JP2004248597A - New elicitor peptide (fructose 1, 6-bisphosphate aldolase homologous protein) obtained from phytophthora - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a protein having activities of inducing the synthesis of phytoalexin of a low molecular antimicrobial agent carrying out the defense reaction against invasion of microorganisms, so-called elicitor activities. <P>SOLUTION: The elicitor peptide is the protein containing a specific amino acid sequence derived from phytophthora infestance, and having the elicitor activities and 38 kDa molecular weight. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【図面の簡単な説明】
【図１】図１はフィトフトラ・インフェスタンスＤＨ１０１ 株の菌体からの粗抽出物のＦＰＬＣにおける溶出プロファイルを示す図である。
【図２】図２は、図１における溶出画物と、抗−ＨＷＣモノクローナル抗体との反応性を示すグラフである。
【図３】図３は、図１における画分の、ポテト塊茎組織に対する褐色化効果を示す写真であり、生物の形態を示す図面代用写真である。
【図４】図４において、Ａは図１における画分Ｆ１７ 〜Ｆ２０ をＳＤＳ−ＰＡＧＥにより分離し、銀染色した結果を示し、Ｂは抗−ＨＷＣモノクローナル抗体により検出した結果を示し、いずれも電気泳動の結果を示す図面代用写真である。
【図５】図５において、Ａは、図１における画分Ｆ１９ 及びＦ２０ を、抗−ＨＷＣモノクローナル抗体Ａｂｓ−２Ａ１１によりアフィニティー精製して銀染色した結果を示し、Ｂはウエスタンブロットにより検出した結果を示し、いずれも電気泳動の結果を示す図面代用写真である。
【図６】図６のＡは、アフィニティー精製した本発明のタンパク質がポテト塊茎組織に対して褐色化作用（ＨＲの誘導）を有することを示す写真であり、生物の形態を示す図面代用写真であり、Ｂはアフィニティー精製した本発明のタンパク質がポテト塊茎組織において活性酵素の発生を誘導することを示すグラフである。
【図７】図７は、３８ｋＤａ の生来のエリシタータンパク質と、そのプロテアーゼ分解物のＮ−末端のアミノ酸配列、及びそれに基いて設計されたプライマー又はプローブ用オリゴヌクレオチドの塩基配列を示す図である。
【図８】図８は、フィトフトラ属の種々の微生物に、３８ｋＤａ エリシタータンパク質をコードする遺伝子が分布していることを示す電気泳動図であり、図面代用写真である。
【図９】図９は、フィトフトラ・インフェスタンス（Ｐｈｙｔｏｐｈｔｈｏｒａ ｉｎｆｅｓｔａｎｓ）の遊走子からの発芽濾液中のタンパク質の、抗−ＨＷＥ抗体による検出を示す電気泳動図であり、図面代用写真である。図中、レーンＡは、フィトフトラ・インフェスタンス（Ｐｈｙｔｏｐｈｔｈｏｒａ ｉｎｆｅｓｔａｎｓ）からの可溶性サンプルの結果を示し、そしてレーンＢは、フィトフトラ・インフェスタンス（Ｐｈｙｔｏｐｈｔｈｏｒａ ｉｎｆｅｓｔａｎｓ）の遊走子からの発芽濾液の結果を示す。
【図１０】図１０は、本発明の精製した組換えタンパク質の、ＳＤＳ−ＰＡＧＥ（パネルＡ）、イムノブロット（パネルＢ）、ポテト塊茎アッセイ（パネルＣ）及び懸濁培養におけるＣＬＡ指数を示す。パネルＡ及びＢは電気泳動の結果を示す図面代用写真であり、パネルＣは生物の形態を表す図面代用写真である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a novel protein having elicitor activity, which is a substance involved in plant defense, and a method for producing the same.
[0002]
[Prior art]
The defense response of higher plants against the invasion of microorganisms includes the synthesis of the low-molecular-weight antibacterial substance phytoalexin, the accumulation of lignin as a physical defense wall in infected tissues and the accumulation of callose, a main component of papillae, and the accumulation of lignin. Accumulation of glycoproteins rich in hydroxyproline in the cell wall, synthesis of chitinase and glucanase having the function of decomposing the cell wall of invading bacteria and causing elicitor molecules from invading bacteria or autologous cell walls, and proteinase inhibition that inhibits nutrient intake of invading bacteria Synthesis of substances.
[0003]
A substance that induces phytoalexin synthesis, which is one of the above defense reactions, is called an elicitor. Elicitors include those derived from phytopathogenic fungi and endogenous elicitors in plants. Examples of elicitors derived from phytopathogens include β-1,3-D-glucan, glycoproteins rich in galactose and mannose, chitosan, arachidonic acid, and the like. The elicitor derived from the genus Phytophthora is 1,3-β-D-glucan (Ayers, AR et al., Plant Physiol., Vol. 57, p. 760-). 765 (1976)), glycoproteins (Keen, P. et al., Physiol. Plant. Pathol. Vol. 26, p. 343-355 (1985)), chitosan (Shibuya, N., et al., Biology of Biology). Plant-Micr be Interaction Vol.2, p.109-113 (1999)), and arachidonic acid (Bastock, R.M.et al., Science Vol.212, p.67-69 (1981)) it is known.
[0004]
A small extracellular protein, produced by many species of Phytophthora and called elicitin, has been identified as exhibiting a protective response in host cells (Ricci, P. et al., Eur. J. Biochem. Vol. 183, p. 555-563 (1989)), which is a highly conserved 10 kDa protein (Ricci P., et al., Plant-Microbe Interactions, Vol. 3, G. Stacey et al., 1989). eds, p.53-75 (1997)).
[0005]
[Non-patent document 1]
Ayers, A. R. et al. , Plant Physiol. , Vol. 57, p. 760-765 (1976)
[Non-patent document 2]
Keen, P .; et al. Physiol. Plant. Pathol. Vol. 26, p. 343-355 (1985)
[Non-Patent Document 3]
Shibuya, N .; , Et al. , Biology of Plant-Microbe Interaction Vol. 2, p. 109-113 (1999)
[Non-patent document 4]
Basstock, R.A. M. et al. , Science Vol. 212, p. 67-69 (1981)
[Non-Patent Document 5]
Ricci, P .; et al. , Eur. J. Biochem. Vol. 183, p. 555-563 (1989)
[Non-Patent Document 6]
Ricci P.C. , Et al. , Plant-Microbe Interactions, Vol. 3, G. Statey et al. , Eds, p. 53-75 (1997)
[0006]
[Problems to be solved by the invention]
An object of the present invention is to provide a novel elicitor protein derived from a microorganism of the genus Phytophthora and a method for producing the same.
[0007]
[Means for Solving the Problems]
As a result of conducting various studies to solve the above problems, the present invention succeeded in isolating a novel protein having a large molecular weight and elicitor activity, and completed the present invention.
Accordingly, the present invention provides a protein having the amino acid sequence shown in SEQ ID NO: 2 and having elicitor activity.
[0008]
Generally, a physiologically active substance is a protein having an amino acid sequence modified by substitution / deletion and / or addition of one to several amino acids in the amino acid sequence of a native protein, while maintaining the same biological activity as the native protein. Is well known in the art. Therefore, the present invention provides a protein comprising an amino acid sequence modified by substitution, deletion and / or addition of one to several amino acids in the amino acid sequence of SEQ ID NO: 2, and maintaining elicitor activity Is also included.
[0009]
Further, as shown in Example 5, a gene that hybridizes to the nucleotide sequence shown in SEQ ID NO: 1 and encodes an elicitor protein is also present in other microorganism strains. Accordingly, the present invention encompasses a protein which is hybridized with a nucleic acid having the nucleotide sequence shown in SEQ ID NO: 1 under high stringency conditions, is encoded by DNA, and has elicitor activity. This DNA is preferably DNA derived from a microorganism of the genus Phytophthora.
[0010]
The present invention further relates to Phytophthora infestans comprising the amino acid sequence shown in SEQ ID NO: 2, having a molecular weight of 38 kD, and having elicitor activity.Phytophthora  infestans) Is provided.
The present invention further comprises culturing Phytophthora infestans capable of producing the above-mentioned protein, and collecting a protein having a molecular weight of 38 kD and having an elicitor activity from the culture. And a method for producing the same.
[0011]
The present invention also provides a method for producing a protein, comprising culturing a host transformed with an expression vector comprising a gene encoding the above protein, and collecting a protein having elicitor activity from the culture. I will provide a.
[0012]
BEST MODE FOR CARRYING OUT THE INVENTION
The method for isolating and purifying the protein having elicitor activity of the present invention from Phytophthora insolens and the method for cloning the gene encoding the protein are specifically described in Examples.
[0013]
The protein having elicitor activity of the present invention has the amino acid sequence shown in SEQ ID NO: 2. However, generally, a biologically active substance also has a protein having an amino acid sequence modified by substitution / deletion and / or addition of one to several amino acids in the amino acid sequence of a native protein, and has a biological activity equivalent to that of the native protein. Maintaining is well known in the art. Therefore, the present invention provides a protein comprising an amino acid sequence modified by substitution, deletion and / or addition of one to several amino acids in the amino acid sequence of SEQ ID NO: 2, and maintaining elicitor activity Is also included.
[0014]
Further, as shown in Example 5, a gene that hybridizes to the nucleotide sequence shown in SEQ ID NO: 1 and encodes an elicitor protein is also present in other microorganism strains. Accordingly, the present invention includes a protein which is hybridized with a DNA having the nucleotide sequence shown in SEQ ID NO: 1 under high stringency conditions, is encoded by the DNA, and has elicitor activity. Examples of the origin of this DNA include DNA derived from the genus Phytophthora.
[0015]
The present invention also provides genes encoding the various proteins described above. The present invention also provides a vector comprising the above gene, for example, an expression vector. The present invention also provides a host, eg, a microbial host, transformed with the vector.
[0016]
【Example】
Next, the present invention will be described more specifically with reference to examples.
Example 1.Isolation of proteins having elicitor activity
Phytophthora infestans (Mont) deBary isolate DH101 (race 0) was stored in the dark at 18 ° C. on rye agar supplemented with 2% sucrose and 0.2% Bacto yeast extract. For liquid cultures, see Furich, N.M. , Et al. , Ann. Phytopathol. Soc. Jpn. Vol. 56,. 457-467) in a synthetic medium described at 18 ° C. in the dark for 2-3 weeks. The mycelium was collected by filtration, washed and frozen at -20 ° C.
[0017]
Protein extraction was performed at 0 ° C. to 4 ° C. on a bath or in a cold room. The frozen mycelium is ground in liquid nitrogen, and the resulting powder is homogenized in a 5-fold amount of 0.2 M phosphate buffer (pH 7.2) containing 1 M NaCl, and phenylmethylsulfonyl is added thereto. Fluocud (PMSF) was added to a final concentration of 25 mM. The homogenate was sonicated for 3 minutes and centrifuged at 20,000 xg for 20 minutes at 4 ° C. The supernatant was dialyzed against cold 25 mM Tris-HCl (pH 8.1) through a porous membrane having a cut-off molecular weight of 1,000.
[0018]
The protein contained in the crude extract thus obtained was subjected to FPLC-anion exchange chromatography on a column (4.6 mm × 100 mm, 1.7 mL, PoRos QE / M; PerSeptine Biosystems) equilibrated with the above buffer. Purified. The flow rate was 5 ml / min and elution was performed with a linear gradient of 0M to 0.5M NaCl in 25mM Tris-HCl (pH 8.1). Elution was performed by absorbance at 280 nm, and fractions of 1 ml were collected. The results are shown in FIG.
[0019]
The above fractions were analyzed by ELISA to estimate the presence of the protein of interest. As an antibody, a monoclonal antibody (anti-HWC monoclonal antibody) (Abs-2A11) against a hyphal wall component (HWC) (Ikeda, R. et al., Annual Meeting of Plant-Microbe Interaction Vol. 3, p. 18). -20 (1993)). This monoclonal antibody is described in Garas, N.W. A. et al. Physiol. Plant Pathol. , Vol. 15, p. 117-126 (1979). It recognizes a proteinaceous epitope present in the HWC elicitor extracted by the method described in 117-126 (1979).
[0020]
The ELISA is described by McLaughlin, R.A. J. et al. , Phyfopathol, Vol. 79, p. 610-613 (1989). The wells of the microplate were coated with Abs-2A11 antibody diluted 1/1000 with 1% bovine serum albumin (BSA) / phosphate buffer (PBS), and the Abs-2A11 binding activity of the FPLC fraction was measured. did. As a secondary antibody, an anti-mouse immunoglobulin-alkaline phosphatase conjugate was used at a dilution of 1 to 2000 times. Absorbance was measured at 595 nm using a Microplate Header (Bio-Rad). The results are shown in FIG. In FIG. 2, C1 is a crude extract before separation by LPLC, C2 is an FLLC elution buffer, C3 is a C1 fraction treated only with the primary antibody Abs-2A11, and C4 is treated only with the secondary antibody. C1 shows the results. Maximal Abs-2A11 binding activity was detected in fractions (F) 17-20 eluted at 0.35 M NaCl. This is the result of four replicate experiments.
[0021]
Measurement of biological activity
The elicitor activity of the FPLC fractions F15 to F21 having Abs-2A11 binding activity obtained by the above method was tested using, as an index, a cell line of potato tuber tissue due to hypersensitive response (HR). Each fraction (F15 to F21) was pooled, concentrated by a Centricon-30 microconcentrator to a final protein concentration of 600 μg / ml, and the concentrated samples were aged at 18 ° C. for 16 hours for the cultivar Potato Eniwa (R).₁  Gene) and Irish cobbler (r gene) were applied in a volume of 30 μl.
[0022]
Induction of a hypersensitivity response (HR) was monitored by tuber tissue browning and cell death after 98 hours. The results are shown in FIG. Water did not induce HR, whereas elicitor-active fractions induced high intensity HR. The HR intensity was much higher in fractions F19 and F20 than in the other fractions. These results revealed that the elicitor protein was contained in fractions F19 and F20.
[0023]
Western immunoblot analysis of FPLC fraction
For FPLC fractions F17-F20 showing high elicitor activity, see Leammmli, U.S. K. et al. , Nature Vol. 227, p. 680-681 (1970) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 12.5% acrylamide for the separating gel and 4.5% acrylamide for the stack gel, according to the method described in 680-681 (1970). (SDS-PAGE).
[0024]
After electrophoresis, the gel was run on a Switcher, R.A. C. et al. , Anal. Biochem. Vol. 98, p. 231-237 (1979), or silver staining, as described in Towbin, H .; et al. , Proc. Natl. Acad. Sci. , USP, Vol. 76, p. 4350-4354 (1979), using a MilliBlot ™ -SDS System (Millipore) at 2 mA / cm.²  Was transferred to a polyvinylidene difluoride (PVDF) membrane (Immbilon-P, pore size 0.45 μM, Millipore Corp.) for 30 minutes. The membrane was then incubated for 1 hour at room temperature in blocking buffer (10 mM Tris-HCl, 150 mM NaCl and 5% skim milk, pH 7.5).
[0025]
This was washed twice in TBS-Tween20 for 5 minutes, incubated in the Abs-2A11 antibody diluted in TBS for 1 hour at room temperature, washed three times in TBS-Tween20 for 20 minutes, and washed with TBS 1/1/3. Diluted to 2000. Incubated in alkaline phosphate conjugated rabbit anti-mouse immunoglobulin G (IgG) (Bio-Rad) for 1 hour at room temperature and washed three times for 10 minutes each in TBS-Tween20. The antigen-antibody complex was detected using an alkaline phosphatase color former (Bio-Rad).
[0026]
FIG. 4 shows the results. FIG. 4A shows the result of silver staining of SDS-PAGE, and FIG. 4B shows the result of western immunoblot. As a result, fractions F17 and F18 showed a sharp band of 47 kDa, and fractions F19 and F20 showed three bands of 47 kDa, 38 kDa and 34 kDa. As a result, the elicitor activity is considered to be related to the major peptide band with an apparent molecular weight of 38 kDa. The three peptides with molecular weights of 47 kDa, 38 kDa and 34 kDa are expected to have the same epitope.
[0027]
Elicitor activity of affinity purified proteins
In order to know whether or not the affinity-purified proteins from fractions F19 and F20 had elicitor activity, affinity purification was performed using Abs-2A11 antibody. The results are shown in FIG. FIG. 5A shows the result of SDS-PAGE silver staining, and FIG. 5B shows the result of Western immunoblot. All three proteins having a molecular weight of 47 kDa, 38 kDa and 34 kDa had Abs-2A11 antibody binding activity.
[0028]
The purified samples were then pooled and concentrated on a Centricon-30 microconcentrator. The concentrated sample (30 μL) was applied to tuber tissues of potato cultivars Eniwa and Irish Cobbler aged at 18 ° C. for 16 hours. After 98 hours, induction of HR was observed by browning and cell death of tuber tissue. The results are shown in FIG. Distilled water and the flow through fraction did not induce HR.
[0029]
Measurement of active enzyme generation
Active enzymes are known to be important for the induction of HR. Elicitors are known to enhance the production of active enzymes in potato tissue. Potato cultivar Eniwa (R₁  500 μL (500 μg / ml) of the affinity-purified sample was added to 5 ml of suspension-cultured cells (cultured for 3 to 4 days) of the gene and Irish Cobbler (r gene), and the cell suspension was collected over time. The supernatant was collected by centrifugation at room temperature.
[0030]
The supernatant was mixed with 426 μL of 39 mM HEPES (pH 7.0) and 5 μL of 10 mM MgCl 2.₂Or 10 mM CaCl₂  Was added to a 15 ml sample tube containing 5 μL of 10 mM EGTA, 1.5 μL of 10 mM guanosine 5′-triphosphate (GTP) -γ-S, and 15 μL of firefly luciferase. Firefly luciferase was added last. The sample tubes were incubated at 37 ° C. for 3 minutes and the emitted light counts were measured with a luminescence reader at 15 minute intervals immediately after the addition of 23 μL of 3.3 mM NADPH.
The results are shown in FIG. Active enzyme production reached a maximum after 30 minutes and then gradually decreased to the same level as the control after 150 minutes.
[0031]
Example 2.  Anti- HWE Immunochemical analysis of germination filtrates using antibodies.
Phytoftra Infestance (Phytophthora  infestans) Germination filtrate from zoospores (Doke et al., Phytopathology Vol. 90, p. 236-242 (1977)) to determine whether it contains a glycoprotein elicitor more recognized by anti-HWE antibodies Therefore, Phytoftra Infestance (Phytophthora  infestansThe germinated filtrate from the zoospores of (1) was subjected to immunoblot. Two bands of 47 kDa and 38 kDa were shown to react with the antibody (FIG. 9). Glycoprotein Elicitor (GE), a glycoprotein, appears to be secreted into the germ filtrate of oomycete species.
[0032]
As shown in FIG. 4B, the 47 kDa protein was detected in fractions 17 and 18, but these fractions did not show elicitor activity when tested on potato tubers (FIG. 3). In addition, when fractions 19 and 20 (FIG. 4B) were affinity purified, three bands of 47, 38 and 34 kDa (FIG. 5) were observed, indicating that these proteins were hypersensitive response (HR). Was induced (FIG. 6A) and AOS production was enhanced (FIG. 6A). On the other hand, immunoblotting of the germination filtrate showed two bands at 47 and 38 kDa (FIG. 9).
[0033]
These results clearly show that the elicitor activity of fractions 19 and 20 is based on the 38 kDa glycoprotein elicitor (GE). Only the 47 kDa protein of F17 and F18 showed no elicitor activity (FIG. 3), and the 34 kDa fragment was phytoftra infestans (Phytophthora  infestans) Was not observed by immunoblotting of the germination filtrate from FIG. 9 (FIG. 9). These results suggested that the 38 kDa protein recognized by the antibody was an elicitor of P1 during the initial infection process.
[0034]
Example 3.38kDa Of the gene encoding the elicitor protein of Escherichia coli  Protein bands from SDS gels stained with Coomassie brilliant blue (CBB) after SDS-PAGE were digested with Staphylococcus aureus V8 protease without eluting from the gel. This was done by placing the gel slice containing the band of interest into a sample well of a second SDS gel, and then covering each mill with Staphylococcus aureus V8 protease. Digestion proceeded directly in the stack gel during the next electrophoresis. Digestion resulted in several bands, but two major bands.
[0035]
For determination of the N-terminal amino acid sequence, the protein was concentrated to 100 pmol on a Centricon-30 microconcentrator and transferred to a PVDF membrane (Souterton, SG et al., Physiol. Mol. Plant Pathol, Vol. 42, p. 97-107 (1993)). Automated Edman degradation of proteins was performed using a Shimazu PPSQ-21 sequencer. The results of determining the N-terminal sequence of the native protein and the shorter peptide fragment generated by digestion with V8 protease are shown in FIG.
[0036]
Based on this sequence, degenerate oligonucleotides were designed and synthesized. Phytophthora, Logmann, J. et al. , Et al. Aval. Biochem. , Vol. 163, p. 16-20 (1987), total RNA was isolated, using this as a template, the degenerate oligonucleotide as a primer, and Ready-To-Go ™ beads (Takara). The cDNA was amplified. That is, 20 ng to 2 μg of total RNA and a final amount of 1 pM oligo d (T) were added to DEPC-treated beads dissolved in water.₁₈The primer was added, and reverse transcription was performed under the conditions of 72 ° C. for 30 minutes and 95 ° C. for 5 minutes. Next, PCR was performed by adding the above-mentioned degenerate primers. Incubation conditions were 94 ° C. for 4 minutes, 35 cycles of (94 ° C. for 40 minutes, 50 ° C. for 1 minute, 27 ° C. for 1.5 minutes), and 72 ° C. for 7 minutes. .
[0037]
As a result, a 659 bp cDNA encoding the protein portion of the 38 kDa elicitor protein was obtained. The nucleotide sequence and deduced amino acid sequence of this cDNA are shown in SEQ ID NO: 1. This sequence had no homology with elicitin and the 42 kDa glycoprotein elicitor from Phytophthora megasperma. This indicates that the elicitor protein of the present invention and the gene encoding it are completely novel.
[0038]
Example 4 .  Expression and purification of elicitor active protein
Protein expression
Cells of the E. coli BL21 pLysS strain transformed with the plasmid vector pCR T7CT TOPO (Invitrogen) into which the cDNA encoding the elicitor protein cloned in Example 3 was inserted were subjected to 100 μg / mL ampicillin and 34 μg / mL chloramphenicol. Was added to LB medium containing 10 mL, and the cells were cultured overnight at 37 ° C. with shaking until the OD (600 nm) reached 0.5. A 50 mL LB medium containing 100 μg / mL ampicillin was inoculated with 2 mL of the culture solution of E. coli cultured above, and cultured at 37 ° C. for 4 hours with shaking until the OD (600 nm) reached 0.5.
[0039]
Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.6 mM and cultured at 25 ° C. for 24 hours. To recover the protein, it was centrifuged at 4000 rpm for 10 minutes at 4 ° C. The resulting cell pellet was resuspended in guanidinium cell lysis buffer (6 M guanidine hydrochloride, 20 mM sodium phosphate, 500 mM sodium chloride, pH 7.8). Samples were incubated with guanidinium cell lysis buffer for 7 minutes at room temperature with gentle rocking and sonicated on ice. Insoluble matter was removed by centrifugation at 4500C for 15 minutes at 6500 rpm. The supernatant was collected and stored at 4 ° C. for further purification.
[0040]
His- Purification of labeled proteins
For purification of His-tagged proteins, the Xpress ™ System protein purification protocol (Invitrogen) was followed. The polyhistidine-labeled fusion protein was applied to a ProBond histidine-conjugated resin column equilibrated with cell lysis buffer. The column was washed with 8 mL of denaturing binding buffer (8 M urea, 20 mM sodium phosphate, 500 mM sodium chloride, pH 7.8). Next, the column was washed with 8 mM denaturing wash buffer (8 M urea, 20 mM sodium phosphate, 500 mM sodium chloride) over pH 6.0 and 5.3. Finally, the protein was eluted with 5 mL of denaturing elution buffer (8 M urea, 20 mM sodium phosphate, 500 mM sodium chloride, pH 4.08). The eluate was dialyzed overnight at 4 ° C. against 10 mM Tris-HCl, pH 8.0, Triton X-100 to remove urea. During this time, the dialysate was changed four times.
[0041]
The result of confirming the purified protein obtained by the above method is shown in FIG. FIG. 10A shows that the protein (lane 2) obtained by the above method and a sample (lane 1) obtained by performing the same experiment using a vector into which cDNA is not inserted are separated by SDS-PAGE. The results of CBB staining are shown. The 27.5 kDa band indicates the resulting expression product. The 38 kDa protein is a glycoprotein, whereas in Example 4, Escherichia coli was used as a host, so the produced protein was not glycosylated, and a protein consisting of 226 amino acids was about 27. It has an apparent molecular weight of 5 kDa. In FIG. 10A, the presence of a 27.5 kDa protein derived from cDNA was confirmed.
[0042]
FIG. 10B is an immunoblot diagram of a protein derived from the inserted cDNA, wherein lane a shows the result of detection with an anti-histidine antibody, and lane b shows the result of detection with an anti-HWE protein antibody. In this example, since the elicitor protein was expressed in a histidine-labeled state, it was detected by both antibodies.
[0043]
FIG. 10C is a photograph showing the browning effect of potatoes (using cultivars “Eniwa” and “Irish Cobbler”) on the tuber tissue, and “Cont. 1” is the result of the water control and “Cont. ".1" is the result of the product from a vector that does not insert the cDNA, and "Fusion Protein" is the result of the protein produced from the expression of the inserted cDNA. In "Fusion Protein", activity was observed.
FIG. 10D shows CLA indices of reactive oxygen species in suspension culture of cells of two kinds of potatoes ([Rishiri] and “Irish Cobbler”) for the same three kinds of samples as in FIG. 10C. Show. Only the protein derived from the vector into which the cDNA was inserted showed activity.
Example 5.38kDa Elicitor gene distribution
To investigate the distribution of the 38 kDa elicitor gene in various species of the genus Phytophthora, Phytophthora infestans isolates DN101 (race 0), E003 (race 0), Pi831 (race 1) and SE401 (race 1), Phytophthora megasperma, Phytophthora nicotiana, Phytophthora cryptogea, Phytophthora cryptogea, and Phytophthora capsici as a new strain of Phytophthora capsici, using the above-described oligonucleotide as a genomic oligonucleotide as a primer and a phytophthora capsici as a lyonucleotide as a primer, and using a phytophthora capsici as a template for the above-mentioned oligonucleotides as a fresh strain of RNA as a primer, RT-PCR was performed to synthesize cDNA. Further, the obtained cDNA band of 659 bp was amplified by PCR. The result is shown in FIG.
In addition, the PCR product (659 bp) was transferred to a nylon membrane and Southern blot hybridization was performed. The 659 bp cDNA cloned in Example 2 was hybridized with cDNAs derived from the various sources described above. The results are shown in FIG. Therefore, it is clear that genes encoding proteins homologous to the elicitor protein of the present invention are widely distributed among Phytophthora microorganisms.
[0044]
【The invention's effect】
The elicitor proteins of the present invention are envisioned as being useful for amplification of plant defense mechanisms.
[0045]
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a diagram showing an elution profile of a crude extract from cells of Phytophthora infestans DH101 strain in FPLC.
FIG. 2 is a graph showing the reactivity between the eluted product in FIG. 1 and an anti-HWC monoclonal antibody.
FIG. 3 is a photograph showing the browning effect of the fraction in FIG. 1 on potato tuber tissue, and is a drawing substitute photograph showing the form of an organism.
In FIG. 4, A shows the result of separating fractions F17 to F20 in FIG. 1 by SDS-PAGE and staining with silver, and B shows the result of detection with an anti-HWC monoclonal antibody. It is a drawing substitute photograph which shows the result of electrophoresis.
FIG. 5A shows the results of affinity purification of fractions F19 and F20 in FIG. 1 using anti-HWC monoclonal antibody Abs-2A11 and silver staining, and FIG. 5B shows the results detected by Western blot. All of which are photographs as substitutes for drawings, showing the results of electrophoresis.
FIG. 6A is a photograph showing that the affinity-purified protein of the present invention has a browning effect (induction of HR) on potato tuber tissue, and is a drawing substitute photograph showing the form of an organism. A and B are graphs showing that the affinity-purified protein of the present invention induces the generation of active enzymes in potato tuber tissue.
FIG. 7 is a diagram showing a 38 kDa native elicitor protein, the N-terminal amino acid sequence of its protease degradation product, and the base sequence of a primer or probe oligonucleotide designed based on the amino acid sequence.
FIG. 8 is an electrophoretogram showing that a gene encoding a 38 kDa elicitor protein is distributed in various microorganisms of the genus Phytophthora, and is a photograph substituted for a drawing.
FIG. 9 shows Phytoftra Infestation (Phytophthora  infestansFIG. 3 is an electrophoretogram showing the detection of a protein in a germination filtrate from a zoospore by an anti-HWE antibody, and a photograph substituted for a drawing. In the figure, lane A is Phytoftra Infestance (Phytophthora  infestans) Shows the results of a soluble sample from) and lane B shows Phytophthora infestans (Phytophthora  infestans3) shows the results of the filtrate germinated from the zoospores.
FIG. 10 shows the CLA index of the purified recombinant protein of the present invention in SDS-PAGE (panel A), immunoblot (panel B), potato tuber assay (panel C) and suspension culture. Panels A and B are drawing substitute photographs showing the results of electrophoresis, and panel C is a drawing substitute photograph showing the form of living things.

Claims (6)

A protein having the amino acid sequence shown in SEQ ID NO: 2 and having elicitor activity. A protein having an amino acid sequence modified by deletion, addition and / or substitution of one to several amino acids in the amino acid sequence shown in SEQ ID NO: 2, and having elicitor activity. A protein encoded by a DNA that hybridizes to a nucleic acid having the nucleotide sequence shown in SEQ ID NO: 1 under high stringency conditions and having elicitor activity. A glycoprotein derived from Phytophthora infestans comprising the amino acid sequence shown in SEQ ID NO: 2, having a molecular weight of 38 kD, and having elicitor activity. A phytoftora infestans capable of producing the protein according to claim 1 is cultured, and a protein having a molecular weight of 38 kD and having elicitor activity is collected from the culture. The method for producing a protein according to the above. A host transformed with an expression vector comprising the gene encoding the protein according to any one of claims 1 to 3, and culturing the host, and collecting a protein having elicitor activity from the culture. A method for producing the protein according to any one of claims 1 to 3.

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