CA2294652A1 - Fungus-resistant plants and method for creating the same - Google Patents

Abstract

DNAs encoding chimeric proteins each having a domain containing at least the elicitor-binding site of an elicitor receptor and another domain containing at least the signal transduction motif of a protein capable of imparting disease resistance to plants; recombinant vectors containing these DNAs; transformed plants prepared by using these vectors or offsprings thereof; and a method for the construction of disease resistant plants characterized by transferring the above vectors into plant hosts.

Description

. ~F'~. C f.P~.~-fN T ~i I ~ ~; ~uE ~ ~- .. .
~-r~i~i~r+iiv' DESCRIPTION
FUNGUS-RESISTANT PLANTS AND METHOD FOR CREATING THE SAME
TECHNICAL FIELD
The present invention relates to a DNA coding for a protein which confers plant disease resistance, plant disease-resistant plants and a method for creating such plants. More specifically, the invention relates to a DNA coding for a chimeric protein (chimeric receptor) comprising a domain containing at least an elicitor binding site of an elicitor receptor and a domain containing a signal transduction motif of a plant-derived resistance gene; plant disease-resistant plants into which the above DNA is transferred; and a method for creating such plants.
BACKGROUND ART
It is known that plants synthesize and accumulate antibiotic agents called phytoalexins in response to infection with pathogenic microorganisms [M. Yoshikawa (1978) Nature 257:
546]. Substances which induce such resistance reactions have been found in plant Phytophthora [N. T. Keen (1975) Science 187:
74] and are called "elicitors".
Various elicitors have been known, including degradation products generated by plant- or fungus-derived hydrolytic enzymes, proteinaceous elicitors produced by fungi and Avr (avirulence) gene products generated by fungi. As examples of the plant-derived degradation products, polygalacturonates produced by polygalacturonases may be given. As examples of the fungus-derived degradation products, oligosaccharides of chitin (N-acetylchitooligosaccharides) produced by chitinases or glucans produced by glucanases may be given. As examples of the proteinaceous elicitors, elicitins derived from fungi belonging to the genus Phytophthora may be given. As examples of the Avr gene products, Avr4 and Avr9 derived from Cladosporium fulvum or nipl derived from Rhynchosporium secalis may be given. These elicitors perform specific interactions with plant resistance gene products including elicitor receptors on plant plasma membranes. Through such interactions, invasion of fungi, etc. is recognized and then signals are generated. These signals are transmitted to nuclei to cause various resistance reactions for preventing the invasion of fungus into plants. These reactions are called the hypersensitive response, and include cell death, production of hydrogen peroxide, expression of PR proteins, expression of lipoxygenases and production of resistant agents called phytoalexins [W. Knogge (1996) Plant Cell 8: 1711; A.F.
Bent (1996) Plant Cell 8: 1757; K.E. Hammond-Kosack and J.D.G.
Jones (1996) Plant Cell 8: 1773].
The biochemical process from the infection of plants with pathogens to the induction of resistance in the plants is believed to proceed as follows, taking the synthesis and accumulation of phytoalexin for example. First, when mycelia of pathogens invade plant cells, glucanases in plant cells work so as to cleave polysaccharides on the surface of the mycelial walls of pathogens, thereby liberating elicitors. Elicitors are polysaccharides composed of glucose. They are a-1, 6-linked or Q-1, 3-linked glucans [J. K. Sharp et al. (1984) J. Biol. Chem.
259: 11321; M. Yoshikawa (1990) Plant Cell Engineering, 1.2:
695; Y. Okinaka et al. (1995) Plant Physiol. 109: 839J. If such an elicitor binds to a receptor in plant cells, a second messenger which performs the role of signal transduction is produced. This signal transducing substance is incorporated into the nuclei of plant cells, and activates the transcription of those genes coding for phytoalexin synthesis enzymes to thereby induce phytoalexin synthesis. At the same time, the phytoalexin degradation system is inhibited. Thus, phytoalexins are efficiently accumulated in plant cells. In the case of soybean, for example, a phytoalexin playing an important role in the resistance of this plant is called glyceollin, the structure of which has been determined [M. Yoshikawa et al. (1978) Physiol.
Plant. Pathol. 12: 73].
Therefore, a receptor specific to a glucan elicitor derived from a soybean pathogenic filamentous fungus Phytophthora megasperma f. sp. glycinea is believed to be a receptor protein which plays an important role in the synthesis and accumulation of the antiboitic agent glyceollin. This glucan elicitor specific receptor has been isolated and purified from a plasma membrane fraction of soybean roots by Kakitani et al. (Japanese Unexamined Patent Publication No. 6-321995). Further, Umemoto et al. [N. Umemoto et al. (1997) Proc. Natl. Acad. Sci. USA 94:
1029] have cloned a gene coding for this glucan elicitor receptor.
Recently, a number of reports have been made on the cloning of plant-derived resistance genes. In these genes, characteristic motifs or domains involved in signal transduction (such as leucine-rich repeats, leucine zippers, nucleotide binding sites (P-loop) and serine/threonine kinase domains) are found in part or in their entirety. As plant-derived disease resistance genes which are expected to contain such motifs or domains involved in signal transduction, the following genes have been reported for example: Arabidopsis thaliana-derived resistance gene RPS2 [M. Mindrinos et al. (1994) Cell 78: 1089;
A.F. Bent et al. (1994) Science 265: 1856], Arabidopsis thaliana-derived resistance gene RPM1 [M. R. Grant et al. (1995) Science 269: 843], tobacco-derived resistance gene N [S. Whitham et al. (1994) Cell 78: 1101], flax-derived resistance gene L6 [G. J. Lawrence et al. (1995) Plant Cell 7: 1195], tomato-derived resistance gene Pto [G. B. Martin et al. (1993) Science 262:
1432], tomato-derived resistance gene Prf [J.M. Salmeron et al.
(1996) Cell 86: 123], tomato-derived resistance gene Cf-9 [D. A.
Jones et al. (1994) Science 266: 789], tomato-derived resistance gene Cf-2 [M. S. Dixon et al. (1996) Cell 84: 451], and rice-derived resistance gene Xa21 [W.-Y. Song et al. (1995) Science 270: 1804].
On the other hand, in contrast to plant cell-derived receptors, a large number of reports have been made on the cloning of animal cell-derived receptors. Also, a large number of researches into chimeric receptors composed of an extracellular domain and an intracellular domain derived from heterologous receptors have been made. For example, an extracellular domain which recognizes a ligand of a certain receptor is replaced with an extracellular domain of a receptor which recognizes a ligand other than the above ligand, and the latter, new extracellular domain is fused to an intracellular domain which transmits a signal to nuclei, thereby preparing a chimeric receptor. When this receptor is expressed in cells, it is reported that the chimeric receptor recognizes the ligand which the new extracellular domain recognizes, to thereby perform signal transduction in cells [H. Riedel et al. (1986) Nature 324: 68, J. Lee et al. (1989) EMBO J. 8: 167; H.
Lehvaslaiho et al. (1989) EMBO J. 8: 159, S. Lev et al. (1990) Mol. Cell. Biol. 10: 6064]. Further, it is also reported that even if a gene coding for a receptor of an animal cell is transferred into a heterologous animal cell and expressed therein, the gene functions in a similar manner; for example, it is reported that when human-derived GM-CSF receptor is expressed in mouse 3T3 cells, the transmission of growth promoting signal is observed [S. Watanabe et al. (1993) Mol. Cell. Biol. 13:
1440] .
However, as far as the present inventors know, no report has been ever made concerning chimeric reporters in plants.
Besides, no report has suggested that such chimeric receptors function in plants.
DISCLOSURE OF THE INVENTION
The glucan elicitor receptor described above has no characteristic motifs or domains involved in signal transduction as found in plant-derived resistance genes. Thus, a possibility has been suggested that this glucan elicitor receptor may lack signal transduction domains. From researches into chimeric receptors using animal cell-derived receptors, the present inventors have considered as follows: if a chimeric receptor obtained by fusing a domain recognizing a certain ligand to a domain involved in signal transduction derived from a different protein (including receptor) is transferred into plant cells and expressed therein, the ligand may be recognized and signal transduction may be performed. The inventors have also considered that even if the cell in which a chimeric receptor is to be expressed and the cell from which a domain involved in signal transduction has been derived do not belong to the same plant species, signal transduction may be performed in a similar manner. In other words, the inventors have considered that if a gene coding for a chimeric receptor in which an elicitor receptor is bound to an intracellular signal transduction domain of a plant-derived receptor; or a gene coding for a chimeric receptor in which an elicitor receptor is bound to a domain that is encoded by a plant-derived resistance gene (e. g. Arabidopsis thaliana-derived resistance gene RPS2 or RPM1, tobacco-derived resistance gene N, flax-derived resistance gene L6, tomato-derived resistance gene Pto, rice-derived resistance gene Xa21) and that is expected to be involved intracellular signal transduction can be synthesized and expressed in plant regardless of plant species, the efficiency in signal transduction may be increased compared to those cases where an elicitor receptor alone is expressed. As a result, it would become possible to create plants having clearly strong resistance to pathogenic fungi, and the productivity of agricultural products would be improved. Further, economical effects from reduced use of agricultural chemicals and reduction of environmental pollution could be expected.
It is the object of the invention to provide a DNA coding for a chimeric protein comprising a domain containing at least an elicitor binding site of an elicitor receptor and a domain containing a signal transduction motif of a plant-derived resistance gene; plant disease-resistant plants into which the above DNA is transferred; and a method for creating such plants.
As a result of intensive and extensive researches toward the solution of the above-described problem, the present inventors have succeeded in conferring disease resistance on plants by synthesizing a gene coding for a chimeric receptor composed of a domain which is encoded by a plant-derived resistance gene and expected to be involved in signal transduction and a domain containing at least an elicitor binding site of an elicitor receptor, transferring the gene into Arabidopsis plants and tobacco plants, and expressing the gene in these plants. Thus, the present invention has been achieved.
The present invention relates to a DNA coding for a chimeric protein comprising a domain containing at least an elicitor binding site of an elicitor receptor and a domain containing at least a signal transduction motif of a protein which confers plant disease resistance. This chimeric protein functions as a receptor, thereby conferring disease resistance on plants.
Specific examples of the above elicitor include a glucan, polygalacturonate, N-acetylchitosaccharide, elicitin, Cladosporium fulvum-derived Avr gene product, or Rhynchosporium secalis-derived niple gene product. Specific examples of the above elicitor receptor include receptors of the above-enumerated elicitors. As the elicitor binding site of a glucan elicitor receptor, a protein comprising at least the amino acid sequence shown in SEQ ID NO: 27, or a protein which comprises the amino acid sequence shown in SEQ ID NO: 27 having deletion, substitution, addition or insertion of at least one amino acid and which has ability to bind to the relevant glucan elicitor may be given. As the domain containing a signal transduction motif, a domain containing at least one motif selected from the group consisting of leucine-rich repeats, leucine zippers, nucleotide binding sites and serine/threonine kinase domains may be given. Specific examples of such a domain include a signal transduction domain of an expression product of tomato-derived Pto, Prf, Cf-2 or Cf-9 gene; a signal transduction domain of an expression product of rice-derived Xa21 gene; a signal transduction domain of an expression product of Arabidopsis thaliana-derived RPS2 or RPMI gene (e. g. the domain represented by SEQ ID NO: 29); a signal transduction domain of an expression product of flax-derived L6 gene; and a signal transduction domain of an expression product of tobacco-derived N gene.
The present invention further relates to a DNA coding for a chimeric protein comprising the amino acid sequence shown in SEQ
ID NO: 5, 7 or 9; or a chimeric protein which comprises the amino acid sequence shown in SEQ ID NO: 5, 7 or 9 having deletion, substitution, addition or insertion of at least one amino acid and which confers plant disease resistance. Specific examples of the DNA include DNAs comprising the nucleotide sequence shown in SEQ ID NO: 6, 8 or 10.
The present invention further relates to a recombinant vector comprising the above-described DNA.
The present invention further relates to a transformed plant transformed with the above recombinant vector, or the progeny of the plant.
The present invention further relates to a plant having plant disease resistance, the plant having the above DNA and expressing the DNA, or the progeny of the plant.

The present invention further relates to a method for creating a plant disease resistant plant, comprising transferring the above recombinant vector into a plant.
The present invention further relates to a method for creating a plant having plant disease resistance or the progeny of this plant, comprising incorporating the above DNA into a chromosome of a plant and expressing the DNA.
Hereinbelow, the present invention will be described in detail.
The DNA of the invention can be obtained by ligating a DNA
coding for a domain containing at least an elicitor binding site of an elicitor receptor to a DNA coding for a domain containing at least a signal transduction motif of a protein which confers plant disease resistance. The DNA of the invention is sometimes called "the DNA coding for a chimeric receptor" or "the chimeric DNA".
Specific examples of the elicitor receptor include receptors which bind to plant degradation product elicitors such as polygalacturonates or fungal cell wall degradation product elicitors such as glucans, N-acetylchitooligosaccharides, and receptors which bind to proteinaceous elicitors such as elicitins produced by fungi, and receptors which bind to such elicitors as Cladosporium fulvum-derived Avr gene products or Rhynchosporium secalis-derived niple gene products.
As a method for cloning cDNAs coding for these elicitor receptors (hereinafter, referred to as "ERDNAs", the method described below may be used.
Briefly, the above elicitor is labelled with an isotope.

Then, an elicitor receptor which specifically binds to the elicitor is purified from plant plasma membrane fractions by determining binding activities of the fractions with the labelled elicitor. Based on the partial amino acid sequence information obtained from the purified elicitor receptor, PCR
primers are designed. Using these primers and a plant cell-derived DNA as a template, a PCR is performed to thereby obtain a probe. With this probe, a cDNA of interest is cloned from a cDNA library.
On the other hand, the DNA coding for a signal transduction motif in the present invention is already known as described above. Thus, the DNA can be cloned by PCR, etc.
Subsequently, ERDNA is ligated to the DNA coding for a signal transduction motif. The ligation of these DNAs are performed by digesting these DNAs with appropriate restriction enzymes and then ligating with ligase. Alternatively, the ligation is performed by binding ERDNA to the DNA coding for a signal transduction motif with an appropriate oligonucleotide.
Alternatively, the above DNAs may be ligated by insertion into an appropriate plasmid. The relative locations of ERDNA and the DNA coding for a signal transduction motif at the time of ligation are not particularly limited, i.e. ERDNA may be located upstream of the DNA coding for a signal transduction motif or vice versa.
Finally, the nucleotide sequence of the resultant chimeric DNA is determined by conventional methods (e. g. the dideoxy method, Maxam-Gilbert method). Usually, the nucleotide sequence is analyzed with an automated DNA sequencer.
Fig. 1 shows schematic representations of the chimeric DNA

of the invention which is composed of, by way of example, a DNA
coding for a glucan elicitor receptor (ER) and RPS2 gene from Arabidopsis thaliana. In Fig. 1, CER is a chimeric DNA composed of a 5' end region of RPS2 gene corresponding to about one third (1/3) of RPS2 (SEQ ID NO: 29) and the entire ERDNA (SEQ ID NO:
2). IER and ISER in Fig. 1 have a truncated ERDNA. In these chimeric DNAs, ERDNA is located upstream of RPS2 gene (i.e.
their locations are exchanged as compared to the locations in CER) .
The elicitor binding site of the glucan elicitor receptor corresponds to the partial sequence from position 239 to position 442 of the amino acid sequence shown in SEQ ID NO: 1, and has the amino acid sequence shown in SEQ ID NO: 27. As long as the elicitor binding site is a protein comprising at least the amino acid sequence shown in SEQ ID NO: 27, or a protein which comprises the amino acid sequence shown in SEQ ID NO: 27 having deletion, substitution, addition or insertion of at least one amino acid and which has ability to bind to the relevant glucan elicitor, the length of the elicitor receptor is not particularly limited; the elicitor receptor may be in a truncated form (see "2-11", "2-16" and "1-5" in Fig. 1). As a DNA coding for the amino acid sequence of the elicitor binding site represented by SEQ ID NO: 27, the nucleotide sequence shown in SEQ ID NO: 28 may be given.
The nucleotide sequence of the above chimeric DNA and the amino acid sequence of a chimeric protein encoded by the DNA are shown in SEQ ID NOS: 5 and 6, respectively, for CER; in SEQ ID
NOS: 7 and 8, respectively, for IER; and in SEQ ID NOS: 9 and 10, respectively, for ISER. The amino acid sequences (SEQ ID NOS: 5, 7 and 8) may have mutation such as deletion, substitution, addition or insertion of at least 1 amino acid, as long as these chimeric proteins have binding function as elicitor receptors and confer disease resistance activity on plants. In addition to those nucleotide sequences coding for the amino acid sequences comprised in the chimeric proteins of the invention, DNAs coding for the same polypeptides differing only in degenerate codons (termed "degenerate isomers") are also included in the DNA of the invention. For example, a DNA in which a codon AAC
corresponding to Asn is changed to a degenerate codon (e. g. AAT) is called herein a degenerate isomer.
The introduction of the above mutation may be performed by site specific mutagenesis (see, for example, D.F. Mark et al., Proc. Natl. Acad. Sci. USA 81: 5662-5666, 1984; PCT WO 85/00817 published on Feb. 28, 1985; R.P. Wharton et al., Nature 316:
601-605, Aug. 15, 1985) which was a well-known technique before the filing of the present application. Once the above nucleotide sequence is determined, the DNA of the invention can be obtained by chemical synthesis, PCR or hybridization using a DNA fragment having the nucleotide sequence as a probe.
The DNA sequence coding for a chimeric receptor used in the invention preferably has at least one stop codon (e. g. TAG) adjacent to its 3' end. Also, if desired, the DNA sequence may optionally have an ATG codon for initiation methionine upstream of its 5' end, the reading frame of which codon coincides with the reading frame of the DNA sequence. Further, the DNA sequence may have other DNAs of appropriate lengths upstream of its 5' end and downstream of its 3' end as non-translated regions.
Typically, the DNA sequence coding for a chimeric receptor used in the invention is present being inserted into plasmid or phage DNA as a part of the constituents thereof; or is present in microorganisms (in particular, bacteria), phage particles or plants while being inserted into plasmid, phage or genomic DNA
as a part of the constituents thereof. Specific examples of the above bacteria include E. coli and Agrobacterium. For stable expression of the DNA sequence coding for a chimeric receptor in plants, a promoter, DNA coding for translation initiation codon (ATG) and terminator may be added to the DNA sequence in an appropriate combination.
The DNA coding for a chimeric receptor of the invention (hereinafter sometimes referred to as the "chimeric DNA") may be incorporated into an appropriate vector to construct the recombinant vector of the invention.
The vector into which the chimeric DNA of the invention is to be incorporated is not particularly limited as long as it is replicable in a host. For example, plasmid DNA, phage DNA and the like may be employed.
A binary vector, which is one of plasmid DNA, can be prepared from E. coli or Agrobacterium by, for example, alkali extraction (Birnboim, H.C. & Doly, J. (1979) Nucleic Acid Res.
7: 1513). Specific examples of binary vectors include pBI121 and pBI101. Alternatively, a commercial plasmid such as pUC118 (Takara Shuzo), pUC119 (Takara Shuzo), pBluescript SK+
(Stratagene) or pGEM-T (Promega) may be used.
Specific examples of phage DNA include M13mp18, M13mp19 and M13tv18.
In order to ligate a chimeric DNA to a vector, a method may be used in which a purified chimeric DNA of the invention is digested with appropriate restriction enzymes and inserted into the relevant restriction site or multicloning site of an appropriate vector DNA for ligation.
The chimeric DNA to be expressed should be incorporated into the vector so that the function of the DNA can be revealed.
For this purpose, signal peptide genes, terminators, drug resistance genes, etc. may be incorporated into the recombinant vector of the invention in addition to promoters and the above-described gene. As signal peptide genes useful in the invention, the signal peptide gene of ~3-1,3 glucanase derived from tomato (Lycopersicon esculentum) or the like may be given. As enhancers useful in the invention, tobacco mosaic virus SZ sequence or the like may be given. As drug resistance genes useful in the invention, kanamycin resistance gene, hygromycin resistance gene, or the like may be given.
Specific examples of the above promoter include the promoter of the gene encoding ribulose-1,5-biphosphate carboxylase small subunit (R. Fluhr et al., Proc. Natl. Acad.
Sci. USA (1986) 83: 2358), the promoter of nopaline synthase (NOS) gene (W.H.R. Langridge et al., Plant Cell Rep. (1985) 4:
355), the promoter generating cauliflower mosaic virus 19S-RNA
(H. Guilley et al., Cell (1982) 30: 763), and the promoter generating cauliflower mosaic virus 35S-RNA (J. T. Odell et al., Nature (1985) 313: 810). Specific examples of the above terminator include the terminator of nopaline synthase gene [A.
Depicker et al., J. Mol. Appl. Gen. (1982) 1: 561] and the terminator of octopine synthase gene [J. Gielen et al., EMBO J.
(1984) 3: 835] .
Specifically, as shown in Fig. 2, cauliflower mosaic virus 35S promoter (p35S) is ligated upstream of the chimeric DNA, and nopaline synthase gene terminator (NosT) is ligated downstream of the chimeric DNA (this construct is called "ER cassette").
Further, a cassette in which kanamycin resistance gene (NPT II) is ligated between nopaline synthase gene promoter and terminator ("NPT II cassette") and a cassette in which hygromycin resistance gene is ligated between p35S and NosT ("Hm cassette") are constructed. Then, NPT II cassette is ligated upstream of ER cassette, followed by ligation of Hm cassette downstream of ER cassette to thereby obtain the recombinant vector of the invention (Fig. 2).
From various researches into elicitors and their receptors, it has been suggested that, in the case of glucan elicitors and their receptors for example, glucan elicitor receptors play an important role in plant resistance to a very wide range of fungi containing glucans in their cell wall components. Further, a gene coding for a glucan elicitor receptor has been cloned and, as a result of analysis of its primary sequence, it has been suggested that the elicitor receptor lacks domains involved in signal transduction. On the other hand, plant-derived resistance genes so far reported are roughly classified into the following 4 classes based on their structures [M. S. Dixon et al. (1996) Cell 84: 451] .
Class 1: Genes of this class have leucine-rich repeats and nucleotide binding sites. Some of them also have leucine zippers.
These genes are believed to be localized probably in cytoplasma.
Specific examples of resistance genes falling under this class include tobacco-derived resistance gene N, Arabidopsis thaliana-derived resistance genes RPS2 and RPM1, flax-derived resistance gene L6, and tomato-derived resistance gene Prf.
Class 2: Genes of this class have leucine-rich repeats and membrane binding sites. These genes are believed to be localized probably in cell surfaces. Tomato-derived resistance genes Cf-2 and Cf-9, and the like fall under this class.
Class 3: Genes of this class have serine/threonine kinase domains. Tomato-derived resistance gene Pto, and the like fall under this class.
Class 4: Genes of this class have leucine-rich repeats, transmembrane domains and serine/threonine-like kinase domains.
Probably, it is believed that the leucine-rich repeats recognize elicitors to thereby activate serine/threonine kinases located in cells. Rice-derived resistance gene Xa-21 and the like fall under this class.
Thus, plant-derived resistance genes contain one or more motifs or domains which are expected to be involved in signal transduction, i.e. leucine-rich repeats, leucine zippers, nucleotide binding sites and serine/threonine kinase domains.
Therefore, in the present invention, a DNA coding for a chimeric protein is prepared by ligating a domain containing at least an elicitor binding site of an elicitor receptor to a domain involved in signal transduction encoded by a plant-derived disease resistance gene (e.g. RPS2 gene, RPM1 gene, N
gene, L6 gene, Pto gene, Xa21 gene, Cf-2 gene, Cf-9 gene, Prf gene), and then expressed.
For example, a chimeric DNA composed of a DNA coding for a domain containing at least an elicitor binding site of a glucan elicitor receptor and a DNA coding for a domain containing a leucine zipper and a nucleotide binding site of Arabidopsis thaliana-derived resistance gene (RPS2), or a fragment of the chimeric DNA, is transferred into higher plants and expressed therein by known methods.
Consequently, it is possible to confer on plants still stronger resistance to fungi, as compared to those cases where a glucan elicitor receptor alone is introduced and expressed.
A theory has been proposed that those fungi infectious to plants generally have suppressors and, thus, have acquired ability to suppress plants' inherent resistance to fungi. Even in these cases, however, it is possible to create a plant having stronger resistance to fungi by transferring the DNA of the invention coding for a chimeric receptor or a fragment of the DNA into the plant and expressing it therein so that the chimeric receptor functions, or by modifying the DNA or regulating the amount of expression of the DNA.
Further, by transferring the DNA of the invention or a fragment thereof into higher plants in combination with a gene or character which enhance resistance to fungi (e. g. a glucanase which exhibits resistance to fungi), it is possible to confer on plants still stronger resistance to fungi than the resistance of those plants into which a glucanase or the like alone has been introduced.
Examples of DNA sequences coding for glucanases include a DNA comprising a nucleotide sequence coding for a protein comprising the amino acid sequence shown in SEQ ID NO: 11 or 13, or protein which comprises the amino acid sequence shown in SEQ
ID NO: 11 or 13 having deletion, substitution, addition or insertion of at least one amino acid and which has glucanase activity. The above-described DNA is intended to include all the degenerate isomers. Specific examples of such isomers include a DNA comprising the nucleotide sequence shown in SEQ ID NO: 12 or 14.
As methods for transferring a gene into plant cells or plant, conventional methods may be used. For example, methods described in "Plant Genetic Transformation and Gene Expression;
A Laboratory Manual", J. Draper, et al. (eds.), Blackwell Scientific Publications (1988) may be used. Examples of methods for gene transfer include biological methods using viruses or Agrobacterium and physicochemical methods such as electroporation, the polyethylene glycol method, microinjection, the particle gun method and the dextran method.
When the plant to be transformed is a dicotyledonous plant, a biological method using Agrobacterium is generally preferable.
When the plant to be transformed is a monocotyledonous plant, or a dicotyledonous plant that is not susceptible to infection with Agrobacterium, a physical/chemical method such as electroporation is preferable. As a plant material into which a DNA of interest is to be transferred, an appropriate material may be selected from leaves, stems, roots, petals, tubers, protoplasts, calli, pollen, seed embryos, shoot primordia, etc.
according to the method of transfer.
When a DNA of interest is to be transferred into cultured plant cells, protoplasts are generally used as a material, and the DNA is transferred thereinto by a physical/chemical method such as electroporation, the polyethylene glycol method or the like. On the other hand, when a DNA of interest is to be transferred into plant tissues, leaves, stems, roots, petals, tubers, protoplasts, calli, pollen, seed embryos, shoot primordia or the like are used as a plant material. Preferably, leaves or stems are used. The DNA is transferred into such plant tissues by a biological method using a virus or Agrobacterium, or a physical/chemical method such as the particle gun method, microinjection or the like. Preferably, a biological method using Agrobacterium is used.
In order to regenerate a plant from those plant tissues or plant cells into which a DNA sequence coding for a chimeric receptor has been transferred, these transformed plant tissues or cells may be cultured in a medium, such as MS medium, containing appropriate plant growth regulators. The resultant seedlings which are rooting may be transferred to soil to give grown-up plants.
By transferring and expressing a DNA sequence coding for a chimeric receptor by the procedures as described above, resistance to pathogenic fungi can be conferred on plants or enhanced in plants. Examples of such plants include those plants which are susceptible to infection with pathogenic fungi containing glucans in their cell walls. Specifically, these plants include, but are not limited to, solanaceous plants, leguminous plants, asteraceous plants, caryophyllaceous plants and gramineous plants. More specific examples include, but are not limited to, tobacco, soybean, potato, rice, chrysanthemum and carnation.
As pathogenic fungi, those containing glucan in cell walls are preferable as targets. Specific examples of such pathogenic fungi include, but are not limited to, the genera Phytophthora, Rhizoctonia, Pyricularia, Puccinia, Fusarium, Uromyces, Botrytis and Alternaria. More specifically, the pathogenic fungi include, but are not limited to, Phytophthora nicotianae, Rhizoctonia solani, Pyricularia oryzae, Puccinia horiana, Fusarium oxysporum, Fusarium roseum, Fusarium tricinctum, Uromyces dianthi, Botrytis cinerea and Alternaria dianthi.
According to the present invention, the DNA sequence coding for a chimeric receptor transferred into a plant can be inherited to subsequent generations through seeds. Thus, the transferred DNA sequence is also present in those seeds which are formed from the pollens or ovaries of the plant of the invention, and the inherited character can be transmitted to the progeny. Accordingly, the plant of the invention into which a DNA sequence coding for a chimeric receptor has been transferred can be propagated through seeds without losing its resistance to pathogenic fungi. The plant of the invention can also be propagated by a mass propagation method using plant tissue culture or by conventional techniques such as cutting, layering, grafting, division, etc. without losing its resistance to pathogenic fungi.
Whether a transformed plant has resistance to fungi or not can be confirmed from the presence or absence of resistance reaction when a glucan elicitor is added, or examined by fungus inoculation tests. Resistance reaction tests by elicitor addition are performed by adding a glucan elicitor solution to the surface of leaves, or infiltrating a glucan elicitor solution into leaves from the back side of leaves with a syringe and, after a specific period of time, observing browning reaction appearing on leaf surfaces or examining the accumulation of fluorescent substances (phytoalexins) excited by UV light. In fungus inoculation tests, resistance can be examined by the following inoculation methods, which are roughly divided into two groups, i.e. inoculation to the aerial part and inoculation to the subterranean part.
I. The following methods may be employed for the inoculation of a pathogen to the aerial part.
(1) Spray inoculation: A suspension of a pathogenic fungi or spores thereof is sprayed onto plants, which are then incubated in an inoculation box at an appropriate temperature under saturation humidity for one hour to examine the severity of the disease.
(2) Dusting inoculation: Spores collected from lesions are directed dusted off onto plants with a writing brush, or inoculated onto plants with a small duster. The inoculated plants are incubated a whole day and night in an inoculation box adjusted to a temperature suitable for infection. Subsequently, the same procedures as in the spray inoculation method are taken.
(3) Wound inoculation: A spore suspension or a piece of cultured mycelial tuft is inoculated into cut ends of branches or those portions where the bark is pressed through with a cork borer.
(4) Needle inoculation: A needle or a bundle of needles dipped in a suspension of a pathogenic fungus is used to scratch plants for inoculation.

(5) Cell adhesion inoculation: When a pathogenic fungus which does not form spores on medium is used, cultured mycelial masses or sclerotia are adhered to the surface of plants for inoculation.

(6) Damaged plant inoculation: In stead of direct inoculation of a pathogenic fungus, plants damaged by the fungus are placed on plants to be tested, or foliage damaged by the fungus is suspended over plants to be tested so that inoculation is performed by mycelia growing from the damaged plant or spores falling off from the foliage.
II. The following methods may be employed for the inoculation of a pathogen to the subterranean part.
(1) Soil drenching inoculation: Soil is drenched with a suspension of spores or disrupted mycelia for inoculation.
(2) Soil mixing inoculation: A filamentous fungus cultured in wheat bran, crop grains, straw of rice, straw of barley, etc. is mixed with soil for inoculation.
(3) Diseased plant burying inoculation: Residue from diseased plants is buried in soil to prepare a soil polluted with the pathogenic fungus. When the residue has been rotten, the soil is mixed well. Then, seeds are sown therein or seedlings are transplanted thereto.
(4) Polluted soil inoculation: If a specific soil disease is always occurring in some area, soil is taken therefrom, mixed well and filled in pots or vats. Then, seeds are sown therein or seedlings are transplanted thereto.
(5) Root dipping inoculation: A suspension of spores from a pathogenic bacterium or filamentous fungus is prepared. Roots of healthy seedlings are dipped in this suspension for 3-5 min and then transplanted to pots.
Resistance tests may be performed according to the above-described inoculation methods. More specifically, resistance to Phytophthora can be assayed by directly inoculating fungal mycelia into plants and observing the expansion of lesions.
Alternatively, the resistance may be assayed by inoculating zoospores from Phytophthora and observing the vicissitudes of lesions. Resistance to soil fungi other than Phytophthora can be assayed by mixing cultured fungal cells with soil, sowing seeds or growing plants on the soil, and observing the phenomenon of damping-off. However, methods for testing resistance to fungi are not limited to these methods.
BRIEF EXPLANATION OF THE DRAWINGS
Fig. 1 shows chimeric DNA constructs of the invention schematically .
Fig. 2 shows an outline of the construction of plasmid pBI(Hm)-ER.
Fig. 3 shows an outline of the construction of plasmid pBI(Hm)-LER.
Fig. 4 shows an outline of a plasmid containing a DNA
coding for an N-terminal region of RPS2 protein corresponding to about 1/3 of this protein.
Fig. 5 shows an outline of the construction of plasmid pBI(Hm)-CER.
Fig. 6 shows an outline of the construction of plasmid pBI (Hm) -IER.
Fig. 7 shows an outline of the construction of plasmid pBI(Hm)-ISER.

BEST MODES FOR CARRYING OUT THE INVENTION
Hereinbelow, the present invention will be described more specifically with reference to the following Examples. However, the technical scope of the invention is not limited by these Examples.
In the following Examples, a glucan elicitor receptor is abbreviated to "ER". Abbreviations used in Figs. 2-7 have the following meanings.
Restriction sites:
B: BamHI; Xb: XbaI; SI: SalI; RI: EcoRI; RV: EcoRV; Xh:XhoI;
Sph: SphI; Sp: SpeI; ScI: SacI; Sm: SmaI; Nsp: NspI; N: NotI;
Nsi: NsiI; Nc: NcoI; H3: HindIII
(Sm): SmaI recognition sequence is deleted by the insertion of DNA into SmaI site.
dScI, dB: Sacl site and BamHI site are deleted, respectively.
L: Leader sequence of ~-1,3-glucanase pNOS: NOS promoter; p35S: Cauliflower mosaic virus 35S promoter;
Nos-T: NOS terminator NPTII: Kanamycin resistance gene; Hm: Hygromycin resistance gene RB, LB: Right border DNA sequence of Ti-plasmid, left border DNA
sequence of Ti-plasmid [EXAMPLE lJ Construction of Vector Plasmids Individual vectors were constructed by the procedures described below. Unless otherwise specified, reagents such as restriction enzymes, linkers, etc. used in plasmid preparation were products manufactured by Takara Shuzo. For the amplification and selection in E. coli, DH5a (BRL) was used.

(1) Construction of pBI(Hm)-ER (see Fig. 2) The BamHI and SalI sites located in the multilinker in plasmid pER23-1 [N. Umemoto et al. (1997) Proc. Natl. Acad. Sci.
USA 279: 141] were digested with restriction enzymes BamHI and SalI to obtain a fragment containing the full length ER gene sandwiched between the two restriction sites. This fragment was separated from agarose gel. On the other hand, a plant expression type binary plasmid pBI121 (Clontech) was digested with BamHI and SacI; and synthetic linker DNAs (SEQ ID NOS: 15 and 16) shown below were synthesized with an automated DNA
synthesizer (Applied Biosystems).
These linker DNAs were annealed and ligated to the digested pBI121 to thereby produce pBI linker. Then, the above-described fragment was cloned between the BamHI and SalI sites of this pBI
linker to thereby obtain pBI-ER.
5'-CTAGAGGATCCGGTACCCCCGGGGTCGACGAGCT-3' (SEQ ID NO: 15) 5'-CGTCGACCCCGGGGGTACCGGATCCT-3' (SEQ ID NO: 16) Subsequently, pBI-ER was digested with BamHI and SacI to obtain a fragment containing the full length ER gene. This fragment was separated from agarose gel and ligated to the plasmid fragment obtained by digesting a plant expression type binary plasmid pIG121-Hm [Nakamura et al. (1991), Plant Biotechnology II (Modern Chemistry Suppl. Vol. 20), pp.123-132;
released from Prof. Kenzo Nakamura of Nagoya University, Japan]
with BamHI and SacI, to thereby obtain pBI(Hm)-ER, the vector of interest.
As a result of DNA sequencing of the resultant vector, the nucleotide sequence shown in SEQ ID NO: 2 was obtained. The amino acid sequence encoded by the nucleotide sequence of SEQ ID
N0: 2 is shown in SEQ ID NO: 1.
E. coli DH5a EKB633 carrying plasmid pER23-1 transferred thereinto was deposited at the National Institute of Bioscience and Human-technology, Agency of Industrial Science and Technology (1-3, Higashi 1-chome, Tsukuba City, Ibaraki Pref., Japan) on June 15, 1994 under the Accession No. FERM BP-4699.
(2) Construction of pBI(Hm)-LER (see Fig. 3) The two HindIII sites in pER23-1 were cleaved with HindIII.
The resultant plasmid was subjected to self-ligation to thereby delete the portion of ER after the HindIII site located in ER.
Subsequently, the remaining two NotI sites were cleaved with NotI and filled in with Klenow fragment. To the resultant plasmid, BamHI linker was introduced to thereby create a BamHI
site. This plasmid was digested with BamHI and HindIII to obtain a fragment containing the portion of ER prior to the HindIII
site. To this fragment, a plasmid fragment containing the portion of ER after the HindIII site obtained by digesting pER23-1 with BamHI and HindIII was ligated.
The resultant plasmid was further digested with Xbal and BamHI. Then, synthetic linker DNAs (SEQ ID NOS: 17 and 18) derived from the untranslated 5'end of soybean ~3-1,3-glucanase and synthesized with an automated DNA synthesizer were annealed and ligated to the digested plasmid, thereby preparing pLER.
5'-CTAGACTTCTTTCCTCAACCTTCTTTCTTCTTATATATTCGAAC-3' (SEQ ID NO: 17) 5'-GATCGTTCGAATATATAAGAAGAAAGAAGGTTGAGGAAAGAAGT-3'(SEQ ID NO: 18) Subsequently, pLER was digested with SalI and filled in with Klenow fragment. Then, Sacl linker was introduced to create a SacI site. The resultant plasmid was further digested with XbaI and SacI to obtain a fragment containing the ER gene with the leader sequence (L). This fragment was separated from agarose gel and then ligated to a plasmid fragment obtained by digesting a plant expression type binary vector plasmid pIG121-Hm with Xbal and SacI, thereby obtaining pBI(Hm)-LER, the vector of interest.
As a result of DNA sequencing of the resultant vector, the nucleotide sequence shown in SEQ ID NO: 4 was obtained. The amino acid sequence encoded by the nucleotide sequence of SEQ ID
NO: 4 is shown in SEQ ID NO: 3.
(3) Acquisition of cDNA Clones Coding for an N-Terminal Region of Arabidopsis thaliana-Derived RPS2 Corresponding to about 1/3 of This Protein Arabidopsis thaliana cDNA was obtained by plating a cDNA
library (Clontech) on ten 15 cm LB plates at 200,000 plaques/plate after infecting E. coli C600hf1, and recovering the phage from the plates. 5 pools of phage library were obtained, the phage recovered from 2 plates being taken as 1 pool. Phage DNA was recovered from each pool according to the method of Ishida ("Gene Expression Experiment Manual", Ishida &
Ando (eds.), Kodansha Scientific Co.) The cDNA of interest encoding the N-terminal region of pRPS2 was amplified by PCR (using EX-Taq kit from Takara Shuzo) using either of the following PCR primer sets and about 1 ~ g of phage DNA (pool #1-#5) as a template, and then cloned into pBluescript SKII(+) plasmid.
(i) Sense primer: 5'-TGGCATGCGATGGATTTCATCTCATCTCTT-3' (SEQ ID N0:19) Antisense primer: 5'-GGGAATTCACTCCGCGAGCCGGCGAAT-3' (SEQ ID N0:20) (ii) Sense primer: 5'-ACAAGTAAAAGAAAGAGCGAGAAATCATCGAAATG-3' (SEQ ID N0:21) Antisense primer: 5'-AGCCATGGCTCCTCCTAAAGTGAT-3' (SEQ ID N0:22) PCR was performed using the above primer set (i) or (ii).
The amplified DNA was phosphorylated and inserted into the SmaI site of pBluescript SKII(+), thereby obtaining the plasmid DNAs shown in Fig. 4 (pRPS2-6, pRPS2-14 and pRPS2-16). It was confirmed by DNA sequencing that these DNAs were identical with the reported sequence [M. Mindrinos et al. (1994) Cell 78: 1089]
except their primer portions.
(4) Construction of pBI(Hm)-CER (see Fig. 5) pRPS2-14 was digested with restriction enzyme XbaI, followed by self-ligation to thereby delete the XbaI fragment.
BamHI linker was inserted into the EcoRV site of the resultant plasmid. Further, SacI linker was inserted into the XhoI site.
The resultant plasmid was digested with BamHI and SalI, and the full-length ER cut out from pER23-1 with BamHI and salI was cloned thereinto. From this plasmid, a chimeric DNA containing a part of RPS2 and the full-length ER was purified. By DNA
sequencing of the junction between RPS2 and ER, it was confirmed that the reading frame of RPS2 coincided with the reading frame of ER.
On the other hand, pSpeI linker was inserted into the RcoRV
site of pRPS2-6. Subsequently, this plasmid was digested with XbaI and SacI, and then the above-described purified DNA
fragment was cloned thereinto. This plasmid containing the chimeric gene composed of a 5' end region of RPS2 corresponding to about 1/3 of RPS2 and the full-length ER was digested with SpeI and SacI to cut out the chimeric gene, which was then cloned into pIG121-Hm plasmid. Thus, pBI(Hm)-CER, the vector of interest, was obtained.
As a result of DNA sequencing of the resultant vector, the nucleotide sequence shown in SEQ ID NO: 6 was obtained. The amino acid sequence encoded by the nucleotide sequence of SEQ ID
NO: 6 is shown in SEQ ID NO: 5.
(5) Construction of pBI(Hm)-IER (see Fig. 6) An ER cDNA fragment coding for the N-terminal 537 amino acid was purified from pER23-1 using BamHI and NspI. On the other hand, a RPS2 cDNA fragment was purified from pRPS2-16 plasmid using EcoRV and SphI. These two DNA fragments were cloned between the BamHI and SmaI sites of pBluescript SKII(+) plasmid. This plasmid was designated pERRPS-8. By DNA
sequencing of the junction between RPS2 and ER, it was confirmed that the reading frame of RPS2 coincided with the reading frame of ER. pERRPS-8 was digested with SpeI and SacI to obtain a fragment containing a fusion gene composed of a domain encoding an elicitor binding region and a domain encoding a partial-length RPS2. This fragment was separated from agarose gel and ligated to a plasmid fragment obtained by digesting a plant expression type binary vector pIG121-Hm with XbaI and SacI, to thereby obtain pBI(Hm)-IER, the vector of interest.
As a result of DNA sequencing of the resultant vector, the nucleotide sequence shown in SEQ ID NO: 8 was obtained. The amino acid sequence encoded by the nucleotide sequence of SEQ ID
NO: 8 is shown in SEQ ID NO: 7.
(6) Construction of pBI(Hm)-ISER (see Fig. 7) pERRPS-8 plasmid was digested with NsiI and NotI. Then, the NotI-NsiI adaptors shown below were inserted into the plasmid. It was confirmed by DNA sequencing that the reading frame of the ATG within the adaptor coincided with the reading frame of ER. The resultant plasmid was digested with EcoRV and SalI, and then the resultant RPS2/ER chimeric DNA was purified.
5'-GGCCGCGATATCATGAATGCA-3' (SEQ ID NO: 23) 5'-TTCATGATATCGC-3' (SEQ ID NO: 24) The synthetic DNAs shown below derived from the untranslated 5' end of ~3-1,3-glucanase were prepared and inserted into the NotI and EcoRI sites of pBluescript SKII(+), thereby preparing plasmid pL.
5'-GGCCGCCTTCTTTCCTCAACCTTCTTTCTTCTTATATATTCGAAC-3' (SEQ ID NO: 25) 5'-AATTGTTCGAATATATAAGAAGAAAGAAGGTTGAGGAAAGAAGGC-3' (SEQ ID NO: 26) Plasmid pL was digested with EcoRV and SalI, and the RPS2/ER chimeric DNA purified above was inserted between the restriction sites. The resultant plasmid is designated pSERRPS-8.
pSERRPS-8 was digested with SpeI and SacI to obtain a fragment containing a fusion gene composed of a domain encoding an elicitor binding site and a domain encoding a partial-length RPS2. This fragment was separated from agarose gel and ligated to a plasmid fragment obtained by digesting a plant expression type binary vector pIG121-Hm with XbaI and SacI, to thereby obtain pBI(Hm)-ISER, the vector of interest.
As a result of DNA sequencing of the resultant vector, the nucleotide sequence shown in SEQ ID NO: 10 was obtained. The amino acid sequence encoded by the nucleotide sequence of SEQ ID
NO: 10 is shown in SEQ ID NO: 9.
[EXAMPLE 2] Transfer of the Chimeric Receptor Gene into Arabidopsis thaliana Plants (1) Preparation of Agrobacterium Infection Liquid Agrobacterium tumefaciens EHA101 into which a transformation vector [pBI(Hm)-CER or pBI(Hm)-ISER] containing the chimeric receptor gene or a transformation vector [pBI(Hm)-ER] containing ER gene as a control had been transferred by electroporation was inoculated into 3 ml of YEB medium and cultured at 28~C for 16 hr. Then, the cells were harvested by centrifugation and suspended in 10 ml of an infection medium to prepare an Agrobacterium infection liquid for transformation.
The compositions of YEB medium and the infection medium are as described below.
YEB medium: 5 g/L beef extract; 1 g/L yeast extract; 5 g/L
peptone; 5 g/L sucrose; 2 mM magnesium sulfate Infection medium: the inorganic salts & vitamins used in MS
[Murashige & Skoog (1962) Physiol. Plant. 15: 473] medium at 0.5-fold concentrations; 15 g/L sucrose; 10 g/L glucose; 10 mM
MES (pH 5.4) (2) Method for Creating Arabidopsis thaliana Transformants Seeds of Arabidopsis thaliana L. ecotypes Landsberg and Columbia were sown in MSHF medium asceptically and cultured for 3 weeks. Roots grown up to 2-3 cm were cut to approximately 5 mm, transferred onto a callus-inducing medium and cultured for 2 days. Roots cut into pieces were dipped in the Agrobacterium infection liquid. After excessive liquid on the root pieces was removed on a paper filter, the root pieces were transferred onto Arabidopsis co-culture medium and cultured in a dark place for 2 days. Then, the root pieces were transferred onto Arabidopsis redifferentiation medium and cultured for 2 days. Then, the root pieces were transferred onto Arabidopsis redifferentiation medium for selection and cultured for 1 week. Thereafter, the root pieces were transferred onto fresh Arabidopsis redifferentiation medium for selection every 1 week and cultured until regenerated seedlings were obtained.
Seedlings whose shoot had grown up to approximately 1 cm were transplanted to Arabidopsis root-inducing medium and cultured further. The presence of a transgene in seedlings whose roots had elongated to 1-2 cm was confirmed by PCR to obtain transformants. Unless otherwise specified, the cultivation was performed at 22~C under a 16 hr illumination/8 hr non-illumination regime. The components of each medium used in the cultivation are as described below.
MSHF medium: the inorganic salts & vitamins used in MS
medium [Murashige & Skoog (1962) Physiol. Plant. 15: 473]; 20 g/L sucrose; 2 g/L gellan gum; 5 mM MES (pH 6.2) Callus-inducing medium: the inorganic salts & vitamins used in MS medium; 20 g/L sucrose; 0.5 mg/L 2,4-dichlorophenoxyacetic acid; 0.1 mg/L kinetin; 2 g/L gellan gum; 5 mM MES (pH 6.2) Arabidopsis co-culture medium: the inorganic salts &
vitamins used in MS medium; 20 g/L sucrose; 0.5 mg/L 2,4-dichlorophenoxyacetic acid; 0.1 mg/L kinetin; 0.2 mM
acetosyringone; 2 g/L gellan gum; 5 mM MES (pH 6.2) Arabidopsis redifferentiation medium: the inorganic salts &
vitamins used in MS medium; 20 g/L sucrose; 1 mg/L 2-isopentenylamine; 0.15 mg/L indoleacetic acid; 250 mg/L
Cefotaxime; 2 g/L gellan gum; 5 mM MES (pH 6.2) Arabidopsis redifferentiation medium for selection: the inorganic salts & vitamins used in MS medium; 20 g/L sucrose; 1 mg/L 2-isopentenylamine; 0.15 mg/L indoleacetic acid; 2 g/L
gellan gum; 50 mg/L kanamycin; 250 mg/L Cefotaxime; 5 mM MES (pH
6.2) Arabidopsis root-inducing medium: the inorganic salts &
vitamins used in MS medium; 20 g/L sucrose; 2 mg/L
naphthaleneacetic acid; 2 g/L gellan gum; 250 mg/L Cefotaxime; 5 mM MES (pH 6.2) [EXAMPLE 3] Transfer of the Chimeric Receptor Gene into Tobacco Plants (1) Preparation of Agrobacterium Infection Liquid Agrobacterium tumefaciens EHA101 into which a transformation vector [pBI(Hm)-IER] containing the chimeric receptor gene or a transformation vector [pBI(Hm)-ER] containing ER gene as a control had been transferred by electroporation was inoculated into 3 ml of YEB medium and cultured at 28~C for 16 hr.
Then, the cells were harvested by centrifugation and suspended in 10 ml of an infection medium to prepare an Agrobacterium infection liquid for transformation. The compositions of YEB
medium and the infection medium are as described below.
YEB medium: 5 g/L beef extract; 1 g/L yeast extract; 5 g/L
peptone; 5 g/L sucrose; 2 mM magnesium sulfate Infection medium: the inorganic salts & vitamins used in MS
[Murashige & Skoog (1962) Physiol. Plant. 15: 473] medium at 0.5-fold concentrations; 15 g/L sucrose; 10 g/L glucose; 10 mM
MES (pH 5.4) (2) Method for Creating Tobacco Transformants Leaves of tobacco (Nicotiana tabacum L.) cv. Samsun were sterilized and cut into pieces about 1 cm square. These leaf pieces were floated on the Agrobacterium infection liquid for 10 min and then excessive liquid on them was removed on a paper filter. Subsequently, the leaf pieces were transferred onto tobacco co-culture medium and cultured in the dark for 2 days.
After the leaf pieces were transferred onto tobacco redifferentiation medium and cultured for 1 week, they were transferred onto tobacco redifferentiation medium for selection and cultured for 2 weeks. Thereafter, they were transferred onto fresh tobacco redifferentiation medium for selection every 2 weeks and cultured until regenerated seedlings were obtained.
Seedlings whose shoot had grown up to approximately 1 cm were transplanted to tobacco root-inducing medium and cultured further. The presence of a transgene in seedlings whose roots had elongated to 1-2 cm was confirmed by PCR to obtain transformants. Unless otherwise specified, the cultivation was performed at 25~C under a 16 hr illumination/8 hr non-illumination regime. The components of each medium used in the cultivation are as described below.
Tobacco co-culture medium: the inorganic salts & vitamins used in MS medium [Murashige & Skoog (1962) Physiol. Plant. 15:
473]; 30 g/L sucrose; 0.1 mg/1 naphthaleneacetic acid; 1 mg/L
benzyladenine; 0.2 mM acetosyringone; 8 g/L agar; 5 mM MES (pH
5.8) Tobacco redifferentiation medium: the inorganic salts &
vitamins used in MS medium; 30 g/L sucrose; 0.1 mg/L
naphthaleneacetic acid; 1 mg/L benzyladenine; 250 mg/L
Cefotaxime; 8 g/L agar; 5 mM MES (pH 5.8) Tobacco redifferentiation medium for selection: the inorganic salts & vitamins used in MS medium; 30 g/L sucrose;
0.1 mg/L naphthaleneacetic acid; 1 mg/L benzyladenine; 100 mg/L
kanamycin; 250 mg/L Cefotaxime; 8 g/L agar; 5 mM MES (pH 5.8) Tobacco root-inducing medium: the inorganic salts &
vitamins used in MS medium; 30 g/L sucrose; 250 mg/L Cefotaxime;
8 g/L agar; 5 mM MES (pH 5.8) [EXAMPLE 4] Hypersensitive Response Test on Transformed Arabidopsis thaliana (1) Hypersensitive Response Test on Transformed Arabidopsis thaliana (Ecotype Landsberg) Induction of the hypersensitive response by a soybean elicitor was examined using leaves of Arabidopsis thaliana transformant (CER) in which high expression of one of the created chimeric receptor genes, i.e. the gene comprising the nucleotide sequence shown in SEQ ID NO: 6 was confirmed, as well as Arabidopsis thaliana transformant (ER; SEQ ID N0: 2) in which high expression of ER protein was confirmed and non-transformed Arabidopsis thaliana (control).
A fungal cell wall-derived glucan elicitor (100,u g/ml) dissolved in 10 mM MgCl2 solution and 10 mM MgCl2 solution alone were infiltrated separately into leaves of Arabidopsis thaliana plants grown up in a greenhouse, from the back side of the leaves using a syringe not equipped with a needle. The plants were cultured at 22 ~C for 7 days under a 16 hr light/8 hr dark regime to observe changes appearing on the leaf surface. The fungal cell wall-derived glucan elicitor used in this experiment was obtained according to the method of Umemoto et al. [N.
Umemoto et al. (1997) Proc. Natl. Acad. Sci. USA 94: 1029].
When the glucan elicitor was infiltrated into non-transformed Arabidopsis thaliana, no changes were observed on the leaf surface. This indicates that Arabidopsis thaliana cannot recognize the glucan elicitor. On the other hand, in the Arabidopsis thaliana plants (ER) expressing a glucan elicitor receptor, 2 clones out of the 6 tested exhibited weak browning reactions (resistance reaction). In the Arabidopsis thaliana plants (CER) expressing a chimeric receptor, 8 clones out of the 12 tested exhibited effective browning reactions (resistance reaction) and 1 clone exhibited weak browning reactions (Table 1) .

Table 1. Hypersensitive Response Test on Transformed Arabidopsis thaliana (Ecotype Landsberg) Fungal cell wall - derived 10 mM MgCl2 glucan elicitor Control 1 - -Control 2 - -Control 3 - -Control 4 - -Control 5 - -Control 6 - -Control 7 - -Control 8 - -Control 9 - -Control 10 - -Control 11 - -Control 12 - -ER 3 + -ER 6 + -CER 1 ++ -CER 2 ++ -CER 3 ++ -CER 4 ++ -C E R 6 +++ -C E R 7 +++ -CER 8 ++ -CER 11 ++ -. no change; +: slight browning reactions;
++; medium browning reactions; +++: strong browning reactions From these results, it has been proved that recognition of a glucan elicitor which a host plant cannot recognize takes place not only when a soybean-derived ER protein is expressed alone but also when a chimeric receptor composed of the ER
protein and an N-terminal domain of Arabidopsis thaliana-derived RPS2 protein which is expected to contain a signal transduction domain is expressed in Arabidopsis thaliana. Furthermore, it has been proved that expressing a chimeric receptor is more advantageous for recognition of a glucan elicitor. This demonstrates that when a chimeric receptor is expressed, the signal transduction after the binding of a glucan elicitor to the receptor is more efficiently performed, as compared to the case where an ER protein alone is expressed.
The present invention provide a DNA and plants which can induce the resistance reaction deeply involved in disease resistance more strongly than an ER protein expressed alone, by recognizing the contact and/or invasion of plant pathogenic microorganisms such as fungi having glucan structures in their cell walls, etc. By creating such plants which strongly induce the resistance reaction caused by a glucan elicitor, it will become possible to breed plants which exhibit strong resistance to plant pathogenic microorganisms such as fungi.
(2) Hypersensitive Response Test on Transformed Arabidopsis thaliana Ecotype Columbia Induction of the hypersensitive response by a soybean elicitor was examined using leaves of Arabidopsis thaliana transformant (ISER) in which high expression of one of the created chimeric receptor genes, i.e. the gene comprising the nucleotide sequence shown in SEQ ID NO: 10 was confirmed, as well as non-transformed Arabidopsis thaliana (control).
A fungal cell wall-derived glucan elicitor (100 g/ml) dissolved in 10 mM MgCl2 solution and 10 mM MgCl2 solution alone were infiltrated separately into leaves of Arabidopsis thaliana plants grown up in a greenhouse from the back side of the leaves using a syringe not equipped with a needle. The plants were cultured at 22~C for 7 days under a 16 hr light/8 hr dark regime to observe changes appearing on the leaf surface. The fungal cell wall-derived glucan elicitor used in this experiment was obtained according to the method of Umemoto et al. [N. Umemoto et al. (1997) Proc. Natl. Acad. Sci. USA 94: 1029].
When the glucan elicitor was infiltrated into non-transformed Arabidopsis thaliana, no changes were observed on the leaf surface. This indicates that Arabidopsis thaliana cannot recognize the glucan elicitor. On the other hand, in the Arabidopsis thaliana plants (ISER) expressing a chimeric receptor, 3 clones out of the 5 tested exhibited effective browning reactions (resistance reaction)(Table 2).
Table 2. Hypersensitive Response Test on Transformed Arabidopsis thaliana (Ecotype Columbia) Fungal cell wall-derived 10 mM MgCl2 glucan elicitor Control 1 - -Control 2 - -Control 3 - -ISER 1 +++ -ISER 3 ++ -ISER 4 +++ -. no change; +: slight browning reactions;
++, medium browning reactions; +++, strong browning reactions From these results, it has been proved that recognition of a glucan elicitor which a host plant cannot recognize takes place by expressing in Arabidopsis thaliana a chimeric receptor composed of an ER from which an N-terminal region corresponding to about 1/3 of this protein has been deleted and an N-terminal domain of Arabidopsis thaliana-derived RPS2 protein, the domain being expected to contain a signal transduction domain. The present invention provides plants which can recognize the contact and/or invasion of plant pathogenic microorganisms such as fungi having glucan structures in their cell walls, etc.
By creating such plants which strongly induce the resistance reaction caused by a glucan elicitor, it will become possible to breed plants which exhibit strong resistance to plant pathogenic microorganisms such as fungi.
[EXAMPLE 5] Hypersensitive Response Test on Transformed Tobacco Induction of the hypersensitive response by a soybean elicitor was examined using leaves of tobacco transformant (IER) in which high expression of one of the created chimeric receptor genes, i.e. the gene comprising the nucleotide sequence shown in SEQ ID NO: 8 was confirmed, as well as tobacco transformant (LER; SEQ ID NO: 4) in which high expression of ER protein was confirmed and non-transformed tobacco (control).
Leaves of tobacco plants grown up in a greenhouse were cut off at the petiole and placed in light-transmittable plastic boxes. Silicone tube pieces 5 mm in diameter and 5 mm in height were put on the upper surface of leaves so that a liquid could be retained. A fungal cell wall-derived glucan elicitor (100 g/ml) dissolved in 10 mM MgCl2 solution, a chemically synthesized glucan elicitor (100 g/ml) dissolved in the same solution, and mM MgCl2 solution alone as a control were retained separately in the silicone tube pieces so that each solution was in contact with the leaf surface. The leaves were cultured under excessive moisture to prevent drying, under a 16 hr light/8 hr dark regime at 25 ~C for 7 days. The chemically synthesized glucan elicitor (a-D-glucohexaoside) [N. Hong and T. Ogawa (1990) Tetrahedron Lett. 31: 3179] was released from Prof. Ogawa (Institute of Physical and Chemical Research, and the University of Tokyo).
After the cultivation, the silicone tube pieces were removed.
Then, the accumulation of tobacco phytoalexins was observed on a UV illuminator (Funakoshi).
When the glucan elicitor was added to non-transformed tobacco, no changes were observed. This indicates that the non-transformed tobacco cannot recognize the glucan elicitor. In the ER-expressing tobacco plants, 1 clone out of the 5 tested only exhibited accumulation of very small amounts of fluorescent substances (phytoalexins). In the IER-expressing tobacco, 3 clones out of the 5 tested exhibited accumulation of remarkable amounts of fluorescent substances (phytoalexins) and 1 clone exhibited accumulation of very small amounts of fluorescent substances (phytoalexins)(Table 3).

Table 3. Hypersensitive Response Test on Transformed Tobacco Fungal cell Chemically wall-derived synthesized lOmM MgCl2 glucan elicitor glucan elicitor Control 1 - - -Control 2 - - -LER 3 + + -I E R 1 +++ ++ -I E R 2 +++ +++ -I E R 3 ++ + -IER 5 + + -- . no change; +: slight accumulation of fluorescent substances;
++: medium accumulation of fluorescent substances;
+++; large accumulation of fluorescent substances From these results, it has been proved that not only when a soybean-derived ER protein is expressed alone but also when a chimeric receptor composed of the ER protein and an N-terminal domain of Arabidopsis thaliana-derived RPS2 protein is expressed in a host plant other than soybean and Arabidopsis thaliana (tobacco), recognition of a glucan elicitor which the host plant cannot recognize takes place. Also, it has been shown that expressing a chimeric receptor is more advantageous for recognition of a glucan elicitor. Thus, it has been shown that even in a plant other than Arabidopsis thaliana, signal transduction is performed more efficiently when a gene coding for a chimeric receptor containing an N-terminal domain of RPS2 is expressed therein, as compared to the case where a gene coding for an ER protein alone is expressed. The present invention provides a DNA coding for a chimeric receptor which can recognize the contact and/or invasion of plant pathogenic microorganisms such as fungi having glucan structures in their cell walls, and plants which have such a chimeric receptor. As a result, resistance reactions deeply involved in disease resistance, such as phytoalexin induction, are induced more strongly than those cases where an ER protein alone is expressed.
By creating such plants which strongly induce the resistance reactions caused by glucan elicitors, it becomes possible to breed plants having strong resistance to plant pathogenic microorganisms such as fungi.
INDUSTRIAL APPLICABILITY
According to the present invention, a DNA coding for a chimeric protein (chimeric receptor) comprising a domain containing at least an elicitor binding site of an elicitor receptor and a domain containing a signal transduction motif of a plant-derived resistance gene; disease resistant plants into which the above DNA is transferred; and a method for creating such plants are provided.
The chimeric receptor of the invention can be used to elucidate the mechanism of resistance to fungi.
Further, the DNA of the invention comprising a nucleotide sequence coding for a chimeric receptor and fragments thereof are useful as materials for establishing techniques to breed fungus-resistant plants. That is, if the DNA of the invention or a fragment thereof is transferred into various plants and expressed therein, the plants' resistance to fungi can be enhanced.

SEQUENCE LISTING
SEQ ID N0: 1 SEQUENCE LENGTH: 667 SEQUENCE TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: protein SEQUENCE DESCRIPTION:
Val Asn Ile Gln Thr Asn Thr Ser Tyr Ile Phe Pro Gln Thr Gln Ser Thr Val Leu Pro Asp Pro Ser Lys Phe Phe Ser Ser Asn Leu Leu Ser Ser Pro Leu Pro Thr Asn Ser Phe Phe Gln Asn Phe Val Leu Lys Asn Gly Asp Gln Gln Glu Tyr Ile His Pro Tyr Leu Ile Lys Ser Ser Asn Ser Ser Leu Ser Leu Ser Tyr Pro Ser Arg Gln Ala Ser Ser Ala Val Ile Phe Gln Val Phe Asn Pro Asp Leu Thr Ile Ser Ala Pro Gln Gly Pro Lys Gln Gly Pro Pro Gly Lys His Leu Ile Ser Ser Tyr Ser Asp Leu Ser Val Thr Leu Asp Phe Pro Ser Ser Asn Leu Ser Phe Phe Leu Val Arg Gly Ser Pro Tyr Leu Thr Val Ser Val Thr Gln Pro Thr Pro Leu Ser Ile Thr Thr Ile His Ser Ile Leu Ser Phe Ser Ser Asn Asp Ser Asn Thr Lys Tyr Thr Phe Gln Phe Asn Asn Gly Gln Thr Trp Leu Leu Tyr Ala Thr Ser Pro Ile Lys Leu Asn His Thr Leu Ser Glu Ile Thr Ser Asn Ala Phe Ser Gly Ile Ile Arg Ile Ala Leu Leu Pro Asp Ser Asp Ser Lys His Glu Ala Val Leu Asp Lys Tyr Ser Ser Cys Tyr Pro Val Ser Gly Lys Ala Val Phe Arg Glu Pro Phe Cys Val Glu Tyr Asn Trp Glu Lys Lys Asp Ser Gly Asp Leu Leu Leu Leu Ala His Pro Leu His Val Gln Leu Leu Arg Asn Gly Asp Asn Asp Val Lys Ile Leu Glu Asp Leu Lys Tyr Lys Ser Ile Asp Gly Asp Leu Val Gly Val Val Gly Asp Ser Trp Val Leu Lys Thr Asp Pro Leu Phe Val Thr Trp His Ser Ile Lys Gly Ile Lys Glu Glu Ser His Asp Glu Ile Val Ser Ala Leu Ser Lys Asp Val Glu Ser Leu Asp Ser Ser Ser Ile Thr Thr Thr Glu Ser Tyr Phe Tyr Gly Lys Leu Ile Ala Arg Ala Ala Arg Leu Val Leu Ile Ala Glu Glu Leu Asn Tyr Pro Asp Val Ile Pro Lys Val Arg Asn Phe Leu Lys Glu Thr Ile Glu Pro Trp Leu Glu Gly Thr Phe Ser Gly Asn Gly Phe Leu His Asp Glu Lys Trp Gly Gly Ile Ile Thr Gln Lys Gly Ser Thr Asp Ala Gly Gly Asp Phe Gly Phe Gly Ile Tyr Asn Asp His His Tyr His Leu Gly Tyr Phe Ile Tyr GIy IIe Ala Val Leu Thr Lys Leu Asp Pro Ala Trp Gly Arg Lys Tyr Lys Pro Gln Ala Tyr Ser Ile Val Gln Asp Phe Leu Asn Leu Asp Thr Lys Leu Asn Ser Asn Tyr Thr Arg Leu Arg Cys Phe Asp Pro Tyr Val Leu His Ser Trp Ala Gly Gly Leu Thr Glu Phe Thr Asp Gly Arg Asn Gln Glu Ser Thr Ser Glu Ala Val Ser Ala Tyr Tyr Ser Ala Ala Leu Met Gly Leu Ala Tyr Gly Asp Ala Pro Leu Val Ala Leu Gly Ser Thr Leu Thr Ala Leu Glu Ile Glu Gly Thr Lys Met Trp Trp His Val Lys Glu Gly Gly Thr Leu Tyr Glu Lys Glu Phe Thr Gln Glu Asn Arg Val Met Gly Val Leu Trp Ser Asn Lys Arg Asp Thr Gly Leu Trp Phe Ala Pro Ala Glu Trp Lys Glu Cys Arg Leu Gly Ile Gln Leu Leu Pro Leu Ala Pro Ile Ser Glu Ala Ile Phe Ser Asn Val Asp Phe Val Lys Glu Leu Val Glu Trp Thr Leu Pro Ala Leu Asp Arg Glu Gly Gly Val Gly Glu Gly Trp Lys Gly Phe Val Tyr Ala Leu Glu Gly Val Tyr Asp Asn Glu Ser Ala Leu Gln Lys Ile Arg Asn Leu Lys Gly Phe Asp Gly Gly Asn Ser Leu Thr Asn Leu Leu Trp Trp Ile His Ser Arg Ser Asp Glu SEQ ID N0: 2 SEQUENCE LENGTH: 2240 SEQUENCE TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear MOLECULE TYPE: cDNA to mRNA
FEATURE

NAME/KEY: CD S

LOCATION: 29 ..2029 SEQUENCE
DESCRIPTION:

C CA ATC ACC

Val AsnIle GlnThr AsnThr Ser TTC

Tyr Ile Pro GlnThr GlnSerThr ValLeu ProAsp ProSer Lys Phe TCC

Phe Phe Ser AsnLeu LeuSerSer ProLeu ProThr AsnSer Phe Ser AAC

Phe Gln Phe ValLeu LysAsnGly AspGln GlnGlu TyrIle His Asn CTC

Pro Tyr Ile LysSer SerAsnSer SerLeu SerLeu SerTyr Pro Leu SerArg GlnAla SerSer Ala ValIle PheGln ValPhe AsnPro Asp LeuThr IleSer AlaPro Gln GlyPro LysGln GlyPro ProGly Lys HisLeu IleSer SerTyr Ser AspLeu SerVal ThrLeu AspPhe Pro SerSer AsnLeu SerPhe Phe LeuVal ArgGly SerPro TyrLeu Thr ValSer ValThr GlnPro Thr ProLeu SerIle ThrThr IleHis Ser IleLeu SerPhe SerSer Asn AspSer AsnThr LysTyr ThrPhe Gln PheAsn AsnGly GlnThr Trp LeuLeu TyrAla ThrSer ProIle Lys LeuAsn HisThr LeuSer Glu IleThr SerAsn AlaPhe SerGly Ile IleArg IleAla LeuLeu Pro AspSer AspSer LysHis GluAla Val LeuAsp Lys TyrSer SerCys TyrPro ValSer GlyLys AlaVal Phe ArgGlu Pro PheCys ValGlu TyrAsn TrpGlu LysLys AspSer Gly AspLeu Leu LeuLeu AlaHis ProLeu HisVal GlnLeu LeuArg Asn GlyAsp Asn AspVal LysIle LeuGlu AspLeu LysTyr LysSer Ile AspGly Asp LeuVal GlyVal ValGly AspSer TrpVal LeuLys Thr AspPro Leu PheVal ThrTrp HisSer IleLys GlyIle LysGlu Glu SerHis Asp GluIle ValSer AlaLeu SerLys AspVal GluSer Leu AspSer Ser SerIle ThrThr ThrGlu SerTyr PheTyr GlyLys Leu IleAla Arg AlaAla ArgLeu ValLeu IleAla GluGlu LeuAsn Tyr ProAsp Val IlePro LysVal ArgAsn PheLeu LysGlu ThrIle Glu ProTrp Leu GluGly ThrPhe SerGly AsnGly PheLeu HisAsp Glu LysTrp Gly GlyIle IleThr GlnLys GlySer ThrAsp AlaGly Gly AspPhe Gly PheGly IleTyr AsnAsp HisHis TyrHis LeuGly Tyr PheIle Tyr GlyIle AlaVal LeuThr LysLeu AspPro AlaTrp Gly ArgLys Tyr LysPro GlnAla TyrSer IleVal GlnAsp PheLeu Asn LeuAsp Thr LysLeu AsnSer AsnTyr ThrArg LeuArg CysPhe Asp ProTyr Val LeuHis SerTrp AlaGly GlyLeu ThrGlu PheThr Asp GlyArg Asn GlnGlu SerThr SerGlu AlaVal SerAla TyrTyr Ser AlaAla Leu MetGly LeuAla TyrGly AspAla ProLeu ValAla Leu GlySer Thr LeuThr AlaLeu GluIle GluGly ThrLys MetTrp Trp His Val Lys Glu Gly Gly Thr Leu Tyr Glu Lys Glu Phe Thr Gln Glu Asn Arg Val Met Gly Val Leu Trp Ser Asn Lys Arg Asp Thr Gly Leu Trp Phe Ala Pro Ala Glu Trp Lys Glu Cys Arg Leu Gly Ile Gln Leu Leu Pro Leu Ala Pro Ile Ser Glu Ala Ile Phe Ser Asn Val Asp Phe Val Lys Glu Leu Val Glu Trp Thr Leu Pro Ala Leu Asp Arg Glu Gly Gly Val Gly Glu Gly Trp Lys Gly Phe Val Tyr Ala Leu Glu Gly Val Tyr Asp Asn Glu Ser Ala Leu Gln Lys Ile Arg Asn Leu Lys Gly Phe Asp Gly Gly Asn Ser Leu Thr Asn Leu Leu Trp Trp Ile His Ser Arg Ser Asp Glu SEQ ID NO: 3 SEQUENCE LENGTH: 667 SEQUENCE TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: protein SEQUENCE DESCRIPTION:
Val Asn Ile Gln Thr Asn Thr Ser Tyr Ile Phe Pro Gln Thr Gln Ser Thr Val Leu Pro Asp Pro Ser Lys Phe Phe Ser Ser Asn Leu Leu Ser Ser Pro Leu Pro Thr Asn Ser Phe Phe Gln Asn Phe Val Leu Lys Asn Gly Asp Gln Gln Glu Tyr Ile His Pro Tyr Leu Ile Lys Ser Ser Asn Ser Ser Leu Ser Leu Ser Tyr Pro Ser Arg Gln Ala Ser Ser Ala Val Ile Phe Gln Val Phe Asn Pro Asp Leu Thr Ile Ser Ala Pro Gln Gly Pro Lys Gln Gly Pro Pro Gly Lys His Leu Ile Ser Ser Tyr Ser Asp Leu Ser Val Thr Leu Asp Phe Pro Ser Ser Asn Leu Ser Phe Phe Leu Val Arg Gly Ser Pro Tyr Leu Thr Val Ser Val Thr Gln Pro Thr Pro Leu Ser Ile Thr Thr Ile His Ser Ile Leu Ser Phe Ser Ser Asn Asp Ser Asn Thr Lys Tyr Thr Phe Gln Phe Asn Asn Gly Gln Thr Trp Leu Leu Tyr Ala Thr Ser Pro Ile Lys Leu Asn His Thr Leu Ser Glu Ile Thr Ser Asn Ala Phe Ser Gly Ile Ile Arg Ile Ala Leu Leu Pro Asp Ser Asp Ser Lys His Glu Ala Val Leu Asp Lys Tyr Ser Ser Cys Tyr Pro Val Ser Gly Lys Ala Val Phe Arg Glu Pro Phe Cys Val Glu Tyr Asn Trp Glu Lys Lys Asp Ser Gly Asp Leu Leu Leu Leu Ala His Pro Leu His Val Gln Leu Leu Arg Asn Gly Asp Asn Asp Val Lys Ile Leu Glu Asp Leu Lys Tyr Lys Ser Ile Asp Gly Asp Leu Val Gly Val Val Gly Asp Ser Trp Val Leu Lys Thr Asp Pro Leu Phe Val Thr Trp His Ser Ile Lys Gly Ile Lys Glu Glu Ser His Asp Glu Ile Val Ser Ala Leu Ser Lys Asp Val Glu Ser Leu Asp Ser Ser Ser Ile Thr Thr Thr Glu Ser Tyr Phe Tyr Gly Lys Leu Ile Ala Arg Ala Ala Arg Leu Val Leu Ile Ala Glu Glu Leu Asn Tyr Pro Asp Val Ile Pro Lys Val Arg Asn Phe Leu Lys Glu Thr Ile Glu Pro Trp Leu Glu Gly Thr Phe Ser Gly Asn Gly Phe Leu His Asp Glu Lys Trp Gly Gly Ile Ile Thr Gln Lys Gly Ser Thr Asp Ala Gly Gly Asp Phe Gly Phe Gly Ile Tyr Asn Asp His His Tyr His Leu Gly Tyr Phe Ile Tyr Gly Ile Ala Val Leu Thr Lys Leu Asp Pro Ala Trp Gly Arg Lys Tyr Lys Pro Gln Ala Tyr Ser Ile Val Gln Asp Phe Leu Asn Leu Asp Thr Lys Leu Asn Ser Asn Tyr Thr Arg Leu Arg Cys Phe Asp Pro Tyr Val Leu His Ser Trp Ala Gly Gly Leu Thr Glu Phe Thr Asp Gly Arg Asn Gln Glu Ser Thr Ser Glu Ala Val Ser Ala Tyr Tyr Ser Ala Ala Leu Met Gly Leu Ala Tyr Gly Asp Ala Pro Leu Val Ala Leu Gly Ser Thr Leu Thr Ala Leu Glu Ile Glu Gly Thr Lys Met Trp Trp His Val Lys Glu Gly Gly Thr Leu Tyr Glu Lys Glu Phe Thr Gln Glu Asn Arg Val Met Gly Val Leu Trp Ser Asn Lys Arg Asp Thr Gly Leu Trp Phe Ala Pro Ala Glu Trp Lys Glu Cys Arg Leu Gly Ile Gln Leu Leu Pro Leu Ala Pro Ile Ser Glu 580 585 ~ 590 Ala Ile Phe Ser Asn Val Asp Phe Val Lys Glu Leu Val Glu Trp Thr Leu Pro Ala Leu Asp Arg Glu Gly Gly Val Gly Glu Gly Trp Lys Gly Phe Val Tyr Ala Leu Glu Gly Val Tyr Asp Asn Glu Ser Ala Leu Gln Lys Ile Arg Asn Leu Lys Gly Phe Asp Gly Gly Asn Ser Leu Thr Asn Leu Leu Trp Trp Ile His Ser Arg Ser Asp Glu SEQ ID N0: 4 SEQUENCE LENGTH: 2291 SEQUENCE TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear MOLECULE TYPE: cDNA
to mRNA

FEATURE

NAME/KEY: CDS

LOCATION: 80..2080 SEQUENCE DESCRIPTION:

TC C

GTT

Val Asn IleGln ThrAsn ThrSer Tyr IlePhe ACT

Pro Gln Thr Gln Ser Val LeuPro AspPro SerLys Phe PheSer Thr AGT

Ser Asn Leu Leu Ser Pro LeuPro ThrAsn SerPhe Phe GlnAsn Ser GGT

Phe Val Leu Lys Asn Asp GlnGln GluTyr IleHis Pro TyrLeu Gly IleLys SerSer AsnSer SerLeu SerLeu SerTyr Pro SerArg Gln AlaSer SerAla ValIle PheGln ValPhe AsnPro Asp LeuThr Ile SerAla ProGln GlyPro LysGln GlyPro ProGly Lys HisLeu Ile SerSer TyrSer AspLeu SerVal ThrLeu AspPhe Pro SerSer Asn LeuSer PhePhe LeuVal ArgGly SerPro TyrLeu Thr ValSer Val ThrGln ProThr ProLeu SerIle ThrThr IleHis Ser IleLeu Ser PheSer SerAsn AspSer AsnThr LysTyr ThrPhe Gln PheAsn Asn GlyGln ThrTrp LeuLeu TyrAla ThrSer ProIle Lys LeuAsn His ThrLeu SerGlu IleThr SerAsn AlaPhe SerGly Ile IleArg Ile AlaLeu LeuPro Asp SerAsp SerLys HisGlu AlaVal LeuAsp Lys TyrSer SerCys Tyr ProVal SerGly LysAla ValPhe ArgGlu Pro PheCys ValGlu Tyr AsnTrp GluLys LysAsp SerGly AspLeu Leu LeuLeu AlaHis Pro LeuHis ValGln LeuLeu ArgAsn GlyAsp Asn AspVal LysIle Leu GluAsp LeuLys TyrLys SerIle AspGly Asp LeuVal GlyVal Val GlyAsp SerTrp ValLeu LysThr AspPro Leu PheVal ThrTrp His SerIle LysGly IleLys GluGlu SerHis Asp GluIle ValSer Ala LeuSer LysAsp ValGlu SerLeu AspSer Ser SerIle ThrThr Thr GluSer TyrPhe TyrGly LysLeu IleAla Arg AlaAla ArgLeu Val LeuIle AlaGlu GluLeu AsnTyr ProAsp Val IlePro LysVal Arg AsnPhe LeuLys GluThr IleGlu ProTrp Leu GluGly ThrPhe Ser GlyAsn GlyPhe LeuHis AspGlu LysTrp Gly GlyIle IleThr Gln LysGly SerThr AspAla GlyGly AspPhe Gly PheGly IleTyr Asn AspHis HisTyr HisLeu GlyTyr PheIle Tyr GlyIle AlaVal Leu ThrLys LeuAsp ProAla TrpGly ArgLys Tyr LysPro GlnAla Tyr SerIle ValGln AspPhe LeuAsn LeuAsp Thr LysLeu AsnSer Asn TyrThr ArgLeu ArgCys PheAsp ProTyr Val LeuHis SerTrp Ala GlyGly LeuThr GluPhe ThrAsp GlyArg Asn GlnGlu SerThr Ser GluAla ValSer AlaTyr TyrSer AlaAla Leu MetGly LeuAla Tyr GlyAsp AlaPro LeuVal AlaLeu GlySer Thr LeuThr AlaLeu Glu IleGlu GlyThr LysMet TrpTrp HisVal Lys GluGly GlyThr Leu TyrGlu LysGlu PheThr GlnGlu AsnArg Val MetGly ValLeu Trp SerAsn LysArg AspThr GlyLeu TrpPhe Ala ProAla GluTrp Lys GluCys ArgLeu GlyIle GlnLeu LeuPro Leu AlaPro IleSer Glu AlaIle PheSer AsnVal AspPhe ValLys Glu LeuVal GluTrp Thr LeuPro AlaLeu AspArg GluGly GlyVal Gly GluGly TrpLys Gly PheVal TyrAla LeuGlu GlyVal TyrAsp Asn GluSer AlaLeu Gln LysIle ArgAsn LeuLys GlyPhe AspGly Gly AsnSer LeuThr Asn LeuLeu TrpTrp IleHis SerArg SerAsp Glu TAGAATGGAT CACTGCTGAT TTGGTTATTA

TTGGTTATTG
AACATGACAA

SEQ ID N0: 5 SEQUENCE LENGTH: 1054 SEQUENCE TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: protein SEQUENCE DESCRIPTION:
Met Asp Phe Ile Ser Ser Leu Ile Val Gly Cys Ala Gln Val Leu Cys Glu Ser Met Asn Met Ala Glu Arg Arg Gly His Lys Thr Asp Leu Arg Gln Ala Ile Thr Asp Leu Glu Thr Ala Ile Gly Asp Leu Lys Ala Ile Arg Asp Asp Leu Thr Leu Arg Ile Gln Gln Asp Gly Leu Glu Gly Arg Ser Cys Ser Asn Arg Ala Arg Glu Trp Leu Ser Ala Val Gln Val Thr Glu Thr Lys Thr Ala Leu Leu Leu Val Arg Phe Arg Arg Arg Glu Gln Arg Thr Arg Met Arg Arg Arg Tyr Leu Ser Cys Phe Gly Cys Ala Asp Tyr Lys Leu Cys Lys Lys Val Ser Ala Ile Leu Lys Ser Ile Gly Glu Leu Arg Glu Arg Ser Glu Ala Ile Lys Thr Asp Gly Gly Ser Ile Gln Val Thr Cys Arg Glu Ile Pro Ile Lys Ser Val Val Gly Asn Thr Thr Met Met Glu Gln Val Leu Glu Phe Leu Ser Glu Glu Glu Glu Arg Gly Ile Ile Gly Val Tyr Gly Pro Gly Gly Val Gly Lys Thr Thr Leu Met Gln Ser Ile Asn Asn Glu Leu Ile Thr Lys Gly His Gln Tyr Asp Val Leu Ile Trp Val Gln Met Ser Arg Glu Phe Gly Glu Cys Thr Ile Gln Gln Ala Val Gly Ala Arg Leu Gly Leu Ser Trp Asp Glu Lys Glu Thr Gly Glu Asn Arg Ala Leu Lys Ile Tyr Arg Ala Leu Arg Gln Lys Arg Phe Leu Leu Leu Leu Asp Asp Val Trp Glu Glu Ile Asp Leu Glu Lys Thr Gly Val Pro Arg Pro Asp Arg Glu Asn Lys Cys Lys Val Met Phe Thr Thr Arg Ser Ile Ala Leu Cys Asn Asn Met Gly Ala Glu Tyr Lys Leu Arg Val Glu Phe Leu Glu Lys Lys His Ala Trp Glu Leu Phe Cys Ser Lys Val Trp Arg Lys Asp Leu Leu Glu Ser Ser Ser Ile Arg Arg Leu Ala Glu Ile Ile Val Ser Lys Cys Gly Gly Leu Pro Leu Ala Leu Ile Thr Leu Gly Gly Ala Met Ala Gly Leu Gln Glu Phe Asp Pro Gly Ser Pro Gly Leu Gln Glu Phe Ala Ala Ala Arg Ile Lys Pro Pro Ser Ala Thr Met Val Asn Ile Gln Thr Asn Thr Ser Tyr Ile Phe Pro Gln Thr Gln Ser Thr Val Leu Pro Asp Pro Ser Lys Phe Phe Ser Ser Asn Leu Leu Ser Ser Pro Leu Pro Thr Asn Ser Phe Phe Gln Asn Phe Val Leu Lys Asn Gly Asp Gln Gln Glu Tyr Ile His Pro Tyr Leu Ile Lys Ser Ser Asn Ser Ser Leu Ser Leu Ser Tyr Pro Ser Arg Gln Ala Ser Ser Ala Val Ile Phe Gln Val Phe Asn Pro Asp Leu Thr Ile Ser Ala Pro Gln Gly Pro Lys Gln Gly Pro Pro Gly Lys His Leu Ile Ser Ser Tyr Ser Asp Leu Ser Val Thr Leu. Asp Phe Pro Ser Ser Asn Leu Ser Phe Phe Leu Val Arg Gly Ser Pro Tyr Leu Thr Val Ser Val Thr Gln Pro Thr Pro Leu Ser Ile Thr Thr Ile His Ser Ile Leu Ser Phe Ser Ser Asn Asp Ser Asn Thr Lys Tyr Thr Phe Gln Phe Asn Asn Gly Gln Thr Trp Leu Leu Tyr Ala Thr Ser Pro Ile Lys Leu Asn His Thr Leu Ser Glu Ile Thr Ser Asn Ala Phe Ser Gly Ile Ile Arg Ile Ala Leu Leu Pro Asp Ser Asp Ser Lys His Glu Ala Val Leu Asp Lys Tyr Ser Ser Cys Tyr Pro Val Ser Gly Lys Ala Val Phe Arg Glu Pro Phe Cys Val Glu Tyr Asn Trp Glu Lys Lys Asp Ser Gly Asp Leu Leu Leu Leu Ala His Pro Leu His Val Gln Leu Leu Arg Asn Gly Asp Asn Asp Val Lys Ile Leu Glu Asp Leu Lys Tyr Lys Ser Ile Asp Gly Asp Leu Val Gly Val Val Gly Asp Ser Trp Val Leu Lys Thr Asp Pro Leu Phe Val Thr Trp His Ser Ile Lys Gly Ile Lys Glu Glu Ser His Asp Glu Ile Val Ser Ala Leu Ser Lys Asp Val Glu Ser Leu Asp Ser Ser Ser Ile Thr Thr Thr Glu Ser Tyr Phe Tyr Gly Lys Leu Ile Ala Arg Ala Ala Arg Leu Val Leu Ile Ala Glu Glu Leu Asn Tyr Pro Asp Val Ile Pro Lys Val Arg Asn Phe Leu Lys Glu Thr Ile Glu Pro Trp Leu Glu Gly Thr Phe Ser Gly Asn Gly Phe Leu His Asp Glu Lys Trp Gly Gly Ile Ile Thr Gln Lys Gly Ser Thr Asp Ala Gly Gly Asp Phe Gly Phe Gly Ile Tyr Asn Asp His His Tyr His Leu Gly Tyr Phe Ile Tyr Gly Ile Ala Val Leu Thr Lys Leu Asp Pro Ala Trp Gly Arg Lys Tyr Lys Pro Gln Ala Tyr Ser Ile Val Gln Asp Phe Leu Asn Leu Asp Thr Lys Leu Asn Ser Asn Tyr Thr Arg Leu Arg Cys Phe Asp Pro Tyr Val Leu His Ser Trp Ala Gly Gly Leu Thr Glu Phe Thr Asp Gly Arg Asn Gln Glu Ser Thr Ser Glu Ala Val Ser Ala Tyr Tyr Ser Ala Ala Leu Met Gly Leu Ala Tyr Gly Asp Ala Pro Leu Val Ala Leu Gly Ser Thr Leu Thr Ala Leu Glu Ile Glu Gly Thr Lys Met Trp Trp His Val Lys Glu Gly Gly Thr Leu Tyr Glu Lys Glu Phe Thr Gln Glu Asn Arg Val Met Gly Val Leu Trp Ser Asn Lys Arg Asp Thr Gly Leu Trp Phe Ala Pro Ala Glu Trp Lys Glu Cys Arg Leu Gly Ile Gln Leu Leu Pro Leu Ala Pro Ile Ser Glu Ala Ile Phe Ser Asn Val Asp Phe Val Lys Glu Leu Val Glu Trp Thr Leu Pro Ala Leu Asp Arg Glu Gly Gly Val Gly Glu Gly Trp Lys Gly Phe Val Tyr Ala Leu Glu Gly Val Tyr Asp Asn Glu Ser Ala Leu Gln Lys Ile Arg Asn Leu Lys Gly Phe Asp Gly Gly Asn Ser Leu Thr Asn Leu Leu Trp Trp Ile His Ser Arg Ser Asp Glu SEQ ID NO: 6 SEQUENCE LENGTH: 3405 SEQUENCE TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear MOLECULE cDNA
TYPE: to mRNA

FEATURE

NAME/KEY: CD S

LOCATION: 33 ..3194 SEQUENCE ION:
DESCRIPT

GAAAGAGCGA
GAAATCATCG
AA

Met AspPhe IleSer SerLeu GGC

IleVal Cys AlaGln ValLeuCys Glu SerMet AsnMet AlaGlu Gly GGA

ArgArg His LysThr AspLeuArg Gln AlaIle ThrAsp LeuGlu Gly ATC

ThrAla Gly AspLeu LysAlaIle Arg AspAsp LeuThr LeuArg Ile CAA

IleGln Asp GlyLeu GluGlyArg Ser CysSer AsnArg AlaArg Gln CTT

GluTrp Ser AlaVal GlnValThr Glu ThrLys ThrAla LeuLeu Leu AGG

LeuVal Phe ArgArg ArgGluGln Arg ThrArg MetArg ArgArg Arg TyrLeu SerCys PheGly Cys AlaAsp TyrLys LeuCys LysLys Val SerAla IleLeu LysSer Ile GlyGlu LeuArg GluArg SerGlu Ala ATCAAA ACAGAT GGCGGG TCA ATTCAA GTAACT TGTAGA GAGATA CCC 4$5 IleLys ThrAsp GlyGly Ser IleGln ValThr CysArg GluIle Pro IleLys SerVal ValGly Asn ThrThr MetMet GluGln ValLeu Glu PheLeu SerGlu GluGlu Glu ArgGly IleIle GlyVal TyrGly Pro GlyGly ValGly LysThr Thr LeuMet GlnSer IleAsn AsnGlu Leu IleThr LysGly HisGln Tyr AspVal LeuIle TrpVal GlnMet Ser ArgGlu PheGly GluCys Thr IleGln GlnAla ValGly AlaArg Leu GlyLeu SerTrp AspGlu Lys GluThr GlyGlu AsnArg AlaLeu Lys IleTyr ArgAla LeuArg Gln LysArg PheLeu LeuLeu LeuAsp Asp ValTrp GluGlu Ile AspLeu GluLys ThrGly ValPro ArgPro Asp ArgGlu AsnLys Cys LysVal MetPhe ThrThr ArgSer IleAla Leu CysAsn AsnMet Gly AlaGlu TyrLys LeuArg ValGlu PheLeu Glu LysLys HisAla Trp GluLeu PheCys SerLys ValTrp ArgLys Asp LeuLeu GluSer Ser SerIle ArgArg LeuAla GluIle IleVal Ser LysCys GlyGly Leu ProLeu AlaLeu IleThr LeuGly GlyAla Met AlaGly LeuGln Glu PheAsp ProGly SerPro GlyLeu GlnGlu Phe AlaAla AlaArg Ile LysPro ProSer AlaThr MetVal AsnIle Gln ThrAsn ThrSer Tyr IlePhe ProGln ThrGln SerThr ValLeu Pro Asp ProSer LysPhe PheSer SerAsn LeuLeu SerSer ProLeu Pro Thr AsnSer PhePhe GlnAsn PheVal LeuLys AsnGly AspGln Gln Glu TyrIle HisPro TyrLeu IleLys SerSer AsnSer SerLeu Ser Leu SerTyr ProSer ArgGln AlaSer SerAla ValIle PheGln Val Phe AsnPro AspLeu ThrIle SerAla ProGln GlyPro LysGln Gly Pro ProGly LysHis LeuIle SerSer TyrSer AspLeu SerVal Thr Leu AspPhe ProSer SerAsn LeuSer PhePhe LeuVal ArgGly Ser Pro TyrLeu ThrVal SerVal ThrGln ProThr ProLeu SerIle Thr Thr IleHis SerIle LeuSer PheSer SerAsn AspSer AsnThr Lys Tyr ThrPhe GlnPhe AsnAsn GlyGln ThrTrp LeuLeu TyrAla Thr SerPro Ile LysLeu AsnHis ThrLeu SerGlu IleThr SerAsn Ala PheSer Gly IleIle ArgIle AlaLeu LeuPro AspSer AspSer Lys HisGlu Ala ValLeu AspLys TyrSer SerCys TyrPro ValSer Gly LysAla Val PheArg GluPro PheCys ValGlu TyrAsn TrpGlu Lys LysAsp Ser GlyAsp LeuLeu LeuLeu AlaHis ProLeu HisVal Gln LeuLeu Arg AsnGly AspAsn AspVal LysIle LeuGlu AspLeu Lys TyrLys Ser IleAsp GlyAsp LeuVal GlyVal ValGly AspSer Trp ValLeu Lys ThrAsp ProLeu PheVal ThrTrp HisSer IleLys Gly IleLys Glu GluSer HisAsp GluIle ValSer AlaLeu SerLys Asp ValGlu Ser LeuAsp SerSer SerIle ThrThr ThrGlu SerTyr Phe TyrGly LysLeu IleAla Arg AlaAla ArgLeu ValLeu IleAla Glu GluLeu AsnTyr ProAsp Val IlePro LysVal ArgAsn PheLeu Lys GluThr IleGlu ProTrp Leu GluGly ThrPhe SerGly AsnGly Phe LeuHis AspGlu LysTrp Gly GlyIle IleThr GlnLys GlySer Thr AspAla GlyGly AspPhe Gly PheGly IleTyr AsnAsp HisHis Tyr HisLeu GlyTyr PheIle Tyr GlyIle AlaVal LeuThr LysLeu Asp ProAla TrpGly ArgLys Tyr LysPro GlnAla TyrSer IleVal Gln AspPhe LeuAsn LeuAsp Thr LysLeu AsnSer AsnTyr ThrArg Leu ArgCys PheAsp ProTyr Val LeuHis SerTrp AlaGly GlyLeu Thr Glu Phe Thr Asp Gly Arg Asn Gln Glu Ser Thr Ser Glu Ala Val Ser Ala Tyr Tyr Ser Ala Ala Leu Met Gly Leu Ala Tyr Gly Asp Ala Pro Leu Val Ala Leu Gly Ser Thr Leu Thr Ala Leu Glu Ile Glu Gly Thr Lys Met Trp Trp His Val Lys Glu Gly Gly Thr Leu Tyr Glu Lys Glu Phe Thr Gln Glu Asn Arg Val Met Gly Val Leu Trp Ser Asn Lys Arg Asp Thr Gly Leu Trp Phe Ala Pro Ala Glu Trp Lys Glu Cys Arg Leu Gly Ile Gln Leu Leu Pro Leu Ala Pro Ile Ser Glu Ala Ile Phe Ser Asn Val Asp Phe Val Lys Glu Leu Val Glu Trp Thr Leu Pro Ala Leu Asp Arg Glu Gly Gly Val Gly Glu Gly Trp Lys Gly Phe Val Tyr Ala Leu Glu Gly Val Tyr Asp Asn Glu Ser Ala Leu Gln Lys Ile Arg Asn Leu Lys Gly Phe Asp Gly Gly Asn Ser Leu Thr Asn Leu Leu Trp Trp Ile His Ser Arg Ser Asp Glu A

SEQ ID N0: 7 SEQUENCE LENGTH: 877 SEQUENCE TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: protein SEQUENCE DESCRIPTION:
Val Asn Ile Gln Thr Asn Thr Ser Tyr Ile Phe Pro Gln Thr Gln Ser Thr Val Leu Pro Asp Pro Ser Lys Phe Phe Ser Ser Asn Leu Leu Ser Ser Pro Leu Pro Thr Asn Ser Phe Phe Gln Asn Phe Val Leu Lys Asn Gly Asp Gln Gln Glu Tyr Ile His Pro Tyr Leu Ile Lys Ser Ser Asn Ser Ser Leu Ser Leu Ser Tyr..Pro Ser Arg Gln Ala Ser Ser Ala Val 65 70 75 gp Ile Phe Gln Val Phe Asn Pro Asp Leu Thr Ile Ser Ala Pro Gln Gly 85 90 g5 Pro Lys Gln Gly Pro Pro Gly Lys His Leu Ile Ser Ser Tyr Ser Asp Leu Ser Val Thr Leu Asp Phe Pro Ser Ser Asn Leu Ser Phe Phe Leu Val Arg Gly Ser Pro Tyr Leu Thr Val Ser Val Thr Gln Pro Thr Pro Leu Ser Ile Thr Thr Ile His Ser Ile Leu Ser Phe Ser Ser Asn Asp Ser Asn Thr Lys Tyr Thr Phe Gln Phe Asn Asn Gly Gln Thr Trp Leu Leu Tyr Ala Thr Ser Pro Ile Lys Leu Asn His Thr Leu Ser Glu Ile Thr Ser Asn Ala Phe Ser Gly Ile Ile Arg Ile Ala Leu Leu Pro Asp Ser Asp Ser Lys His Glu Ala Val Leu Asp Lys Tyr Ser Ser Cys Tyr Pro Val Ser Gly Lys Ala Val Phe Arg Glu Pro Phe Cys Val Glu Tyr Asn Trp Glu Lys Lys Asp Ser Gly Asp Leu Leu Leu Leu Ala His Pro Leu His Val Gln Leu Leu Arg Asn Gly Asp Asn Asp Val Lys Ile Leu Glu Asp Leu Lys Tyr Lys Ser Ile Asp Gly Asp Leu Val Gly Val Val Gly Asp Ser Trp Val Leu Lys Thr Asp Pro Leu Phe Val Thr Trp His Ser Ile Lys Gly Ile Lys Glu Glu Ser His Asp Glu Ile Val Ser Ala Leu Ser Lys Asp Val Glu Ser Leu Asp Ser Ser Ser Ile Thr Thr Thr Glu Ser Tyr Phe Tyr Gly Lys Leu Ile Ala Arg Ala Ala Arg Leu Val Leu Ile Ala Glu Glu Leu Asn Tyr Pro Asp Val Ile Pro Lys Val Arg Asn Phe Leu Lys Glu Thr Ile Glu Pro Trp Leu Glu Gly Thr Phe Ser Gly Asn Gly Phe Leu His Asp Glu Lys Trp Gly Gly Ile Ile Thr Gln Lys Gly Ser Thr Asp Ala Gly Gly Asp Phe Gly Phe Gly Ile Tyr Asn Asp His His Tyr His Leu Gly Tyr Phe Ile Tyr Gly Ile Ala Val Leu Thr Lys Leu Asp Pro Ala Trp Gly Arg Lys Tyr Lys Pro Gln Ala Tyr Ser Ile Val Gln Asp Phe Leu Asn Leu Asp Thr Lys Leu Asn Ser Asn Tyr Thr Arg Leu Arg Cys Phe Asp Pro Tyr Val Leu His Ser Trp Ala Gly Gly Leu Thr Glu Phe Thr Asp Gly Arg Asn Gln Glu Ser Thr Ser Glu Ala Val Ser Ala Tyr Tyr Ser Ala Ala Leu Met Gly Leu Ala Tyr Gly Asp Ala Pro Leu Val Ala Leu Gly Ser Thr Leu Thr Ala Leu Glu Ile Glu Gly Thr Lys Met Trp Trp His Ala Met Asp Phe Ile Ser Ser Leu Ile Val Gly Cys Ala Gln Val Leu Cys Glu Ser Met Asn Met Ala Glu Arg Arg Gly His Lys Thr Asp Leu Arg Gln Ala Ile Thr Asp Leu Glu Thr Ala Ile Gly Asp Leu Lys Ala Ile Arg Asp Asp Leu Thr Leu Arg Ile Gln Gln Asp Gly Leu Glu Gly Arg Ser Cys Ser Asn Arg Ala Arg Glu Trp Leu Ser Ala Val Gln Val Thr Glu Thr Lys Thr Ala Leu Leu Leu Val Arg Phe Arg Arg Arg Glu Gln Arg Thr Arg Met Arg Arg Arg Tyr Leu Ser Cys Phe Gly Cys Ala Asp Tyr Lys Leu Cys Lys Lys Val Ser Ala Ile Leu Lys Ser Ile Gly Glu Leu Arg Glu Arg Ser Glu Ala Ile Lys Thr Asp Gly Gly Ser Ile Gln Val Thr Cys Arg Glu Ile Pro Ile Lys Ser Val Val Gly Asn Thr Thr Met Met Glu Gln Val Leu Glu Phe Leu Ser Glu Glu Glu Glu Arg Gly Ile Ile Gly Val Tyr Gly Pro Gly Gly Val Gly Lys Thr Thr Leu Met Gln Ser Ile Asn Asn Glu Leu Ile Thr Lys Gly His Gln Tyr Asp Val Leu Ile Trp Val Gln Met Ser Arg Glu Phe Gly Glu Cys Thr Ile Gln Gln Ala Val Gly Ala Arg Leu Gly Leu Ser Trp Asp Glu Lys Glu Thr Gly Glu Asn Arg Ala Leu Lys Ile Tyr Arg Ala Leu Arg Gln Lys Arg Phe Leu Leu Leu Leu Asp Asp Val Trp Glu Glu Ile Asp Leu Glu Lys Thr Gly Val Pro Arg Pro Asp Arg Glu Asn Lys Cys Lys Val Met Phe Thr Thr Arg Ser Ile Ala Leu Cys Asn Asn Met Gly Ala Glu Tyr Lys Leu Arg Val Glu Phe Leu Glu Lys Lys His Ala Trp Glu Leu Phe Cys Ser Lys Val Trp Arg Lys Asp Leu Leu Glu Ser Ser Ser Ile Arg Arg Leu Ala Glu SEQ ID NO: 8 SEQUENCE LENGTH: 2666 SEQUENCE TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear MOLECULE TYPE: cDNA to mRNA
FEATURE
NAME/KEY: CDS
LOCATION: 29..2659 SEQUENCE DESCRIPTION:

Val Asn Ile Gln Thr Asn Thr Ser Tyr Ile Phe Pro Gln Thr Gln Ser Thr Val Leu Pro Asp Pro Ser Lys PhePhe Ser SerAsn LeuLeu SerSer ProLeu ProThr AsnSer Phe PheGln Asn PheVal LeuLys AsnGly AspGln GlnGlu TyrIle His ProTyr Leu IleLys SerSer AsnSer SerLeu SerLeu SerTyr Pro SerArg Gln AlaSer SerAla ValIle PheGln ValPhe AsnPro Asp LeuThr Ile SerAla ProGln GlyPro LysGln GlyPro ProGly Lys HisLeu Ile SerSer TyrSer AspLeu SerVal ThrLeu AspPhe Pro SerSer Asn LeuSer PhePhe LeuVal ArgGly SerPro TyrLeu Thr ValSer Val ThrGln ProThr ProLeu SerIle ThrThr IleHis Ser IleLeu Ser PheSer SerAsn AspSer AsnThr LysTyr ThrPhe Gln PheAsn Asn GlyGln ThrTrp LeuLeu TyrAla ThrSer ProIle Lys LeuAsn HisThr Leu SerGlu IleThr SerAsn AlaPhe SerGly Ile IleArg IleAla Leu LeuPro AspSer AspSer LysHis GluAla Val LeuAsp LysTyr Ser SerCys TyrPro ValSer GlyLys AlaVal Phe ArgGlu ProPhe Cys ValGlu TyrAsn TrpGlu LysLys AspSer Gly AspLeu LeuLeu Leu AlaHis ProLeu HisVal GlnLeu LeuArg Asn GlyAsp AsnAsp Val LysIle LeuGlu AspLeu LysTyr LysSer Ile AspGly AspLeu Val GlyVal ValGly AspSer TrpVal LeuLys Thr AspPro LeuPhe Val ThrTrp HisSer IleLys Glytle LysGlu Glu SerHis AspGlu Ile ValSer AlaLeu SerLys AspVal GluSer Leu AspSer SerSer Ile ThrThr ThrGlu SerTyr PheTyr GlyLys Leu IleAla ArgAla Ala ArgLeu ValLeu IleAla GluGlu LeuAsn Tyr ProAsp ValIle Pro LysVal ArgAsn PheLeu LysGlu ThrIle Glu ProTrp LeuGlu Gly ThrPhe SerGly AsnGly PheLeu HisAsp Glu LysTrp GlyGly Ile IleThr GlnLys GlySer ThrAsp AlaGly Gly AspPhe GlyPhe Gly IleTyr AsnAsp HisHis TyrHis LeuGly Tyr PheIle TyrGly Ile AlaVal LeuThr LysLeu AspPro AlaTrp Gly ArgLys TyrLys Pro GlnAla TyrSer IleVal GlnAsp PheLeu Asn LeuAsp ThrLys Leu AsnSer AsnTyr ThrArg LeuArg CysPhe Asp ProTyr ValLeu His SerTrp AlaGly GlyLeu ThrGlu PheThr Asp Gly ArgAsn GlnGlu SerThr SerGlu AlaVal SerAla TyrTyr Ser Ala AlaLeu MetGly LeuAla TyrGly AspAla ProLeu ValAla Leu Gly SerThr LeuThr AlaLeu GluIle GluGly ThrLys MetTrp Trp His AlaMet AspPhe IleSer SerLeu IleVal GlyCys AlaGln Val Leu CysGlu SerMet AsnMet AlaGlu ArgArg GlyHis LysThr Asp Leu ArgGln AlaIle ThrAsp LeuGlu ThrAla IleGly AspLeu Lys Ala IleArg AspAsp LeuThr LeuArg IleGln GlnAsp GlyLeu Glu Gly ArgSer CysSer AsnArg AlaArg GluTrp LeuSer AlaVal Gln Val ThrGlu ThrLys ThrAla LeuLeu LeuVal ArgPhe ArgArg Arg Glu GlnArg ThrArg MetArg ArgArg TyrLeu SerCys PheGly Cys AlaAsp TyrLys Leu CysLys LysVal SerAla IleLeu LysSer Ile GlyGlu LeuArg Glu ArgSer GluAla IleLys ThrAsp GlyGly Ser IleGln ValThr Cys ArgGlu IlePro IleLys SerVal ValGly Asn ThrThr MetMet Glu GlnVal LeuGlu PheLeu SerGlu GluGlu Glu ArgGly IleIle Gly ValTyr GlyPro GlyGly ValGly LysThr Thr LeuMet GlnSer Ile AsnAsn GluLeu IleThr LysGly HisGln Tyr AspVal LeuIle Trp ValGln MetSer ArgGlu PheGly GluCys Thr IleGln GlnAla Val GlyAla ArgLeu GlyLeu SerTrp AspGlu Lys GluThr GlyGlu Asn ArgAla LeuLys IleTyr ArgAla LeuArg Gln LysArg PheLeu Leu LeuLeu AspAsp ValTrp GluGlu IleAsp Leu GluLys ThrGly Val ProArg ProAsp ArgGlu AsnLys CysLys Val MetPhe ThrThr Arg SerIle AlaLeu CysAsn AsnMet GlyAla Glu TyrLys LeuArg Val GluPhe LeuGlu LysLys HisAla TrpGlu Leu PheCys SerLys Val TrpArg LysAsp LeuLeu GluSer SerSer Ile ArgArg LeuAla Glu SEQ ID N0: 9 SEQUENCE LENGTH: 683 SEQUENCE TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: protein SEQUENCE DESCRIPTION:
Asn Ala Phe Ser Gly Ile Ile Arg Ile Ala Leu Leu Pro Asp Ser Asp Ser Lys His Glu Ala Val Leu Asp Lys Tyr Ser Ser Cys Tyr Pro Val Ser Gly Lys Ala Val Phe Arg Glu Pro Phe Cys Val Glu Tyr Asn Trp Glu Lys Lys Asp Ser Gly Asp Leu Leu Leu Leu Ala His Pro Leu His Val Gln Leu Leu Arg Asn Gly Asp Asn Asp Val Lys Ile Leu Glu Asp Leu Lys Tyr Lys Ser Ile Asp Gly Asp Leu Val Gly Val Val Gly Asp Ser Trp Val Leu Lys Thr Asp Pro Leu Phe Val Thr Trp His Ser Ile Lys Gly Ile Lys Glu Glu Ser His Asp Glu Ile Val Ser Ala Leu Ser Lys Asp Val Glu Ser Leu Asp Ser Ser Ser Ile Thr Thr Thr Glu Ser Tyr Phe Tyr Gly Lys Leu Ile Ala Arg Ala Ala Arg Leu Val Leu Ile Ala Glu Glu Leu Asn Tyr Pro Asp Val Ile Pro Lys Val Arg Asn Phe Leu Lys Glu Thr Ile Glu Pro Trp Leu Glu Gly Thr Phe Ser Gly Asn Gly Phe Leu His Asp Glu Lys Trp Gly Gly Ile Ile Thr Gln Lys Gly Ser Thr Asp Ala Gly Gly Asp Phe Gly Phe Gly Ile Tyr Asn Asp His His Tyr His Leu Gly Tyr Phe Ile Tyr Gly Ile Ala Val Leu Thr Lys Leu Asp Pro Ala Trp Gly Arg Lys Tyr Lys Pro Gln Ala Tyr Ser Ile Val Gln Asp Phe Leu Asn Leu Asp Thr Lys Leu Asn Ser Asn Tyr Thr Arg Leu Arg Cys Phe Asp Pro Tyr Val Leu His Ser Trp Ala Gly Gly Leu Thr Glu Phe Thr Asp Gly Arg Asn Gln Glu Ser Thr Ser Glu Ala Val Ser Ala Tyr Tyr Ser Ala Ala Leu Met Gly Leu Ala Tyr Gly Asp Ala Pro Leu Val Ala Leu Gly Ser Thr Leu Thr Ala Leu Glu Ile Glu Gly Thr Lys Met Trp Trp His Ala Met Asp Phe Ile Ser Ser Leu Ile Val Gly Cys Ala Gln Val Leu Cys Glu Ser Met Asn Met Ala Glu Arg Arg Gly His Lys Thr Asp Leu Arg Gln Ala Ile Thr Asp Leu Glu Thr Ala Ile Gly Asp Leu Lys Ala Ile Arg Asp Asp Leu Thr Leu Arg Ile Gln Gln Asp Gly Leu Glu Gly Arg Ser Cys Ser Asn Arg Ala Arg Glu Trp Leu Ser Ala Val Gln Val Thr Glu Thr Lys Thr Ala Leu Leu Leu Val Arg Phe Arg Arg Arg Glu Gln Arg Thr Arg Met Arg Arg Arg Tyr Leu Ser Cys Phe Gly Cys Ala Asp Tyr Lys Leu Cys Lys Lys Val Ser Ala Ile Leu Lys Ser Ile Gly Glu Leu Arg Glu Arg Ser Glu Ala Ile Lys Thr Asp Gly Gly Ser Ile Gln Val Thr Cys Arg Glu Ile Pro Ile Lys Ser Val Val Gly Asn Thr Thr Met Met Glu Gln Val Leu Glu Phe Leu Ser Glu Glu Glu Glu Arg Gly Ile Ile Gly Val Tyr Gly Pro Gly Gly Val Gly Lys Thr Thr Leu Met Gln Ser Ile Asn Asn Glu Leu Ile Thr Lys Gly His Gln Tyr Asp Val Leu Ile Trp Val Gln Met Ser Arg Glu Phe Gly Glu Cys Thr Ile Gln Gln Ala Val Gly Ala Arg Leu Gly Leu Ser Trp Asp Glu Lys Glu Thr Gly Glu Asn Arg Ala Leu Lys Ile Tyr Arg Ala Leu Arg Gln Lys Arg Phe Leu Leu Leu Leu Asp Asp Val Trp Glu Glu Ile Asp Leu Glu Lys Thr Gly Val Pro Arg Pro Asp Arg Glu Asn Lys Cys Lys Val Met Phe Thr Thr Arg Ser Ile Ala Leu Cys Asn Asn Met Gly Ala Glu Tyr Lys Leu Arg Val Glu Phe Leu Glu Lys Lys His Ala Trp Glu Leu Phe Cys Ser Lys Val Trp Arg Lys Asp Leu Leu Glu Ser Ser Ser Ile Arg Arg Leu Ala Glu SEQ ID N0: 10 SEQUENCE LENGTH: 2117 SEQUENCE TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear MOLECULE TYPE: cDNA to mRNA

FEATURE

NAME/KEY: CDS

LOCATION: 62..2110 SEQUENCE ION:
DESCRIPT

TCTTTCCTCA
ACCTTCTTTC
TTCTTA

AAT TCT TTG T
GCA GGC TTG
TT ATA CCG
ATC G
CGG
ATA
G

Asn e e le la sp er Ala Ser I Arg Leu S
Ph Gly Ile Leu Il A Pro A

AspSer Lys HisGlu AlaValLeu Asp LysTyr SerSer CysTyrPro ValSer Gly LysAla ValPheArg Glu ProPhe CysVal GluTyrAsn TrpGlu Lys LysAsp SerGlyAsp Leu LeuLeu LeuAla HisProLeu HisVal Gln LeuLeu ArgAsnGly Asp AsnAsp ValLys IleLeuGlu AspLeu Lys TyrLys SerIleAsp Gly AspLeu ValGly ValValGly AspSer Trp ValLeu LysThrAsp Pro LeuPhe ValThr TrpHisSer IleLys Gly IleLys GluGluSer His AspGlu IleVal SerAlaLeu SerLys Asp ValGlu SerLeu AspSer SerSer IleThr ThrThr Glu SerTyr Phe TyrGly LysLeu IleAla ArgAla AlaArg LeuVal Leu IleAla Glu GluLeu AsnTyr ProAsp ValIle ProLys ValArg Asn PheLeu Lys GluThr IleGlu ProTrp LeuGlu GlyThr PheSer Gly AsnGly Phe LeuHis AspGlu LysTrp GlyGly IleIle ThrGln Lys GlySer Thr AspAla GlyGly AspPhe GlyPhe GlyIle TyrAsn Asp HisHis Tyr HisLeu GlyTyr PheIle TyrGly IleAla ValLeu Thr LysLeu Asp ProAla TrpGly ArgLys TyrLys ProGln AlaTyr Ser IleVal Gln AspPhe LeuAsn LeuAsp ThrLys LeuAsn SerAsn Tyr ThrArg Leu ArgCys PheAsp ProTyr ValLeu HisSer TrpAla Gly GlyLeu Thr GluPhe ThrAsp GlyArg AsnGln GluSer ThrSer Glu AlaVal Ser AlaTyr TyrSer AlaAla LeuMet GlyLeu AlaTyr Gly AspAla Pro LeuVal AlaLeu GlySer ThrLeu ThrAla LeuGlu Ile GluGly Thr LysMet TrpTrp HisAla MetAsp PheIle SerSer Leu IleVal Gly CysAla GlnVal LeuCys GluSer MetAsn MetAla Glu ArgArg Gly HisLys ThrAsp LeuArg GlnAla IleThr AspLeu Glu ThrAla Ile GlyAsp LeuLys AlaIle ArgAsp AspLeu ThrLeu Arg IleGln Gln AspGly LeuGlu GlyArg SerCys SerAsn ArgAla Arg GluTrp Leu SerAla ValGln ValThr GluThr LysThr AlaLeu Leu LeuVal Arg PheArg ArgArg GluGln ArgThr ArgMet ArgArg Arg TyrLeu Ser CysPhe GlyCys AlaAsp TyrLys LeuCys LysLys Val SerAla Ile LeuLys SerIle GlyGlu LeuArg GluArg SerGlu Ala IleLys Thr AspGly GlySer IleGln ValThr CysArg GluIle Pro IleLys Ser ValVal GlyAsn ThrThr MetMet GluGln ValLeu Glu PheLeu Ser GluGlu GluGlu ArgGly IleIle GlyVal TyrGly Pro GlyGly Val GlyLys ThrThr LeuMet GlnSer IleAsn AsnGlu Leu IleThr Lys GlyHis GlnTyr AspVal LeuIle TrpVal GlnMet Ser ArgGlu Phe GlyGlu CysThr IleGln GlnAla ValGly AlaArg Leu GlyLeu Ser TrpAsp GluLys GluThr GlyGlu AsnArg AlaLeu Lys IleTyr ArgAla LeuArg Gln LysArg PheLeu LeuLeu LeuAsp Asp ValTrp GluGlu IleAsp Leu GluLys ThrGly ValPro ArgPro Asp ArgGlu AsnLys CysLys Val MetPhe ThrThr ArgSer IleAla Leu CysAsn AsnMet GlyAla Glu TyrLys LeuArg ValGlu PheLeu Glu LysLys HisAla TrpGlu Leu PheCys SerLys ValTrp ArgLys Asp LeuLeu GluSer SerSer Ile ArgArg LeuAla Glu SEQ ID N0: 11 SEQUENCE LENGTH: 347 SEQUENCE TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: protein SEQUENCE DESCRIPTION:
Met Ala Lys Tyr His Ser Ser Gly Lys Ser Ser Ser Met Thr Ala Ile Ala Phe Leu Phe Ile Leu Leu Ile Thr Tyr Thr Gly Thr Thr Asp Ala Gln Ser Gly Val Cys Tyr Gly Arg Leu Gly Asn Asn Leu Pro Thr Pro Gln Glu Val Val Ala Leu Tyr Asn Gln Ala Asn Ile Arg Arg Met Arg Ile Tyr Gly Pro Ser Pro Glu Val Leu Glu Ala Leu Arg Gly Ser Asn 65 70 75 g0 Ile Glu Leu Leu Leu Asp Ile Pro Asn Asp Asn Leu Arg Asn Leu Ala Ser Ser Gln Asp Asn Ala Asn Lys Trp Val Gln Asp Asn Ile Lys Asn Tyr Ala Asn Asn Val Arg Phe Arg Tyr Val Ser Val Gly Asn Glu Val Lys Pro Glu His Ser Phe Ala Gln Phe Leu Val Pro Ala Leu Glu Asn Ile Gln Arg Ala Ile Ser Asn Ala Gly Leu Gly Asn Gln Val Lys Val Ser Thr Ala Ile Asp Thr Gly Ala Leu Ala Glu Ser Phe Pro Pro Ser Lys Gly Ser Phe Lys Ser Asp Tyr Arg Gly Ala Tyr Leu Asp Gly Val Ile Arg Phe Leu Val Asn Asn Asn Ala Pro Leu Met Val Asn Val Tyr Ser Tyr Phe Ala Tyr Thr Ala Asn Pro Lys Asp Ile Ser Leu Asp Tyr Ala Leu Phe Arg Ser Pro Ser Val Val Val Gln Asp Gly Ser Leu Gly Tyr Arg Asn Leu Phe Asp Ala Ser Val Asp Ala Val Tyr Ala Ala Leu Glu Lys Ala Gly Gly Gly Ser Leu Asn Ile Val Val Ser Glu Ser Gly Trp Pro Ser Ser Gly Gly Thr Ala Thr Ser Leu Asp Asn Ala Arg Thr Tyr Asn Thr Asn Leu Val Arg Asn Val Lys Gln Gly Thr Pro Lys Arg Pro Gly Ala Pro Leu Glu Thr Tyr Val Phe Ala Met Phe Asp Glu Asn Gln Lys Gln Pro Glu Phe Glu Lys Phe Trp Gly Leu Phe Ser Pro Ile Thr Lys Gln Pro Lys Tyr Ser Ile Asn Phe Asn SEQ ID NO:

SEQUENCE LENGTH: 1044 SEQUENCE TYPE:nu cleic cid a STRANDEDNESS: double TOPOLOGY: linear MOLECULE TYPE:cDNA o t mRNA

SEQUENCE DESCRIPT ION:

TAT

Met Ala Lys HisSer Ser GlyLys SerSer SerMet ThrAla Ile Tyr TTT

Ala Phe Leu IleLeu Leu IleThr TyrThr GlyThr ThrAsp Ala Phe GTA

Gln Ser Gly CysTyr Gly ArgLeu GlyAsn AsnLeu ProThr Pro Val GTG

GlnGlu ValVal AlaLeu TyrAsn GlnAla AsnIle Arg ArgMet Arg IleTyr GlyPro SerPro GluVal LeuGlu AlaLeu Arg GlySer Asn IleGlu LeuLeu LeuAsp IlePro AsnAsp AsnLeu Arg AsnLeu Ala SerSer GlnAsp AsnAla AsnLys TrpVal GlnAsp Asn IleLys Asn TyrAla AsnAsn ValArg PheArg TyrVal SerVal Gly AsnGlu Val LysPro GluHis SerPhe AlaGln PheLeu ValPro Ala LeuGlu Asn IleGln ArgAla IleSer AsnAla GlyLeu GlyAsn Gln ValLys Val SerThr AlaIle AspThr GlyAla LeuAla GluSer Phe ProPro Ser LysGly SerPhe LysSer AspTyr ArgGly AlaTyr Leu AspGly Val IleArg PheLeu ValAsn AsnAsn AlaPro LeuMet Val AsnVal Tyr SerTyr PheAla TyrThr Ala AsnPro LysAsp IleSer LeuAsp Tyr AlaLeu PheArg SerPro Ser ValVal ValGln AspGly SerLeu Gly TyrArg AsnLeu PheAsp Ala SerVal AspAla ValTyr AlaAla Leu GluLys AlaGly GlyGly Ser LeuAsn IleVal ValSer GluSer Gly TrpPro SerSer GlyGly Thr AlaThr SerLeu AspAsn AlaArg Thr TyrAsn ThrAsn LeuVal Arg AsnVal LysGln GlyThr ProLys Arg ProGly AlaPro LeuGlu Thr TyrVal PheAla MetPhe AspGlu Asn GlnLys GlnPro GluPhe Glu LysPhe TrpGly LeuPhe SerPro Ile ThrLys GlnPro LysTyr Ser IleAsn PheAsn SEQ ID NO: 13 LENGTH:

SEQUENCE amino aci d TYPE:

TOPOLOGY: linear MOLECULE pr otein TYPE:

SEQUENCE ION:
DESCRIPT

MetSer AlaLeu LeuLeu Leu LeuGly ValLeu SerSer ThrGly Val LeuLeu ThrGly ValGlu Ser ValGly ValCys TyrGly GlyAsn Gly AsnAsn LeuPro ThrLys Gln AlaVal ValAsn LeuTyr LysSer Asn GlyIle GlyLys IleArg Leu TyrTyr ProAsp GluGly AlaLeu Gln AlaLeu ArgGly SerAsn Ile GluVal IleLeu AlaVal ProAsn Asp GlnLeu GlnSer ValSer Asn AsnGly GlyAla ThrAsn TrpVal Asn LysTyr ValLys ProTyr Ala GlyAsn ValLys LeuLys TyrIle Ala ValGly AsnGlu ValHis Pro GlyAsp AlaLeu AlaGly SerVal Leu ProAla LeuGln SerIle Gln AsnAla IleSer AlaAla AsnLeu Gln ArgGln IleLys ValSer Thr AlaIle AspThr ThrLeu LeuGly Asn SerTyr ProPro LysAsp Gly ValPhe SerAsn SerAla SerSer Tyr IleThr ProIle IleAsn Phe LeuAla LysAsn GlyAla ProLeu Leu Ala Asn Val Tyr Pro Tyr Phe Ala Tyr Val Asn Asn Gln Gln Asn Ile Gly Leu Asp Tyr Ala Leu Phe Thr Lys Gln Gly Asn Asn Glu Val Gly Tyr Gln Asn Leu Phe Asp Ala Leu Val Asp Ser Leu Tyr Ala Ala Leu Glu Lys Val Gly Ala Ser Asn Val Lys Val Val Val Ser Glu Ser Gly Trp Pro Ser Gln Gly Gly Val Gly Ala Thr Val Gln Asn Ala Gly Thr Tyr Tyr Arg Asn Leu Ile Lys His Val Lys Gly Gly Thr Pro Lys Arg Pro Asn Gly Pro Ile Glu Thr Tyr Leu Phe Ala Met Phe Asp Glu Asn Gln Lys Gly Gly Ala Glu Thr Glu Lys His Phe Gly Leu Phe Arg Pro Asp Lys Ser Pro Lys Tyr Gln Leu Ser Phe Asn SEQ ID N0: 14 SEQUENCE LENGTH: 993 SEQUENCE TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear MOLECULE TYPE: cDNA to mRNA
SEQUENCE DESCRIPTION:

Met Ser Ala Leu Leu Leu Leu Leu Gly Val Leu Ser Ser Thr Gly Val LeuLeu ThrGly Val GluSer ValGly ValCys TyrGly GlyAsn Gly AsnAsn LeuPro Thr LysGln AlaVal ValAsn LeuTyr LysSer Asn GlyIle GlyLys Ile ArgLeu TyrTyr ProAsp GluGly AlaLeu Gln AlaLeu ArgGly Ser AsnIle GluVal IleLeu AlaVal ProAsn Asp GlnLeu GlnSer Val SerAsn AsnGly GlyAla ThrAsn TrpVal Asn LysTyr ValLys Pro TyrAla GlyAsn ValLys LeuLys TyrIle Ala ValGly AsnGlu Val HisPro GlyAsp AlaLeu AlaGly SerVal Leu ProAla LeuGln Ser IleGln AsnAla IleSer AlaAla AsnLeu Gln ArgGln IleLys Val SerThr AlaIle AspThr ThrLeu LeuGly Asn SerTyr ProPro Lys AspGly ValPhe SerAsn SerAla SerSer Tyr IleThr ProIle IleAsn PheLeu Ala LysAsn GlyAla ProLeu Leu AlaAsn ValTyr ProTyr PheAla Tyr ValAsn AsnGln GlnAsn Ile GlyLeu AspTyr AlaLeu PheThr Lys GlnGly AsnAsn GluVal Gly TyrGln AsnLeu PheAsp AlaLeu Val AspSer LeuTyr AlaAla Leu GluLys ValGly AlaSer AsnVal Lys ValVal ValSer GluSer Gly TrpPro SerGln GlyGly ValGly Ala ThrVal GlnAsn AlaGly Thr TyrTyr ArgAsn LeuIle LysHis Val LysGly GlyThr ProLys Arg ProAsn GlyPro IleGlu ThrTyr Leu PheAla MetPhe AspGlu Asn GlnLys GlyGly AlaGlu ThrGlu Lys HisPhe GlyLeu PheArg Pro Asp Lys.Ser Pro Lys Tyr Gln Leu Ser Phe Asn SEQ ID N0: 15 SEQUENCE LENGTH: 34 SEQUENCE TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:

SEQ ID N0: 16 SEQUENCE LENGTH: 26 SEQUENCE TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:

SEQ ID N0: 17 SEQUENCE LENGTH: 44 SEQUENCE TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:

SEQ ID NO: 18 SEQUENCE LENGTH: 44 SEQUENCE TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:

SEQ ID N0: 19 SEQUENCE LENGTH: 30 SEQUENCE TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:

SEQ ID NO: 20 SEQUENCE LENGTH: 27 SEQUENCE TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:

SEQ ID N0: 21 SEQUENCE LENGTH: 35 SEQUENCE TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:

SEQ ID N0: 22 SEQUENCE LENGTH: 24 SEQUENCE TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:

SEQ ID N0: 23 SEQUENCE LENGTH: 21 SEQUENCE TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:

SEQ ID NO: 24 SEQUENCE LENGTH: 13 SEQUENCE TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:

SEQ ID N0: 25 SEQUENCE LENGTH: 45 SEQUENCE TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:

SEQ ID N0: 26 SEQUENCE LENGTH: 45 SEQUENCE TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: other nucleic acid (synthetic DNA) SEQUENCE DESCRIPTION:

SEQ ID N0: 27 SEQUENCE LENGTH: 204 SEQUENCE TYPE: amino acid TOPOLOGY: linear MOLECULE TYPE: protein SEQUENCE DESCRIPTION:
Glu Tyr Asn Trp Glu Lys Lys Asp Ser Gly Asp Leu Leu Leu Leu Ala His Pro Leu His Val Gln Leu Leu Arg Asn Gly Asp Asn Asp Val Lys Ile Leu Glu Asp Leu Lys Tyr Lys Ser Ile Asp Gly Asp Leu Val Gly Val Val Gly Asp Ser Trp Val Leu Lys Thr Asp Pro Leu Phe Val Thr Trp His Ser Ile Lys Gly Ile Lys Glu Glu Ser His Asp Glu Ile Val Ser Ala Leu Ser Lys Asp Val Glu Ser Leu Asp Ser Ser Ser Ile Thr Thr Thr Glu Ser Tyr Phe Tyr Gly Lys Leu Ile Ala Arg Ala Ala Arg Leu Val Leu Ile Ala Glu Glu Leu Asn Tyr Pro Asp Val Ile Pro Lys Val Arg Asn Phe Leu Lys Glu Thr Ile Glu Pro Trp Leu Glu Gly Thr Phe Ser Gly Asn Gly Phe Leu His Asp Glu Lys Trp Gly Gly Ile Ile Thr Gln Lys Gly Ser Thr Asp Ala Gly Gly Asp Phe Gly Phe Gly Ile Tyr Asn Asp His His Tyr His Leu Gly Tyr Phe Ile Tyr Gly Ile Ala Val Leu Thr Lys Leu Asp Pro Ala Trp Gly Arg Lys SEQ ID N0: 28 LENGTH:

SEQUENCE nu cleic cid TYPE: a STRANDEDNESS: double TOPOLOGY: linear MOLECULE cD NA o RNA
TYPE: t m SEQUENCE ION:
DESCRIPT

GluTyr AsnTrp GluLys Lys AspSer GlyAsp LeuLeu LeuLeu Ala HisPro LeuHis ValGln Leu LeuArg AsnGly AspAsn AspVal Lys IleLeu GluAsp LeuLys Tyr LysSer IleAsp GlyAsp LeuVal Gly ValVal GlyAsp SerTrp Val LeuLys ThrAsp ProLeu PheVal Thr TrpHis SerIle LysGly Ile LysGlu GluSer HisAsp GluIle Val SerAla LeuSer LysAsp Val GluSer LeuAsp SerSer SerIle Thr ThrThr GluSer TyrPhe Tyr GlyLys LeuIle AlaArg AlaAla Arg LeuVal LeuIle AlaGlu Glu LeuAsn TyrPro AspVal IlePro Lys ValArg AsnPhe LeuLys GluThr IleGlu ProTrp LeuGlu Gly Thr PheSer GlyAsn GlyPhe LeuHis AspGlu LysTrp GlyGly Ile Ile ThrGln LysGly SerThr AspAla GlyGly AspPhe GlyPhe Gly Ile TyrAsn AspHis HisTyr HisLeu GlyTyr PheIle TyrGly Ile Ala ValLeu ThrLys LeuAsp ProAla TrpGly ArgLys SEQ ID N0: 29 SEQUENCE LENGTH: 1080 SEQUENCE TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear MOLECULE TYPE: cDNA to mRNA
SEQUENCE DESCRIPTION:

Met Asp Phe Ile Ser Ser Leu Ile Val Gly Cys Ala Gln Val Leu Cys Glu Ser Met Asn Met Ala Glu Arg Arg Gly His Lys Thr Asp Leu Arg GlnAla IleThr AspLeu GluThr AlaIle GlyAsp Leu LysAla Ile ArgAsp AspLeu ThrLeu ArgIle GlnGln AspGly Leu GluGly Arg SerCys SerAsn ArgAla ArgGlu TrpLeu SerAla Val GlnVal Thr GluThr LysThr AlaLeu LeuLeu ValArg PheArg Arg ArgGlu Gln ArgThr ArgMet ArgArg ArgTyr LeuSer CysPhe Gly CysAla Asp TyrLys LeuCys LysLys ValSer AlaIle LeuLys Ser IleGly Glu LeuArg GluArg SerGlu AlaIle LysThr AspGly Gly SerIle Gln ValThr CysArg GluIle ProIle LysSer ValVal Gly AsnThr Thr MetMet GluGln ValLeu GluPhe LeuSer GluGlu Glu GluArg Gly Ile IleGly ValTyr GlyPro GlyGly ValGly LysThr Thr LeuMet Gln SerIle AsnAsn GluLeu IleThr LysGly HisGln Tyr AspVal Leu IleTrp ValGln MetSer ArgGlu PheGly GluCys Thr IleGln Gln AlaVal GlyAla ArgLeu GlyLeu SerTrp AspGlu Lys GluThr Gly GluAsn ArgAla LeuLys IleTyr ArgAla LeuArg Gln LysArg Phe LeuLeu LeuLeu AspAsp ValTrp GluGlu IleAsp Leu GluLys Thr GlyVal ProArg ProAsp ArgGlu AsnLys CysLys Val MetPhe Thr ThrArg SerIle AlaLeu CysAsn AsnMet GlyAla Glu TyrLys Leu ArgVal GluPhe LeuGlu LysLys HisAla TrpGlu Leu PheCys Ser LysVal TrpArg LysAsp LeuLeu GluSer SerSer Ile ArgArg Leu Ala Glu Ile Ile Val Ser Lys Cys Gly Gly Leu Pro Leu Ala Leu Ile Thr Leu Gly Gly Ala Met Ala

Claims (13)

1. A DNA coding for a chimeric protein comprising a domain containing at least an elicitor binding site of an elicitor receptor and a domain containing at least a signal transduction motif of a protein which confers plant disease resistance.

2. The DNA of claim 1, wherein the elicitor is a glucan, polygalacturonate, N-acetylchitosaccharide, elicitin, Cladosporium fulvum-derived Avr gene product, or Rhynchosporium secalis-derived nip1 gene product.

3. The DNA of claim 1, wherein the elicitor binding site comprises the following recombinant protein (a) or (b):
(a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 27 (b) a protein which consists of the amino acid sequence shown in SEQ ID NO: 27 having deletion, substitution, addition or insertion of at least one amino acid, and which has ability to bind to a glucan elicitor.

4. The DNA of any one of claims 1 to 3, wherein the domain containing a signal transduction motif contains at least one motif selected from the group consisting of leucine-rich repeats, leucine zippers, nucleotide binding sites and serine/threonine kinase domains.

5. The DNA of any one of claims 1 to 4, wherein the domain containing a signal transduction motif is a signal transduction domain of an expression product of tomato-derived Pto, Prf, Cf-2 or Cf-9 gene; a signal transduction domain of an expression product of rice-derived Xa21 gene; a signal transduction domain of an expression product of Arabidopsis thaliana-derived RPS2 or RPM1 gene; a signal transduction domain of an expression product of flax-derived L6 gene; or a signal transduction domain of an expression product of tobacco-derived N gene.

6. The DNA of claim 5, wherein RPS2 gene comprises the nucleotide sequence shown in SEQ ID NO: 29.

7. A DNA coding for the following protein (c) or (d):
(c) a chimeric protein comprising the amino acid sequence shown in SEQ ID NO: 5, 7 or 9;
(d) a chimeric protein which comprises the amino acid sequence shown in SEQ ID NO: 5, 7 or 9 having deletion, substitution, addition or insertion of at least one amino acid, and which confers plant disease resistance.

8. A DNA coding for a chimeric protein which confers plant disease resistance, said DNA comprising the nucleotide sequence shown in SEQ ID NO: 6, 8 or 10.

9. A recombinant vector comprising the DNA of any one of claims 1 to 8.

10. A transformed plant transformed with the recombinant vector of claim 9, or the progeny of said plant.

11. A method for creating a plant disease resistant plant, comprising transferring the recombinant vector of claim 9 into a plant.

12. A plant having plant disease resistance, said plant having the DNA of any one of claims 1 to 8 transferred thereinto and expressing said DNA, or the progeny of said plant.

13. A method for creating a plant having plant disease resistance or the progeny of said plant, comprising incorporating the DNA of any one of claims 1 to 8 into a chromosome of a plant and expressing said DNA.