arg Ser Glu Asp Asp Glu Phe Lys lie Leu Ser 465 470 475 480 j Ser Leu His Pro Thr Pro Ala Val Cys Gly Phe Pro Met Glu Asp Ala 485 490 495 Arg Lys Phe lie Ala Glu Asn Glu Met Phe Asp Arg Gly Leu Tyr Ala 500 505 510 Gly Pro Val Gly Phe Phe Gly Gly Ala Gin Ser Asp Phe Ser Val Gly 515 520 525 lie Arg Ser Ala Leu lie Gly Lys Asp Ala Gly Ala Leu lie Tyr Ala 530 535 540 Gly Leu Gly Val Val Glu Gly Ser Asp Pro Ala Leu Glu Trp Gin Glu 545 550 555 560 Leu Glu Leu Lys Ala Ser Gin Phe Met Lys Leu Met Lys Leu Glu Ala 565 570 575 Pro Ala Leu Lys 580 <210> <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer i: <400> tggtgatcca agagctccgg <210> 21 <211> 19 <212> DNA to. <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> 21 cctggttgaa aggtctgtg 19 <210> 22 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> 22 gcaacacaat gccctgtg 18 <210> 23 <21 1> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> 23 gcaagcttca tgtaccttat cttggcc 27 <210> 24 <21 1 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> 24 tagatgccat gggatgggag <210> <21 1> 3279 <212> DNA <213> Catharanthus roseus C <220> <221 promoter <222> (1)..(3275) <220> <221 promoter <222> (1118)..(3275) <400> aagcttcatg taccttatct tggccttact gataattaaa ctaaaatatt caatttattt atcacatgac catatttttc ctgttccata ttatggtgaa ttaaaaatat attaactgaa 120 aattattcag atcatacact aataaatggt tagatatgtg atttatttgt taatttaaat 180 taacttttaa aattaattaa ttaaaaattt attaaattaa gtatatttga taacttattc 240 agtcttatat ttaataatga fttttacaaa tcacttaatt atttaatata cataattata 300 aattttaagt gaaattattt tactaaactt atgtcaaatt aaaatacttt tttgcatata 360 attactactt tcg attataa atataaaggg ttttagcact atatatatta agggtcttg a 420 aaataaaata attttttaac atagtatctt ttttatcctt tataagtgag aaaagaaaga 480 gatagtagaa gaaaaataaa tgaataaaaa ttatgggtaa atatgaaaag agttcaataa 540 atgctcattg tgagttgcaa aacgcttttt agtttttacc atcgttgatg tctaaaattc 600 cttatacttt tgactggaga tagtatcact tattaattaa tttagtattt aatttcagat 660 taagggtcag acttaagatc aaaattcata aatctaaaca attcaaattt gctactttag 720 tttcaatttt atcaaataga gctataattt aaataagttc atattctcat atataattta 780 ttcaacactc aattaaaaca atctagtaag taatcttttt taatgattat gatgtcctag 840 ggtgacaaat atatattaaa acgttacagt tcttattcag cgacatagaa gtcgattaac 900 aactaaaaca tcgacaatat acctaggcca ttcaatgggt tggtttaaca tatattccag 960 aactgattaa tttggacaat ttatatagtt taaataatca aaatattata taaaatttaa 1020 aattttatat gaaattatta aatctaaact aatttaggaa aatttacggt gcacaaaatt 1080 actgcagcat gcgattgaaa tacaacataa aatcactctc gagataaaag gagctattca 1140 tagtggtgat ttctaggagg attgaatgaa tgcattgaca gttcgatccg atatcgaatt 1200 caaaactttt tacttgataa aattggtgga ggtacttaat agaggagtgo tttactatta 1260 gactatacgt tcgttgctct acaaagcact attttgattg gtattftag taaagtgtaa 1320 ttcttttaga agttcataaa aaaattaaaa ttttgattaa aaaataagtt gaactcaaaa 1380 gaatattgag taaatattga aaaaacaaca aaccaaggaa agttctcgtt tcttcata 1440 tcttcaagag tgcttcgatg ttcaaacttc aaacacagac catccatatt tagaattata 1500 tttaaatcat ctaatgataa atttttttc ctttttggg aaacctaaaa tatatttttc 1560 gaagatcaaa taaaatagta gggactacat caaaactaat ttgtctaatc tgttccaaaa 1620 ttacatcacg cttacattag ataagttatt ggtcacatgt tcaatcaaat ttatatatac 1680 tataaacaaa aaagttttat tattcttgct tacataaaga actattotag attgttggaa 1740 cttttaaagt tataaaaaat acttttaaat aagttgtttt gcaaaaaaat aatataaaca 1800 atttcatttt taatcccaca aataaatagt ftttaaaaaac acctttaaaa taaattttca 1860 ataaattttc tttaaagtta aaactaatt attttagagt caacaaataa attaagaagt 1920 aaacatttt tttgaataaa aatttgtt aagacaacag gaaataaatt acactaatga 1980 gtaggaaata ggactcctgc aactcacggt aataataaac acaagtctaa tagttttaac 2040 tggatataat acaataaaca aatgtatcgt aactctcttt tagcacttct aaccctaact 2100 :.*caacactatc agtaattagt atagaacaaa ctcagtttga acaactcgac cgtaaaaatc 2160 ::::gaccatctaa aagaatctga acgaattaga ttttttgaag atagtttagg acagagatga 2220 acagttgaag aggggttcaa atttgctcaa ggttgaaaga gaccttctca aaagagaaga 2280 accaaactcc gatttaaaga aatcgaagag aaactatgcc aatagatttt agacctagta 2340 aaaaaaaact ctaaagagaa gaactaaaat gatttgtaaa caaaataaga cttggagaaa 2400 taaaagaaat agaagataga ttttcagatc aagataaaca ctctagtgta aatcaaggat 2460 ccattttggt cgaaaggacg gacagagaaa gaggagaggt ggtttggcac aagtaaggga :2520 ggaagaagag aagaaggata aaattcaacg aacatttaat tcatacataa tgaatattat 2580 ttatcaaaag aaaaataata gtaagaacaa agatgatgga ataagtgaga aagtaataat 2640 ttattaataa aaatatcttt tattatgtca gatatttcat ttatatcaca tcctctctct 2700 aataataatg aaacaaaaga atatcatgaa aatattaaga aaaagaaaaa aaatcatgaa 2760 atactctttc cag aagttg a tgcattag at tag atg gatg actattttat tcatatg aac 2820 ttgaattaat aaaagtaaac tttgacaaaa aaattatcaa agtttttgac tatactttcc 2880 attcacacgc tcattttctc cctttcttgc ctccttgttt gttgggtcaa aattgtaatc 2940 gcactacaca aaatggcctt aaggtaccat tcatttccaa gaaccaagca atcgtggaat 3000 tctatattag taccactttg actg acgg at attataaaat ttccacg cat ttcataaaag 3060 tcctctggaa aataaataaa tatatataac tcctcctcct cctattttca ctattattat 3120 aaataaacct tcaaataata tattatatat aagaaaattc ctcttagtct gtgtacatgt 3180 ataaataaac ctagagactt ccccttcatg tttgcatcgc ttacaaagtt caccaatcag 3240 tctcattctc tctctctctc tctctctccc atcccatgg 3279 a a a a a a a a -0c

Claims (22)

1. Method to induce pathogen resistance in plants, wherein the plants are transformed with an expression cassette harboring a gene coding for an isochorismate synthase, wherein the gene coding for isochorismate synthase is the ICS gene from Catharantus roseus.

2. Method according to claim 1, wherein 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.

3. Method according to claim 1 or 2, wherein 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.

4. Method according to any one of claims 1 to 3, wherein the plants are additionally transformed with an expression cassette harboring a gene coding S* for an isochorismate pyruvate lyase. 20

5. Method according to claim 4, wherein the gene coding for isochorismate synthase and the gene coding for isochorismate pyruvate lyase both are present on the same vector.

6. Method according to claim 4 or 5, wherein the gene coding for 25 isochorismate pyruvate lyase is selected from the group consisting of orfD and pchB.

7. Method according to claim 6, wherein the gene coding for isochorismate synthase is entC and the gene coding for isochorismate pyruvate lyase is orfD.

8. A pathogen-inducible promoter, wherein it comprises the 5' regulatory region which is naturally found to regulate the expression of the ICS gene in Catharantus roseus. W:llona\Sharn\SJJspecAsp36025 41

9. A pathogen-inducible promoter according to claim 8, wherein it comprises a nucleotide sequence from nucleotide 1118 to nucleotide 3275 as depicted in SEQ ID No:

10. A pathogen-inducible promoter according to claim 9, wherein it comprises a nucleotide sequence from nucleotide 1 to nucleotide 3275 as depicted in SEQ ID No:

11. A pathogen-inducible promoter according to claim 8, wherein it comprises a nucleotide sequence from plasmid pMOG1431 (deposited under no. 101670 at the Centraal Bureau voor Schimmelcultures, Baarn, The Netherlands) located between the restriction sites HindIII and Ncol as shown in fig. 3.

12. Use of a pathogen-inducible promoter according to any one of claims 8 to 11 to drive expression of a heterologous protein.

13. Use according to claim 12, wherein the heterologous protein is an antipathogenic protein selected from the group consisting of chitinase, 20 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 25 wheat-alpha-amylase.

14. Use according to claim 12, wherein 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, Rpml and Rps2 from Arabidopsis thaliana, N-protein from tobacco, avr proteins from Cladosporium fulvum, harpins from Erwinia and elicitor proteins (avrBs3, avrRpml, avrRpt2) from Pseudomonas or Xanthomonas.

W:llona\Sharon\SJJspeci\sp36025 Vector comprising a nucleotide sequence, wherein the nucleotide sequence is capable of encoding a protein having isochorismate synthase activity which is isolated from Catharantus roseus (and has an MW of about 67 kD).

16. A vector according to claim 15, wherein the protein comprises the amino acid sequence of SEQ ID No: 19.

17. A vector according to claim 15 or 16, wherein the nucleotide sequence includes SEQ ID No: 18.

18. Agrobacterium strain comprising a vector according to any one of claims to 17.

19. Plant cells capable of overexpression of isochorismate synthase, wherein the cells have been transformed with a gene coding for isochorismate synthase, wherein the gene coding for isochorismate synthase is the ICS gene from Catharantus roseus.

20. Plant cells according to any one of claim 19, wherein the cells additionally comprise a gene coding for isochorismate pyruvate lyase.

21. Plant cells according to claim 19 or 20, wherein the gene for isochorismate pyruvate lyase is selected from the group consisting of orfD and pchB. C W:\llona\Sharon\SJJspecsp36025 43

22. Plant cells according to claim 20 or 21, wherein the gene coding for isochorismate synthase is ICS and the gene coding for isochorismate pyruvate lyase is orfD. DATED: 27 February, 2002 PHILLIPS ORMONDE FITZPATRICK Attorneys for: RIJKSUNIVERSITEIT LEIDEN and SYNGENTA MOGEN B. V. and KATHOLIEKE UNIVERSITEIT NIJMEGEN