AP605A - Glucan elicitor receptor and DNA molecule coding therefor. - Google Patents

Abstract

A glucan elecitor receptor

Description

DESCRIPTION

GLUCAN ELICITOR RECEPTOR AND DNA MOLECULE CODING THEREFOR

TECHNICAL FIELD

The present invention relates to glucan elicitor receptor (hereinafter sometimes referred to as ER), DNA molecules coding for ER, vectors containing the DNA molecules and plant cells transformed with the DNA molecules. The present invention relates more specifically to ER derived from a soybean root plasma membrane fraction, DNA molecules coding for ER, vectors containing the DNA molecules and plant cells transformed with the DNA molecules.

BACKGROUND ART

It is known that plants synthesize and accumulate an antibiotic agent called phytoalexin in response to infection with pathogens (M. Yoshikawa (1978) Nature 257: 546). Some plant pathogens were found to have the substances that induce them to perform such a resistance reaction (N.T. Keen (1975) Science 187: 74), which are called elicitors. The biochemical process from the infection of plants with pathogens to the synthesis and accumulation of phytoalexin is believed to be as follows:

When the mycelium of a pathogen invades a plant cell, glucanase in the plant cell works so as to cleave polysaccharides on the surface of the pathogen mycelial wall, thereby liberating an elicitor. If the elicitor binds to a receptor in the plant cell, a second messenger which plays a role in signal transduction is produced. The signal transduction substance is incorporated in the nucleus of the plant cell and activates the transcription of the genes coding for phytoalexin synthesize enzymes to induce a phytoalexin synthesis. At the same

AP/P/ 9 6 / 0 0 7 8 9

AP.00605 time, the phytoalexin degradation is inhibited. As a result, phytoalexin is efficiently accumulated.

Phytoalexin playing an important role in the resistance of soybean is called glyceollin and its structure has been determined (M. Yoshikawa et al. (1978) Physiol. Plant. Pathol. 12: 73). Elicitor is a polysaccharide of glucose and reported to be a glucan having £-1,6 and £ -1,3 linkages (J.K. Sharp et al. (1984) J. Biol. Chem. 259: 11321, M. Yoshikawa (1990) Plant Cell Technology 1.2: 695). A specific receptor for glucan elicitor derived from a soybean pathogenic mold fungus Phytophthora megasperma f. sp. glycinea is believed to be a protein which plays an important role in the synthesis and accumulation of the antibiotic agent glyceollin. The method for the purification of the ER specific to this elicitor has been disclosed (E.G. Cosio et al., (1990) FEBS 264: 235, E.G. Cosio et al. (1992) Eur. J. Biochem. 204: 1115, T.Frey et al. (1993) Phytochemistry 32: 543). However, the amino acid sequence of the ER has not been determined and the gene coding therefor is not yet known.

An object of the present invention is to provide a glucan elictor receptor.

Another object of the present invention is to provide DNA molecules coding for the glucan elicitor receptor.

A further object of the present invention is to provide vectors containing DNA molecules coding for the glucan elicitor receptor.

A still further object of the present invention is to provide plant cells transformed with DNA molecules coding for the glucan elicitor receptor.

DISCLOSURE OF THE INVENTION

As a result pf the various studies conducted to solve the above

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AP.00605 problems, the inventors succeeded in purifying a soybean root-derived ER and cloning the ER gene from a soybean cDNA library, thereby accomplishing the present invention. The present invention provides a glucan elicitor receptor having an amino acid sequence as substantially shown in SEQ ID NO:1. The present invention also provides DNA molecules containing nucleotide sequences coding for a glucan elicitor receptor having an amino acid sequence as substantially shown in SEQ ID NO:1, and fragments thereof. The present invention further provides DNA molecules containing nucleotide sequences coding for the glucan elicitor receptor which are incorporated in plasmid pER23-l, and fragments thereof. The present invention still further provides vectors containing DNA molecules coding for the glucan elicitor receptor and plant cells transformed with DNA molecules coding for the glucan elicitor receptor.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows SDS-polyacrylamide gel electrophoresis patterns of three purification steps.

Figure 2 shows the maps of plasmids pER23-l and pER23-2.

Figure 3 shows the procedure for constructing plasmid pKVl-ER23. Figure 4 shows a transient increase in intracellular Ca^J* concentration after the addition of an elicitor to cultured soybean cells.

Figure 5 shows a transient increase in intracellular Ca¹* concentration after the addition of an elicitor to transformed cultured tobacco cells.

Figure 6 shows elicitor-binding activities of full- or partial length ER expressed in E. coli.

Figure 7 shows the inhibition of the binding of an elicitor with an

68/00/96 /d/dV

AP.00605 elicitor binding protein in a soybean cotyledon membrane fraction by an antibody against an elicitor-binding domain.

Figure 8 shows the inhibition of an elicitor-induced phytoalexin accumulation in soybean cotyledons by an antibody against an elicitorbinding domain.

PREFERRED EMBODIMENTS OF CARRYING OUT THE INVENTION

The glucan elicitor receptor of the present invention is a protein having a function as a receptor for glucan elicitors derived from plant phatogens, particularly Phytophthora. More specifically, it is a protein which binds to the glucan elicitor produced by the cleavage of parts of the pathogen mycelial wall with β -1,3-glucanase in plant cells at the time of invasion of plant pathogens, particularly microorganisms belonging to genus Phytophthora and which subsequently orders the microsomes and nucleus to produce an increased amount of phytoalexin in the plant cells. The glucan elicitor receptor of the present invention have an amino acid sequence as substantially shown in SEQ ID NO:1. The amino acid sequence as substantially shown in SEQ ID NO:1 includes amino acid sequences as shown in SEQ ID NO: 1 in which ./ there may be the deletion, replacement or addition of an amino acid(s), provided that they maintain the function of a glucan elicitor receptor.

The glucan elicitor receptor of the present invention can be produced, for example, by a partially modified Cosio's method (E.J.B. (1992) 204: 1115). Briefly, the roots, leaves and stems of soybean, preferably variety green homer are homogenized and a membrane fraction is collected from the resulting slurry, purified by ion-exchange chromatography and further purified by affinity chromatography using an elicitor as a ligand. The elicitor used in the affinity chromatography is preferably derived from Phytophthora megasperma f. sp. glycinea

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AP.00605 race 1 (ATCC34566) because it shows incompatibility for green homer (i.e., resistance to the pathogen).

The amino acid sequence of the glucan elicitor receptor thus prepared can be determined as follows:

The purified ER is transferred on a PVDF membrane (Millipore Co.) by electroblotting and digested with lysylendopeptidase (AP-I). The fragmented peptides are recovered from the PVDF membrane and fractionated by reversed-phase HPLC ( μ -Bondasphere 5 μ C8). The peak fractions are analyzed with a gas-phase protein sequencer (Applied Biosystems Co.).

The ER of the present invention is useful in the elucidation of resistance mechanism of plants to fungi and the development of elicitor derivatives capable of inducing resistance to fungi, and it can be used as an antigen for the production of antibodies against ERs.

The present invention encompasses DNA molecules containing nucleotide sequences coding for a glucan elicitor receptor, and fragments thereof. The DNA molecules of the present invention have preferably at least one stop codon (e.g., TAG) adjacent to the 3' end.

More specifically, the present invention encompasses DNA molecules containing nucleotide sequences coding for a glucan elicitor receptor having an amino acid sequence as substantially shown in SEQ ID NO: 1, and fragments thereof. The DNA molecules containing nucleotide sequences coding¹for a glucan elicitor receptor include all degenerate isomers. The term degenerate isomers means DNA molecules coding for the same polypeptide with different degenerate codons. If a DNA molecule having the nucleotide sequence of SEQ ID NO:2 is taken as an example, a DNA molecule in which a codon for any amino acid, e.g., AAC for Asn is changed to a degenerate codon AAT is called a degenerate isomer. Examples of such degenerate isomers include DNA molecules

8 L 0 0 / 9 6 /d/dV (ί

AP. Ο Ο 6 Ο 5 containing the nucleotide sequence shown in SEQ ID NO: 2.

In another aspect, the present invention provides DNA molecules containing nucleotide sequences coding for a glucan elicitor receptor, which are incorporated in plasmid pER23-l, and fragments thereof. E. coli DH5a EKB633 transformed with plasmid PER23-1 was deposited with the National Institute of Bioscience and Human-Technology, the Agency of Industrial Science and Technology, on June 15, 1994 under Accession

Number FERM BP-4699.

The fragments of the DNA molecules containing nucleotide sequences coding for the glucan elicitor receptor of the present invention may contain a nucleotide sequence encoding the amino acid sequence of SEQ

ID NO:1.

The DNA molecules of the present invention which contain nucleotide sequences coding for a glucan elicitor receptor and fragments thereof may optionally bind to an ATG codon for initiation methionine together with a translation frame in the upstream portion toward the 5' end and also bind to other DNA molecules having appropriate lengths as nontranslation regions in the upstream portion toward the 5' end and the downstream portion toward the 3' end.

The DNA molecules of the present invention which contain nucleotide sequences coding for a glucan elicitor receptor and fragments thereof can be present typically in the form of parts of constituents of plasmid or phage DNA molecules or in the form of parts of constituents of plasmid, phage or genomic DNA molecules which are introduced into microorganisms (particularly, bacteria including E. coli and Agrobacterium), phage particles or plants.

In order to express stably the DNA molecules coding for a glucan elicitor receptor and fragments thereof in plants, a promoter, a DNA molecule (ATG) encoding the initiation codon and a terminator may be

8 Ζ υ υ / 9 6 /d/dV

AP .0 0 6 0 5

added to the DNA molecules of the present invention and fragments thereof in appropriate combinations. Examples of the promoter include the promoter of genes encoding ribulose-1,5-biphosphate carboxylase small subunit (Fluhr et al., Proc. Natl. Acad. Sci. USA (1986) 83: 2358), the promoter of a nopaline synthase gene (Langridge et al., Plant Cell Rep. (1985) 4:355), the promoter for the production of cauliflower mosaic virus 19S-RNA (Guilley et al., Cell (1982) 30:763), the promoter for the production of cauliflower mosaic virus 35S-RNA (Odell et al., Nature (1985) 313:810) and the like. Examples of the terminator include the terminator of a nopaline synthase gene (Depicker et al., J. Mol. Appl. Gen. (1982) 1:561) and the terminator of an octopine synthase gene (Gielen et al., EMBO J. (1984) 3:835).

The DNA molecule containing a nucleotide sequence coding for a glucan elicitor receptor can be obtained by a method comprising the steps of chemically synthesizing at least a part of the DNA molecule according to a conventional procedure of synthesis of nucleic acids and obtaining a desired DNA molecule from an appropriate cDNA library using the synthesized DNA molecule as a probe by a conventional method, for example, an immunological method or a hybridization method. Some plasmids, various kinds of restriction enzymes, T4DNA ligase and other enzymes for use in the above method are commercially available. The DNA cloning, the construction of plasmids, the transfection of a host, the cultivation of the transfectant, the recovery of DNA molecules from the culture and other steps can be performed by the methods described in Molecular Cloning, J. Sambrook et al., CSH Laboratory (1989), Current Protocols in Molecular Biology, F. M. Ausubel et al., John Wiley & Sons (1987) and others.

More specifically, the DNA molecules of the present invention which contain nucleotide sequences coding for a glucan elicitor receptor can

AP/P/ 9 6 / 0 0 7 8 9 (

AP.00605 be obtained as follows:

Two kinds of partial amino acid sequences are selected from the amino acid sequences of a glucan elicitor receptor. Primers consisting of combinations of all bases which can encode the C terminus of the selected partial sequence and primers consisting of combinations of all bases which can encode the N terminus of the selected partial sequence are prepared. These synthesized primers are used as mixed primers to perform two PCR using DNA molecules of an appropriate soybean cDNA library as a template. Subsequently, two amplified fragments of given lengths whose amplification is expected (these fragments correspond to DNA molecules encoding the above two partial amino acid sequences) are picked up and the nucleotide sequences thereof are determined. On the basis of the determined nucleotide sequences, a primer having nucleotide sequences coding for the C terminus of an amino acid partial sequence positioned at the C terminal side of the glucan elicitor receptor and a primer having nucleotide sequences coding for the N terminus of an amino acid partial sequence positioned at the N terminal side of the glucan elicitor receptor are synthesized. These two synthesized primers are used to perform a PCR using the DNA molecules of the above soybean cDNA library as a template. The resulting amplified fragments are used as probes to hybridize the aforementioned soybean cDNA library, thereby yielding DNA molecules containing nucleotide sequences coding for the glucan elicitor receptor.

The obtained DNA molecules containing nucleotide sequences coding for the glucan elicitor receptor can be sequenced by any known methods, for example, the Maxam-Gilbert method (Methods Enzymol., 65:499, 1980), a dideoxynucleotide chain termination method using M13 phage (J. Messing, et al., Gene, 19:269, 1982) and the like.

Since the results of various studies on glucan elicitor suggest

AP/P/ 96/00789

AP. Ο Ο 6 Ο 5 that a glucan elicitor receptor plays an important role in resistance to fungi in plants, it is expected that the DNA molecules coding for glucan elicitor receptor of the present invention and fragments thereof can impart fungal resistance to plants if they are introduced and expressed in plant cells (particularly higher plant cells) which have no glucan elicitor receptor according to a conventional procedure. It has been proposed that fungi capable of infecting plants have generally suppressors, thereby acquiring an ability to suppress the fungal resistance of the plants. It is expected that new plants having resistance to fungi can be developed by introducing and expressing the DNA molecules coding for glucan elicitor receptor of the present invention and fragments thereof such that the ER works or by modifying the DNA molecules and fragments thereof or by regulating their expression amounts. Moreover, if the DNA molecules of the present invention and fragments thereof are introduced and expressed in plant cells, particularly in higher plant cells, together with fungal resistance enhancing genes or characters such as the gene of glucanase which imparts fungal resistance to plants, it is expected that higher fungal resistance can be imparted to plants than in the case where the gene of glucanase is introduced.

Vectors used for introducing the DNA molecules coding for the glucan elicitor receptor and fragments thereof may be constructed such that the glucan elicitor receptor can be stably expressed in plants. More specifically, a promoter, a DNA molecule encoding the initiation codon (ATG) and a terminator may be added to the DNA molecules coding for the glucan elicitor receptor and fragments thereof in appropriate combinations. Examples of the promoter include the promoter of genes encoding ribulose-l,5-biphosphate carboxylase small subunit (Fluhr et al., Proc. Natl. Acad. Sci. USA (1986) 83:2358), the promoter of a

AP/P/ 96/00789

AP . 0 0 6 0 5 nopaline synthase gene (Langridge et al., Plant Cell Rep. (1985) 4: 355), the promoter for the production of cauliflower mosaic virus 19SRNA (Guilley et al., Cell (1982) 30:763), the promoter for the production of cauliflower mosaic virus 35S-RNA (Odell et al., Nature (1985) 313:810) and the like. Examples of the terminator include the terminator of a nopaline synthase gene (Depicker et al., J. Mol. Appl. Gen. (1982) 1:561) and the terminator of an octopine synthase gene (Gielen et al., EMBO J. (1984) 3:835).

The DNA molecules coding for a glucan elicitor receptor and fragments thereof can be introduced into plant cells by any usual known methods, for example, the method described in Plant genetic transformation and gene expression; a laboratory manual, J. Draper, et al. eds., Blackwell Scientific Publications, 1988. Examples of the methods include biological methods such as those using viruses or Agrobacteria and physicochemical methods such as electroporation, a polyethylene glycol method, microinjection and the like.

The present invention will now be explained in greater detail with reference to the-following examples which are by no means intended to limit the scope of the present invention.

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Example 1

Purification of soybean root-derived ER

1) Measurement of glucan elicitor binding activity of ER

A complex of an elicitor (average molecular weight: 10,000) and tyramine (TOKYO KASEI KOGYO CO., LTD.) was synthesized by the method of Jong-Joo Cheong (The Plant Cell (1991) 3: 127). The elicitor-tyramine complex was labelled with iodine using chloramine T.

A sample (protein amount < 500μ g) was suspended in 500 μ 1 of an assay buffer (50 mM Tris-HCl pH7.4, 0.1 M saccharose, 5 mM MgCli , ImM

0

AP.00605

PMSF and 5 mM EDTA) and incubated at 0°C for 2 hours. The iodinelabelled elicitor-tyramine complex in an amount of 7.0 nM (70 Ci/mmol, the number of moles is calculated on the assumption that the molecular weight of the elicitor is 10,000, and this applies to the following description) was added to the suspension and the mixture was incubated at 4 °C for 2 hours. The reaction solution was filtered through Whatman GF/B as treated with a 0.3% aqueous solution of polyethylene imine for at least 1 hour. The residue was washed 3 times with 5 ml of an ice-cold buffer (10 mM Tris-HCl pH 7.0, 1 M NaCl, 10 mM MgCli ). The radio activity retained on the filter was counted with a gamma counter (count A). In order to eliminate the effect of non-specific binding, the same procedure as above was performed except that 17 μ M of the elicitor was added to the same sample, the mixture was suspended in the assay buffer and the suspension was incubated at 0 “C for 2 hours. The obtained count was subtracted from the count A to give a count ( Δ cpm) of elicitor-specific binding. The resulting count (Acpm) was divided by the total number of counts and then multiplied by the total amount of elicitor used in the experiment to calculate the amount of the elicitor-binding protein (in moles).

The purity of ER was checked by the above method.

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2) Purification of soybean root-derived ER

Soybean (Glycine max cv. Green Homer) seeds (Takayama Seed Co. ) ' were cultured on vermiculite for 1 week and then aquicultured for 15 days to harvest roots (about 40 kg, wet weight). The harvested roots were stored at -80 °C until they were used for the purification of ER. An ice-cold buffer (25 mM Tris-HCl pH 7.0, 30 mM MgClj , 2 mM dithiothreitol, 2.5 mM potassium metabisulfite and 1 mM PMSF) was added to the roots (2 kg, wet weight) in an amount of 1.25 L and the mixture

AP.00605 was homogenized with a Waring Blender for 2 minutes.

The resulting slurry was filtered through a Miracloth (Calbiochem

Co.) and the filtrate was centrifuged at 9,000 rpm at 4 °C for 15 minutes. The supernatant was ultracentrifuged at 37,000 rpm at 4°C for 20 minutes. The precipitate was suspended in 160 ml of an ice-cold buffer (25 mM Tris-HCl pH 7.4, 0.1 M sucrose, 5 mM MgClj , 1 mM PMSF and 5 mM EDTA) to give a membrane fraction. An ampholytic detergent ZW3-12 (Boehringer Co.) was added to the membrane fraction to give a final concentration of 0.25% for solubilization of ER from the membrane fraction and the mixture was stirred at 8 °C for 30 minutes. The resulting mixture was ultracentrifuged at 37,000 rpm at 4 °C for 20 minutes to collect the supernatant containing the solubilized ER (soluble fraction). The soluble fraction (165 ml) was dialyzed against 2 1 of a buffer (50 mM Tris-HCl pH 8.0, 0.2% ZW3-12, 4 °C ) 4 times. Five milliliters of Protrap (TAKARA SHUZO CO., LTD.) was added to the sample and the mixture was stirred at 8 °C for 30 minutes to remove proteases from the sample and to stabilize ER. The resulting mixture was centrifuged at 2,800 rpm at 4 °C for 2 minutes to collect the supernatant. The obtained supernatant (160 ml) was concentrated to about 50 ml using an ultrafiltration membrane YM-10 (Amicon Co.) and the concentrate was dialyzed against an A buffer (50 mM Tris-HCl pH 8.0, 0.1 M sucrose, 5 mM MgCl, , 1 mM PMSF, 5 mM EDTA and 0.2% ZW3-12, 4 °C )·

The dialysate was applied to Q-Sepharose HP 26/10 (Pharmacia Co.) and ER was eluted in a linear gradient of 0-1 M NaCl (Q-Sepharose active fraction). The ER was eluted at a NaCl concentration of about

0.45 M. The Q-Sepharose active fraction was diluted 3 folds with A buffer and the diluted fraction was applied to Mono Q 10/10 (Pharmacia

Co.). The ER was eluted in a linear gradient of 0-1 M NaCl (Mono Q active fraction,, 8 ml). The ER was eluted at a NaCl concentration of

AP/P/ 96/00789

2

AP.00605 about 0.25 M.

The ER was purified with an affinity gel using an elicitor as a ligand as follows:

Elicitor was prepared according to N.T. Keen with some modifications (Plant Physiol. (1983) 71: 460, Plant Physiol. (1983) 71: 466). Briefly, the mycelial wall of pathogenic Phytophthora megasperma f. sp. glycinea race 1 (ATCC34566) was treated with zymolyase 100T (KIRIN BREWERY CO., LTD.) to liberate an elicitor. After the treatment, Zymolyase 100T was eliminated by the adsorption on CMcellulose packed in a column. The resulting passage-through fraction was purified with a gel permeation chromatography G-75 (Pharmacia Co.) to collect an elicitor fraction whose average molecular weight was 10,000 Da. The glyceollin-inducing elicitor activity of the collected fraction was determined by the method of M. Yoshikawa (Nature (1978) 257:546). The addition of 8//g of the elicitor to soybean cotyledons resulted in the induction of about 550 χζ g of glyceollin after 24 hours incubation.

In order to eliminate non-specific adsorption on the gel carrier, Mono Q active fraction was collected and stirred with about 33 mg of maltose-coupled glass gel (bed volume: about 100μ 1) at 8 °C for 1 hour. The gel was precipitated by centrifugation (1,000 rpm, 4 °C , 2 minutes) to collect the supernatant (maltose-coupled glass gel passagethrough fraction). The maltose-coupled glass gel was prepared by the method of A.M. Jeffrey et al. (Biochem. Biophys. Res. Commun. (1975) 62: 608). Briefly, 120 mg of maltose and 6 g of Glass Aminopropyl (Sigma Co.) were suspended in 36 ml of H>0 and the suspension was stirred at room temperature overnight. To the resulting suspension was added 36 ml of ethanol. Immediately thereafter a solution of sodium borohydride (864,mg) in ethanol (72 ml) was added to the mixture. The

AP/P/ 96/00789

AP.00605 resulting mixture was sonicated for 2 minutes and stirred at room temperature for 5 hours. Water (288 ml) was added to the reaction mixture and the resulting mixture was cooled with ice and adjusted to pH 5.6 with acetic acid. The gel was washed with about 1.8 L of Hj0 to remove the free maltose. Maltose contained in the washing solution was determined quantitatively by the method of J. H. Roe (J. Biol. Chem. (1955) 212:335) using an anthrone reagent. An amount of the gel-coupled maltose was estimated from the amount of the maltose contained in the washing solution. As a result, it was found that 60 mg of maltose was coupled to 6 g of Glass Aminopropyl.

About 17 mg of the elicitor-coupled glass gel (bed volume: about 50 μ 1) was added to 8 ml of the maltose-coupled glass gel passagethrough fraction and the mixture was stirred gently at 8 °C overnight. The gel was collected by centrifugation (1,000 rpm, 4 °C , 2 minutes) and washed with 2 bed volumes of A buffer 2 times. The gel was washed additionally with 4 bed volumes of 0.1% SDS 3 times to collect gelcoupled ER (elicitor-coupled glass gel eluted fraction). The elicitorcoupled glass gel was prepared by the method of A.M. Jeffrey et al. (Biochem. Biophys. Res. Commun. (1975) 62: 608). Briefly, elicitor (37 mg) and Glass Aminopropyl (490 mg) were suspended in 6 ml of Hj0 and stirred at room temperature overnight. Ethanol (6 ml) was added to the suspension and a solution of sodium borohydride (144 mg) in ethanol (12 ml) was added immediately thereafter. The mixture was sonicated for 2 minutes and stirred at room temperature for 5 hours. To the resulting mixture was added 48 ml of HjO. The mixture was cooled with ice and adjusted to pH 5.6 with acetic acid. The free elicitor was determined quantitatively with an anthrone reagent. The amount of the gel-coupled elicitor was estimated from the amount of the free elicitor contained in the washing solution. As a result, it was found that 34 mg of the

AP/P/ 96/00789

4 elicitor was coupled to 490 mg of Glass Aminopropyl.

The protein and ER amounts in the above steps for purification are summarized in Table 1.

Table 1. Protein and ER Amounts in the Steps for Purification (Soybean roots weighing 40 kg on a wet basis were used as a starting material)

	Protein (mg)	ER (pmol)
Membrane Fraction	17900	30
Soluble Fraction	2000	214
Q-Sepharose Active Fraction	190	205
Mono Q Active Fraction	49	233
Maltose-Coupled Glass Gel Passage-through Fraction	45	220
Elicitor-Coupled Glass Gel Eluted Fraction	0.004*	45

• : Estimated from the band intensity obtained by silver stain after

SDS-PAGE.

The Mono Q active fraction, passage-through fraction from maltosecoupled glass gel and eluted fraction from elicitor-coupled glass gel ' (10μ 1 each) were electrophoresed on an electrophoretic gradient gel, SDS-PAGE plate 10/20 (Daiich Kagaku Yakuhin Co.) and stained with silver (Daiich Kagaku Yakuhin Co.) The electrophoresis patterns are shown in Figure 1. In Figure 1, lane 1 is the Mono Q active fraction, lane 2; the passage-through fraction from the maltose-coupled glass gel and lane 3; the .eluted fraction from the elicitor-coupled glass gel.

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AP.00605

Figure 1 shows that the ER bands were detected at a molecular weight of about 70,000 Da.

The protein of about 70,000 in molecular weight was labelled with iodine-125 by using an *¹ ’ I-labelled complex of a photoaffinity reagent SASD (Pierce Co.) and the elicitor. The SDS-PAGE band of the membrane fraction was transferred on a PVDF membrane by western blotting and incubated with the same ¹ ” I-labelled elicitor as used in measuring the elicitor-binding activity of ER on the PVDF membrane so that the protein of about 70,000 Da in molecular weight was labeled with iodine-125. These facts reveal that the protein of about 70,000 Da in molecular weight had an elicitor-binding activity.

About 4 μ g of ER was purified from about 40 kg by wet weight of the soybean root by the above method.

3) Analysis of ER-fragmented peptides

The ER was fragmented by protease digestion to peptides. The amino acid sequences of the fragmented peptides were determined by the method of Iwamatsu (Akihiro Iwamatsu, Seikagaku (1991) 63: 139, A. Iwamatsu, Electrophoresis (1992) 13: 142). A solution of the ER purified by the above method was concentrated to about 100 μ 1 with Centricon-30 (Amicon Co.) and subjected to a 10-20% polyacrylamide SDS electrophoresis. The resulting protein bands were transferred on a PVDF membrane (Millipore Co.) with an electroblotting apparatus (Sartrius Co.). The bands transferred on the PVDF membrane were stained with 0.1% Ponceau S (Sigma Co.)/1% acetic acid. The main band of 70,000 Da in molecular weight was sectioned and decolored with 0.5 mM NaOH. This band was reductively S-carboxymethlated. Lysylendopeptidase (AP-1) was added to the resulting band at an enzyme: substrate (mol:mol) ratio of 1:100 and the mixture was reacted at 30 “C for 16 hours. The

8 Z u U / 9 6 /d/dV t

AP.00605 resulting fragmented peptides were applied to a μ -Bondasphere 5 μ C8300 A (2.1 x 150 mm, Waters) column equilibrated with 98% solvent A and 2% solvent B and eluted in a 2-50% linear gradient of solvent B for 30 minutes at a flow rate of 0.25 ml/minute (solvent A: 0.05% TFA solution, solvent B: 0.02% TFA in 2-propanol:acetonitrile=7:3 (v/v)). Eluted peptides were detected by absorbance at 214 nm and each peak fraction was collected manually. The obtained peak fractions were analyzed with a gas-phase protein sequencer (Model 470A of Applied Biosystems). As a result of analysis of all the peak fractions obtained, the following amino acid sequences of the fragmented peptides were clearly determined.

fl:Val

Asn

lie

Gin

Thr

Asn

Thr

Ser

Asn

He

Ser

Pro

Gin

(N-terminus)

#5:Lys

Ser

He

Asp

Gly

Asp

Leu

Val

Gly

Val

Val

Gly

Asp

Ser

#6:Lys

Tyr

Lys

Pro

Gin

Ala

Tyr

Ser

lie

Val

Gin

Asp

Phe

Leu Asn Leu Asp

#7:Lys

Thr

Asp

Pro

Leu

Phe

Val

Thr

Trp

His

Ser

lie

Lys

(mix sequence)

AP/P/ 96/00789

AP.00605 €

Example 2

Cloning of soybean ER gene

1) Preparation of soybean mRNA

Soybean (Glycine max cv. Green Homer) seeds (Takayama Seed Co.) were cultured on vermiculite for 1 week and aquicultured for 15 days to harvest roots (about 40 kg, wet weight). A portion of the harvested roots was stored at -80°C until it was used. Total RNA was obtained by the method of Ishida (Cell Technology Laboratory Manipulation Manual, Kodansha Scientific). Briefly, the stored roots (28.5 g, wet weight) were ground on a mortar while adding liquid nitrogen. To the obtained powder, 35.6 ml of a GTC solution held at 65°C was added and the mixture was homogenized with a Waring Blender. The resulting suspension was centrifuged at 6,000 rpm at room temperature for 15 minutes to collect 40 ml of the supernatant. The supernatant was layered gently on a cushion solution of cesium in a centrifuge tube and centrifuged at 35,000 rpm at 25 °C for 20 hours. The resulting precipitate was dissolved in 9 ml of TE/0.2% SDS. After phenol/chloroform extraction was conducted 2 times, total RNA (4.37 mg) was recovered by ethanol precipitation.

C The obtained total RNA (2.2 mg) was purified with oligotex dT30 (Japan Roche Co.) according to the manual to yield 60μ g of poly(A) +RNA.

2) Preparation of soybean cDNA library cDNA molecules were synthesized from 5 μ g of the poly(A)+RNA with a cDNA synthesis kit (Pharmacia Co.) according to the manual. The synthesized cDNA fragments were ligated to lambda phage vector λ gtlO (Stratagene Co.) with T4 ligase (TAKARA SHUZO CO., LTD.). Gigapack (Stratagene Co.) was used to package a DNA mixture into the phage

AP/P/ 96/00789

AP.00605 particles to prepare a soybean cDNA library of about 1.5 x 10* pfu. The library was amplified to 160 ml of a soybean cDNA library of 1.6 x 10¹¹ pfu/ml.

Total DNA in the cDNA library was prepared as follows:

Chloroform/isoamylalcohol (24:1) was added to 500 μ 1 of a phage solution (1.6 x 10‘¹ pfu/ml) in an equal amount. The mixture was shaken for 30 seconds and centrifuged to collect the aqueous layer. The aqueous layer was extracted again with chloroform/isoamylalcohol (24:1). To the resulting aqueous layer were added 5 μ 1 of a 3 M sodium acetate solution (pH 5.4) and 125 μ 1 of ethanol and the mixture was centrifuged to collect the precipitate. The precipitate was washed with a 70% ethanol solution and dissolved in a 10 mM Tris-HCl solution (pH 8) containing 1 μ g/ml RNase A (Sigma Co.). This solution was used as a PCR template.

3) Amplification and Cloning of Soybean ER cDNA Fragments by PCR

The following four oligodeoxynucleotides (mixed primers 05, 07, 010 and 012) were synthesized with an automatic nucleic acid synthesizer (Model 394 of Applied Biosystems Co.) on the basis of the amino acid sequences of the fragmented peptides obtained in Example 1 (#5 and #6):

Primer 05 5’-AARAGYATHGAYGGNGA-3 ’

Primer 07 5'-WRTCNCCNACNAC-3'

Primer 010 5'-GTNAAYAARATNCARAC-3’

Primer 012 5'-ARRTTNAGRAARTCYTC-3' (R:A/G, Y:C/T, W:A/T, H:A/C/T, N:A/G/T/C)

The total DNA in 0.5 μ g of the cDNA library was dissolved in 79 μ 1 of distilled water. Either a combination of primers 05 and 07 or a combination of primers U10 and 012 (100 pmol each) and 0.5# 1 of Taq DNA polymerase (TAKAJRA SHUZO CO., LTD.) were added to 8 μ 1 of 2.5 mM dNTP bbZUU/96 /d/dV

AP.00605 in 10 μ 1 of a 10 x PCR buffer (attached to Taq DNA polymerase of TAKARA SHUZO CO., LTD.) to give a final amount of 100 μ 1. PCR reaction was performed with a Gene Amp PCR System 9600 (Perkin-Elmer Co.) by 50 cycles of 1) denaturation at 94 °C x 30 seconds, 2) renaturation at 47°C x 30 seconds and 3) elongation at 72 °C x 1 minute. After the reaction, 15//1 of the reaction solution was electrophoresed on a 15% polyacrylamide gel. The gel was stained with a 0.5 μ g/ml ethidium bromide solution for 10 minutes. The bands showing specifically amplified fragments of 40 bp and 47 bp whose amplification was expected were sectioned while observing under UV light. The gel sections were ground with a plastic bar and eluted with an elution buffer (0.5 M ammonium acetate, 10 mM magnesium acetate, 1 mM EDTA and 0.1% SDS) overnight to collect a DNA-containing solution.

The collected DNA fragments were cloned into plasmid pT7Blue(R) with a pT7Blue T-Vector Kit (Novagene Co.). The obtained plasmids p#51, 2, and p#6-l, 2, 3, 4, 5, 6, 7, 8 and 9 were sequenced with a fluorescence automatic DNA sequencer (Model 373A of Applied Biosystems Co.). The results showed that the resulting amplified DNA fragments other than the primers also encoded the amino acid sequences of fragmented peptides #5 and f6.

The following two oligodeoxynucleotides (mixed primers U18 and U19) were synthesized with an automatic nucleic acid synthesizer on the basis of the results of the DNA sequencing.

Primer U18 5'-AAGTAYAAGCCRCAAGCCTATTCA-3'

Primer U19 5'-ATCGCCRACAACMCCAA-3' (Y and R are as defined above, M:A/C)

The total DNA in 0.5 μ g of the cDNA library was dissolved in 79 μ 1 of distilled water. A combination of primers U18 and U19 (100 pmol each) and 0.5 /2,1 of Taq DNA polymerase were added to 8 μ 1 2.5 mM

8 Ζ υ u / y 6 /d/dV

AP.00605 dNTP in 10 μ 1 of a 10 x PCR buffer to give a final amount of 100 μ 1. PCR reaction was performed by 40 cycles of 1) denaturation at 94°C x 30 seconds, 2) renaturation at 52°C x 30 seconds and 3) elongation at 72 °C x 1 minute. Fifteen microliters of the reaction solution was electrophoresed on a 1% agarose gel.

The gel was stained with a 0.5 μ g/ml ethidium bromide solution for 15 minutes. The band showing a specifically amplified fragment of about 540 bp was sectioned while observing under UV light. The gel section was treated with Gene Clean II (BiolOl Co.) to collect a DNAcontaining solution.

The collected DNA fragment was cloned into plasmid pT7Blue(R) with a pT7Blue T-Vector Kit. The obtained plasmid p#5-#6 was sequenced with a fluorescence sequencer. The results showed that the amplified DNA fragment consisted of 539 bp and encoded not only the amino acid sequences of fragmented peptides #5 and #6 at the both sides, but also peptide #7 in the amplified portion.

4) Screening and Cloning of Library by Hybridization

Plasmid #5-#6 into which the ER cDNA fragment was cloned was digested with restriction enzymes BamHI and Pstl. A DNA fragment of about 540 bp was recovered and used as a probe. The recovered DNA fragment was labelled with (a -**P] dCTP using a Megaprime DNA labelling system (Amersham Co.) according to the manual and the reaction solution was used in a hybridization experiment.

A phage of the cDNA library was infected with E. coli C600 hfl (Invitrogen Co.) and inoculated in a 10 mg/ml MgCli-containing L medium on plates of about 15 cm in diameter to form a total of about 1 x 10‘ of plaques. The plaques were blotted on a nylon membrane (Hybond-N; Amersham Co.). The membrane was reacted with the ’’P-dCTP labeled ER cn oo r-.

co co to cn

CL

CL <

C

AP. 00605 cDNA fragment and positive phages detected by autoradiography were screened again in the same way to give about 30 phage clones having different signal intensities. Clone λ ER23 having the longest inserted DNA fragment was selected.

The λ phage DNA molecule was purified with a LambdaSorb (Promega Co.) from a solution of the positive clone λ ER23 isolated in the hybridization experiment. Ten microliters of a 10 x EcoRI cleavage buffer (restriction enzyme EcoRI 10 U) was added to 5 μ g of the DNA solution to give a total amount of 100 μ 1 and the mixture was reacted at 37 °C overnight. The reaction solution was electrophoresed on a 1% agarose gel. A band of about 2.3 kb was sectioned and treated with a Gene Clean II (BiolOl Co.) to collect a DNA-containing solution. Vector pBluescriptll KS- (0.02 μ g) (Stratagene Co.) was cleaved with restriction enzyme EcoRI.

After the two DNA solutions were mixed, 2 μ 1 of a 10 x ligase buffer and 0.2 μΐ of T4 DNA ligase (TAKARA SHUZO CO., LTD) were added to give a total amount of 20//1. The mixture was reacted at 16 °C for 4 hours and the reaction mixture solution was used to transform E. coli

DH5a (Gibco BRL Co.). A 2% agar plate medium was prepared with 25 ml of an L medium containing 50 μ g/ ml ampicillin, 40 μ q/ ml IPTG and 40 μ q/ ml X-gal. The transformed E. coli was inoculated on the agar plate medium and grown at 37°C overnight. White colonies were selected from the formed colonies and cultured in 3 ml of an L medium containing 50 μ q/ ml ampicillin at 37 °C for 8 hours. Plasmids were recovered from these bacterial cells by an alkali method and determined whether they were clones into which a desired fragment was cloned with the restriction enzyme, thereby giving plasmids pER23-l and pER23-2 (5225 bp) which had opposite orientations to the vector. The maps of plasmids PER23-1 and pER23-2 are shown in Figure 2.

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AP.00605

5) Determination of the Nucleotide Sequence of the ER-encoding Clone

The DNA nucleotide sequences of plasmids pER23-l and pER23-2 were determined in both orientations with a fluorescence sequencer by 1) using plasmids pER23-l and pER23-2 digested by appropriate restriction enzymes, 2) using appropriate primers synthesized on the basis of the information about already determined nucleotide sequences, or 3) cleaving pER23-l with restriction enzymes Kpnl and Xhol and pER23-2 with restriction enzymes Kpnl and Clal and then using a kilosequence deletion kit (TAKARA SHUZO CO., LTD) to prepare plasmids having a deletion at intervals of about 200-300 bp. The DNA nucleotide sequence is shown in SEQ ID NO: 2 of the SEQUENCE LISTING. The results showed that the DNA fragment contained a 667 amino acid-encoding open reading frame of 2001 bp beginning at a nucleotide sequence corresponding to the N-terminal sequence (fragmented peptide #1) sequenced with the amino acid sequencer. The amino acid sequence is shown in SEQ ID NO: 1 of the SEQUENCE LISTING. The amino acid sequence deduced from the resulting DNA nucleotide sequence was consistent with the previously determined amino acid sequence of the soybean ER.

In addition, highly homologous amino acid sequences were searched for with a nucleic acid and amino acid sequence analysis software package (MacVector: Kodak Co.) using a nucleic acid and amino acid sequence data base (Entrez: NCBI). However, no amino acid sequences were found to be highly homologous to the heretofore known sequences. Hence, it is clear that the prepared ER is a novel protein.

AP/P/ 96/00789

Example 3

Expression of the Soybean ER in Tobacco Plants 1) Construction of Plant Expression Plasmid pKVl-ER23

3

AP.00605

As shown in Figure 3, a plant expression vector pKVl to be used in this example was prepared from cauliflower mosaic virus 35S promotercontaining plasmid pCaP35J (J. Yamaya et al. (1988) Mol. Gen. Genet. 211:520) as follows:

Plasmid pCaP35J was digested completely with restriction enzyme BamHI to delete a multi-cloning site present upstream of the 35S promoter. Following partial digestion with Pvull, a treatment was conducted with Klenow fragments (TAKARA SHUZO CO., LTD) to make blunt ends. The resulting plasmid DNA was circularized by ligation and introduced into E. coli DH5a . A desired plasmid was selected from the

C -resulting clones. The selected plasmid was digested with restriction enzyme Pstl to insert a multi-cloning site present downstream of the 35S promoter. A treatment was conducted with Klenow fragments to make blunt ends. The resulting plasmid DNA was digested with Hindlll. The following synthetic linker DNAs were synthesized with an automatic nucleic acid synthesizer, annealed and ligated to the Hindi!I-digested plasmid. The resulting plasmid DNA was introduced into E. coli DH5a · Desired plasmid pCaP35Y (2837 bp) was selected from the obtained clones.

C 5'-GGAATTCGAGCTCGGTACCCGGGGGATCCTCTAGAGTCGACCTGCAGGCATGCA-3'(SEQ ID NO:

3)

5¹-CCTTAAGCTCGAGCCATGGGCCCCCTAGGAGATCTCAGCTGGACGTCCGTACGTTCGA-3’(SEQ ID

NO: 4)

In order to introduce a terminator of nopaline synthase into the pCaP35Y, plasmid pBI121 (Clontech Co.) was digested with Sacl and EcoRI and the SacI-EcoRI fragment was treated with Klenow fragments to make blunt ends; then, the resulting fragment of pBI121 was ligated to plasmid pCaP35Y in which blunt ends were made at a Hindlll site downstream of the 35S promoter. The resulting plasmid DNA was

8 / 0 0 / 9 5 /d/dV

AP. Ο Ο 6 Ο 5 introduced into Ε. coli DH5 a - A desired plasmid was selected from the obtained clones. In order to introduce a kanamycin-resistance cassette into the selected plasmid, the latter was digested with PvuII and ligated to a fragment (about 1620 bp) of pLGVneoll03 (R. Hain et al. (1985) Mol. Gen. Genet. 199: 161) that was obtained by the steps of cleavage at a PvuII site present downstream of the octopine synthase terminator, treatment with Bal31 (TAKARA SHUZO CO., LTD) to make a deletion, cleavage at a EcoRl site upstream of a nopaline synthase promoter, and the creation of a blunt end at both ends. The resulting plasmid DNA was introduced into E. coli DH5 a · Desired plasmid, or plant expression vector pKVl (4828 bp), was selected from the obtained clones.

The prepared pKVl was digested at unique sites by restriction enzymes BamHI and Sail and ligated to the ER gene-containing fragment (i.e., the BamHI-Sall fragment of pEB23-l, about 2.3 kbp). The resulting plasmid DNA was introduced into E. coli DH5a . Desired ER-expression plasmid pKVl-ER23 (about 7.1 kbp) was selected from the obtained clones.

c, (-U 2) Transient Expression of ER in Cultured Tobacco Cells

The ER gene was introduced into cultured tobacco cells by electroporation for transient ER expression by a partial modification of Watanabe's method (Y. Watanabe (1987) FEBS 219: 65). The DNA molecules of the plasmid pKVl-ER23 were purified by an alkali method. Cultured tobacco cells were obtained by the method of Hirai et al. (Plant Cell Cultivation Manual, Gakkai Shuppan Center, 1982) for use in the transient expression of ER. Tobacco seeds (variety Bright Yellow, provided by Professor Hirofumi Dchimiya of University of Tokyo) were sterilized with a 1% sodium hypochlorite solution and then germinated.

8 L 0 U / 9 6 /d/dV

AP. Ο Ο 6 Ο 5

The tobacco juvenile tissues just after the germination were transplanted in a tobacco cultivation agar medium (Murashige-Skoog medium (Flow Laboratories Co.) supplemented with 2 ppm 2,4dichlorophenoxyacetic acid, 3% sucrose and 8% agar) to induce calluses after 3 weeks. About 1 g of callus masses were suspended in 50 ml of a tobacco cultivation medium (Murashige-Skoog medium (Flow Laboratories Co.) supplemented with 2 ppm 2,4-dichlorophenoxyacetic acid and 3% sucrose) to prepare cultured cells. These tobacco cells were cultured until they entered a logarithmic growth phase. The cultured cells were collected by centrifugation (600 rpm, 3 minutes) and suspended in a solution consisting of 1% cellulase Onozuka (Yakult Co.), 1% Dricelase (Kyowa Hakko Co., Ltd.), 0.1% Pectriase (Seishin Seiyaku Co.) and 0.4 M D-mannitol (Wako Pure Chemicals Co., Ltd.) and which was adjusted to pH 5.7 with HCI. Reaction was performed at 30 “C for 90 minutes to prepare protoplasts. The reaction solution was washed with 0.4 M Dmannitol at 4 °C by 3 cycles of centrifugation to remove the enzyme solution. The operation of electroporation consisted of suspending 1 x 10* cells in 0.8 ml of an electroporation solution (70 mM KC1, 5 mM MES and 0.3 M mannitol), mixing the suspension with 10 μ g of the DNA molecules of pKVl-ER23 and treating the mixture with a genepluser (Biorad Co.) at 125μΡ and 300 V in an electroporation cuvette (Biorad Co., electrode spacing: 0.4 cm). After the treatment, the solution was collected with a pasteur pipet and left to stand on ice for 30 minutes. Reaction was performed at 30 °C for 5 minutes and the reaction solution was resuspended in a protoplast medium (Murashige-Skoog medium (Flow Laboratories Co.) supplemented with 0.2 ppm 2,4-dichlorophenoxyacetic acid, 1% sucrose and 0.4 M mannitol and adjusted to pH 5.7). The cells were left to stand in the dark at 25°C overnight and collected by centrifugation (8,000 rpm, 3 minutes). Sixty microliters of a

8 / 0 0 / 9 6 /d/dV

AP. Ο Ο 6 Ο 5 suspension buffer (25 mM Tris-HCl pH7.0, 30 mM MgClj , 2 mM dithiothreitol, 2.5 mM potassium metabisulfite and 1 mM PMSF) were added to the cells and the mixture was stirred on a vortex for 3 minutes.

The resulting sample was stored at -80°C until an elicitor-binding experiment was conducted.

For control, the above procedure was repeated except that the DNA molecule of pKVl instead of pKVl-ER23 was introduced into tobacco cells.

3) Stable Expression of ER in Tobacco Suspension Cultured Cells

Transformed cultured tobacco cells capable of constant ER gene retention were selected as follows from the cultured tobacco cells capable of transient ER expression:

The protoplasts obtained in the preparation of the cultured tobacco cells capable of transient ER expression were suspended in a 1% agarose-containing protoplast medium (Murashige-Skoog medium (Flow Laboratories Co.) supplemented with 0.2 ppm 2,4-dichlorophenoxyacetic acid, 1% sucrose and 0.4 M mannitol and adjusted to pH 5.7). The suspension was dropped on a plate with a dropping pipet before the agarose solidified, whereby the protoplasts were fixed in the bead-like solid medium. After the agarose solidified, an agarose-free protoplast medium was added to the plate, thereby immersing the protoplast-fixing agarose medium in the liquid medium. After the protoplasts were cultured in the dark for 1 week, kanamycin was added to a final concentration of 100 μ g/ml and the cultivation was continued. Transformants selected from the grown colonies were transferred in a kanamycin-containing liquid medium and cultured.

Two clones (I 1 and I 6) of cultured tobacco cells stably transformed by pKVl-ER23 and two clones (C 2-1 and C 2-4) of cultured

AP/P/ 9 6 / 0 0 7 8 9

7

AP.00605 tobacco cells stably transformed by pKVl were obtained.

4) Elicitor-binding Activity Experiment

The elicitor-binding activity was measured as follows:

A complex of an elicitor and tyramine (TOKYO KASEI KOGYO CO., LTD.) was synthesized by the method of Jong-Joo Cheong (The Plant Cell (1991) 3: 127). The elicitor-tyramine complex was labelled with iodine-125 using chloramine T. The resulting sample (protein amount < 500^ g) was suspended in 500 μ 1 of an assay buffer (50 mM Tris-HCl pH7.4, 0.1 M saccharose, 5 mM MgCli , ImM PMSF and 5 mM EDTA) and incubated at 0°C for 2 hours. The iodine-labelled elicitor-tyramine complex in an amount of 100 nM (70 Ci/mmol) was added to the suspension and the mixture was incubated at 4 °C for 2 hours. The reaction solution was filtered through Whatman GF/B (as treated with a 0.3% aqueous solution of polyethylenimine for at least 1 hour) and washed 3 times with 5 ml of an ice-cold buffer (10 mM Tris-HCl pH 7.0, 1 M NaCl, 10 mM MgClj ). The radio activity retained on the filter membrane was counted with a gamma counter (count A). In order to eliminate the effect of non-specific binding, the same procedure as above was performed except that 17 μ M of the elicitor was added to the same sample, the mixture was suspended in the assay buffer and the suspension was incubated at 0 'C for 2 hours. The obtained count was subtracted from the count A to give a count ( Δ cpm) of elicitor-specific binding. The resulting count (Acpm) was divided by the total number of counts and then multiplied by the total amount of elicitor used in the experiment to calculate the amount of the elicitor-binding protein (in moles).

As a result, a specific binding to the elicitor was observed in the tobacco cells transformed with the DNA molecule of pKVl-ER23, whereas no specific binding to the elicitor was observed in the control tobacco

AP/P/ 96/00789 e

AP. Ο Ο 6 Ο 5 cells in which the DNA molecule of pKVl was introduced (Table 2). This fact reveals that the gene obtained above encodes a protein having the elicitor-binding activity.

Table 2. Elicitor-binding Activity of Cultured Tobacco Cells

Fraction Transfoming DNA	Binding Activity ( fmol/mg)
Transient Expression	pKVl	< 0
	PKV1-ER23	90.5
Stable Expression
C2-1	pKVl	< 0
C2-4	pKVl	< 0
I 1	PKV1-ER23	150
I 6	PKV1-ER23	196

5) Transient Increase in Intracellular Ca^{1 +} Concentration in Transformed Tobacco Cultured Cells by Addition of Glucan Elicitor

Plants recognize the elicitor at a receptor specific thereto and then promote the accumulation of phytoalexin or induce hypersensitive reaction to prevent fungus invasion. It has been reported for some plants that the inflow of calcium ion into cells in the early phase of such resistance reactions is important (U. Conrath et al. (1991) FEBS LETTERS 279: 141, Μ. N. Zook et al. (1987 ) Plant Physiol. 84: 520, F. Kurosaki et al. (1987 ) Phytochemistry 26: 1919; C. L. Preisig and R. A. Moreau (1994) Phytochemistry 36: 857). A report has also been made suggesting that the inflow of calcium ion into cells triggers the promotion of the,phytoalexin accumulation in soybean which the present

AP/P/ 9 6 / 0 0 7 8 9

9

AP.00605 inventors used to obtain ER (M. R. Stab and J. Ebel (1987) Archi. Biochem. Biophys. 257: 416). Hence, if a transformed cultured tobacco cell is prepared by introducing the ER gene into an ER-free tobacco cultured cell to express the ER and if the intracellular calcium ion concentration is changed by the addition of a glucan elicitor, the change is anticipated to trigger a resistance reaction by the glucan elicitor in plants other than soybean (e.g., tobacco), thereby allowing them to show resistance to a wide variety of fungi which use glucan as a mycelial wall component.

The change in intracellular Ca’⁺ concentration of transformed cultured tobacco cells by the addition of the elicitor was examined.

In this experiment, the transformed cultured tobacco cells (I 6) obtained by the kanamycin selection and the plasmid-containing cultured tobacco cells (C 2-4) were used.

The intracellular Ca¹⁺ concentrations of the cultured cells were measured as follows with an acetoxymethyl derivative (Fura-2 AM) of a fluorescence chelator (Fura-2) for Ca’* measurement:

Cells were harvested from about 2 ml of the transformed tobacco cell culture (corresponding to a cell volume of about 250 ml after standing for 10 minutes) by centrifugation (600 rpm, 30 seconds) and the supernatant was removed. To the cells was added 2 ml of a tobacco cultivation medium and the mixture was stirred gently and centrifuged (600 rpm, 30 seconds) to remove the supernatant. The same operations were repeated to wash the cultured cells. The washed cultured cells was suspended homogeneously in 2 ml of the medium. To 1 ml of the suspension of the cultured cells in the medium, 1 ml of the medium and 4 μ 1 of 1 mM Fura-2 AM (final concentration: 2 μ M, Dojin Chemical Co.) were added and the mixture was incubated in the dark for 30 minutes with occasionally stirring. Subsequently, the cells were washed 2

AP/P/ 96/00789

AP.00605 times with 2 ml of the medium by centrifugation (600 rpm, 30 seconds) to eliminate the free Fura-2 AM which was not incorporated into the cells.

The washed cultured cells were suspended in 2 ml of the medium homogeneously and the suspension (2 ml) was transferred into a fluorescence-measurement cell. The incorporated Fura-2 AM should be changed to Fura-2 by hydrolysis with intracellular esterase. The fluorescence produced by the binding of Fura-2 to intracellular Ca* ⁺ was measured at a fluorescence wavelength of 505 nm under exciting light of 335 nm with the cultured cells being stirred to ensure against precipitation of the cultured cells. The change in intracellular Ca’⁺ concentration was examined by measuring the fluorescence intensity at specified intervals of time after the addition of 50 μ 1 of glucan elicitor (1 mg/ml) or deionized water to the cultured cells. For control, the change in intracellular Ca** concentration was examined on the plasmid-containing cultured tobacco cells by the same method as above. For another control, the change in intracellular Ca’* concentration was examined on cultured soybean cells by the same method as above, except that the cultured cells were washed with a medium for soybean cells having the following formulation.

NaHi PO« · Hi 0 75mg/ml, KH₂ PO, 170mg/ml, KNOi 2,200mg/ml, NH, NOi 600mg/ml, (NH, )jSO, 67mg/ml, MgSO, · 7H, O 310mg/ml, CaCli · 2Hj O 295mg/ml, FeSO««7HiO 28mg/ml, EDTA«Na₂ 37.3mg/ml, KI 0.75mg/ml, MnSO, ·4Η₂Ο lO.Omg/ml, Hi BOi 3.0mg/ml, ZnSO, ·7Η₂Ο 2mg/ml, Na_a MoO, · 2H₂ 0 0.25mg/ml, CuSO, ·5Hi0 0.025mg/ml, CoCl_a*6H_aO 0.025mg/ml, Inositol lOOmg/ml, Nicotinic acid l.Omg/ml, Pyridoxine·HCI l.Omg/ml, Thiamine·HCI lO.Omg/ml, Glucose 5g/ml, Sucrose 25g/ml, Xylose 250mg/ml, Sodium pyruvate 5.0mg/ml, Citric acid lO.Omg/ml, Malic acid lO.Omg/ml, Fumaric acid lO.Omg/ml, N-Z-amine 500.0mg/ml, 2,4-dichlorophenoxyacetic acid l.Omg/ml and Zeatine riboside O.lmg/ml, adjusted to pH 5.7 with KOH.

AP/PZ 96/00789

1

AP.00605

As a result of this experiment, about 7% transient increase in fluorescence intensity was observed in the cultured soybean cells 3 minutes after the addition of the elicitor, whereas no such change was observed after the addition of deionized water (Figure 4). The results suggest that the phenomenon in which the binding of the ER to the glucan elicitor caused a transient inflow of Ca’* into cells could be observed in this experiment, thereby supporting the report that calcium ion plays an important role in the resistance reaction caused by the elicitor in cultured soybean cells. In the transformed cultured tobacco cells, about 10% transient increase in fluorescence intensity was observed 3 minutes after the addition of the elicitor, whereas no such change was observed after the addition of deionized water.

In the plasmid-containing cultured tobacco cells, none of the changes in fluorescence intensity that occurred in the transformed cultured tobacco cells was observed after the addition of the elicitor (Figure 5).

These results show that plants other than soybean (e.g., tobacco), which are not reactive with the glucan elicitor acquire the reactivity by introducing the gene of the soybean-derived glucan elicitor receptor for ER expression. Although the signal transduction pathway of each plant has not been completely explicated, it is expected that plants other than tobacco will acquire the reactivity with the glucan elicitor (i.e., a transient increase in intracellular Ca’⁺ concentration) by introducing the gene of the present ER for ER expression, thereby enabling the development of plants having resistance to a wide variety of fungi which use glucan as a mycelial wall component.

AP/P/ 96/00789 c··

AP. Ο Ο 6 Ο 5

Example 4

Expression of Soybean ER in E. coli and Determination of Elicitorbinding Domain

1) Expression of Elicitor-binding Domain in E. coli

A fused protein of a partial fragment of the soybean ER with a maltose-binding protein (MBP) was prepared with a Protein Fusion & Purification System (New England Biolabs Co.) in order to express the partial fragment of the soybean ER in E. coli. PCR was performed using pER23-l as a template to give DNA fragments of various lengths. The primers were designed to produce the MBP and fused protein in cloning into plasmid pMAL-c2 (New England Biolabs Co.) by adding a BamHI site on the 5' side and a Sail site on the 3' side exterior to the DNA molecule encoding the full-length portion and fragments of soybean ER shown in Figure 6. These primers were synthesized with an automatic nucleic acid synthesizer (Model 394 of Applied Biosystems Co.). The following primers were used in the amplification of the DNA chain.

Primer U35 5'-ATGGATCCATGGTTAACATCCAAACC-3';

Primer 036 5'-ATGGATCCGAATATAACTGGGAGAAG-3';

Primer 037 5'-ATGGATCCCCAGCATGGGGTAGGAAG-3';

Primer U38 5'-TAGTCGACTACTTCTCCCAGTTATATTC-3';

Primer 039 5'-TAGTCGACTACTTCCTACCCCATGCTGG-3’;

Primer 040 5'-TAGTCGACTATTCATCACTTCTGCTATG-3’;

Primer 041 5'-ATGGATCCGCCCCACAAGGTCCCAAA-3'; and

Primer U42 5'-ATGGATCCAATGACTCCAACACCAAG-3'

The DNA molecule of pER23-l (0.01 μ g) was dissolved in 79 μ 1 of distilled water. Either a combination of primers 05 and 07 or a combination of primers 010 and U12 (100 pmol each) and 0.5μ 1 of Taq DNA polymerase (TAKARA SHOZO CO., LTD.) were added to Βμ 1 of 2.5 mM dNTP

AP/P/ 96/00789

3

AP.00605 in 10££ 1 of a 10 x PCR buffer (attached to Taq DNA polymerase of TAKARA SHUZO CO., LTD.) to give a final amount of 100 μ 1. PCR reaction was performed with a Gene Amp PCR System 9600 (Perkin-Elmer Co.) by 30 cycles of 1) denaturation at 94 °C x 30 seconds, 2) renaturation at 55°C x 30 seconds and 3) elongation at 72 °C χ 1 minute. After the reaction, 15//1 of the reaction solution was digested with restriction enzymes BamHI and Sail and electrophoresed on a 1% agarose gel.

The gel was stained with a 0.5 μ g/ml ethidium bromide solution for 15 minutes. The band showing the expected specific amplification was sectioned while observing under UV light. The gel section was treated with Gene Clean II (BiolOl Co. ) to collect a DNA-containing solution. The collected DNA fragments were cloned into the BamHI-Sall site of plasmid pMAL-c2 and the clones were introduced into E. coli DH5a ·

2) Preparation of Soluble Protein Fraction from E. coli

The E. coli cells into which the plasmids were introduced were precultured in an expression medium [10g/l tryptone (Gibco Co.), 5 g/1 yeast extract (Gibco Co.), 5 g/1 NaCl, 2 g/1 glucose and 100 μ g/ml ampicillin]. The precultured solution (0.4 ml) was added to 40 ml of the expression medium and cultured at 37 °C with shaking until OD»οo of 0.55 was reached. Isopropylthiogalactoside was added to the culture solution to give a final concentration of 0.3 mM and the shakeculture was continued for an additional 4 hoursto induce expression. The E. coli was collected by centrifugation and the E. coli cells were washed with a washing buffer (20 mM Tris-HCl, pH 7.4, 200 mM NaCl and 1 mM EDTA). The cells were sonicated for a total of 2 minutes (15 sec x 8). ZW3-12 was added to the sonicated cells to give a final concentration of 0.25% and the mixture was incubated at 4 *C for 30 minutes. The sui>ernatant was collected by centrifugation (10,000 rpm, 5

AP/P/ 96/00789 ν

AP.00605 minutes) to give an E. coli soluble protein fraction. The expression of the fused protein was confirmed by an immunoblotting technique using an anti-maltose-binding protein antibody (New England Biolabs Co.).

3) Elicitor-binding Experiment

The elicitor-binding activity was determined as follows:

A complex of an elicitor and tyramine (TOKYO KASEI KOGYO CO., LTD.) was synthesized by the method of Jong-Joo Cheong (The Plant Cell (1991) 3: 127). The elicitor-tyramine complex was labelled with iodine-125 using chloramine T. The resulting sample (protein amount < 800/z g) was suspended in 500 μ 1 of an assay buffer (50 mM Tris-HCl pH7.4, 0.1 M saccharose, 5 mM MgCl, , ImM PMSF and 5 mM EDTA) and incubated at 0’C for 2 hours. The iodine-labelled elicitor-tyramine complex in an amount of 100 nM (70 Ci/mmol) was added to the suspension and the mixture was incubated at 4 °C for 2 hours. The reaction solution was filtered through Whatman GF/B (as treated with a 0.3% aqueous solution of polyethylenimine for at least 1 hour) and washed 3 times with 5 ml of an ice-cold buffer (10 mM Tris-HCl pH 7.0, 1 M NaCl, 10 mM MgCl, ). The radio activity retained on the filter membrane was counted with a gamma counter (count A). In order to eliminate the effect of non-specific binding, the same procedure as above was performed except that 17 μ M of the elicitor was added to the same sample, the mixture was suspended in the assay buffer and the suspension was incubated at 0 °C for 2 hours. The obtained count was subtracted from the count A to give a count ( Δ cpm) of elicitor-specific binding. The resulting count (Acpm) was divided by the total number of counts and then multiplied by the total amount of the elicitor used in the experiment to calculate the amount of the elicitor-binding protein (in moles).

As a result, a specific binding to the elicitor was observed in the

AP/P/ 96/00789

5

AP. Ο Ο 6 Ο 5

Ε. coli transformed with the DNA molecule encoding the ER (Figure 6). Hence, it was reconfirmed that the obtained gene encoded a protein having the elicitor-binding activity and it was revealed that there was an elicitor-binding domain in the 239-442 amino acid sequence of SEQ ID

NO:1.

Example 5

Inhibition of Binding of Glucan Elicitor to Elicitor-binding Protein in Soybean Cotyledon Membrane Fraction and Inhibition of Accumulation of Phytoalexin in Soybean Cotyledon by Antibody against Elicitor-binding

Domain

1) Expression of Elicitor-binding Domain in E. coli

A fused protein of an elicitor-binding domain derived from the ER with a maltose-binding protein (MBP) was prepared with a Protein Fusion & Purification System (New England Biolabs Co.) in order to express a large amount of the elicitor-binding domain in E. coli. PCR was performed to produce a DNA molecule encoding the elicitor-binding domain. The following primers were synthesized with an automatic nucleic acid synthesizer (Model 394 of Applied Biosystems Co.):

O Primer U36 5'-ATGGATCCGAATATAACTGGGAGAAG-3 ¹ ; and

Primer U 3 9 5'-TAGTCGACTACTTCCTACCCCATGCTGG-3 '

The DNA molecule of pER23-l (0.01 μ g) was dissolved in 79 μ 1 of distilled water. Either a combination of primers U5 and 07 or a combination of primers U10 and 012 (100 pmol each) and 0.5/z 1 of Taq DNA polymerase (TAKARA SHUZO CO., LTD.) were added to 8μ 1 of 2.5 mM dNTP in 10 μ 1 of a 10 x PCR buffer (attached to Taq DNA polymerase of TAKARA SHUZO CO., LTD.) to give a final amount of 100 μ 1. PCR was performed with a Gene Amp PCR System 9600 (Perkin-Elmer Co.) by 30 cycles of 1) denaturation at 94‘C x 30 seconds, 2) renaturation at 55‘C

AP/P/ 9 6 / 0 0 7 8 9

AP. Ο Ο 6 Ο 5 χ 30 seconds and 3) elongation at 72 °C x 1 minute. After the reaction, 15μ 1 of the reaction solution was digested with restriction enzymes BamHI and Sail and electrophoresed on a 1% agarose gel.

The gel was stained with a 0.5 μ g/ml ethidium bromide solution for 15 minutes. The band showing specific amplification was sectioned while observing under UV light. The gel section was treated with Gene Clean II (BiolOl Co.) to collect a DNA-containing solution. The collected DNA fragments were cloned into the BamHI-Sall site of plasmid pMAL-c2 and the clones were introduced into E. coli DH5a .

2) Purification of the Fused Protein Expressed in E. coli and Production of Antibody

The E. coli cells transformed with the plasmids were precultured in an expression medium (10g/l tryptone (Gibco Co.), 5 g/1 yeast extract (Gibco Co.), 5 g/1 NaCl, 2 g/1 glucose and 100 μ g/ml ampicillin) overnight. The precultured solution (150 ml) was added to 1.5 L of the expression medium and cultured in a Sakaguchi flask at 37°C with shaking until OD««_g of 0.55 was reached. Isopropylthiogalactoside was added to the culture solution to give a final concentration of 0.3 mM and the shakeculture was continued for an additional 4 hours to induce expression. The E. coli was collected by centrifugation and the E. coli cells were washed with a washing buffer (20 mM Tris-HCl, pH 7.4, 200 mM NaCl and 1 mM EDTA). The cells were sonicated for a total of 2 minutes (15 sec x 8). A soluble protein fraction was obtained by centrifugation. From this fraction, a MBP-fused protein was purified with an amylose resin. A MBP- and an elicitor-binding domain were cleaved with factor Xa and the elicitor-binding domain was purified by gel filtration column chromatography. The purified protein was injected twice into a mouse at the abdominal cavity for immunization by

P/ 96 /00789

7

AP.00605 the method of E. Harlow and D. Lane (Antibody (1988) Cold Spring Harbor

Co., pp. 53-137). After the increase in titer was confirmed by an

ELISA method, the ascites was obtained and subjected to precipitation with 50% saturated ammonium sulfate and treatment with Protein A Sepharose (Pharmacia Co.) to produce a purified antibody. In the treatment with Protein A Sepharose, the antibody was bound to Protein A Sepharose with 0.1 M sodium phosphate (pH 8.0) and eluted with 0.1 M citric acid (pH 3.5). It was confirmed by an immnoblotting that the obtained antibody recognized only the ER protein in soybean.

©

3) Preparation of Soybean Cotyledon Membrane Fraction

A soybean cytoledon membrane fraction was prepared as follows: Soybean seeds were cultured on soil for 9 days and the cytoledons were harvested (36 g, wet weight). The cytoledons were suspended in 47 ml of an ice-cold buffer (25 mM Tris-HCl pH 7.0, 30 mM MgCli , 2 mM dithiothreitol, 2.5 mM sodium metabisulfite and 1 mM PMSF). A cytoledon membrane fraction was prepared by the same method as the one for preparing the membrane fraction from the soybean roots. The resulting cytoledon membrane fraction was suspended in 1 ml of an ice-cold buffer o (10 mM Tris-HCl pH 7.4, 0.1 M sucrose, 5 mM MgCli, 1 mM PMSF and 5 mM

EDTA) and stored at -80 °C .

4) Measurement of Inhibition of Glucah Elicitor Binding to Elicitorbinding Protein of Soybean Cotyledon Membrane Fraction

The elicitor-binding activity was determined as follows:

A complex of an elicitor and tyramine (TOKYO KASEI KOGYO CO., LTD.) was synthesized by the method of Jong-Joo Cheong (The Plant Cell (1991)

3: 127). The elicitor-tyramine complex was labelled with iodine-125 using chloramine ,T. The soybean cotyledon membrane faction (100μ 1, 820

AP/P/ 96/00789

AP. Ο Ο 6 Ο 5

μ g) was suspended in 500 μ 1 of an assay buffer (50 mM Tris-HCl pH7.4, 0.1 M saccharose, 5 mM MgClj , ImM PMSF and 5 mM EDTA) and incubated at O’C for 2 hours. The iodine-labelled elicitor-tyramine complex in an amount of 714 ng (143 nM; 70 Ci/mmol) was added to the suspension and the mixture was incubated at 4 ‘C for 2 hours. The reaction solution was filtered through Whatman GF/B (as treated with a 0.3% aqueous solution of polyethylenimine for at least 1 hour) and washed 3 times with 5 ml of an ice-cold buffer (10 mM Tris-HCl pH 7.0, 1 M NaCl, 10 mM MgClj ). The radio activity retained on the filter membrane was counted with a gamma counter (count A). In order to eliminate the effect of non-specific binding, the same procedure as above was performed, except that 100 times mole (75# g, 15 μ M) of a cold elicitor was added to the the sample, the mixture was suspended in the assay buffer and the suspension was incubated at O’C for 2 hours. The obtained count was subtracted from the count A to give a count ( Δ cpm) of elicitor-specific binding. Counts of binding obtained by adding 3.6, 7.1, 10.8, 14.4 and 28.8 μ g of the purified antibody rather than the cold elicitor were subtracted from the count A. The resulting values were compared with that for the cold elicitor and expressed as the percentage, with the count ( Δ cpm) of elicitor-specific binding being taken as 100% (Figure 7). The addition of 28.8 μ g of the antibody resulted in the inhibition of the binding of elicitor by about 51%. The results confirmed that the antibody against the elicitorbinding domain inhibited the binding of the elicitor to the elicitorbinding protein.

AP/P/ 96/00789

5) Inhibition of Accumulation of Phytoalexin by Antibody against Elicitor-binding Domain

The amount of phytoalexin accumulated by the action of glucan

AP. Ο Ο 6 Ο 5 elicitor was measured with soybean cotyledons by the method of M.G. Hahn et al. ((1992) Molecular Plant Pathology Volume II A Practical Approach, IRL Press, pp. 117-120).

A purified antibody against the elicitor-binding domain (0, 1, 2, 3, 4, 10 and 20 μ g/Σδμ 1/cotyledon) or a purified antibody against yeast-derived dsRNAse, pac 1 (4, 10 and 20 μ g/25μ 1/cotyledon) as a control was added to soybean cotyledons and the mixture was incubated for 1 hour. Glucan elicitor (200 ng/25 μ 1/cotyledon) was added to the soybean cotyledons and the mixture was incubated for 20 hours to determine whether the accumulation of phytoalexin by the action of glucan elicitor was inhibited by the antibody. The amount of phytoalexin accumulation induced by the addition of elicitor subsequent to the addition of the antibody was expressed as the percentage, with the amount of phytoalexin accumulation by the sole addition of the elicitor being taken as 100% (Figure 8). When the antibody against the elicitor-binding domain was added in an amount of 20.0 μ g per soybean cotyledon, the amount of phytoalexin accumulation decreased by about 53%. In the control, the amount of phytoalexin accumulation changed little even when the antibody against pac 1 was added in an amount of 2O.C^g per soybean cotyledon. These results showed that the obtained gene did not encode a mere elicitor-binding protein but encoded the ER inducing a resistance reaction in soybean.

AP/P/ 96/00789

INDUSTRIAL APPLICABILITY

According to the present invention, a glucan elicitor receptor, DNA molecules coding for the glucan elicitor receptor and fragments thereof, vectors containing the DNA molecules or fragments thereof, and plant cells transformed with the DNA molecules or fragments thereof are provided.

0

AP·00605

The ER of the present invention is useful in the elucidation of resistance to fungi and the development of elicitor derivatives capable of inducing fungal resistance, and it can be used as an antigen for the production of antibodies against ERs.

The DNA molecules of the present invention which contain nucleotide sequences coding for the glucan elicitor receptor and fragments thereof are useful as materials for establishing techniques for developing fungi-resistant plants. In other words, the DNA molecules of the present invention and fragments thereof may be introduced and expressed in various plants to enhance their fungal resistance.

Antibodies against the glucan elicitor receptor of the present invention, the DNA molecules of the present invention which contain nucleotide sequences coding for the glucan elicitor receptor, their mutants and anti-sense DNAs can be used in the studies of the elicitorbinding site of ER and signal transduction.

Further, the information on the amino acid sequence of ER and the nucleotide sequence coding therefor can be used in the studies of the elicitor-binding site of ER and the signal transduction in which ER is f involved.

o

AP/P/ 96/00789

AP. Ο Ο 6 Ο 5

Sequence Listing

INFORMATION FOR SEQ ID NO:1

LENGTH: 667 amino acids

TYPE: amino acid

TOPOLOGY: linear

MOLECULE TYPE: peptide

SEQUENCE DESCRIPTION: SEQ ID NO:1:

Val

Asn

He

Gin

Thr

Asn

Thr

Ser

Tyr

He

Phe

Pro

Gin

Thr

Gin

Ser

Thr

Val

1

5

10

15

Leu

Pro

Asp

Pro

Ser

Lys

Phe

Phe

Ser

Ser

Asn

Leu

Leu

Ser

Ser

Pro

Leu

Pro

20

25

30

35

Thr

Asn

Ser

Phe

Phe

Gin

Asn

Phe

Val

Leu

Lys

Asn

Gly

Asp

Gin

Gin

Glu

Tyr

40

45

50

cz>

OO

lie

His

Pro

Tyr

Leu

He

Lys

Ser

Ser

Asn

Ser

Ser

Leu

Ser

Leu

Ser

Tyr

Pro

55

60

65

70

co co

Ser

Arg

Gin

Ala

Ser

Ser

Ala

Val

He

Phe

Gin

Val

Phe

Asn

Pro

Asp

Leu

Thr

(O

75

80

85

90

cn

lie

Ser

Ala

Pro

Gin

Gly

Pro

Lys

Gin

Gly

Pro

Pro

Gly

Lys

His

Leu

lie

Ser

£:

95

100

105

CL <

Ser

Tyr

Ser

Asp

Leu

Ser

Val

Thr

Leu

Asp

Phe

Pro

Ser

Ser

Asn

Leu

Ser

Phe

110

115

120

125

Phe

Leu

Val

Arg

Gly

Ser

Pro

Tyr

Leu

Thr

Val

Ser

Val

Thr

Gin

Pro

Thr

Pro

130

135

140

Leu

Ser

He

Thr

Thr

lie

His

Ser

lie

Leu

Ser

Phe

Ser

Ser

Asn

Asp

Ser

Asn

145

150

155

160

Thr

Lys

Tyr

Thr

Phe

Gin

Phe

Asn

Asn

Gly

Gin

Thr

Trp

Leu

Leu

Tyr

Ala

Thr

165

170

175

180

Ser

Pro

lie

Lys

Leu

Asn

His

Thr

Leu

Ser

Glu

lie

Thr

Ser

Asn

Ala

Phe

Ser

185

190

195

2

AP .0 0 6 0 5

Gly He 200

He Arg

He Ala

Leu 205

Leu Pro Asp Ser Asp Ser Lys His Glu Ala

Val

210

215

Leu

Asp

Lys

Tyr

Ser

Ser

Cys

Tyr

Pro

Val

Ser

Gly

Lys

Ala

Val

Phe

Arg

Glu

220

225

230

Pro

Phe

Cys

Val

Glu

Tyr

Asn

Trp

Glu

Lys

Lys

Asp

Ser

Gly

Asp

Leu

Leu

Leu

235

240

245

250

Leu

Ala

His

Pro

Leu

His

Val

Gin

Leu

Leu

Arg

Asn

Gly

Asp

Asn

Asp

Val

Lys

255

260

265

270

lie

Leu

Glu

Asp

Leu

Lys

Tyr

Lys

Ser

He

Asp

Gly

Asp

Leu

Val

Gly

Val

Val

275

280

285

Gly

Asp

Ser

Trp

Val

Leu

Lys

Thr

Asp

Pro

Leu

Phe

Val

Thr

Trp

His

Ser

He

290

295

300

305

Lys

Gly

He

Lys

Glu

Glu

Ser

His

Asp

Glu

He

Val

Ser

Ala

Leu

Ser

Lys

Asp

310

315

320

Val

Glu

Ser

Leu

Asp

Ser

Ser

Ser

lie

Thr

Thr

Thr

Glu

Ser

Tyr

Phe

Tyr

Gly

325

330

335

340

Lys

Leu

He

Ala

Arg

Ala

Ala

Arg

Leu

Val

Leu

He

Ala

Glu

Glu

Leu

Asn

Tyr

345

350

355

360

Pro

Asp

Val

He

Pro

Lys

Val

Arg

Asn

Phe

Leu

Lys

Glu

Thr

He

Glu

Pro

Trp

365

370

375

Leu

Glu

Gly

Thr

Phe

Ser

Gly

Asn

Gly

Phe

Leu

His

Asp

Glu

Lys

Trp

Gly

Gly

380

385

390

395

lie

He

Thr

Gin

Lys

Gly

Ser

Thr

Asp

Ala

Gly

Gly

Asp

Phe

Gly

Phe

Gly

He

400

405

410

Tyr

Asn

Asp

His

His

Tyr

His

Leu

Gly

Tyr

Phe

lie

Tyr

Gly

He

Ala

Val

Leu

415

420

425

430

Thr

Lys

Leu

Asp

Pro

Ala

Trp

Gly

Arg

Lys

Tyr

Lys

Pro

Gin

Ala

Tyr

Ser

lie

435

440

445

450

Val

Gin

Asp

Phe

Leu

Asn

Leu

Asp

Thr

Lys

Leu

Asn

Ser

Asn

Tyr

Thr

Arg

Leu

AP/P/ 9 6 / 0 0 7 8 9

AP . 0 0 6 0 5

455 460 465

Arg Cys 470

Phe Asp Pro

Tyr

Val 475

Leu His

Ser

Trp Ala Gly Gly Leu Thr Glu

Phe

480

485

Thr

Asp

Gly

Arg

Asn

Gin

Glu

Ser

Thr

Ser

Glu

Ala

Val

Ser

Ala

Tyr

Tyr

Ser

490

495

500

Ala

Ala

Leu

Met

Gly

Leu

Ala

Tyr

Gly

Asp

Ala

Pro

Leu

Val

Ala

Leu

Gly

Ser

505

510

515

520

Thr

Leu

Thr

Ala

Leu

Glu

lie

Glu

Gly

Thr

Lys

Met

Trp

Trp

His

Val

Lys

Glu

525

530

535

540

Gly

Gly

Thr

Leu

Tyr

Glu

Lys

Glu

Phe

Thr

Gin

Glu

Asn

Arg

Val

Met

Gly

Val

545

550

555

Leu

Trp

Ser

Asn

Lys

Arg

Asp

Thr

Gly

Leu

Trp

Phe

Ala

Pro

Ala

Glu

Trp

Lys

cn

co

560

565

570

575

Γ.

Glu

Cys

Arg

Leu

Gly

Ile

Gin

Leu

Leu

Pro

Leu

Ala

Pro

Ile

Ser

Glu

Ala

Ile

o

580

585

590

o

<£>

Phe

Ser

Asn

Val

Asp

Phe

Val

Lys

Glu

Leu

Val

Glu

Trp

Thr

Leu

Pro

Ala

Leu

cn

595

600

605

610

a

Asp

Arg

Glu

Gly

Gly

Val

Gly

Glu

Gly

Trp

Lys

Gly

Phe

Val

Tyr

Ala

Leu

Glu

ix

-s

615

620

625

630

<

0

Gly

Val

Tyr

Asp

Asn

Glu

Ser

Ala

Leu

Gin

Lys

Ile

Arg

Asn

Leu

Lys

Gly

Phe

635

640

645

Asp

Gly

Gly

Asn

Ser

Leu

Thr

Asn

Leu

Leu

Trp

Trp

Ile

His

Ser

Arg

Ser

Asp

650

655

660

665

Glu

667

INFORMATION

FOR

SEQ

ID NO: 2

LENGTH: 2004 base pairs

TYPE: nucleic acid

AP. Ο Ο 6 Ο 5

Ε ·1

STRANDEDNESS: double

TOPOLOGY: linear

MOLECULE TYPE: CDNA

ORIGINAL SOURCE:

ORGANISM: Soybean (Glycine max L.)

STRAIN: Green Homer

SEQUENCE DESCRIPTION: SEQ ID NO: 2:

		9			18			27
GTT	AAC	ATC	CAA	ACC	AAT	ACA	TCT	TAC	ATC
Val	Asn	lie	Gin	Thr	Asn	Thr	Ser	Tyr	He
		63			72			81
CTT	CCT	GAT	CCC	TCC	AAA	TTC	TTC	TCC	TCA
Leu	Pro	Asp	Pro	Ser	Lys	Phe	Phe	Ser	Ser
		117			126			135
ACA	AAC	TCT	TTC	TTC	CAA	AAC	TTT	GTC	CTA
Thr	Asn	Ser	Phe	Phe	Gin	Asn	Phe	Val	Leu
		171			180			189
ATT	CAT	CCT	TAC	CTC	ATC	AAA	TCC	TCC	AAC
He	His	Pro	Tyr	Leu	He	Lys	Ser	Ser	Asn
		225			234			243
TCT	CGC	CAA	GCC	AGT	TCA	GCT	GTC	ATA	TTC
Ser	Arg	Gin	Ala	Ser	Ser	Ala	Val	He	Phe

	36			45			54
TTC	CCT	CAA	ACA	CAA	TCC	ACT	GTT
Phe	Pro	Gin	Thr	Gin	Ser	Thr	Val

	90	99	108	CD
AAC	CTT	CTC	TCA	AGT	CCA	CTC	CCC	OO
								r-.
Asn	Leu	Leu	Ser	Ser	Pro	Leu	Pro	c

CS

AAA Lys	144 AAT Asn 198	153 GGT GAC CAA CAA GAA	162 TAC Tyr 216	Cn £ £ <
Gly Asp	Gin 207	Gin	Glu
TCT	TCC	CTC	TCT	CTC	TCA	TAC	CCT
Ser	Ser	Leu	Ser	Leu	Ser	Tyr	Pro

	252			261			270
CAA	GTC	TTC	AAT	CCT	GAT	CTT	ACC
Gin	Val	Phe	Asn	Pro	Asp	Leu	Thr

279 288 297 306 315 324

ATT TCA GCC CCA CAA GGT CCC AAA CAA GGT CCC CCT GGT AAA CAC CTT ATC TCC

AP . 0 0 6 0 5

lie

Ser

Ala

Pro

Gin

Gly Pro Lys

Gin

Gly

Pro

Pro

Gly

Lys

His

Leu

Ile

Ser

333

342

351

360

369

378

TCC

TAC

AGT

GAT

CTC

AGT GTC ACC

TTG

GAT

TTC

CCT

TCT

TCC

AAT

CTG

AGC

TTC

Ser

Tyr

Ser

Asp

Leu

Ser Val Thr

Leu

Asp

Phe

Pro

Ser

Ser

Asn

Leu

Ser

Phe

387

396

405

414

423

432

TTC

CTT

GTT

AGG

GGA

AGC CCC TAT

TTG

ACT

GTG

TCT

GTG

ACT

CAA

CCA

ACT

CCT

Phe

Leu

Val

Arg

Gly

Ser Pro Tyr

Leu

Thr

Val

Ser

Val

Thr

Gin

Pro

Thr

Pro

441

450

459

468

477

486

CTT

TCA

ATT

ACC

ACC

ATC CAT TCC

ATT

CTC

TCA

TTC

TCT

TCA

AAT

GAC

TCC

AAC

Leu

Ser

Ile

Thr

Thr

Ile His Ser

Ile

Leu

Ser

Phe

Ser

Ser

Asn

Asp

Ser

Asn

495

504

513

522

531

540

ACC

AAG

TAC

ACC

TTT

CAG TTC AAC

AAT

GGT

CAA

ACA

TGG

CTT

CTT

TAT

GCT

ACC

Thr

Lys

Tyr

Thr

Phe

Gin Phe Asn

Asn

Gly

Gin

Thr

Trp

Leu

Leu

Tyr

Ala

Thr

549

558

567

576

585

594

TCC

CCC

ATC

AAG

TTG

AAC CAC ACC

CTT

TCT

GAG

ATA

ACT

TCT

AAT

GCA

TTT

TCT

Ser

Pro

Ile

Lys

Leu

Asn His Thr

Leu

Ser

Glu

Ile

Thr

Ser

Asn

Ala

Phe

Ser

603

612

621

630

639

648

GGC

ATA

ATC

CGG

ATA

GCT TTG TTG

CCG

GAT

TCG

GAT

TCG

AAA

CAC

GAG

GCT

GTT

Gly

Ile

Ile

Arg

Ile

Ala Leu Leu

Pro

Asp

Ser

Asp

Ser

Lys

His

Glu

Ala

Val

657

666

675

684

693

702

CTT

GAC

AAG

TAT

AGT

TCT TGT TAC

CCC

GTG

TCA

GGT

AAA

GCT

GTG

TTC

AGA

GAA

Leu

Asp

Lys

Tyr

£er

Ser Cys Tyr

Pro

Val

Ser

Gly

Lys

Ala

Val

Phe

Arg

Glu

ΑΡ/Ρ/ 96/00789

6

AP. Ο Ο 6 Ο 5

711

720

729

738

747

756

CCT

TTC

TGT

GTG

GAA

TAT

AAC

TGG

GAG

AAG

AAA

GAT

TCA

GGG

GAT

TTG

CTA

CTC

Pro

Phe

Cys

Val

Glu

Tyr

Asn

Trp

Glu

Lys

Lys

Asp

Ser

Gly

Asp

Leu

Leu

Leu

765

774

783

792

801

810

TTG

GCT

CAC

CCT

CTC

CAT

GTT

CAG

CTT

CTT

CGT

AAT

GGA

GAC

AAT

GAT

GTC

AAA

Leu

Ala

His

Pro

Leu

His

Val

Gin

Leu

Leu

Arg

Asn

Gly

Asp

Asn

Asp

Val

Lys

819

828

837

846

855

864

ATT

CTT

GAA

GAT

TTA

AAG

TAT

AAA

AGC

ATT

GAT

GGG

GAT

CTT

GTT

GGT

GTT

GTC

lie

Leu

Glu

Asp

Leu

Lys

Tyr

Lys

Ser

He

Asp

Gly

Asp

Leu

Val

Gly

Val

Val

873

882

891

900

909

918

GGG

GAT

TCA

TGG

GTT

TTG

AAA

ACA

GAT

CCT

TTG

TTT

GTA

ACA

TGG

CAT

TCA

ATC

Gly

Asp

Ser

Trp

Val

Leu

Lys

Thr

Asp

Pro

Leu

Phe

Val

Thr

Trp

His

Ser

He

927

936

945

954

963

972

AAG

GGA

ATC

AAA

GAA

GAA

TCC

CAT

GAT

GAG

ATT

GTC

TCA

GCC

CTT

TCT

AAA

GAT

Lys

Gly

Xie

Lys

Glu

Glu

Ser

His

Asp

Glu

He

Val

Ser

Ala

Leu

Ser

Lys

Asp

981

990

999

1008

1017

1026

GTT

GAG

AGC

CTA

GAT

TCA

TCA

TCA

ATA

ACT

ACA

ACA

GAG

TCA

TAT

TTT

TAT

GGG

Val

Glu

Ser

Leu

Asp

Ser

Ser

Ser

lie

Thr

Thr

Thr

Glu

Ser

Tyr

Phe

Tyr Gly

1035

1044

1053

1062

1071

1080

AAG

TTG

ATT

GCA

AGG

GCT

GCA

AGG

TTG

GTA

TTG

ATT

GCT

GAG

GAG

TTG

AAC

TAC

Lys

Leu

He

Ala

Arg

Ala

Ala

Arg

Leu

Val

Leu

He

Ala

Glu

Glu

Leu

Asn Tyr

ΑΡ/Ρ/ 9 6 / 0 0 7 8 9

AP.0 0 6 0 5

1089

1098

1107

1116

1125

1134

CCT

GAT

GTG ATT

CCA

AAG

GTT

AGG

AAT

TTT

TTG AAA

GAA

ACC ATT

GAG

CCA TGG

Pro

Asp

Val lie

Pro

Lys

Val

Arg

Asn

Phe

Leu Lys

Glu

Thr lie

Glu

Pro Trp

1143

1152

1161

1170

1179

1188

TTG

GAG

GGA ACT

TTT

AGT

GGG

AAT

GGA

TTC

CTA CAT

GAT

GAA AAA

TGG

GGT GGC

Leu

Glu

Gly Thr

Phe

Ser

Gly

Asn

Gly

Phe

Leu His

Asp

Glu Lys

Trp

Gly Gly

1197

1206

1215

1224

1233

1242

ATT

ATT

ACC CAA

AAG

GGG

TCC

ACT

GAT

GCT

GGT GGT

GAT

TTT GGA

TTT

GGA ATT

lie

He

Thr Gin

Lys

Gly

Ser

Thr

Asp

Ala

Gly Gly

Asp

Phe Gly

Phe

Gly He

1251

1260

1269

1278

1287

1296

TAC

AAT

GAT CAC

CAC

TAT

CAT

TTG

GGG

TAC

TTC ATT

TAT

GGA ATT

GCG

GTG CTC

Tyr

Asn

Asp His

His

Tyr

His

Leu

Gly

Tyr

Phe He

Tyr

Gly He

Ala

Val Leu

1305

1314

1323

1332

1341

1350

ACT

AAG

CTT GAT

CCA

GCA

TGG

GGT

AGG

AAG

TAC AAG

CCT

CAA GCC

TAT

TCA ATA

Thr

Lys

Leu Asp

Pro

Ala

Trp

Gly

Arg

Lys

Tyr Lys

Pro

Gin Ala

Tyr

Ser He

1359

1368

1377

1386

1395

1404

GTG

CAA

GAC TTC

TTG

AAC

TTG

GAC

ACA

AAA

TTA AAC

TCC

AAT TAC

ACA

CGT TTG

Val

Gin

Asp Phe

Leu

Asn

Leu

Asp

Thr

Lys

Leu Asn

Ser

Asn Tyr

Thr

Arg Leu

1413

1422

1431

1440

1449

1458

AGG

TGT

TTT GAC

CCT

TAT

GTG

CTT

CAC

TCT

TGG GCT

GGA

GGG TTA

ACT

GAG TTC

Arg

Cys

Phe Asp

Pro

Tyr

Val

Leu

His

Ser

Trp Ala

Gly

Gly Leu

Thr

Glu Phe

1467

1476

1485

1494

1503

1512

AP/P/ 9 6 / 0 0 7 8 9

AP.00605

ACA GAT GGA	AGG AAT CAA Arg Asn Gin	GAG AGC ACA AGT GAG GCT GTG AGT GCA TAT TAT TCT
Thr	Asp	Gly	Glu Ser Thr	Ser Glu Ala Val	Ser Ala	Tyr Tyr Ser
		1521	1530	1539	1548	1557	1566
GCT	GCT	TTG	ATG GGA TTA	GCA TAT GGT	GAT GCA CCT CTT	GTT GCA	CTT GGA TCA
Ala	Ala	Leu	Met Gly Leu	Ala Tyr Gly	Asp Ala Pro Leu	Val Ala	Leu Gly Ser
		1575	1584	1593	1602	1611	1620
ACA	CTC	ACA	GCA TTG GAA	ATT GAA GGG	ACT AAA ATG TGG	TGG CAT	GTG AAA GAG
Thr	Leu	Thr	Ala Leu Glu	He Glu Gly	Thr Lys Met Trp	Trp His	Val Lys Glu
		1629	1638	1647	1656	1665	1674
GGA	GGT	ACT	TTG TAT GAG	AAA GAG TTT	ACA CAA GAG AAT	AGG GTG	ATG GGT GTT
Gly	Gly	Thr	Leu Tyr Glu	Lys Glu Phe	Thr Gin Glu Asn	Arg Val	Met Gly Val
		1683	1692	1701	1710	1719	1728
CTA	TGG	TCT	AAC AAG AGG	GAC ACT GGA	CTT TGG TTT GCT	CCT GCT	GAG TGG AAA
Leu	Trp	Ser	Asn Lys Arg	Asp Thr Gly	Leu Trp Phe Ala	Pro Ala	Glu Trp Lys
		1737	1746	1755	1764	1773	1782
GAG	TGT	AGG	CTT GGC ATT	CAG CTC TTA	CCA TTG GCT CCT	ATT TCT	GAA GCC ATT
Glu	Cys	Arg	Leu Gly lie	Gin Leu Leu	Pro Leu Ala Pro	lie Ser	Glu Ala He
		1791	1800	1809	1818	1827	1836
TTC	TCC	AAT	GTT GAC TTT	GTA AAG GAG	CTT GTG GAG TGG	ACT TTG	CCT GCT TTG
Phe	Ser	Asn	Val Asp Phe	Val Lys Glu	Leu Val Glu Trp	Thr Leu	Pro Ala Leu
		1845	1854	1863	1872	1881	1890
GAT	AGG	GAG	GGT GGT GTT	GGT GAA GGA	TGG AAG GGG TTT	GTG TAT	GCC CTT GAA

AP/P/ 96/00789

AP . Ο Ο β Ο 5

Asp

Arg

Glu

Gly

Gly

Val

Gly

Glu Gly

Trp

Lys

Gly

Phe

Val

Tyr

Ala

Leu

Glu

1899

1908

1917

1926

1935

1944

GGG

GTT

TAT

GAC

AAT

GAA

AGT

GCA CTG

CAG

AAG

ATA

AGA

AAC

CTG

AAA

GGT

TTT

Gly

Val

Tyr

Asp

Asn

Glu

Ser

Ala Leu

Gin

Lys

Ile

Arg

Asn

Leu

Lys

Gly

Phe

1953

1962

1971

1980

1989

1998

GAT

GGT

GGA

AAC

TCT

TTG,

ACC

AAT

CTC

TTG

TGG

TGG

ATT

CAT

AGC

AGA

AGT GAT

Asp

Gly

Gly

Asn

Ser

Leu

Thr

Asn

Leu

Leu

Trp

Trp

lie

His

Ser

Arg

Ser Asp

C

2004

GAA TAG

Glu

INFORMATION FOR SEQ ID NO: 3

LENGTH: 54 base pairs

TYPE: nucleic acid

STRANDEDNESS: single

TOPOLOGY: linear

MOLECULE TYPE: Other nucleic acid, synthetic DNA

SEQUENCE DESCRIPTION: SEQ ID NO: 3:

GGAATTCGAG CTCGGTACCC GGGGGATCCT CTAGAGTCGA CCTGCAGGCA TGCA

AP/P/ 96/00789

INFORMATION FOR SEQ ID NO: 4

LENGTH: 58 base pairs

TYPE: nucleic acid

STRANDEDNESS: single

TOPOLOGY: linear

MOLECULE TYPE: Other nucleic acid, synthetic DNA

AP.00605

SEQUENCE DESCRIPTION: SEQ ID NO: 4:

CCTTAAGCTC GAGCCATGGG CCCCCTAGGA GATCTCAGCT GGACGTCCGT ACGTTCGA

Claims (6)

1. A glucan elicitor receptor having an amino acid sequence as substantially shown in SEQ ID NO:1.

2. A DNA molecule containing a nucleotide sequence coding for a glucan elicitor receptor having an amino acid sequence as substantially shown in SEQ ID NO:1, or fragments thereof.

3. The DNA molecule of claim 2, wherein the nucleotide sequence coding for a glucan elicitor receptor is as shown in SEQ ID NO:2, or fragments thereof.

4. A DNA molecule containing a nucleotide sequence coding for a glucan elicitor receptor, which is incorporated in plasmid pER23-l, or fragments thereof.

5. A vector containing the DNA molecule coding for a glucan elicitor receptor of any one of claims 2-4 or a fragment thereof.

6. A plant cell transformed with the DNA molecule coding for a glucan elicitor receptor of any one of claims 2-4 or a fragment thereof.