Antifungal proteins, DNA coding therefor, and hosts incorporating same

The present invention provides an isolated protein obtainable from a plant source which has anti-Phytophthora activity and a molecular weight of about 60.+-.5 kDa as judged by SDS PAGE-electrophoresis, an isolated DNA sequence comprising an open reading frame capable of encoding a protein according to the invention, preferably characterized in that it comprises an open reading frame which is capable of encoding a protein as represented by amino acids 1 to 540 of SEQ ID NO: 6, or the precursor of said protein, and DNA capable of hybridising therewith under stringent conditions. The invention further comprises plants incorporating chimeric DNA capable of encoding a protein according to the invention, and wherein the protein is expressed. Also methods are provided for combatting fungi, especially Phytophthora infestans, using a protein or a host cell capable of producing the protein.

FIELD OF THE INVENTION
 The present invention relates to antifungal proteins, DNA coding therefor
 and hosts incorporating the DNA, as well as methods of combating fungal
 pathogens by causing said fungal pathogens to be contacted with said
 protein or proteins.
 The invention further relates to plants, incorporating and expressing DNA
 coding for antifungal proteins, and to plants which as a result thereof
 show reduced susceptibility to fungal pathogens, in particular to the
 Oomycete Phytophthora infestans.
 BACKGROUND ART
 Phytophthora infestans belongs to the group of fungi referred to as
 Oomycetes. Phytophthora infestans infects various members of Solanaceae,
 such as potato, tomato and some ornamentals. It causes late blight of
 potatoes and tomatoes affecting all parts except roots. Geographically,
 the fungus is widely distributed, and it can be found in all
 potato-producing countries. Economically late blight in potatoes is of
 major importance, as infection early in the season can severely reduce
 crop yield. Currently the disease is controlled by spraying chemical
 fungicides (dithiocarbamates, such as mancozeb, manec and zineb)
 regularly. (For a review, vide: European Handbook of Plant Diseases, ed.
 by I. M. Smith et al., 1988, Blackwell Scientific Publications, Ch.8).
 Both from an environmental and economical point of view, biological
 control of diseases caused by Phytophthora infestans could have advantages
 over the use of chemical fungicides. A protein with antifungal activity,
 isolated from TMV-induced tobacco leaves, which is capable of causing
 lysis of germinating spores and hyphal tips of Phytophthora infestans and
 which causes the hyphae to grow at a reduced rate, was disclosed in
 WO91/18984 A1. This protein has an apparent molecular weight of about 24
 kDa and was named AP24. Comparison of its complete amino acid sequence, as
 deduced from the nucleic acid sequence of the AP24 gene, with proteins
 known from a database revealed that the protein was an osmotin-like
 protein.
 Experiments are in progress to evaluate plants which relatively
 over-express AP24 for resistance against various other fungal pathogens.
 Despite initial success in combating fungal pathogens, such as Phytophthora
 infestans, and the genetic engineering of plants capable of producing
 these antifungal proteins with activity against this fungal pathogen there
 remains a need to identify and isolate other proteins with antifungal
 activity against this fungus.
 SUMMARY OF THE INVENTION
 The present invention provides an isolated protein obtainable from a plant
 source which has anti-Phytophthora activity and a molecular weight of
 about 60.+-.5 kDa as judged by SDS PAGE-electrophoresis. A more preferred
 protein is one that is obtainable from tobacco or tomato plants. A still
 more preferred isolated protein is characterised in that it is selected
 from the group of proteins having the amino acid sequence extending from:
 (a) amino adds 1 to 540 of SEQIDNO: 6, or
 (b) amino adds 23 to 540 of SEQIDNO: 6,
 as well as muteins thereof which have anti-Phytophthora activity. A still
 further preferred protein according to the invention is one characterised
 in that it is capable of being encoded by the open reading frame
 represented by SEQIDNO: 6, or by part of said open reading frame by virtue
 of an in-frame translational stop codon introduced in the 3' end of said
 open reading frame, causing the protein encoded thereby to be C-terminally
 truncated.
 The invention also embraces an isolated DNA sequence comprising an open
 reading frame capable of encoding a protein according to the invention,
 preferably characterised in that it comprises an open reading frame which
 is capable of encoding a protein as represented by amino acids 1 to 540 of
 SEQIDNO: 6, or the precursor of said protein, and DNA capable of
 hybridising therewith under stringent conditions.
 The invention also provides a chimeric DNA sequence according to the
 invention further comprising a transcriptional initiation region and,
 optionally, a transcriptional termination region, so linked to said open
 reading frame as to enable the DNA to be transcribed in a living host cell
 when present therein, thereby producing RNA which comprises said open
 reading frame. A preferred chimeric DNA sequence according to the
 invention is one, wherein the RNA comprising said open reading frame is
 capable of being translated into protein in said host cell, when present
 therein, thereby producing said protein.
 The invention also embraces a chimeric DNA sequence comprising a DNA
 sequence according to the invention, which may be selected from replicons,
 such as bacterial cloning plasmids and vectors, such as a bacterial
 expression vector, a (non-integrative) plant viral vector, a Ti-plasmid
 vector of Agrobacterium, such as a binary vector, and the like, as well as
 a host cell comprising a replicon or vector according to the invention,
 and which is capable of maintaining said replicon once present therein.
 Preferred according to that embodiment is a host cell which is a plant
 cell, said vector being a non-integrative viral vector.
 The invention further provides a host cell stably incorporating in its
 genome a chimeric DNA sequence according to the invention, such as a plant
 cell, as well as multicellular hosts comprising such cells, or essentially
 consisting of such cells, such as plants. Especially preferred are plants
 characterised in that the chimeric DNA according to the invention is
 expressed in at least a number of the plant's cells causing the said
 anti-Phytophthora protein to be produced therein.
 According to yet another embodiment of the invention a method for producing
 a protein with anti-Phytophthora activity is provided, characterised in
 that a host cell according to the invention is grown under conditions
 allowing the said protein to be produced by said host cell, optionally
 followed by the step of recovering the protein from the host cells.
 The invention provides also for the use of a protein according to the
 invention for retarding the growth of Phytophthora infestans, preferably
 characterised in that spores of the said fungus are caused to be contacted
 with said protein. According to yet another embodiment, retarding the
 growth of the fungus Phytophthora infestans is on plant leaves,
 characterised in that hyphae thereof, or spores thereof, are caused to be
 contacted with a protein produced from a host cell according to the
 invention capable thereof.
 The invention also provides a method for obtaining plants with reduced
 susceptibility to Phytophthora infestans, comprising the steps of
 (a) introducing into ancestor cells which are susceptible of regeneration
 into a whole plant,
 a chimeric DNA sequence comprising an open reading frame capable of
 encoding a protein according to claim 1, said open reading frame being
 operatively linked to a transcriptional and translational region and,
 optionally, a transcriptional termination region, allowing the said
 protein to be produced in a plant cell that is susceptible to infection by
 Phytophthora infestans, and
 a chimeric DNA sequence capable of encoding a plant selectable marker
 allowing selection of transformed ancestor cell's when said selectable
 marker is present therein, and
 (b) regenerating said ancestor cells into plants under conditions favouring
 ancestor cells which have the said selectable marker, and
 (c) identifying a plant which produces a protein according to claim 1,
 thereby reducing the susceptibility of said plant to infection by
 Phytophthora infestans. Preferred according to the invention is a method
 characterised in that step (a) is performed using an Agrobacterium
 tumefadens strain capable of T-DNA transfer to plant cells and which
 harbours the said chimeric DNA on binary vector pMOG841, step (b) being
 performed in the presence of an antibiotic favouring cells which have a
 neomycin phosphotransferase.
 The invention further provides an antifungal composition comprising a
 protein according to the invention and a suitable carrier.
 An antibody, capable of reacting with an N-terminal fragment of a protein
 according to the invention, preferably to the peptide represented by
 SEQIDNO: 17, is also provided. The antibody is suitably used to detect
 expression levels of chimeric DNA according to the invention in host cells
 and multicellular hosts, preferably plants, capable of producing a protein
 according to the invention.
 The invention also provides a nudeic acid sequence obtainable from a gene
 encoding a protein according to the invention, said nudeic acid sequence
 having tissue-specific transcriptional regulatory activity in a plant. The
 invention specifically provides a nucleic acid sequence obtainable from
 the region upstream of the translational initiation site of said gene,
 preferably at least 780 nucleotides immediately upstream of the
 translational initiation site of said gene. This nudeic acid sequence is
 preferably used to make a tissue-specific plant expressible gene construct
 which is preferentially expressed in vascular tissue of the plant, and/or
 preferentially expressed in the style and stigma of the plant.

DETAILED DESCRIPTION OF THE INVENTION
 The protein according to the present invention may be obtained by isolating
 it from any suitable plant source material containing it. A particularly
 suitable source comprises leaves of TMV-induced plants, more in particular
 Solanaceae, such as tobacco (e.g. Nicotiana tabacum Samsun NN), tomato
 (Lycopersicon esculentum var. Moneymaker) and the like. The presence of
 anti-Phytophthora proteins according to the invention in plant source
 material can readily be determined for any plant species by making plant
 extracts from those species and testing those extracts for the presence of
 anti-Phytophthora activity using an in vitro antifungal assay as described
 herein, further fractionating the obtained samples by any suitable protein
 fractionation technique in conjunction with the in vitro assay until an
 antifungal fraction is obtained which comprises an approximately 60 kDa
 protein, which in isolated form shows anti-Phytophthora activity.
 Similarly, fractions may be tested for antifungal activity on other
 Oomycetes, for example, Pythium ultimum and the like, or other fungi, such
 as the Basidiomycetes, or other taxons.
 Alternatively, anti-Phytophthora proteins according to the invention may be
 obtained by cloning DNA comprising an open reading frame capable of
 encoding said protein, or the precursor thereof, linking said open reading
 frame to a transcriptional, and optionally a translational initiation and
 transcriptional termination region, inserting said DNA into a suitable
 host cell and allowing said host cell to produce said protein.
 Subsequently, the protein may be recovered from said host cells,
 preferably after secretion of the protein into the culture medium by said
 host cells.
 Alternatively, said host cells may be used directly in a process of
 combating fungal pathogens according to the invention as a pesticidally
 acceptable composition.
 Host cells suitable for use in a process of obtaining a protein according
 to the invention may be selected from eprokaryotic microbial hosts, such
 as bacteria e.g. Agrobacterium, Bacillus, Cyanobacteria, E.coli,
 Pseudomonas, and the like, as well as eukaryotic hosts including yeasts,
 e.g. Saccharomyces cerevisiae, fungi, e.g. Trichoderma and plant cells,
 including protoplasts.
 In a method of retarding the growth of the fungus Phytophthora infestans on
 plant leaves, characterised in that hyphae thereof, or spores thereof, are
 caused to be contacted with a protein produced from a host cell, host
 cells may suitably be selected from any species routinely used as
 biological fungicides.
 Although the invention is set out in more detail using
 Phytophthora infestans as an example, it will be clear that proteins
 according to the invention may be tested for antifungal activity other
 than anti-Phytophthora activity using an antifungal assay similar to that
 described in the present specification. Suitable antifungal assays have
 been described for several other fungi, such as Fusarium solani, in
 European patent application 440 304 A1, Alternaria alternate, Fusarium
 oxysporum, Rhizoctonia solani, sclerotinia and the like.
 The activity of the proteins according to the invention may be investigated
 further by altering the amino acid sequence to obtain muteins, i.e.
 proteins derived from proteins with anti-Phytophthora activity wherein one
 or more amino acid residues have been replaced, added or removed. Such
 muteins can readily be made by protein engineering in vivo, e.g. by
 changing the open reading frame capable of encoding the antifungal protein
 such that the amino acid sequence is thereby affected. As long as the
 changes in amino acid sequence do not altogether abolish anti-Phytophthora
 activity such muteins are embraced by the present invention.
 The present invention provides a chimeric DNA sequence which comprises an
 open reading frame capable of encoding a protein according to the
 invention. The expression 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 said
 open reading frame in a non-natural location of the plant genome,
 notwithstanding the fact that said plant genome normally contains a copy
 of the said open reading frame in its natural chromosomal location.
 Similarly, the said open reading frame may be incorporated in the plant
 genome wherein it is not naturally found, or in a replicon or vector where
 it is not naturally found, such as a bacterial plasmid or a viral vector.
 Chimeric DNA shall not be limited to DNA molecules which are replicable in
 a host, but shall also mean to comprise DNA capable of being ligated into
 a replicon, for instance by virtue of specific adaptor sequences,
 physically linked to the open reading frame according to the invention.
 The open reading frame may or may not be linked 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. 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 provided be 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 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 Ti-plasmid vectors are the so-called binary vectors as claimed in
 (EP 0 120 516 B1 and U.S. Pat. No. 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
 vital vectors, such as derivable from the double stranded plant viruses
 (e.g. CaMV) and single stranded viruses, gemini viruses and the like. The
 use of such vectors may be advantageous, particularly when it is difficult
 to stably transform the plant host. Such may be the case with woody
 species, especially trees and vines.
 The expression "host cells incorporating a chimeric DNA sequence according
 to the invention in their genome" shall mean to comprise cells, as well as
 multicellular organisms comprising such cells, or essentially consisting
 of such cells, which stably incorporate said chimeric DNA into their
 genome thereby maintaining the chimeric DNA, and preferably transmitting a
 copy of such chimeric DNA to progeny cells, be it through mitosis or
 meiosis. According to a preferred embodiment of the invention plants are
 provided, which essentially consist of cells which incorporate one or more
 copies of said chimeric DNA into their genome, and which are capable of
 transmitting a copy or copies to their progeny, preferably in a Mendelian
 fashion. By virtue of the transcription and translation of the chimeric
 DNA according to the invention in some or all of the plant's cells, those
 cells as produce the anti-Phytophthora protein will show enhanced
 resistance to fungal infections, especially to Phytophthora infections.
 Although the principles as 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. Alternatively, pathogen-inducible promoters may be used such as
 the PRP1 promoter obtainable from tobacco (Martini N. et al. (1993), Mol.
 Gen. Genet 263, 179-186). The choice of the promoter is not essential,
 although it must be said that constitutive high-level 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.
 (1987), Science 236, 1299-1302: the duplication of the sequence between
 -343 and -90 of the CaMV 35S promoter increases the activity of that
 promoter). Generally, high-level expression is desired. In addition to the
 35S promoter, singly or doubly enhanced, examples of high-level promoters
 are the light-inducible ribulose bisphosphate carboxylase small subunit
 (rbcSSU) promoter and the chlorophyl 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 (U.S. Pat. No. 5,106,739), which comprises elements of the
 mannopine synthase promoter linked to the CaMV enhancer.
 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.
 Another aspect of gene expression in transgenic plants concerns the
 targeting of antifungal proteins to the extracellular space (apoplast).
 Naturally intracellularly occurring proteins, among which proteins
 according to the present invention, may be caused to be targeted to the
 apoplast by removal of the C-terminal propeptide, e.g. by modifying the
 open reading frame at its 3' end such that protein is caused to be
 C-terminally truncated. A certain number of amino acids of the C-terminal
 part of the protein was found to be responsible for targeting of the
 protein to the vacuole (e.g. vide WO91/18984 A1). By introducing a
 translational stopcodon in the open reading frame the truncated protein is
 caused to be targeted to the apoplast.
 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 tomato and tobacco 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 fungal attack,
 in particular by Phytophthora infestans, may be treated with proteins
 according to the invention, or preferably, be provided with a chimeric DNA
 sequence according to the invention, allowing the protein to be produced
 in some or all of the plant's cells.
 Although some of the embodiments of the invention may not be practicable at
 present, e.g. because some plant species are as yet recalcitrant to
 genetic transformation, the practicing of the invention in such plant
 species is merely a matter of time and not a matter of principle, because
 the amenability to genetic transformation as such is of no relevance to
 the underlying embodiment of the invention.
 Transformation of plant species is now routine for an impressive number of
 plant species, including both the Dicotyledoneae as well as the
 Monocotyledoneae. In principle any transformation method may be used to
 introduce chimeric DNA according to the invention into a suitable ancestor
 cell, as long as the cells are capable of being regenerated into whole
 plants. Methods may suitably be selected from the calcium/polyethylene
 glycol method for protoplasts (Krens, F. A. et al., 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
 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. Pat. No. 4,940,838).
 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 phosphinothridn acetyltransferase (an enzyme which
 inactivates the herbicide phosphinothridn), 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
 corn are also amenable to DNA transfer by Agrobacterium strains (vide EP 0
 159 418 B1; Gould J, Michael D, Hasegawa O, Ulian E C, Peterson G, Smith R
 H, (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 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 a pathogen susceptible to the protein according
 to the invention, such as Phytophthora infestans. Alternatively, the
 selected plants may be subjected to another round of transformation, for
 instance to introduce further genes, such as genes encoding chitinases,
 glucanases or the like, in order to enhance resistance levels, or broaden
 the resistance to other fungi found not to be susceptible to the protein
 according to the invention in an in vitro assay as described herein.
 Other evaluations may include the testing of fungal 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.
 Many plant proteins exhibit antifungal effects, some however do not do so
 as such, but yield a significant synergistic antifungal effect if used in
 combination with other plant proteins. In European Patent Application 440
 304 A1 it was disclosed that simultaneous relative over-expression of a
 plant expressible glucanase gene in conjunction with an intracellular
 class-I chitinase from tobacco in transgenic plants results in a higher
 level of resistance to fungi than in plants expressing a plant expressible
 class-I chitinase alone.
 Both chitinases, glucanases and the new anti-Phytophthora protein according
 to the invention accumulate in infected plant tissues upon an incompatible
 pathogen-plant interaction. From this observation and the fact that
 several proteins are found to synergise each others antifungal effects, we
 envision, that the anti-Phytophthora protein according to the invention
 may be suitably used in conjunction with other proteins that are
 associated with pathogen resistance.
 Examples of proteins that may be used in combination with the proteins
 according to the invention include, but are not limited to,
 .beta.-1,3-glucanases and chitinases which are obtainable from barley
 (Swegle M. et al., 1989, Plant Mol. Biol. 12, 403-412; Balance G. M. et
 al., 1976, Can. J. Plant Sci. 56, 459-466; Hoj P. B. et al., 1988, FEBS
 Lett. 230, 67-71; Hoj P. B. et al., 1989, Plant Mol. Biol. 13, 31-42
 1989), bean (Boller T. et al, 1983, Planta 157, 22-31; Broglie K. E. et
 al. 1986, Proc. Nat. Acad. Sci. USA 83, 6820-6824; V_geli U. et al., 1988
 Planta 174. 364-372); Mauch F. & Staehelin L. A., 1989, Plant Cell 1,
 447-457); cucumber (M_traux J. P. & Boller T. (1986), Physiol. Mol. Plant
 Pathol. 28, 161-169); leek (Spanu P. et al., 1989, Planta 177, 447-455);
 maize (Nasser W. et al., 1988, Plant Mol. Biol. 11, 529-538), oat (Fink W.
 et al., 1988, Plant Physiol. 88, 270-275), pea (Mauch F. et al. 1984,
 Plant Physiol. 76, 607-611; Mauch F. et al., 1988, Plant Physiol. 87,
 325-333), poplar (Parsons, T. J. et al, 1989, Proc. Natl. Acad. Sci. USA
 86, 7895-7899), potato (Gaynor J. J. 1988, Nucl. Acids Res. 16, 5210;
 Kombrink E. et al. 1988, Proc. Natl. Acad. Sci. USA 85, 782-786; Laflamme
 D. and Roxby R., 1989, Plant Mol. Biol. 13, 249-250), tobacco (e.g.
 Legrand M. et al. 1987, Proc. Natl. Acad. Sci. USA 84, 6750-6754; Shinshi
 H. et al. 1987, Proc. Natl. Acad. Sci. USA 84, 89-93), tomato (Joosten
 M.H.A. & De Wit P.J.G.M. 1989, Plant Physiol. 89, 945-951), wheat (Molano
 J. et al., 1979, J. Biol. Chem. 254, 4901-4907), and the like.
 To obtain transgenic plants capable of constitutively expressing more than
 one chimeric gene, a number of alternatives are available including the
 following:
 A. The use of DNA, e.g a T-DNA on a binary plasmid, with a number of
 modified genes physically coupled to a selectable marker gene. The
 advantage of this method is that the chimeric genes are physically coupled
 and therefore migrate as a single Mendelian locus.
 B. Cross-pollination of transgenic plants each already capable of
 expressing one or more chimeric genes, preferably coupled to a selectable
 marker gene, with pollen from a transgenic plant which contains one or
 more chimeric genes coupled to another selectable marker. Afterwards the
 seed, which is obtained by this crossing, maybe selected on the basis of
 the presence of the two selectable markers, or on the basis of the
 presence of the chimeric genes themselves. The plants obtained from the
 selected seeds can afterwards be used for further crossing. In principle
 the chimeric genes are not on a single locus and the genes may therefore
 segregate as independent loci.
 C. The use of a number of a plurality chimeric DNA molecules, e.g.
 plasmids, each having one or more chimeric genes and a selectable marker.
 If the frequency of co-transformation is high, then selection on the basis
 of only one marker is sufficient. In other cases, the selection on the
 basis of more than one marker is preferred.
 D. Consecutive transformation of transgenic plants already containing a
 first, second, (etc), chimeric gene with new chimeric DNA, optionally
 comprising a selectable marker gene. As in method B, the chimeric genes
 are in principle not on a single locus and the chimeric genes may
 therefore segregate as independent loci.
 E. Combinations of the above mentioned strategies.
 The actual strategy may depend on several considerations as maybe easily
 determined such as the purpose of the parental lines (direct growing, use
 in a breeding programme, use to produce hybrids) but is not critical with
 respect to the described invention.
 In this context it should be emphasised that plants already containing
 chimeric DNA capable of encoding antifungal proteins may form a suitable
 genetic background for introducing chimeric DNA according to the
 invention, for instance in order to enhance resistance levels, or broaden
 the resistance. 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.
 The obtention of transgenic plants capable of expressing, or relatively
 over-expressing, proteins according to the invention is a preferred method
 for counteracting the damages caused by fungi, such as Phytophthora
 infestans, as will be clear from the above description. However, the
 invention is not limited thereto. The invention clearly envisions also the
 use of the proteins according to the invention as such, preferably in the
 form of a fungicidal composition. Fungicidal composition include those in
 which the protein is formulated as such, but also in the form of host
 cells, such as bacterial cells, capable of producing the protein thereby
 causing the pathogen to be contacted with the protein. Suitable host cells
 may for instance be selected from harmless bacteria and fungi, preferably
 those that are capable of colonising roots and/or leaves of plants.
 Example of bacterial hosts that may be used in a method according to the
 invention are strains of Agrobacterium, Arthrobacter, Azospyillum,
 Pseudomonas, Rhizobacterium, and the like, optionally after having been
 made suitable for that purpose.
 Compositions containing anti-Phytophthora proteins according to the
 invention may comprise in addition thereto, osmotin-like proteins as
 defined in (WO91/18984). Independently, the invention provides antifungal
 compositions which further comprise inhibitory agents such as classical
 fungal antibiotics, SAFPs and chemical fungicides such as polyoxines,
 nikkomycines, carboxymides, aromatic carbohydrates, carboxines,
 morpholines, inhibitors of sterol biosynthesis, organophosphorus
 compounds, enzymes such as glucanases, chitinases, lysozymes and the like.
 Either per se, or in combination with other active constituents, the
 anti-Phytophthora protein should be applied in concentrations between 0.1
 .mu.g/ml and 100 mg/ml, preferably between 5 .mu.g/ml and 5 mg/ml, within
 pH boundaries of 3.0 and 9.0. In general it is desired to use buffered
 preparations, e.g. phosphate buffers between 1mM and 1M, preferably
 between 10 mM and 100 mM, in particular between 15 and 50 mM, whereby in
 case of low buffer concentrations it is desired to add a salt to increase
 ionic strength, preferably NaCl in concentrations between 1 mM and 1 M,
 preferably 10 mM and 100 mM.
 Plants, or parts thereof, which relatively over-express a protein according
 to the invention, including plant varieties, with improved resistance
 against Phytophthora diseases 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 fungicide 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 fungal disease. They shall at
 least include angiosperms as well as gymnosperms, monocotyledonous as well
 as dicotyledonous plants.
 The phrase "plants which relatively over-express a protein" shall mean
 plants which contain cells expressing a transgene-encoded protein which is
 either not naturally present in said plant, or if it is present by virtue
 of an endogenous gene encoding an identical protein, not in the same
 quantity, or not in the same cells, compartments of cells, tissues or
 organs of the plant. It is known for instance that normally intracellular
 proteins may be targeted to the apoplastic space.
 According to another aspect of the invention the regulatory region of an
 AP60 protein coding plant gene may be used to express other heterologous
 sequences under the control thereof in a tisue-specific manner. The use of
 a 780 bp region directly upstream of the AP60 gene coding region is
 sufficient for obtaining expression of any heterologous sequence
 preferentially in vascular tissue of roots, stem and leaves of plants, as
 well as the trichomes (leaf hairs) and the stigma of the female
 reproductive organ.
 In the vascular tissue expression of the heterologous gene is dearly
 visible in the phloem; it is not excluded that expression also occurs in
 other cells of the vascular tissue, such as cambium and/or xylem.
 Heterologous sequencer in this respect means gene regions not naturally
 associated to said regulatory region, and they comprise both different
 gene coding regions, as well as antisense antisense gene regions.
 Heterologous coding sequences that may be advantageously expressed in the
 vascular tissue comprise those coding for antipathogenic proteins, e.g.
 insecticidal, bacteriddal, fungicidal, and nematicidal proteins. In such a
 strategy it may prove exceptionally advantageous to select a protein with
 activity against a pathogen or pest which has a preference for phloem as
 source of nutrients (e.g. aphids), or as entrance to invade the plant.
 Examples are extensin, lectin, or lipoxidase against aphids (See
 WO93/04177). Assuming that the regulatory region according to the
 invention is active in xylem, chitinases and glucanases may be expressed
 under the control of said regulatory region to combat Fusarium,
 Vertidilium and Ceratocystus species.
 The use of the regulatory region according to the invention may also be
 used advantageously to regulate or control phloem transport processes.
 Numerous other applicaions will readily occur to those of skill in the
 art.
 The expression of part of (part of) an endogenous gene in the antisense
 orientation (such as disclosed in EP 0 233 399 A), can effectively
 down-regulate expression of said endogenous gene, with interesting
 aplications. Moreover, the AP60 gene itself may be down-regulated using
 the antisense approach which may help establishing the nature and function
 of the AP60 protein. The regions responsible for tissue-specific
 expression may be unravelled further using the GUS-marker in a way
 analogous to the way illustrated herein.
 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 A1;
 EP-A 460 753 A2;
 WO90/07001 A1;
 U.S. Pat. No. 4,940,840.
 EXPERIMENTAL
 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
 Isolation and Characterization of an Anti-Phytophthora Protein from Tobacco
 Leaves
 Leaves of 7 to 8 weeks old Samsun NN tobacco plants were inoculated with
 tobacco mosaic virus (TMV). Seven days after inoculation leaves (400 gram)
 were harvested and homogenized at 4.degree. C. in 500 ml 0.5 M NaOAc
 pH5.2, 15 mM 2-mercapto-ethanol, and 4 gram active carbon, using a Waring
 blender. The homogenate was filtered over four layers of cheese cloth and
 subsequently the filtrate was centrifuged for 50 minutes at 20,000g at
 4.degree. C. and desalted by passage through a Sephadex G25 column (medium
 course; Pharmacia), length 60 cm, diameter 11.5 cm, equilibrated in 40 mM
 NaOAc pH5.2. The desalted protein solution was stored overnight at
 4.degree. C. and subsequently centrifuged for 45 minutes at 20,000 g at
 4.degree. C. The supernatant was passed through a S-sephadex (Fast-flow,
 Pharmacia) column, length 5 cm, diameter 5 cm, which was equilibrated with
 40 mM NaOAc pH 5.2. The column was washed with the above mentioned buffer
 (flow rate 400 to 500 ml/hr) until the OD.sub.280 dropped to zero.
 The bound proteins were eluted using an increasing linear NaCl gradient (0
 to 300 mM) in 500 ml of the above mentioned buffer, and a flow rate of 3
 ml per minute; fractions of approximately 5 ml were collected. All
 fractions were analyzed by electrophoresis (Laemmli (1970), Nature
 227:680-685) using a 12.5% polyacrylamide gel in the presence of sodium
 dodecyl sulphate (SDS), using prestained molecular weight markers (15-105
 kDa) as reference.
 After dialysis against 15 mM potassium phosphate pH 6.0, 20 mM NaCl, the
 fractionated eluate was analyzed for antifungal activity. Antifungal
 activity was monitored in a microtiter plate assay using the fungus
 Phytophthora infestans. In each well of a 24-well microtiter dish 250
 .mu.l potato dextrose agar (PDA) was pipetted. Fungal spores were
 suspended in water and 400-600 spores in 50 .mu.l were added to the wells.
 Subsequently 100 .mu.l filter sterilized (0.22 .mu.m filter) protein
 solution was added. Microtiter dishes were wrapped with Parafilm and
 incubated at room temperature in the dark. At several timepoints after the
 initiation of incubation the fungus was monitored microscopically for
 effects of the added protein. After 2-3 days the mycelium of the growing
 fungus in the wells was stained with lactophenol cotton blue and the
 extent of growth was estimated.
 Two distinct inhibitory activities were detectable in the fractions eluted
 at around 300 mM NaCl. One caused lysis of germinating spores and hyphal
 tips and reduced the growth rate of the hyphae. This activity was caused
 by the protein AP24, or osmotin, disclosed in WO 91/18984 A1. The second
 did not cause lysis, but inhibited the germination of spores and reduced
 hyphal growth rate. We call this latter activity AP60. Neither activity
 appeared to be solely due to either chitinase or .beta.-1,3-glucanase, or
 to a combination of those two hydrolytic enzymes. To separate AP60 from
 AP24 the S-sepharose fractions containing the antifungal activity were
 chromatographed on a FPLC Superdex 75 HR 10/30 column (Pharmacia).
 Proteins elute from this column according to their molecular size. AP60
 eluted at the 55-60 kDa range and AP24 at the 20-25 kDa range. AP60 was
 further purified by rechromatographing on the FPLC Superdex 75 HR 10/30
 column.
 To characterize AP60 further its amino acid sequence was partially
 determined. Therefore, AP60 was separated in the presence of 0.1 mM
 thioglycolate in the upper reservoir buffer and SDS on a 15%
 polyacrylamide gel, which was prerun for 2 hours at 50 V with 0.05 mM
 glucolate in the upper reservoir buffer. The protein was blotted onto PVDF
 membrane as described by Matsudaira et al. (1987, J.Biol. Chem.
 262:10035-10038). After blotting the blot was washed in water and stained
 with 0.1% (w/v) Coomassie Blue R-250 in 50% (v/v) methanol for 5 minutes
 and destained in 50% (v/v) methanol containing 10% (v/v) acetic acid for 5
 to 10 minutes. Finally the blot was rinsed with water and the 60 kDa band
 was cut out and sequenced using Edman degradation on an Applied Biosystems
 477A protein sequencer according to the protocol provided by the
 manufacturer. N-terminal amino acid sequencing of AP60 resulted in the
 following 32 amino adds:
EQU E-D-P-Y-R-F-F-E-(R)-(N)-V-T-Y-G-T-I-Y-P-L-G-V-P-Q-Q-(L/
 G)-I-L-I-(N)-(G)-(Q)-(F) (SEQIDNO: 1).
 The amino acid sequence is given using the one-letter code. Amino acid
 residues between brackets could not be identified unambiguously.
 To obtain internal sequences, AP60 was digested with endoproteinase Glu-C
 (V.sub.8 protease) from Staphylococcus aureus. Therefore, AP60 was run
 over a 12.5% polyacrylamide gel in the presence of SDS. The protein was
 stained in 0.1% Coommassie Brillant Bluein 50% methanol and 10% acetic
 add, destained in 5% methanol and 10% acetic acid and cut out. Several of
 such AP60 bands were collected and applied to a 15% polyacrylamide gel,
 which was prerun for 2 hours at 50 V with 0.05 mM glucolate and 1 mM
 EDTA,in the presence of SDS and digested with Glu-C according to Cleveland
 et al (1977, J. Biol. Chem. 252: 102-1106). V.sub.8 protease cuts proteins
 at glutamic acid residues. The digestion products were electroblotted onto
 a PVDF membrane as described by Matsudaira et al. (1987, J. Biol. Chem.
 262 10035-10038). After blotting the blot was washed in water and stained
 with 0.1% (w/v) Coomassie Blue R-250 in 50% (v/v) methanol for 5 minutes
 and destained in 50% (v/v) methanol containing 10% (v/v) acetic acid for 5
 to 10 minutes. Finally the blot was rinsed with water and the protein band
 migrating as a polypeptide of 12 kDa was cut out of the gel and sequenced.
 Edman degradation revealed 32 amino acids:
EQU (E)-(K)-(G)-V-Y-G-T-T-?-P-(I/
 S)-P-P-G-K-(R)-F-T-Y-I-L-Q-M-K-D-Q-(I)-?-(S)-(H/Y)-?-(Y) (SEQIDNO: 2).
 The amino acid sequence is given using the one-letter code. Amino acid
 residues between brackets could not be identified unambiguously. The first
 amino acid (E) was not determined, but since in the method used Glu-C cuts
 proteins at glutamic acid residues, it was placed at that position.
 To obtain further internal sequence information AP60 was run over a 12.5%
 polyacrylamide gel (Laemmli (1970), Nature 227: 680-685) in the presence
 of sodum dodecyl sulphate (SDS), using prestained molecular weight markers
 (15-105 kDa) as reference. The protein was visualized and cut out. The
 protein was cleaved in situ with trypsin. Trypsin cleaves protein at
 arginine and lysine residues. The digestion products were separated on a
 reversed-phase column and analyzed by Edman degradation. Two additional
 sequences were obtained:
 (T)-?-L-S-A-S-G-P-R-P-N-?-(Q)-(G)-?-(Y)-(G)-(G/R) (SEQIDNO: 15)
EQU I-P-V-P-F-P-D-P-A-D-D-Y-T-L-L-I-G-D-(W)-Y-K (SEQIDNO: 16)
 An inhibitory activity similar to the tobacco AP60 was extracted from
 tomato plants treated with an arachidonic acid solution. Leaves of two
 month old tomato plants (Moneymaker) were treated with a 250 .mu.g/ml
 arachidonic acid solution. After four days of incubation, the leaves were
 harvested and the proteins extracted and purified as described for tobacco
 AP60. Several characteristics (antifungal activity, chromatographical
 properties, molecular mass) of the tobacco and tomato AP60 protein
 indicate that the two proteins are very similar. We predict similar
 proteins to occur in other plant species as well.
 Polyclonal antibodies were raised against AP60. Firstly, the whole AP60
 protein was used to raise antibodies. Therefore, AP60 was separated on
 SDS-polyacrylamide gels and the AP60 band was cut out after visualization.
 The band (approximately 100 .mu.g protein) was dehydrated in EtOH,
 dialyzed extensively against phosphate buffered saline (PBS), ground in a
 mortar and injected into a rabbit. After one month the rabbit was
 boostered every 2 weeks with 50 .mu.g AP60 protein. After 15 weeks the
 rabbit was sacrificed. The antiserum was further purified on a horse
 radish peroxidase (HRP) column, since the antiserum was cross-reactive
 with a lot of probably glycosylated proteins. After purification the
 antiserum recognized besides a protein band of 60 kDa also a protein band
 of approximately 35 kDa.
 In a second approach the N-terminal protein sequence of AP60:
 E-D-P-Y-R-F-F-E, (SEQIDNO: 17) coupled to bovine serum albumine was used
 to raise antiserum. This antiserum, which did not need a purification on a
 HRP column, also recognized two proteins, one of 60 kDa and one of 35 kDa.
 EXAMPLE 2
 In vitro Antifungal Assays
 Purified AP60, isolated from TMV inoculated Samsun NN tobacco leaves, was
 tested on Phytophthora infestans in an in vitro assay. A concentration
 range from 0 to 35 .mu.g/ml was tested by incubating spores with the
 protein on a solid nutrient medium (PDA). Growth of the emerging germtubes
 was severely inhibited in the presence of AP60, resulting in a growth
 inhibition of around 50 per cent after 3 to 4 days when tested at 5
 .mu.g/ml. (The denatured controls (10 minutes boiling) also showed some
 growth inhibition, although less than the non denatured, indicating that
 the protein is somewhat heat stable. Storage of at least three months in
 the cold room does not affect antifungal activity.)
 EXAMPLE 3
 Identification of Genes Homologous to the Deduced AP60 Nucleotide Sequence
 A PCR-A fragment (SEQIDNO: 5) (hereinafter "PCR-A") was cleaved by the
 restriction enzyme Pstl resulting in a 500 bp 5' subfragment and in a 176
 bp 3' subfragment.
 A genomic library of tobacco var. Samsun NN (Cornelissen B.J.C. et al.
 (1987) Nucl. Acids Res. 15, 6799-6811) was screened for AP60 homologous
 recombinant phages using the 3' subfragment as a hybridizing probe. Ten
 positive clones were selected and rescreened with the 5' subfragment as
 probe. Six of the ten clones, lambda 3, 4, 5, 6, 7 and 8, were also
 positive in the second screening and were selected for further sequence
 analysis.
 The lambda clones were digested with the restriction enzymes HindIII or
 Sstl and fragments hybridizing to PCR-A were subcloned in the cloning
 vector pBS (Stratagene).
 Restriction and sequence analysis on these clones indicated that clones
 lambda 3 and 4 as well as clones lambda 6 and 7 were identical. Comparison
 of the deduced amino acid sequence with the AP60 protein (ie. the
 sequenced parts thereof) revealed that lambda 3 and lambda 4 represented
 the genomic sequence encoding AP60 with the highest homology to the AP60
 protein, with only one amino acid different out of 60 amino acids which
 had been determined by protein sequencing. The difference was a
 conservative Lys to Arg exchange at position 15 in SEQIDNO: 2. This clone
 (lambda 3) was sequenced and characterized in detail.
 Alignment of the nucleotide and the deduced amino acid sequence with a
 related gene from tobacco, which is specifically expressed in pollen
 (Weterings K. et al. (1992) Plant Mol. Biol. 18, 1101-1111), and
 comparison with PCR-derived partial AP60 CDNA fragments indicated the
 exon-intron structure shown in SEQIDNO: 6. The open reading frame (ORF) is
 78% homologous to PCR-A (SEQIDNO: 5) on the nucleotide level.
 SEQIDNO: 6 includes a 769 bp sequence containing promoter and 5'
 untranslated leader upstream from the translation initiation codon. The
 coding region contains 7 introns. The supposed poly-adenylation signal is
 found 351 bp downstream of the coding region suggesting an unusually long
 trailer sequence. The deduced amino acid sequence for the lambda 3 protein
 is shown in SEQIDNO: 6 and SEQIDNO: 7. Comparison with the N-terminal AP60
 peptide sequence (SEQIDNO: 1) suggests the presence of a hydrophobic 22
 amino acid N-terminal leader peptide which is cleaved off during
 processing between Ala (22) and Glu (23).
 The lambda 3 protein shows homology to a group of enzymes called blue
 copper oxidases which include L-ascorbate oxidases from plants, laccases
 and ceruloplasmins from vertebrate animals. Laccases have been suggested
 to play a major role in lignification (O'Malley D.M. et al. (1993) The
 Plant J. 4, 751-757). Since lignin formation is closely correlated with
 pathogen induced defense responses (Keen N.T. (1992) Plant Mol. Biol. 19,
 109-122), we propose that in planta AP60 might act against fungal
 penetration in part by reinforcing structural cell wall components.
 EXAMPLE 4
 Tailoring a Lambda 3 Expression Construct
 In order to obtain high constitutive expression of the cloned AP60 gene in
 plants the entire coding region of lambda 3 was cloned into an expression
 cassette between the cauliflower mosaic virus (CaMV) 35S promoter and a
 transcription terminator (PPI-II) from the potato proteinase inhibitor II
 gene (Keil M. et al. (1986) Nucl. Acids Res. 14 5641-5650).
 The expression cassette was constructed from the expression vector pMOG180,
 a pUC18 derived plasmid containing the CaMV 35S promoter and a "leader"
 sequence from the alfalfa mosaic virus RNA4 on an EcoRI-BamHI fragment and
 the nopaline synthase (nos) transcription terminator on a BamHI-HindIII
 fragment. Construction of this vector has been described in detail in
 International patent application (WO91/18984 A1), the description whereof
 being herein incorporated by reference. (pMOG180 is freely available upon
 request to the applicant).
 Construction of the expression cassette and insertion of the lambda3 coding
 region into it involved the following steps:
 The EcoRI restriction site in pMOG180 was changed into an Xbal site by
 digesting the vector with EcoRI and ligating an adaptor into this site
 which converts the EcoRi site into an Xbal site, resulting in clone 1. The
 adapter was made by self-annealing the oligonucleotide 5' AATTGTCTAGAC 3'
 (SEQIDNO: 8).
 Using a similar adapter strategy the HindIII site in clone 1 was replaced
 by the restriction sites Xbal, EcoRI, Xbal, resulting in clone 2. The
 adaptor was obtained by self-annealing the oligonucleotide 5'
 AGCTCTAGAATTCTAG 3' (SEQIDNO: 9) (new restriction sites underlined).
 In order to replace the nos terminator in clone 2 by the PPI-II terminator
 the clone was digested with BamHI and EcoRI. A 241 bp terminator fragment
 from the PPI-II gene (GenEMBL accession nr. X04118, position 1520-1760)
 was modified by standard cloning techniques known to the researchers in
 this area to add a HindIII site (the sequence AAGCTT) at the 5' end and an
 EcoRI site (the sequence GAGCTGGAATTC) (SEQIDNO: 10) at the 3' end. This
 HindIII-EcoRI fragment was ligated together with a BamHI-HindIII adaptor
 into the BamHI/EcoRI digested clone 2, resulting in clone 3.
 The adaptor, which introduces a unique Kpnl site (underlined) 5' to the
 HindIII site, was obtained by annealing the oligonucleotides 5'
 GATCGGTACCGCGA 3' (SEQIDNO:11) and 5' AGCTTCGCGGTACC 3' (SEQIDNO: 12). The
 entire coding region of lambda 3 is found on two HindIII fragments
 (SEQIDNO: 6, 1-5065 and 5060-5752) which were subcloned from the lambda
 clone into the HindIII site of the cloning vector pBS, resulting in clone
 4 (5 kb HindIII fragment) and clone 5 (0.7 kb HindIII fragment). In order
 to obtain a clean fusion of the coding region to the CaMV 35S promoter a
 701 bp fragment was amplified in a PCR reaction from lambda 3 using the
 oligonudeotides 5' CGCGGTACCAAAGGGAAGAAACAATGGTGCCGCTAAAACTCGC 3'
 (SEQIDNO: 13) and 5' CTAAAACAATGGAAATGAATGGAC 3' (SEQIDNO: 14) as primers.
 These primers anneal at position 756-789 (top strand) and position
 1479-1502 (bottom strand), respectively. The top strand primer introduces
 the underlined KpnI site and the plant consensus sequence 5' AACA 3'
 (Lutcke et al. (1987) EMBO J. 6, 43-48) upstream of the translation
 initiation codon (ATG).
 In order to remove an Aval restriction site in the polylinker of clone 4,
 this clone was digested with the restriction enzyme Xmal, the ends were
 filled-in with the Klenow fragment of DNA polymerase I and re-ligated,
 resulting in clone 6.
 The PCR fragment was digested with the restriction enzymes Kpnl and Aval,
 the latter cleaving dose to the 3' end of the fragment, and cloned between
 the Kpnl site and the Aval site in clone 6, resulting in clone 7.
 The integrity of the PCR-derived fragment was verified by sequencing.
 Subsequently the KpnI-HindIII fragment from clone 7 was ligated into the
 corresponding sites in the expression cassette (clone 3), resulting in
 clone 8.
 The remaining 3' part of the lambda 3 coding region (the 0.7 kb HindIII
 fragment in clone 5) could then be ligated into the HindIII site of clone
 8. The resulting clones were screened for insertion of the fragment in the
 correct orientation and a proper clone was selected as clone 9. This clone
 contains the entire lambda 3 coding region between a 35S promoter and the
 PPI-II terminator on an SstI-KpnI fragment *** Clone 9 was renamed
 pMOG841, and was deposited in E. coli DH5.alpha. at the Centraal Bureau
 voor Schimmelcultures, Baarn, The Netherlands, under CBS115.94, on Feb. 8,
 1994. The Xbal fragment was cloned into the respective sites in the binary
 vector pMOG800 (deposited at the Centraal Bureau voor Schimmelcultures,
 Baarn, The Netherlands, under CBS 414.93, on Aug. 12, 1993); FIG. 1. The
 resulting clone, pMOG846, was transferred to Agrobacterium tumefaciens
 strain EHA105 by direct DNA transfer.
 EXAMPLE 5
 Preparation and Analysis of Transgenic Plants
 Cotyledon explants of tomato, Lycopersicon esculentum cv. Moneymaker were
 infected with the Agrobacterium tumefaciens strain EHA105 (McCormick et
 al. (1986) Plant Cell Rep. 5, 81) and primary transformants resistant to
 kanamycin were regenerated. Fifty transgenic plants were selected for
 expression analysis using the so called Western blotting technique.
 According to this method, the AP60 protein was visualized from crude plant
 extracts by virtue of its binding to an AP60 specific polyclonal antibody
 (Example 1). The protein amounts were quantified by comparison with a
 standard range of purified AP60 protein. The transgenic plants were
 grouped according to their expression levels in four categories as shown
 in Table 1.
 TABLE 1
 expression level 0-0.25 0.25-0.5 0.5-1 .gtoreq.1
 % of soluble
 protein
 number of 13 22 11 4
 transgenic
 plants
 EXAMPLE 6
 Preparation and Analysis of Transgenic Tomato Plants
 Transgenic tomato plants where created expressing AP60 constitutively.
 Levels of expression were determined using Western analysis. Extracts of
 the transgenic material were assayed for in vitro growth inhibitory
 activity against Phytophthora infestans. The extracts were made by
 grinding up leaf tissue from transgenic plants in 50 mM NaOAc, pH=5.2.
 After repeated centrifugation, overnight incubation on ice and an
 additional centrifugation step, the supernatant was dialysed to 15 mM
 potassium phosphate+20 mM sodium chloride, pH=6. After filter
 sterilisation, 100 .mu.g protein in 100 .mu.l dialysis buffer was added
 per well containing 250 .mu.l PDA and 50 .mu.l water containing 400-600
 spores. Growth inhibition was scored after 3 to 4 days, resulting in the
 following scores for growth inhibition:
 TABLE 2
 class % AP60 expr. % growth inhib. GI-values *)
 A .sup. 0 0 0
 B 0.25-0.5 0-20 0-1
 C 0.5-1 25-40 1-2
 D .gtoreq.1 .gtoreq.50-75.sup. .gtoreq.2-3.sup.
 *) Growth inhibition (GI) is expressed on a scale from 0 to 4 where:
 0 = no growth inhibition
 1 = 0-30% inhibition
 2 = 30-60% inhibition
 3 = 60-90% inhibition
 4 = 100% inhibition
 The amount of growth inhibition in this experiment is similar to the levels
 found if purified AP60 isolated from TMV inoculated tobacco leaves was
 used.
 EXAMPLE 7
 Analysis of Transgenic Tobacco Plants Transformed With pMOG907
 Using primers LS209 (5'-AAGGGAACAAAAGTCTAGATCTTGCTCCATT-3') (SEQ ID NO:18)
 and LS210 (5'-TTTAGCGGCCATGGCTTCTTCCCTAGGGAAGAAGCCATGGTGCCGCTAAA-3') a 787
 bp fragment was amplified from a genomic AP60 clone (sequence depicted in
 SEQIDNO: 6). Due to the primers an Xbal site was created upstream of the
 fragment and a Ncol fragment was created downstream of the fragment. Using
 these sites this fragment was cloned in front of a GUS-intron gene,
 flanked by the terminator of the Potpill terminator (Keil et al, 1986,
 Nucleic Acids Research 14 5641-5650). The structure of this construct is
 shown in FIG. 3. The entire fragment was cloned into binary vector pMOG800
 (deposited at the Centraal Bureau voor Schimmelcultures, Baarn, The
 Netherlands, under CBS 414.93, on Aug. 12, 1993); FIG. 1.
 The resulting binary vector was transferred to Agrobacterium tumefaciens
 strain EHA105 by direct DNA transfer.
 Tobacco (Nicotiana tabacum Samsun NN) plants transformed with this strain
 were analysed for expression of the GUS gene as described in Jefferson,
 1987, Plant. Mol. Biol. Reporter 5, 387-405) in leaves, roots, and female
 reproductive organs. FIGS. 4, 5 and 6 show the results. The GUS gene was
 preferentially expressed in the vascular tissue of the leaves, roots and
 stigma. In the vascular tissue, detection of GUS could positively be
 determined inside the phloem throughout the whole plant. Also the
 trichomes (leaf hairs) showed blue staining.
 Experiments are under way to show that the GUS gene is also expressed
 inside the vascular tissue of the stem.
 It can thus be concluded that the upstream regulatory region of the AP60
 gene (SEQIDNO: 6, nucleotides upstream of the translational start site) is
 sufficient and suitable for the tissue-preferential expression of
 heterologous genes in vascular tissue, trichomes and the stigma of the
 transgenic plants.
 SEQUENCE LISTING
 (1) GENERAL INFORMATION:
 (iii) NUMBER OF SEQUENCES: 20
 (2) INFORMATION FOR SEQ ID NO: 1:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 32 amino acids
 (B) TYPE: amino acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: peptide
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: Nicotiana tabacum
 (B) STRAIN: Samsun NN
 (F) TISSUE TYPE: Leaf TMV-induced
 (ix) FEATURE:
 (A) NAME/KEY: Peptide
 (D) OTHER INFORMATION: /label= uncertain
 (ix) FEATURE:
 (A) NAME/KEY: Peptide
 (B) LOCATION: 10
 (D) OTHER INFORMATION: /label= residue /note= "uncertain"
 (ix) FEATURE:
 (A) NAME/KEY: Peptide
 (B) LOCATION: 25
 (D) OTHER INFORMATION: /label= residue /note= "Leucin or
 Glycin"
 (ix) FEATURE:
 (A) NAME/KEY: Peptide
 (B) LOCATION: 29..32
 (D) OTHER INFORMATION: /label= residue /note= "uncertain"
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1
 Glu Asp Pro Tyr Arg Phe Phe Glu Arg Asn Val Thr Tyr Gly Thr Ile
 1 5 10 15
 Tyr Pro Leu Gly Val Pro Gln Gln Xaa Ile Leu Ile Asn Gly Gln Phe
 20 25 30
 (2) INFORMATION FOR SEQ ID NO: 2:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 32 amino acids
 (B) TYPE: amino acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: peptide
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: Nicotiana tabaum
 (B) STRAIN: Samsunn NN
 (F) TISSUE TYPE: Leaf TMV-induced
 (ix) FEATURE:
 (A) NAME/KEY: Protein
 (D) OTHER INFORMATION: /label= residue /note= "unknown"
 (ix) FEATURE:
 (A) NAME/KEY: Protein
 (B) LOCATION: 11
 (D) OTHER INFORMATION: /label= residue /note= "isoleucin or
 serine"
 (ix) FEATURE:
 (A) NAME/KEY: Peptide
 (B) LOCATION: 28
 (D) OTHER INFORMATION: /label= residue /note= "uncertain"
 (ix) FEATURE:
 (A) NAME/KEY: Protein
 (D) OTHER INFORMATION: /label= residue /note= "uncertain"
 (ix) FEATURE:
 (A) NAME/KEY: Protein
 (D) OTHER INFORMATION: /label= residue /note= "uncertain"
 (ix) FEATURE:
 (A) NAME/KEY: Protein
 (D) OTHER INFORMATION: /label= residue /note= "uncertain"
 (ix) FEATURE:
 (A) NAME/KEY: Protein
 (B) LOCATION: 16
 (D) OTHER INFORMATION: /label= residue /note= "uncertain"
 (ix) FEATURE:
 (A) NAME/KEY: Protein
 (B) LOCATION: 27
 (D) OTHER INFORMATION: /label= residue /note= "uncertain"
 (ix) FEATURE:
 (A) NAME/KEY: Protein
 (B) LOCATION: 28
 (D) OTHER INFORMATION: /label= residue /note= "unknown"
 (ix) FEATURE:
 (A) NAME/KEY: Protein
 (B) LOCATION: 29
 (D) OTHER INFORMATION: /label= residue /note= "uncertain"
 (ix) FEATURE:
 (A) NAME/KEY: Protein
 (B) LOCATION: 30
 (D) OTHER INFORMATION: /label= residue /note= "his or tyr"
 (ix) FEATURE:
 (A) NAME/KEY: Protein
 (B) LOCATION: 31
 (D) OTHER INFORMATION: /label= residue /note= "unknown"
 (ix) FEATURE:
 (A) NAME/KEY: Protein
 (B) LOCATION: 32
 (D) OTHER INFORMATION: /label= residue /note= "uncertain"
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2
 Glu Lys Gly Val Tyr Gly Thr Thr Xaa Pro Xaa Pro Pro Gly Lys Arg
 1 5 10 15
 Phe Thr Tyr Ile Leu Gln Met Lys Asp Gln Ile Xaa Ser Xaa Xaa Tyr
 20 25 30
 (2) INFORMATION FOR SEQ ID NO: 3:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 40 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: double
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: cDNA
 (iii) HYPOTHETICAL: YES
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (ix) FEATURE:
 (A) NAME/KEY: misc_feature
 (D) OTHER INFORMATION: /label= inosine
 (ix) FEATURE:
 (A) NAME/KEY: misc_feature
 (B) LOCATION: 15
 (D) OTHER INFORMATION: /label= inosine
 (ix) FEATURE:
 (A) NAME/KEY: misc_feature
 (B) LOCATION: 27
 (D) OTHER INFORMATION: /label= inosine
 (ix) FEATURE:
 (A) NAME/KEY: misc_feature
 (B) LOCATION: 33
 (D) OTHER INFORMATION: /label= inosine
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3
 GAGGATCCNT AYAGNTTYTT YGARAGNAAY GTNACTAYGG 40
 (2) INFORMATION FOR SEQ ID NO: 4:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 26 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: double
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: cDNA
 (iii) HYPOTHETICAL: YES
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (ix) FEATURE:
 (A) NAME/KEY: misc_feature
 (D) OTHER INFORMATION: /label= inosine
 (ix) FEATURE:
 (A) NAME/KEY: misc_feature
 (B) LOCATION: 13
 (D) OTHER INFORMATION: /label= inosine
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4
 TTYACNTAYA TYNTSCARAT GAARGA 26
 (2) INFORMATION FOR SEQ ID NO: 5:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 676 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: double
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: DNA (genomic)
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: Nicotiana tabacum
 (B) STRAIN: Samsun NN
 (vii) IMMEDIATE SOURCE:
 (B) CLONE: PCR-A
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5
 GATCCGTACA GGTTTTTCGA GAGGAACGTG ACTTATGGCA CCACTTATCC TCTTGGTGTT 60
 CCCCAACAGG TTGGTTGCTT TTTTCATGTT GTGTTCAAGT AATATGTGAA AAGATATATT 120
 GCGCTTTAAT TTGATCATGG TTTATTTGTG ATAAGCTTAC AGTACCGTGT CCTGTGTATA 180
 GGGTATTCTG ATCAATGGCC AATTCCCTGG TCCTGACATT TACTCTCACC AACGAGAATA 240
 TTATTATCAA CGCTTCAACG GCTTGGATGA ACCTTTTCTT CTTTCTTGGT AATTTCCTTC 300
 CTTGAGAGGC AGATTCAGAA TTTTAAACTT ATGGGTTCCT ATAACAATCT TAAGTTAATT 360
 ATATGATAAC TTGACCAAAC GAATTGTGTT CCAAGCTAAA TATTCTTATA CTTGTAATGA 420
 AATTTTTAAT ACAAATTAAA TACAGAGTGT ATGCAAAAGT AACTGGGGAA CCCGTAATGT 480
 TTGCTCTATA TGCGCTCCTG CAGTTTCATT ATCTTTCTTT GTGTTCAGAC AATCAGTTTT 540
 CGGGAGATAA TATTTTGATA AACACATGCA GGAATGGAGT GCAAAATAGG AGAAACTCAT 600
 ACGAAGACGG AGTATGGGGA ACGACGTGCC CAATACCACC GGGAAAAANN TTCACCTACA 660
 TTCTCCAAAT GAAGGA 676
 (2) INFORMATION FOR SEQ ID NO: 6:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 6305 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: double
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: DNA (genomic)
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: Nicotiana tabacum
 (B) STRAIN: Samsun NN
 (F) TISSUE TYPE: leaf
 (vii) IMMEDIATE SOURCE:
 (B) CLONE: lambda 3
 (ix) FEATURE:
 (A) NAME/KEY: exon
 (B) LOCATION: 774..911
 (ix) FEATURE:
 (A) NAME/KEY: exon
 (B) LOCATION: 1045..1154
 (ix) FEATURE:
 (A) NAME/KEY: exon
 (B) LOCATION: 3105..3375
 (ix) FEATURE:
 (A) NAME/KEY: exon
 (B) LOCATION: 4226..4328
 (ix) FEATURE:
 (A) NAME/KEY: exon
 (B) LOCATION: 4432..4777
 (ix) FEATURE:
 (A) NAME/KEY: exon
 (B) LOCATION: 4891..5253
 (ix) FEATURE:
 (A) NAME/KEY: exon
 (B) LOCATION: 5336..5414
 (ix) FEATURE:
 (A) NAME/KEY: exon
 (B) LOCATION: 5531..5743
 (ix) FEATURE:
 (A) NAME/KEY: intron
 (B) LOCATION: 912..1044
 (ix) FEATURE:
 (A) NAME/KEY: intron
 (B) LOCATION: 1155..3104
 (ix) FEATURE:
 (A) NAME/KEY: intron
 (B) LOCATION: 3376..4225
 (ix) FEATURE:
 (A) NAME/KEY: intron
 (B) LOCATION: 4329..4431
 (ix) FEATURE:
 (A) NAME/KEY: intron
 (B) LOCATION: 4778..4890
 (ix) FEATURE:
 (A) NAME/KEY: intron
 (B) LOCATION: 5254..5335
 (ix) FEATURE:
 (A) NAME/KEY: intron
 (B) LOCATION: 5415..5530
 (ix) FEATURE:
 (A) NAME/KEY: CDS
 (B) LOCATION: join(774..911, 1045..1154, 3105..3375,
 4226..4328, 4432..4777, 4891..5253, 5336..5414,
 5531..5743)
 (ix) FEATURE:
 (A) NAME/KEY: sig_peptide
 (B) LOCATION: 774..839
 (ix) FEATURE:
 (A) NAME/KEY: polyA_site
 (B) LOCATION: 6095..6100
 (D) OTHER INFORMATION: /label= putative
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6
 AAGCTTGGAT CTTGCTCCAT TTCATCATGG TCATTCTATA GTTGAAATAT GTGATTAATG 60
 GTGAAGGGAA TTCGTCTATT TGATCATTAT CATTCTAAAG CTCGAATATT AGACATTTAA 120
 ATAACCGTGA AGGGTTCTCG TCCATTTCAT CATTATTATT CTGAGCCTCG TATTTTAGAT 180
 ATCTAATTAA TGATAAAGGG ATCCCGTACG TTCATCACCT TCATTCTCAT GTTCAAGCCT 240
 TAGATATCTA ATTAACGGTG AAAGGAACTT GTTTATTTAA TCATTGTCAT TCTAAAGCTC 300
 GAATCTTAGA CATTTGATTA ACAATCAAGT GATCTTGTCC ATTTCATCAT GATCATTCTA 360
 AGGTTGAAAT ACGTGACTAA TGGTGAAGGG AGCTCGTCAT TGTCATTCTA AAGCTGGAAT 420
 ATTAGACATC TAAATAACCG CGAAGGATTC TCGTCCAGTT TATCACTATC ATTCTAAGTT 480
 AAATATCTAA TTAAAATATG ATAAAGAGAT CTCGTCCATT TAATCACATA TCTAGCGTAG 540
 CGATGTGTTA GTAGCCGCAT ACTGCTAGGC CAACTTGACT TTTAGTAGTA GTCTGTACCG 600
 CACTTTTAAA TATACCATGC CTTTTTCATC ACCTTCACTT TAATTCCCTC CAACCCACTC 660
 CCCCCCCCCC CCACCTCTTG TTCCACAACT CACTCTTTCT TCTTTTAAAT CCTTCTATTT 720
 CCAGTTTATT TCTTAATATA GCAAATTTCA GTCTAACAAA AAGGGAAGAA GAA ATG 776
 Met
 1
 GTG CCG CTA AAA CTC GCA GTA GCG GCA TTT TTA GTG GTA GGA TTA ATT 824
 Val Pro Leu Lys Leu Ala Val Ala Ala Phe Leu Val Val Gly Leu Ile
 5 10 15
 GCG AAT ACA TTA GCA GAG GAT CCG TAT AGA TTC TTC GAG TGG AAT GTT 872
 Ala Asn Thr Leu Ala Glu Asp Pro Tyr Arg Phe Phe Glu Trp Asn Val
 20 25 30
 ACT TAT GGC ACT ATT TAT CCT CTT GGA GTT CCT CAA CAG GTTGGTTTCT 921
 Thr Tyr Gly Thr Ile Tyr Pro Leu Gly Val Pro Gln Gln
 35 40 45
 TTTCCTCAGT TTCTTAAAAT AGAATGTGTG TGTGTGTGTG TTTTCTCTTC TGGATCTGGT 981
 TTTTTCGGGT TGTTTTGATC ATATATGCTC ATCAAATTGT GATTTTGTTT TCTGTTTGTG 1041
 TAG GGA ATT TTG ATC AAT GGC CAA TTT CCT GGT CCT GAT ATT TAC TCC 1089
 Gly Ile Leu Ile Asn Gly Gln Phe Pro Gly Pro Asp Ile Tyr Ser
 50 55 60
 GTT ACT AAT GAC AAT CTC ATT ATC AAC GTT TTC AAC AGC TTG GAC GAG 1137
 Val Thr Asn Asp Asn Leu Ile Ile Asn Val Phe Asn Ser Leu Asp Glu
 65 70 75
 CCT TTT CTT CTT TCC TG GTAATTTACT AATCTTCCAT TTTCTCACAT 1184
 Pro Phe Leu Leu Ser Trp
 80
 TTCAAACATT AATTAAGTAA TTAGTACATA ATATATCATG TGTCATATTG AAAAATGCCT 1244
 TGCAAAATGA TAATGCTGGT CTTCAAAGTA TCACGATTTG AATACATTTT GGGTCCTGAA 1304
 TTCCAGGGCC TGAGCTAGTG CATAGGTTTC GGATTTGGCC ATTATTAATT TAAAATCTAA 1364
 TAACTTAAAA TAATTAAAAT TTCGAATCCA TAAATTTTAA ATCCAGTCTC CGTCTCTTAA 1424
 TTCAACTCCA TTCCCTATAT GTAAGTTTTG ATATGCCTCG GGGCTTTACA ATTCTCATGT 1484
 CCATTCATTT CCATTGTTTT AGGTATATTG TTTTTAATTT TTTTCGATCT AGTTAGGTAG 1544
 GTGTCTAGTT CTAAAGTTTC TGTAGTCAGA TCTCACTCTT TAGTTAAAGG CGGTTTTAGG 1604
 ACGAGTTATT TAAGTTCAAC TAAACTTTTT ACTTTTTGCT GAAAAATAAA TATATATAAA 1664
 AGCAAAAAAT TTAATATATA TATATATTCA ATAAGATAGA CGTATTTTCA AACCAACTGA 1724
 CTTTAGATCA TGTATTCTTA TATGTCTGTC AACCTTTTAA TACGAGAGGC AGACTGGAGA 1784
 TTCTTAAATT AGTGGATATA AATTGTTTTG TATAGCGACT GTGCTAGTTT ATGGTAATGG 1844
 ATATTAGTAT AGATCTACAT TTTTCTAAAT ATTGTTCAAA TAAGTAAATG TGGTTAAAGT 1904
 CGAAAATGGT ACACTCTGCC CAAATACTAG CAAAAAAGAG TCTAGTTGGA TATAAGCGAG 1964
 TCTTTCGTTT TTCTTTTCAT TTGATTCCTT CTCATTTAGC TGATTATTAT TGCGTCATTA 2024
 TAAAGAAATA ATTTGATTTG GGTAAACTTC AATCTCTTGT AGTTTAGTAG ATTTAGTACT 2084
 AGTGAATAAG TTCAAAATAG AAGAAGTGAC ATACAAGTTA GAACAAGTAG TAGATCTGAT 2144
 CTTAAAATCG ATTGATTTGG TGACAATGAA ATTCACTGGT GCAGAAAATT TGGTGTGGAA 2204
 TTAGTTATTT TGTAGTTTAG ATAGGATTTG GAAGACAAGT TCACCACTTG CTTCACTGCT 2264
 GGATTCAACT AACAACGAAA ATAGAAAGTA CCACATGGGC TTGACATCAA CATTGTCGGA 2324
 TTATGAATTG GTTTTTGGAT CTTGACTTTT GTCTAAATTT ATACGCCACG CATCGTGTTT 2384
 AGTGATTTTT GGCCACATTT TAGTCACTTA TCCAGCTGTA TAATTTTCAA ATTATTTTTC 2444
 ATTTATACCG CCTCTTTGCC CTTACGTGGA AGGGTAAGGT TGCGTATGTC CTACCCTCCC 2504
 CAAATATCAC TAGTAGAAAT TCACTGGATT GTTTTGTTGT TGTAAACAGC GATGCAAGCA 2564
 ACAATGTTTT TGATCCCATA ATTGAAATGT GGTGTGATCT CAAATTCGAG CCGTAAAAAT 2624
 ATAAATTTTA ATATCAGAGA GTGTTATATT CCCGTAACAA CTTATTTAGC ATGAATTCAG 2684
 ATTAATCCAG CAAGTGAATT TTTCAAATAT TGGACGCTAA AGGAAAAAAT AAAATTGAAT 2744
 AACGTGGATT TTTTCTCATT ATATATGCAA AATCACATGG TTATTTTCGT CTGCTGAGCA 2804
 AATGATGAGT GTTTGAAATA TTAGATTGAA TATAACTAGT CGGTAGATCA TGTGATATCC 2864
 ATGTGAAAAG TTGCTGCTAA TTCTGTGTCA AATACATCTT CCTCTGGTCT TTTTGAGGAT 2924
 TGTGCTTTTG TCCTTGAAAT GTGTTGGCCA TTTCAGCTCT TTTGTTTCTA TTATTTTCTG 2984
 ACCCTACAAT CTTATTAAAT ATTAAATATT AGATAAATCA ATGCCCTTCT ACTATATTAC 3044
 AATATTTTCT GAATTACCTT ATTTAAAGAT TTAAATTGAC ATCTTGGAAC ATACATGTAG 3104
 G AAT GGA ATA CAA AAC AGG AGA AAC TCA TTT GAA GAT GGA GTA TAC 3150
 Asn Gly Ile Gln Asn Arg Arg Asn Ser Phe Glu Asp Gly Val Tyr
 85 90 95
 GGT ACA ACC TGC CCA ATA CCA CCA GGA AGG AAT TTC ACA TAC ATT TTA 3198
 Gly Thr Thr Cys Pro Ile Pro Pro Gly Arg Asn Phe Thr Tyr Ile Leu
 100 105 110
 CAA ATG AAA GAT CAA ATA GGG AGT TAT TAC TAT TTT CCT TCT CTT GCA 3246
 Gln Met Lys Asp Gln Ile Gly Ser Tyr Tyr Tyr Phe Pro Ser Leu Ala
 115 120 125 130
 TTT CAC AAA GCT GCT GGT GGT TTT GGA GGA ATT AAA ATT CTC AGC AGA 3294
 Phe His Lys Ala Ala Gly Gly Phe Gly Gly Ile Lys Ile Leu Ser Arg
 135 140 145
 CCA AGA ATC CCC GTC CCT TTT CCG GAT CCC GCA GAC GAT TAC ACT CTC 3342
 Pro Arg Ile Pro Val Pro Phe Pro Asp Pro Ala Asp Asp Tyr Thr Leu
 150 155 160
 CTC ATT GGA GAT TGG TAC AAA AAG AAT CAT ACG GTACGAATAT TTATTTTATA 3395
 Leu Ile Gly Asp Trp Tyr Lys Lys Asn His Thr
 165 170
 CTGTTAACGT ATAAAAAGTT AAACTCGTTA AAATGCATTT AATTCTTGTA AGTACATAGG 3455
 GACACTTGTA TTTGGACACT TGTATATTGT ATTTACAAAT AGTTATGTGG ACAAGGGGTT 3515
 GTCTAATGTC TTGAGTGTCG GTAAGGTAAA GTAGTAATGT TGTTTGGCTT GTTGCATTTT 3575
 TGGAATATAG TGTGCTTTTT CTCGTGGGCC ATTACTTGCT TCGTCTGTCC TTGACTAAGT 3635
 CTTTTATTAT TGCGGGCTCC ATAGGAGACA TCAACAGGAA ATTATACCTT TTCTTTTAAT 3695
 TCTCAAACCA AACTTCTTAT ACTGAGTTAA ATGATTGAGC AGAGTCAAAA AGATGTAAAT 3755
 CTCTACAATT ACTTTAAACA CAAAAAGAAG TATGGTTTTG TAATTTTCAT CTGATCCTAG 3815
 ACTTGTCAAC TAAGTTATAA CTTCCAATAC TTAAGGGCTA GGGTTATTGT TTTGGAAAAA 3875
 AGAAATGTAG ATATATTAGT CTTGAGTTGA GTTAAATCAA ATAAAAATAT AGCCATTGTT 3935
 CAGTGATAGA AACTACAACA ACCACAGCAG CTATTCATTA GTTATAAATG AGTTGTCGGC 3995
 TATATGAATT CTCGCTTCCT ATTTAAGCTC AACTCATGTT ATCACAATAC CAAATTAAAT 4055
 AAATTGAAAA AATAAGTTAC ATATAATCCC ACAAGTCTGA GGAGCGTAGT GAATACGCAG 4115
 ACCTTACCCA TATAGTGAAG GTAGAGAGGT TGTTTTCAGT AGACCCTCTG CACGAGGAAA 4175
 AATTCAGTGA TAGAAACTCA AAATTATTAT TTGAATATCC TATTATACAG GCC TTG 4231
 Ala Leu
 175
 AAA GCA ATT CTT GAT GGA GGA AAG AAG TTG CCT TTC CCT GAT GGC ATT 4279
 Lys Ala Ile Leu Asp Gly Gly Lys Lys Leu Pro Phe Pro Asp Gly Ile
 180 185 190
 CTT ATC AAT GGA CGT GGA CCT AAT GGT GTT TCT TTC ACA GTT GAG CAA G 4328
 Leu Ile Asn Gly Arg Gly Pro Asn Gly Val Ser Phe Thr Val Glu Gln
 195 200 205
 GTAAAATAAT TTGATGAATA TTGGTTACTA AAATCTGTGA AAATTCATAG CCTAATGTTA 4388
 TCGCATTTCT GAAAAATCTA ACATTGAGTT TTTCTTATAA CAG GG AAA ACT TAT 4442
 Gly Lys Thr Tyr
 210
 AGA CTG AGG ATA TCC AAT GTT GGA TTA CAA AAT TCA CTT AAC TTC CGT 4490
 Arg Leu Arg Ile Ser Asn Val Gly Leu Gln Asn Ser Leu Asn Phe Arg
 215 220 225
 ATT GAA GGA CAC AGG ATG AAA TTA GTT GAA GTA GAG GGA ACA CAC ACA 4538
 Ile Glu Gly His Arg Met Lys Leu Val Glu Val Glu Gly Thr His Thr
 230 235 240
 TTG CAA ACT ACC TAT TCC TCA CTT GAT GTT CAT GTT GGG CAA ACC TAC 4586
 Leu Gln Thr Thr Tyr Ser Ser Leu Asp Val His Val Gly Gln Thr Tyr
 245 250 255
 TCT GTC CTC ATT ACA GCT GAT CAA GAA GCT AAA GAC CAC TAC ATT GTT 4634
 Ser Val Leu Ile Thr Ala Asp Gln Glu Ala Lys Asp His Tyr Ile Val
 260 265 270 275
 GTT TCG TCG CGT TTT ACA TCT CAA GTC CTG ACC ACC ACC GGT GTA CTT 4682
 Val Ser Ser Arg Phe Thr Ser Gln Val Leu Thr Thr Thr Gly Val Leu
 280 285 290
 CAC TAT AGC AAC TCT AAC ACC CCC GTC TCC GGT CCT CCT CCT GGT GGT 4730
 His Tyr Ser Asn Ser Asn Thr Pro Val Ser Gly Pro Pro Pro Gly Gly
 295 300 305
 CCT ACC ATC CAA ATT GAT TGG TCC CTT AAC CAA GCC CGC TCC ATC AG 4777
 Pro Thr Ile Gln Ile Asp Trp Ser Leu Asn Gln Ala Arg Ser Ile Arg
 310 315 320
 GTATGGCCAA CTTCCACTGA GCCTATATGT GGAAGCTGTT TGGCTTAGCT GATTAAAAGT 4837
 AGCTGATAAG CATTAACTGT TTGTATAACT TGGTTATTGA TTGTGTAATT TAG G ACG 4894
 Thr
 AAC TTG TCA GCA AGT GGA CCA AGG CCA AAT CCA CAA GGT TCA TAC CAT 4942
 Asn Leu Ser Ala Ser Gly Pro Arg Pro Asn Pro Gln Gly Ser Tyr His
 325 330 335 340
 TAT GGT ATG ATC AAC ACA ACC CGA ACC ATC AGA CTT GCT AGC TCA GCT 4990
 Tyr Gly Met Ile Asn Thr Thr Arg Thr Ile Arg Leu Ala Ser Ser Ala
 345 350 355
 GGT CAA GTG AAT GGC AAA CAG AGA TAT GCA GTC AAC AGC GTG TCG TTT 5038
 Gly Gln Val Asn Gly Lys Gln Arg Tyr Ala Val Asn Ser Val Ser Phe
 360 365 370
 GTG CCA CTT GAT ACT CCT CTC AAG CTT CTG GAC TAC TTC AAA GTT GGT 5086
 Val Pro Leu Asp Thr Pro Leu Lys Leu Leu Asp Tyr Phe Lys Val Gly
 375 380 385
 GGA TTC CGC GTT GGA AGC ATA TCT GAT GCT CCA AGT GGT GGA GGA ATT 5134
 Gly Phe Arg Val Gly Ser Ile Ser Asp Ala Pro Ser Gly Gly Gly Ile
 390 395 400
 TTC CTA GAC ACG TCT GTT CTA GGC GCT GAT TAC AGG CAA TTC ATT GAG 5182
 Phe Leu Asp Thr Ser Val Leu Gly Ala Asp Tyr Arg Gln Phe Ile Glu
 405 410 415 420
 ATT GTA TTC GAG AAC ACT GAG GAC ATC GTC CAA AGC TGG CAT CTT AAT 5230
 Ile Val Phe Glu Asn Thr Glu Asp Ile Val Gln Ser Trp His Leu Asn
 425 430 435
 GGC TAC TCT TTT TGG GTT GTA GG GTATGTAGTG ATCAATGATT TTTGTTATCA 5283
 Gly Tyr Ser Phe Trp Val Val Gly
 440
 TATGCGTGTT ATTTGAATCT TGTTTTTGAT TTAATTTTGA TGTTATATGC AG G ATG 5339
 Met
 445
 GAT GGA GGG CAT TGG ACT CAA GCT AGT AGA AAC GGG TAC AAT CTT CGT 5387
 Asp Gly Gly His Trp Thr Gln Ala Ser Arg Asn Gly Tyr Asn Leu Arg
 450 455 460
 GAT GCA GTT GCA CGT TAC ACA ACT CAG GTAACATTAA AAAGAACAAA 5434
 Asp Ala Val Ala Arg Tyr Thr Thr Gln
 465 470
 AAAAATCAAA GAATTTGAAG TCACTTGTTT AGGGGCAAGG AACACTAGTT AATTTCACAT 5494
 ATGATCACTG GAACTTTACT CGCTATAATT TCACAG GTG TAT CCC AAG TCA TGG 5548
 Val Tyr Pro Lys Ser Trp
 475
 ACT GCA ATA TAT ATT GCA TTG GAC AAT GTA GGA ATG TGG AAC CTA AGG 5596
 Thr Ala Ile Tyr Ile Ala Leu Asp Asn Val Gly Met Trp Asn Leu Arg
 480 485 490
 ACT GAA TTT TGG GCA CGA CAA TAC CTT GGA CAA CAA TTA TAC ATG AGA 5644
 Thr Glu Phe Trp Ala Arg Gln Tyr Leu Gly Gln Gln Leu Tyr Met Arg
 495 500 505
 GTT TAC ACT ACA TCA ACG TCT TTG AGA GAC GAA TAT CCA ATT CCA AGG 5692
 Val Tyr Thr Thr Ser Thr Ser Leu Arg Asp Glu Tyr Pro Ile Pro Arg
 510 515 520
 AAC GCT CGT CTC TGT GGC AAA GTG GCT GGC CGG CAC ACA CGA CCA CTT 5740
 Asn Ala Arg Leu Cys Gly Lys Val Ala Gly Arg His Thr Arg Pro Leu
 525 530 535 540
 TAAGCAAAGC TTGCAATTCT AATAGAGAGG AATTTTATTT ATACAGTCCT TATTATTGCG 5800
 AGTTTTAGAA GTAATATCTT ATTACTACTA CTACTATGGC CATTGGTGTA CTACTAGCTG 5860
 CTAATTTGAA TGACCTTAAC TCAAGGTCTC ATTCTTTCTT TCCACGGAAC TTTGTATTTG 5920
 GTTCTTTCCT CATTGTTTTT AGTTGTATTA AAATGAAGTG TTTTTTTTCC ATTACTATAT 5980
 GCAGATTTGA TGCTTTCTAC ATTTACTTCC TTTTCTTTTT CGGTATTTGC ATTGTTGGCC 6040
 AACTAAATTT AGATTCACGT TGGAAAATCT TATATTACGG GTAAAACAAT TCCTAATAAA 6100
 AGCGACTTCG TATGACTAGA AGTTATGGTC AAAACATATG GTTTACCGAT TTTTGGGCTC 6160
 CAATAGTCCA ACATATGTTT CAAGATTTGA AAAGAGAAAA AGAGATGAAA ACAGCAAATT 6220
 AACTCTCTTG TTACTTCCTA TTAGAGGATG AATCGCTCGT TGACAAATCT GCTTTACTTT 6280
 TTATTGGTAA ATTCAAAAGT TCTAA 6305
 (2) INFORMATION FOR SEQ ID NO: 7:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 540 amino acids
 (B) TYPE: amino acid
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: protein
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7
 Met Val Pro Leu Lys Leu Ala Val Ala Ala Phe Leu Val Val Gly Leu
 1 5 10 15
 Ile Ala Asn Thr Leu Ala Glu Asp Pro Tyr Arg Phe Phe Glu Trp Asn
 20 25 30
 Val Thr Tyr Gly Thr Ile Tyr Pro Leu Gly Val Pro Gln Gln Gly Ile
 35 40 45
 Leu Ile Asn Gly Gln Phe Pro Gly Pro Asp Ile Tyr Ser Val Thr Asn
 50 55 60
 Asp Asn Leu Ile Ile Asn Val Phe Asn Ser Leu Asp Glu Pro Phe Leu
 65 70 75 80
 Leu Ser Trp Asn Gly Ile Gln Asn Arg Arg Asn Ser Phe Glu Asp Gly
 85 90 95
 Val Tyr Gly Thr Thr Cys Pro Ile Pro Pro Gly Arg Asn Phe Thr Tyr
 100 105 110
 Ile Leu Gln Met Lys Asp Gln Ile Gly Ser Tyr Tyr Tyr Phe Pro Ser
 115 120 125
 Leu Ala Phe His Lys Ala Ala Gly Gly Phe Gly Gly Ile Lys Ile Leu
 130 135 140
 Ser Arg Pro Arg Ile Pro Val Pro Phe Pro Asp Pro Ala Asp Asp Tyr
 145 150 155 160
 Thr Leu Leu Ile Gly Asp Trp Tyr Lys Lys Asn His Thr Ala Leu Lys
 165 170 175
 Ala Ile Leu Asp Gly Gly Lys Lys Leu Pro Phe Pro Asp Gly Ile Leu
 180 185 190
 Ile Asn Gly Arg Gly Pro Asn Gly Val Ser Phe Thr Val Glu Gln Gly
 195 200 205
 Lys Thr Tyr Arg Leu Arg Ile Ser Asn Val Gly Leu Gln Asn Ser Leu
 210 215 220
 Asn Phe Arg Ile Glu Gly His Arg Met Lys Leu Val Glu Val Glu Gly
 225 230 235 240
 Thr His Thr Leu Gln Thr Thr Tyr Ser Ser Leu Asp Val His Val Gly
 245 250 255
 Gln Thr Tyr Ser Val Leu Ile Thr Ala Asp Gln Glu Ala Lys Asp His
 260 265 270
 Tyr Ile Val Val Ser Ser Arg Phe Thr Ser Gln Val Leu Thr Thr Thr
 275 280 285
 Gly Val Leu His Tyr Ser Asn Ser Asn Thr Pro Val Ser Gly Pro Pro
 290 295 300
 Pro Gly Gly Pro Thr Ile Gln Ile Asp Trp Ser Leu Asn Gln Ala Arg
 305 310 315 320
 Ser Ile Arg Thr Asn Leu Ser Ala Ser Gly Pro Arg Pro Asn Pro Gln
 325 330 335
 Gly Ser Tyr His Tyr Gly Met Ile Asn Thr Thr Arg Thr Ile Arg Leu
 340 345 350
 Ala Ser Ser Ala Gly Gln Val Asn Gly Lys Gln Arg Tyr Ala Val Asn
 355 360 365
 Ser Val Ser Phe Val Pro Leu Asp Thr Pro Leu Lys Leu Leu Asp Tyr
 370 375 380
 Phe Lys Val Gly Gly Phe Arg Val Gly Ser Ile Ser Asp Ala Pro Ser
 385 390 395 400
 Gly Gly Gly Ile Phe Leu Asp Thr Ser Val Leu Gly Ala Asp Tyr Arg
 405 410 415
 Gln Phe Ile Glu Ile Val Phe Glu Asn Thr Glu Asp Ile Val Gln Ser
 420 425 430
 Trp His Leu Asn Gly Tyr Ser Phe Trp Val Val Gly Met Asp Gly Gly
 435 440 445
 His Trp Thr Gln Ala Ser Arg Asn Gly Tyr Asn Leu Arg Asp Ala Val
 450 455 460
 Ala Arg Tyr Thr Thr Gln Val Tyr Pro Lys Ser Trp Thr Ala Ile Tyr
 465 470 475 480
 Ile Ala Leu Asp Asn Val Gly Met Trp Asn Leu Arg Thr Glu Phe Trp
 485 490 495
 Ala Arg Gln Tyr Leu Gly Gln Gln Leu Tyr Met Arg Val Tyr Thr Thr
 500 505 510
 Ser Thr Ser Leu Arg Asp Glu Tyr Pro Ile Pro Arg Asn Ala Arg Leu
 515 520 525
 Cys Gly Lys Val Ala Gly Arg His Thr Arg Pro Leu
 530 535 540
 (2) INFORMATION FOR SEQ ID NO: 8:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 12 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: cDNA
 (iii) HYPOTHETICAL: YES
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8
 AATTGTCTAG AC 12
 (2) INFORMATION FOR SEQ ID NO: 9:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 16 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: cDNA
 (iii) HYPOTHETICAL: YES
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9
 AGCTCTAGAA TTCTAG 16
 (2) INFORMATION FOR SEQ ID NO: 10:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 12 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: cDNA
 (iii) HYPOTHETICAL: YES
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10
 GAGCTGGAAT TC 12
 (2) INFORMATION FOR SEQ ID NO: 11:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 14 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: cDNA
 (iii) HYPOTHETICAL: YES
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11
 GATCGGTACC GCGA 14
 (2) INFORMATION FOR SEQ ID NO: 12:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 14 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: cDNA
 (iii) HYPOTHETICAL: YES
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12
 AGCTTCGCGG TACC 14
 (2) INFORMATION FOR SEQ ID NO: 13:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 43 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: cDNA
 (iii) HYPOTHETICAL: YES
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13
 CGCGGTACCA AAGGGAAGAA ACAATGGTGC CGCTAAAACT CGC 43
 (2) INFORMATION FOR SEQ ID NO: 14:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 24 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: cDNA
 (iii) HYPOTHETICAL: YES
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14
 CTAAAACAAT GGAAATGAAT GGAC 24
 (2) INFORMATION FOR SEQ ID NO: 15:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 20 amino acids
 (B) TYPE: amino acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: protein
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (ix) FEATURE:
 (A) NAME/KEY: Protein
 (D) OTHER INFORMATION: /label= residue /note= "unknown"
 (ix) FEATURE:
 (A) NAME/KEY: Protein
 (B) LOCATION: 12
 (D) OTHER INFORMATION: /label= residue /note= "unknown"
 (ix) FEATURE:
 (A) NAME/KEY: Protein
 (B) LOCATION: 15
 (D) OTHER INFORMATION: /label= residue /note= "unknown"
 (ix) FEATURE:
 (A) NAME/KEY: Protein
 (B) LOCATION: 17
 (D) OTHER INFORMATION: /label= residue /note= "unknown"
 (ix) FEATURE:
 (A) NAME/KEY: Protein
 (B) LOCATION: 18
 (D) OTHER INFORMATION: /label= residue /note= "uncertain"
 (ix) FEATURE:
 (A) NAME/KEY: Protein
 (B) LOCATION: 19
 (D) OTHER INFORMATION: /label= residue /note= "uncertain"
 (ix) FEATURE:
 (A) NAME/KEY: Protein
 (B) LOCATION: 20
 (D) OTHER INFORMATION: /label= residue /note= "uncertain"
 (ix) FEATURE:
 (A) NAME/KEY: Protein
 (B) LOCATION: 21
 (D) OTHER INFORMATION: /label= residue /note= "Gly or Arg"
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15
 Thr Xaa Leu Ser Ala Ser Gly Pro Arg Pro Asn Xaa Gln Gly Xaa Tyr
 1 5 10 15
 Xaa Tyr Gly Xaa
 20
 (2) INFORMATION FOR SEQ ID NO: 16:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 21 amino acids
 (B) TYPE: amino acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: protein
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: Nicotiana tabacum
 (B) STRAIN: Samsun NN
 (F) TISSUE TYPE: leaf TMV-induced
 (ix) FEATURE:
 (A) NAME/KEY: Protein
 (B) LOCATION: 19
 (D) OTHER INFORMATION: /label= residue /note= "uncertain"
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16
 Ile Pro Val Pro Phe Pro Asp Pro Ala Asp Asp Tyr Thr Leu Leu Ile
 1 5 10 15
 Gly Asp Trp Tyr Lys
 20
 (2) INFORMATION FOR SEQ ID NO: 17:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 8 amino acids
 (B) TYPE: amino acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: peptide
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17
 Glu Asp Pro Tyr Arg Phe Phe Glu
 1 5
 (2) INFORMATION FOR SEQ ID NO: 18:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 31 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: cDNA
 (iii) HYPOTHETICAL: YES
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18
 AAGGGAACAA AAGTCTAGAT CTTGCTCCAT T 31
 (2) INFORMATION FOR SEQ ID NO: 19:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 50 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (ii) MOLECULE TYPE: cDNA
 (iii) HYPOTHETICAL: YES
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19
 TTTAGCGGCC ATGGCTTCTT CCCTAGGGAA GAAGCCATGG TGCCGCTAAA 50
 (2) INFORMATION FOR SEQ ID NO: 20:
 (i) SEQUENCE CHARACTERISTICS:
 (A) LENGTH: 49 base pairs
 (B) TYPE: nucleic acid
 (C) STRANDEDNESS: single
 (D) TOPOLOGY: linear
 (iii) HYPOTHETICAL: YES
 (vi) ORIGINAL SOURCE:
 (A) ORGANISM: not provided
 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20
 GGAATTCTGG TACCTCCCGG GAGGATCCAT CTAGAGCTCG AGTAAGCTT 49