WO2003048365A1

WO2003048365A1 - Fungal elicitor

Info

Publication number: WO2003048365A1
Application number: PCT/NL2002/000783
Authority: WO
Inventors: Maarten Hendrik Stuiver; Johanna Pieternella Els Van Deventer-Troost; Eugène Joannes Augustinus ROEBROECK
Original assignee: Syngenta Mogen B.V.
Priority date: 2001-12-04
Filing date: 2002-12-03
Publication date: 2003-06-12
Also published as: AU2002347667A1

Abstract

The invention comprises a new elicitor protein form Cercospora zeae maydis, its amino acid sequence and the polynucleotide sequence coding for it. Further described is a method for increasing pathogen resistance in plants by transforming them with said polynucleotide under control of a pathogen inducible promotor.

Description

FUNGAL ELICITOR

Background

Plants resistant to pathogens often are found to evoke their resistance through a mechanis which eventually yields a hypersensitive response (HR) resulting in rapid cell death of the infected plant cells. This rapid cell death or necrosis inhibits the pathogen from further growth and thus stops the infection. This mechanism is known already for a long time (Klement, Z., In: Phytopathogenic Prokaryotes, Vol. 2, eds . : Mount, M.S. and Lacy, G.H., New York, Academic Press, 1982, pp. 149-177) . The HR is often confused with other lesion-like phenomena, but a typical HR gives local cell death and is associated with secondary responses such as callus deposition, generation of active oxygen species, induction of phytoalexins, changes in ion fluxes across membranes and induction of acquired resistance (AR) (Hammond-Kosack, K.E., et al . , Plant Physiol. 110, 1381-1394, 1996) .

The pathogen resistance is elicited by response to elicitor compounds, which is frequently found to be of proteinaceous nature (Arlat, M., et al . , EMBO J., 13, 543-553, 1994; Baker, C.J. et al . , Plant Physiol. 102, 1341-1344, 1993; Staskawicz, B.J. et al . , Proc. Natl. Acad. Sci. USA 81, 6024-6028, 1984; Vivian, A. et al . , Physiol. Mol . Plant Pathol. 35, 335-344, 1989; Keen, N.T., Ann. Rev. Gen. 24, 447-463, 1990; Ronald, P.C. et al . , J. Bacteriol. 174, 1604-1611, 1992; Whitha ,S. et al., Cell 78, 1-20, 1994;Kobe, B. and Deisenhofer, J., Trends Biochem. Sci. 19, 415, 1994; and Honee G. et al . , Plant Mol. Biol . 29, 909-920, 1995) . These elicitor proteins (encoded by avirulence genes) are produced by the pathogen and are thought to signal through a resistance protein available in the plant, therewith starting a cascade of events resulting in the HR-response. The elicitor proteins are characterized by that they are race-specific and only are able to elicit the response with a corresponding (also specific) resistance protein. The concept of avirulence-gene based resistance is also known under the name of the gene-for-gene response. Avirulence genes have been cloned from bacterial and viral pathogens (such as TMV, Pseudomonas and Xanthomonas) and from fungal pathogens (such as Cladosporiuπt fulvum, Rhynchosporium secalls and Phytophthora parasitica) . Also plant genes coding for some of the corresponding resistance genes have been cloned (such as the tomato gene Cf9 corresponding to the avirulence gene avr9 from Cladosporlum fulvum, RPM1 from Arabidopsls corresponding to the avirulence gene avrRPMl from Pseudomonas syringae pv. Macullcola, Pi-ta from Oryza satlva corresponding to AvrPita from Magnaporthe grlsea and the N-gene from Nicotiana tabacum which corresponds with TMV-helica from Tobacco Mosaic Virus) .

The fungus Cercospora zeae maydis causes gray leaf spot on maize and is now recognized as one of the most significant yield-limiting diseases of maize worldwide. Documented losses range from 10 to 60% of grain yield depending on the resistance level and year.

Mature foliar lesions symptomatic of gray leaf spot are gray to tan in color, long, narrow, rectangular, and run parallel to the leaf veins. Under heavy disease pressure these lesions may coalesce and blight the entire leaf. Early symptoms of infection include pinpoint lesions surrounded by yellow halo. Generally within about two weeks these pinpoint lesions elongate and develop into their distinctive rectangular shape. Severe blighting not only causes premature death of leaves but also reduces the amount of photosynthate (sugars) required for ear fill.

Cercospora zeae maydis, like many other foliar fungal pathogens of maize, is a poor competitor in the soil and can survive only as long as infested maize debris is present. Infested maize debris on the soil surface is the source of primary inoculum for the next maize crop. The fungus colonizing this debris produces conidia (spores) as early as May. These airborne spores are the means by which the fungus infects the new maize crop.

Gray leaf spot is a highly weather-dependent disease. The pathogen requires long periods of high relative humidity and free moisture (dew) on the leaves for infection to occur. The lower leaves of the maize plant are most often the sites of initial infections. When conditions are favorable for disease development, conidia are produced in lesions on the lower leaves and serve as inoculum for the upper leaves. If conditions are not favorable for disease, the fungus can remain "dormant" during the dry part of summer and then become active when favorable conditions return. Under periods of prolonged favorable conditions, severe blighting can occur. This blighting may extend to the leaf sheath, which remains on the cut stalk after harvest. Sheath lesions are likely to serve as a source of fungal inoculum the following spring.

Very little is known about the mechanisms of resistance in maize or about the mechanisms of virulence in the pathogen. Some research is focused on determining the pathological importance of cercosporin, a phytotoxic polyketide that is thought to be a virulence factor in other species of Cercospora .

Methods to use resistance genes to confer pathogen resistance to plants are often hampered by the fact that the resistance is only limited to a few specific pathotypes

Thus there is still need for a system which can convert a fast and general pathogen resistance to plants upon start of infection and which is silent when no pathogens are infecting.

Summary of the invention

The invention now provides a method for the induction of pathogen resistance in plants which is characterized by transforming a plant with a polynucleotide sequence comprising a pathogen-inducible promoter which regulates the expression of a Cercospora zeae maydis elicitor protein or a homologue thereof which when constitutively expressed gives rise to a hypersensitive response.

A specific embodiment of the invention is a method according to claim 1 wherein the Cercospora zeae maydis elicitor is a peptide of about 24 kDa, which isolated from Cercospora zeae ^'maydis by ultrafiltration, cation exchange, hydrophobic interaction and gelfiltration chromatography.

A more specific embodiment is a method characterized in that the Cercospora zeae maydis elicitor comprises the amino acid sequence as depicted in SEQ ID NO: 2. Obviously part of the invention is the Cercospora zeae maydis elicitor itself and the nucleotide sequence coding for it.

Also part of the invention is a method according to any of the methods described above to make a plant resistant against plant pathogens.

Legend to the figures

Figure 1. SDS-PAGE gel showing the enrichment for ED24 at each stage of purification.

Figure 2. Assessment of the threshold of necrosis inducing activity in potato cv Bintje (A), wheat cv Bobwhite (B) and maize cv Granat (C) .

The concentration of the undiluted sample (0) is 500 nM and each infiltration represents a two fold dilution. The threshold for potato is 62.5 nM, wheat 100 nM and maize 16.7 to 25 nM.

Figure 3. Necrosis induced by ED24 (I), Harpin (II), Cercosporin (III) and Avr9 (IV) viewed with both white (panel A and C) and UV light

(panel B and D) , displaying the autofluorescence (marked with an arrow) around the infiltrated patch of ED24, Harpin and Avr9.

Detailed description

Although the invention is illustrated in detail by activity in potato, wheat and maize plants, it should be understood that any plant species that contains the mechanism to recognize the Cercospora zeae maydis elicitor of the invention may be provided with one or more plant expressible gene constructs, which when expressed are capable of inducing a HR-response. The invention can even be practiced in plant species that are presently not amenable for transformation, as the amenability of such species is just a matter of time and because transformation as such is of no relevance for the principles underlying the invention. Hence, plants for the purpose of this description shall include angiosperms as well as gymnosperms, monocotyledonous as well as dicotyledonous plants, be they for feed, food or industrial processing purposes; included are plants used for any agricultural or horticultural purpose including forestry and flower culture, as well as home gardening or indoor gardening, or other decorative purposes . In order to provide a quick and simple test if a new plant species indeed can yield a hypersensitive response upon presentation of the Cercospora zeae maydis elicitor the person skilled in the art can perform one of two tests. One of the most reliable is the infiltration of the elicitor protein in the leaves, and scoring for the HR. Another one is a rapid transient expression test known under the name of ATTA (Agrobacterium tumefaciens Transient expression Assay) . In this assay (of which a detailed description can be found in Van den Ackerveken, G., et al . , Cell 87, 1307-1316, 1996) the nucleotide sequence coding for the Cercospora zeae maydis elicitor is placed under control of the CaMV 35S promoter and introduced into an Agrobacterium strain which is also used in protocols for stable transformation. After incubation of the bacteria with acetosyringon or any other phenolic compound which is known to enhance Agrobacterium T- DNA transfer, 1 ml of the Agrobacterium culture is infiltrated into an in si tu plant by injection after which the plants are placed in a greenhouse. After 2-5 days the leaves can be scored for occurrence of HR symptoms .

Overexpression of proteins.

Proteins of the invention, also denominated Cercospora zeae maydis elicitor, include all proteins comprising the amino acid sequence of the mature protein of SEQ ID NO: 2 (amino acids 1-226) and muteins thereof. Preferably, the protein also includes the putative protein leader of amino acids -45 to -1 of SEQ ID NO: 2.

The word protein means a sequence of amino acids connected trough peptide bonds. Polypeptides or peptides are also considered to be proteins. A protein leader comprises the protein sequences encoded in the open reading frame which are not present in the mature protein. It may comprise a signal peptide needed for translocation to the ER and a propeptide, which is cleaved off during the posttranslational processing.

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 HR-response inducing activity. Such muteins can readily be made by protein engineering in vivo, e.g. by changing the open reading frame capable of encoding the protein so that the amino acid sequence is thereby affected. As long as the changes in the amino acid sequences do not altogether abolish the activity of the protein such muteins are embraced in the present invention. Further, it should be understood that muteins should be derivable from the proteins depicted in the sequence listing while retaining biological activity, i.e. all, or a great part of the intermediates between the mutein and the protein depicted in the sequence listing should have HR-response inducing activity. A great part would mean 30% or more of the intermediates, preferably 40% of more, more preferably 50% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90% or more, more preferably 95% or more, more preferably 99% or more.

The present invention also provides the nucleotide sequence coding for the Cercospora zeae maydis elicitor of which the amino acid sequence is depicted in SEQ ID NO: 2. Preferably, the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:l, more preferably a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 288 to nucleotide 965 and most preferably a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:l from nucleotide 153 to nucleotide 965 (coding for the complete protein inclusive protein leader sequence) . Also part of the invention are nucleotide sequences which are conservatively modified variants of the above mentioned sequences or polymorphic variants thereof. Those of skill in the art will recognise that the degeneracy of the genetic code allows for a plurality of polynucleotides to encode for the identical amino acid. Such "silent variations" can be used, for example, to selectively hybridise and detect allelic variants of the nucleotide sequences of the present invention. Additionally, the present invention provides isolated nucleotide sequences comprising one or more polymorphic (allelic) variants of the above nucleotide sequences. Further part of the invention are polynucleotides still coding for a protein which has a biological function identical to the function of the Cercospora zeae maydis elicitor, which are the product of amplification from a nucleotide library using primer pairs which selectively hybridise under stringent conditions to loci within the above mentioned nucleotide sequences. The primer length in nucleotides is selected from the group of integers consisting of from at least 15 to 50. Those of skill in the art will recognise that a lengthened primer sequence can be employed to increase specificity of binding (i.e. annealing) to a target sequence. Stringent conditions in this respect means a reaction at a temperature of between 60°C and 65°C in 0.3 strength citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1% SDS.

Thus, also part of the invention are polynucleotides which selectively hybridise, under selective hybridisation conditions, to one or more of the above discussed nucleotide sequences, and which code for an amino acid sequence which has a biological function similar to the function of the Cercospora zeae maydis elicitor of the invention. Another way to indicate hybridisation potential is on sequence identity. In this sense, the present invention provides also for nucleotide sequences which have a percentage of identity related to the above mentioned sequences of 60% to 95%. Thus, for example, the percentage of identity can be at least, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. Sequence identity on nucleotide sequences can be calculated by using the BLASTN computer program (which is publicly available, for instance through the National Center for Biotechnological Information, accessible via the internet on http: //www. ncbi .nlm. nih. gov/ ) using the default settings of 11 for wordlength (W) , 10 for expectation (E) , 5 as reward score for a pair of matching residues (M) , -4 as penalty score for mismatches (N) and a cutoff of 100.

The present invention provides a chimeric DNA sequence which comprises a pathogen inducible promoter which regulates the expression of the Cercospora zeae maydis elicitor which is capable of eliciting a hypersensitive response. The expression chimeric DNA sequence shall mean to comprise any DNA sequence which comprises DNA sequences not naturally found in nature. The 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 nucleotide sequence according to the invention. The open reading frame coding for the Cercospora zeae maydis elicitor 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 the protein. The open reading frame may also be encoded by one uninterrupted exon, or by a cDNA to the mRNA encoding the Cercospora zeae maydis elicitor. 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.

Pathogen inducible promoters are known in the art and are responsive to a large number of pathogens and to aspecific elicitors produced by these pathogens. Examples of such pathogen inducible promoters are: the prpl promoter (Martini, N., et al . , Mol. Gen. Genet. 236, 179-186, 1993), the Fisl promoter (WO 96/34949), the Bet v 1 promoter (Swoboda, I., et al., Plant, Cell and Env. 18_, 865-874, 1995), the Vstl promoter (Fischer, R., Dissertation, Univ. of Hohenheim, 1994; Schubert, R. , et al . Plant Mol. Biol. 34, 417-426, 1997), the sesquiterpene cyclase promoter (Yin, S., et al . , Plant Physiol. 115, 437-451, 1997), the MS-59 promoter (WO 99/50428), the ICS promoter from Ca tharantus roseus (WO 99/50423) , the #488 promoter from Arabidopsis thaliana (WO 00/60086) and the gstAl promoter (Mauch, F. and Dudler, R. , Plant Physiol. 102, 1193-1201, 1993). Several other promoters are known in the art and can be used to drive expression of the nucleotide sequences of this invention.

In eukaryotic cells, an expression cassette usually further comprises a transcriptional termination region located downstream of the 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. The principles governing the expression of a chimeric DNA construct in a chosen host cell are commonly understood by those of ordinary skill in the art and the construction of expressible chimeric DNA constructs is now routine for any sort of host cell, be it prokaryotic or eukaryotic.

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

The expression "host cells incorporating a chimeric DNA sequence according to the invention in their genome" shall mean to comprise cells, as well as multicellular organisms comprising such cells, or essentially consisting of such cells, which stably incorporate said chimeric DNA into their genome thereby maintaining the chimeric DNA, and preferably transmitting a copy of such chimeric DNA to progeny cells, be it through mitosis or meiosis. According to a preferred embodiment of the invention plants are provided, which essentially consist of cells which incorporate one or more copies of said chimeric DNA into their genome, and which are capable of transmitting a copy or copies to their progeny, preferably in a Mendelian fashion. By virtue of the transcription and translation of the chimeric DNA according to the invention in some or all of the plant's cells, those cells that are capable of producing the Cercospora zeae maydis elicitor upon infection with a pathogen will show enhanced resistance to fungal infections .

Transformation of plant species is now routine for an impressive number of plant species, including both the Dicotyledoneae as well as the Monocotyledoneae. In principle any transformation method may be used to introduce chimeric DNA according to the invention into a suitable ancestor cell, as long as the cells are capable of being regenerated into whole plants . Methods may suitably be selected from the calcium/polyethylene glycol method for protoplasts (Krens, F.A. et al . , 1982, Nature 296, 72-74; Negrutiu I. et al, June 1987, Plant Mol. Biol. _8, 363-373), electroporation of protoplasts (Shillito R.D. et al . , 1985 Bio/Technol. 3, 1099-1102), microinjection into plant material (Crossway A. et al . , 1986, Mol. Gen. Genet. 202, 179-185), (DNA or RNA-coated) particle bombardment of various plant material (Klein T.M. et al . , 1987, Nature 327, 70), infection with (non- integrative) viruses and the like. A preferred method according to the invention comprises Agrojbacterium-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 . (Van Roekel, J.S.C., Dam , B., Melchers, L.S., Hoekerαa, A. (1993) . Factors influencing transformation frequency of tomato (Lycopersicon esculentum) . Plant Cell Reports, 12, 644-647) . Potato transformation is preferably done essentially as described by Hoekema et al. (Hoekema, A., Huisman, M.J., Molendijk, L., van den Elzen, P.J.M., and Cornelissen, B.J.C. (1989). The genetic engineering of two commercial potato cultivars for resistance to potato virus X. Bio/Technology 7, 273-278) .

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 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, 1989, Nature 338, 274-276) . Transgenic maize plants have been obtained by introducing the Streptomyces hygroscopicus bar-gene, which encodes phosphinothricin acetyltransferase (an enzyme which inactivates the herbicide phosphinothricin) , into embryogenic cells of a maize suspension culture by microprojectile bombardment (Gordon-Kamm, 1990, Plant Cell, 2 , 603-618). The introduction of genetic material into aleurone protoplasts of other monocot crops such as wheat and barley has been reported (Lee, 1989, Plant Mol. Biol . 13, 21-30). Wheat plants have been regenerated from embryogenic suspension culture by selecting only the aged compact and nodular embryogenic callus tissues for the establishment of the embryogenic suspension cultures (Vasil, 1990 Bio/Technol. 8_, 429-434). The combination with transformation systems for these crops enables the application of the present invention to monocots.

Monocotyledonous plants, including commercially important crops such as rice and corn are also amenable to DNA transfer by Agrobacterium strains { vide WO 94/00977; EP 0 159 418 Bl; Gould J, Michael D, Hasegawa 0, Ulian EC, Peterson G, Smith RH, (1991) Plant. Physiol. 95, 426-434).

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 geno ic organization. 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 a pathogen.

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. These plants, 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 pesticide treatment, thus lowering costs of material, labour, and environmental pollution, or prolonging shelf-life of products (e.g. fruit, seed, and the like) of such plants . Plants for the purpose of this invention shall mean multicellular organisms capable of photosynthesis, and subject to some form of pathogen induced disease. They shall at. least include angiosperms as well as gymnosperms, monocotyledonous as well as dicotyledonous plants.

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

EXAMPLE 1 Growth of fungi and isolation of culture filtrate

The culture filtrate (clarified culture medium) of fungi were screened for their ability to induce necrosis following infiltration into the apoplast of flag leaves from 8 weeks old wheat plants (cv Bobwhite) . Necrosis-inducing activity was observed from the culture filtrate of a 7 day old Cercospora zeae maydis culture, which was grown and isolated using the following conditions . Spores were harvested from a 2 week old PDA culture and used to inoculate a flask of Gamborg-B5 medium (supplemented with 20g/l sucrose) to a final concentration of 3 x 10⁸ spores/1. The fungus was grown at room temperature on an orbitary shaker (120 rprα) . After 7 days, the culture medium was isolated by filtering the culture through 3 layers of miracloth, followed by centrifugation (10,000 rpm in a Sorvall GS3 rotor) to remove any remaining debris.

EXAMPLE 2 Assessment ofthe nature ofthe necrosis inducing component

To assess whether the necrosis-inducing component was proteinaceous, the crude C. zeae maydis culture filtrate was subjected to protease digestion and boiling. Samples were boiled for 10 and 30 minutes, cooled on ice and spun down to remove any debris. The protease treatment was performed using proteinase K coated acrylic beads

(Sigma) . The beads were resuspended in 50 mM Tris-HCl pH 8 (0.5 g/ml) . The Czm extract was diluted 1:1 with the suspension or in buffer

(control) and incubated in a rotating incubator at 40°C for 1 hour. The supernatants of both treatments were infiltrated into wheat flag leaves. Boiling (10 and 30 minutes) as well as the proteinase K treatment completely abolished cell death inducing activity, indicating that the cell death eliciting factor (s) were most likely proteins .

EXAMPLE 3 Purification

The clarified culture medium of a 7 day old culture of C. zeae maydis was concentrated by ultrafiltration across a 10 kDa filter. The retentate was dialyzed against 50 mM NaAc pH 4.0 and subsequently applied to a cation exchange column (Resource S column, Pharmacia) , equilibrated in the same buffer. The bound proteins were eluted using a gradient from 0 - 1 M NaCl and collected in 1 ml fractions. All fractions plus the flow through were dialysed to water and infiltrated into wheat flag leaves and potato leaves ( Solanum tuberosum cv Bintje) to identify the necrosis-inducing component. Necrosis-inducing activity fell into one major peak eluting at approximately 200 mM NaCl (Figures 1) .

Active fractions were pooled, adjusted to 1 M ammonium sulphate and applied to a hydrophobic interaction column (Source 15PHE column, Pharmacia) equilibrated with 50 mM potassium phosphate buffer pH 7 and 1 M ammonium sulphate. The bound proteins were eluted using a decreasing gradient from 1 - 0 M ammonium sulphate and collected in 1.5 ml fractions. The fractions and flow through of this run were dialysed to water and assessed for necrosis inducing activity in wheat and potato. Activity fell in one major peak eluting at approximately 500 mM ammonium sulphate. Analysis of the profile by SDS-PAGE indicated that necrosis-inducing activity corresponded with a 24 kDa band (Figure 1) .

To allow purification to homogeneity active fractions were pooled, concentrated using a centricon ultrafiltration device (10 kDa cutoff) and applied to a gel filtration column (SD75, Pharmacia) . Pooled fractions were applied to the column, equilibrated in 50 mM Mes pH 6 and 150 mM NaCl. Proteins were eluted using an isocratic gradient in the same buffer. Fractions were again tested for necrosis inducing activity in wheat and potato and again activity coincided with the 24 kDa band which was purified to apparent homogeneity and designated ED24 (Figure 1) .

EXAMPLE 4 Determination and characterisation of the sequence of ED24

The pure ED24 was resolved by SDS-PAGE and the band (24 kDa) was excised and submitted for sequencing by Ed an degradation. Sequence was obtained from both the N-terminus and an internal fragment . The internal fragment was generated via an in-gel trypsin digestion followed by RP-HPLC purification of the resulting fragments . Sequence analysis was performed by Edman degradation on an Applied Biosystems 477A protein sequencer according to manufacturers instructions. The N-terminal and internal amino acid sequence allowed degenerate primers to be made and a fragment of the cDNA sequence was amplified. The remaining sequence was obtained by performing 5' and 3' RACE reactions using primers designed from the isolated cDNA fragment. This allowed the isolation of the full length cDNA sequence (SEQ ID NO:l)

The full predicted amino acid sequence of ED24 was obtained following translation of the cDNA sequence. Analysis of this sequence reveals that there is a 45 amino acid sequence that precedes the mature protein. The role of this leader sequence in the formation of the mature protein is currently unclear. As the protein was found to be secreted into the culture medium one function is at least to act as a signal peptide, however this sequence is significantly larger than typical signal peptides (von Heijne, 1983) . The putative mature ED24 protein is 227 amino acids (or 24.7 kDa), consistent with the observed size on SDS gels. It is therefore presumed that there are no posttranslational modifications made to the protein.

When subjected to BLAST analysis neither the amino acid or nucleotide sequence displayed any significant homology to any previous reported sequence.

EXAMPLE 6 Activity spectrum

Necrosis-inducing activity of ED24 was assessed in a range of crops and a threshold for the activity in potato, wheat and maize was assessed (Figure 2) . ED24 displays necrosis-inducing activity in the majority of dicot and monocots crops tested, which included potato, tomato, tobacco, wheat, maize and banana. No activity was observed following infiltration into rice leaves at the concentrations tested. The threshold concentrations for the necrosis-inducing activity of ED24 in potato, wheat and maize are 62.5,- 100 and 16.7 to 25 nM, respectively.

EXAMPLE 7 Partial dissection of the mechanism of ED24-induσed necrosis To further characterise the necrosis-inducing activity of ED24, the protein was infiltrated together with a range of signal transduction inhibitors. A selection of signal transduction inhibitors, thought to be involved in the hypersensitive response, was used for this characterisation. The experiment was performed in wheat (cv Bobwhite) . Vanadate (an inhibitor of ATPase and phosphatase) and lanthanum (a calcium channel blocker) completely abolished the cell death induced by ED24. cPTIO (a scavenger of nitric oxide) was able to reduce the ED24-induced necrosis in wheat by approximately 50%. These observations clearly show that the necrosis induced by ED24 is an active process and may require important plant signalling processes which for ED24 include ATPase activity, nitric oxide and calcium flux across membranes.

Various elicitors of the HR have been shown to depend on active plant metabolism. The induction of HR by avirulent Pseudomonas syringae in soybean cell cultures is inhibited by cPTIO (Delledonne et al . (1998) Nature 394:585-588). Futhermore, the HR induced in tobacco by harpinPss, an elicitor from P. syringae, is inhibited by vanadate and lanthanum (He et al . (1993) Cell 73:1255-1266), similarly lanthanum inhibits the induction of kinase activation and AOS production in tobacco cell cultures treated with Avr9, an elicitor from the fungal pathogen Cladosporium fulvum (Romeis et al . (1999) Plant Cell 11:273- 287) .

Another marker of the hypersensitive response that was examined was the ability of ED24 to induce components of local resistance known to be associated with a hypersensitive response (Dorey et al . , 1997). These responses include the deposition of phenolics around the necrotic patch in the form of lignin, which can be viewed via its autofluorescence. Autofluorescence following the infiltration of protein was examined in potato. Avr9 and harpin_EA (from Erwinia amylovora) were used as positive controls for the induction of an HR and cercosporin as a necrosis-inducing toxin which does not trigger an HR. Cercosporin is a non-specific toxin, produced by members of the fungal genus Cercospora . It is a photosensitising compound that, in the presence of light, transforms light energy to oxygen, producing a highly toxic activated singlet form of oxygen. In plants the toxin causes the peroxidation of membrane lipids, leading to membrane breakdown and cell death (Daub & Ehrenshaft (2000) Annu. Rev. Phytopathol . 38:461-490). As can be seen in Figure 3 ED24 as well as harpin_EA and Avr9 display clear autofluorescence around the necrotic patch whereas no autofluorescence is observed around the necrosis induced by cercosporin. Considering these data it is very likely that ED24 acts as an elicitor of the hypersensitive response, rather than as a toxin.

SEQUENCE LISTING

<110> Syngenta Mogen B.V.

<120> Novel fungal elicitor

<130> ED24

<140> <141>

<160> 2

<170> Patentln Ver. 2.1

<210> 1

<211> 1138

<212> DNA

<213> Cercospora zeae maydis

<220>

<221> CDS

<222> (153) .. (965)

<220>

<221> sig_peptide

<222> (153) .. (288)

<220>

<221> mat_peptide

<222> (288) .. (965)

<400> 1 acgcggggaa acaccctcaa gcagctcttt agcaactctc aacagaaaca cttctctgct 60 gtcttctctc ttcttgagtc tactctctcc atcttgccat catgaagttc acttcaagct 120 atcttgccgc cctggcactt gttcccctgg cc atg get get ccg cag ccc gag 173

Met Ala Ala Pro Gin Pro Glu -45 -40 cac cgc gat gtc etc ace gac ttg ttc aag gtc gac ggc gag cac tgg 221 His Arg Asp Val Leu Thr Asp Leu Phe Lys Val Asp Gly Glu His Trp -35 -30 -25 etc caa gcc gtc aag gag gag get get cgt cag gag gee tct get cct 269 Leu Gin Ala Val Lys Glu Glu Ala Ala Arg Gin Glu Ala Ser Ala Pro -20 -15 -10 aag ggc etc cag gcg cgt aat gat gga aag ttt aag gtg cat gcg atg 317

Lys Gly Leu Gin Ala Arg Asn Asp Gly Lys Phe Lys Val His Ala Met

-5 -1 1 5 10 tac ace gat aat atg ate aac ctt ggt gac gtg gac tac ttc cat gcc 365 Tyr Thr Asp Asn Met lie Asn Leu Gly Asp Val Asp Tyr Phe His Ala

15 20 ^' 25 etc tgg cag cgt atg tac gac gtt agt aac gat aga ggc ggt etc tct 413

Leu Trp Gin Arg Met Tyr Asp Val Ser Asn Asp Arg Gly Gly Leu Ser 30 35 40 gac ace act ace ggc get tgg cat aaa ttc tgc cag aaa cct aac gag 461

Asp Thr Thr Thr Gly Ala Trp His Lys Phe Cys Gin Lys Pro Asn Glu 45 50 55 ggt cct aat att gag gac cgc ttt ate etc gac ggc cag tgg ggt get 509 Gly Pro Asn lie Glu Asp Arg Phe lie Leu Asp Gly Gin Trp Gly Ala 60 65 70 gtc tct ggt gtc age ggc tgg cag atg cgc gac gcc eta ate cac tct 557 Val Ser Gly Val Ser Gly Trp Gin Met Arg Asp Ala Leu lie His Ser

75 80 85 90 atg tgg gag acg gcc agg act ate ggc ace acg ggc agt aat gcc tac 605

Met Trp Glu Thr Ala Arg Thr He Gly Thr Thr Gly Ser Asn Ala Tyr 95 100 105 act gtc tac age gac tge tac ggc tgg acg tgg cag gag tec gtg ccg 653

Thr Val Tyr Ser Asp Cys Tyr Gly Trp Thr Trp Gin Glu Ser Val Pro 110 115 120 aat aat aag aat gcc gcc tge ggt ccc tct get aga gtc caa tge ccc 701

Asn Asn Lys Asn Ala Ala Cys Gly Pro Ser Ala Arg Val Gin Cys Pro 125 130 135 aag aac gac gac tge cct gca cac ggt atg gaa tge gaa cac tct aag 749

Lys Asn Asp Asp Cys Pro Ala His Gly Met Glu Cys Glu His Ser Lys 140 145 150 cct gga gcc tgg tta ccc agt att ate cgt ate aat gtt tat aac cct 797 Pro Gly Ala Trp Leu Pro Ser He He Arg He Asn Val Tyr Asn Pro

155 160 165 170 gat gga tct etc cgt gcc gat gcc tac cag gca cgc att tec tec gag 845

Asp Gly Ser Leu Arg Ala Asp Ala Tyr Gin Ala Arg He Ser Ser Glu 175 180 185 ggt tta ggt ggc aag ggc tge gac aag ctt ace cag gtt get gcc get 893

Gly Leu Gly Gly Lys Gly Cys Asp Lys Leu Thr Gin Val Ala Ala Ala 190 195 200 gtt tct gga ttc ctt ccc ggt gcc ggt cag tac ttt get get ggc ate 941 Val Ser Gly Phe Leu Pro Gly Ala Gly Gin Tyr Phe Ala Ala Gly He 205 210 215 agt gtc cag tge gtc ttc cgt tec taagtgaget ecgaaaggaa atggagtgaa 995

Ser Val Gin Cys Val Phe Arg Ser 220 225 aagaaaggag gagagaaggg tetatccaca tgceaateat atagatctat ttacgtctct 1055 ttacetgtag tttatatata tggactagta atataaatta aattttactc ctagtcccaa 1115 aaaaaaaaaa aaaaaaaaaa aaa 1138

<210> 2

<211> 271

<212> PRT

<213> Cercospora zeae maydis

<400> 2

Met Ala Ala Pro Gin Pro Glu His Arg Asp Val Leu Thr Asp Leu Phe

-45 -40 -35 -30 Lys Val Asp Gly Glu His Trp Leu Gin Ala Val Lys Glu Glu Ala Ala

-25 -20 -15

Arg Gin Glu Ala Ser Ala Pro Lys Gly Leu Gin Ala Arg Asn Asp Gly -10 -5 -1 1

Lys Phe Lys Val His Ala Met Tyr Thr Asp Asn Met He Asn Leu Gly 5 10 15

Asp Val Asp Tyr Phe His Ala Leu Trp Gin Arg Met Tyr Asp Val Ser 20 25 30 35

Asn Asp Arg Gly Gly Leu Ser Asp Thr Thr Thr Gly Ala Trp His Lys 40 45 50

Phe Cys Gin Lys Pro Asn Glu Gly Pro Asn He Glu Asp Arg Phe He 55 60 65

Leu Asp Gly Gin Trp Gly Ala Val Ser Gly Val Ser Gly Trp Gin Met 70 75 80 Arg Asp Ala Leu He His Ser Met Trp Glu Thr Ala Arg Thr He Gly 85 90 95

Thr Thr Gly Ser Asn Ala Tyr Thr Val Tyr Ser Asp Cys Tyr Gly Trp 100 105 110 115

Thr Trp Gin Glu Ser Val Pro Asn Asn Lys Asn Ala Ala Cys Gly Pro 120 125 130

Ser Ala Arg Val Gin Cys Pro Lys Asn Asp Asp Cys Pro Ala His Gly 135 140 145

Met Glu Cys Glu His Ser Lys Pro Gly Ala Trp Leu Pro Ser He He 150 155 160 Arg He Asn Val Tyr Asn Pro Asp Gly Ser Leu Arg Ala Asp Ala Tyr 165 170 175

Gin Ala Arg He Ser Ser Glu Gly Leu Gly Gly Lys Gly Cys Asp Lys 180 185 190 195

Leu Thr Gin Val Ala Ala Ala Val Ser Gly Phe Leu Pro Gly Ala Gly 200 205 210

Gin Tyr Phe Ala Ala Gly He Ser Val Gin Cys Val Phe Arg Ser 215 220 225

Claims

1. Elicitor protein from Cercospora zeae maydis characterized in that it comprises the amino acid sequence of amino acids 1 to 226 of SEQ ID NO: 2 or muteins thereof.

2. Elicitor protein according to claim 1 characterized in that it comprises amino acids -45 to 226 of SEQ ID NO: 2 or muteins thereof.

3. A polynucleotide comprising a sequence coding for a protein according to claim 1 or claim 2.

4. A polynucleotide which can hybridise under stringent conditions to a polynucleotide according to claim 3 and which codes for a protein that upon introduction into a plant causes a hypersensitive reaction.

5. A polynucleotide according to claim 3 characterised in that it comprises the nucleotide sequence of nucleotides 288-965 of SEQ ID N0:1.

6. A polynucleotide according to claim 5 characterised in that it comprises the nucleotide sequence of nucleotides 153-965 of SEQ ID NO:l.

7. A ehimaerie nucleotide sequence comprising a polynucleotide according to any of claims 3 to 6 characterised in that the open reading frame coding for the elicitor protein is under control of a plant pathogen inducible promoter.

8. A chimearic nucleotide according to claim 7, characterised in that the pathogen inducible promoter is selected from the group consisting essentially of the prpl-promoter, the Vstl-promoter, the ICS- promoter, the #488-promoter, the MS59-promoter, the Fisl-promoter, the Bet v 1 promoter, the sequiterpene cyclase promoter and the gs Al-promoter .

9. Method for the induction of pathogen resistance in plants characterized by transforming a plant with a ehimaerie nucleotide sequence according to claim 7 or 8.

10. Plant transformed with a ehimaerie nucleotide according to claim 7 or