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\title{A Novel Elicitor PiPE from Phytophthora Infestans Induces Active Oxygen Species and the Hypersensitive Response in Potato Strictly as per the compliance and regulations of}
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\begin{document}

             \author[1]{Naotaka  Furuichi}

             \affil[1]{  Niigata University}

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\date{\small \em Received: 12 December 2012 Accepted: 5 January 2013 Published: 15 January 2013}

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\begin{abstract}
        


A novel elicitor (PiPE) from the oomycete Phytophthora infestans (Pi) stimulates the hypersensitive response (HR) in potato. The PiPE, purified by anion-exchange chromatography from a water-soluble extract of Pi caused cell death, characteristic of HR, and enhanced active oxygen species (AOS) generation in tuber tissues. The partial amino acid sequence, and the sequence of the PiPE cDNA derived by PCR had homologous domain to fructose 1,6 bisphosphate aldolase (FBA) genes. To demonstrate that the PiPE cDNA encodes an active elicitor, we expressed PiPE in Echerichia coli, high five insect cells and purified the recombinant protein.His-PiPE induced HR, browning and generation of AOS in potato tissues. The PiPE was produced in the germination fluid from Pi and was existing in the cell wall of Pi. The role of PiPE peptides in the induction of HR in an incompatible interaction between Pi and potato cells is a prerequisite for the AOS and HR induction.

\end{abstract}


\keywords{hypersensitive cell death, PiPE elicitor, potato, Phytophthora infestans, signal transduction of HR, fructose 1, 6-bisphosphate aldolase.}

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\let\tabcellsep& 	 	 		 
\section[{Introduction}]{Introduction}\par
n incompatible interaction between plants and pathogens often leads to rapid and localized plant cell death, termed the hypersensitive response (HR), at the infection site. Induction of biochemical defense responses in the host cells \hyperref[b10]{(Ebel and Scheel, 1992}\hyperref[b11]{, Furuichi, 1993}\hyperref[b51]{, Xu and Heath, 1998}) likely involves recognition events for both elicitor and suppressor molecules from the pathogen at the host plasma membrane \hyperref[b10]{(Ebel and Scheel, 1992}\hyperref[b14]{, Furuichi and Tomiyama, 1980} . Subsequently Ca 2+ influx increases, pH decreases in the cytoplasm, and the cytosolic kinases are activated in the plant cells \hyperref[b10]{(Ebel and Scheel, 1992)}   \hyperref[b12]{Furuichi et al., 1997)} \hyperref[b21]{(Hamel et al., 2011)}. Electrolyte leakage contributing to host cell death \hyperref[b20]{(Goodman, 1968}\hyperref[b35]{, Pavlovkin and Novacky, 1986}\hyperref[b48]{, Tomiyama et al., 1983)}, resulting from the activation of a K + efflux across the plasma membrane \hyperref[b49]{(Tomiyama et al., 1968}\hyperref[b48]{, Tomiyama et al., 1983}\hyperref[b0]{, Atkinson et al., 1985}\hyperref[b3]{, Baker et al., 1987}\hyperref[b47]{, Tomiyama and Okamoto, 1989)}. Active oxygen species (AOS) also are generated rapidly at the plasma membrane of host cells during the incompatible A Novel Elicitor (PiPE) from Phytophthora Infestans Induces Active Oxygen Species and the Hypersensitive Response in Potato Abstract -A novel elicitor (PiPE) from the oomycete Phytophthora infestans (Pi) stimulates the hypersensitive response (HR) in potato. The PiPE, purified by anion-exchange chromatography from a water-soluble extract of Pi caused cell death, characteristic of HR, and enhanced active oxygen species (AOS) generation in tuber tissues. The partial amino acid sequence, and the sequence of the PiPE cDNA derived by PCR had homologous domain to fructose 1,6 bisphosphate aldolase (FBA) genes. To demonstrate that the PiPE cDNA encodes an active elicitor, we expressed PiPE in Echerichia coli, high five insect cells and purified the recombinant protein.\par
His-PiPE induced HR, browning and generation of AOS in potato tissues. The PiPE was produced in the germination fluid from Pi and was existing in the cell wall of Pi. The role of PiPE peptides in the induction of HR in an incompatible interaction between Pi and potato cells is a prerequisite for the AOS and HR induction. of these responses associated with HR are duplicated by treatment of tissues with factors derived from pathogenic pathogens termed elicitors \hyperref[b27]{(Keen, 1975}\hyperref[b40]{, Scheel et al., 1999)}. Elicitors from the oomycete pathogens, Phytophthora include 1,3-and 1,6-ß-Dglucans \hyperref[b1]{(Ayers et al., 1976}\hyperref[b42]{, Sharp et al., 1984)}, glycoproteins \hyperref[b28]{(Keenan et al., 1985}\hyperref[b33]{, Parker et al., 1988)}, and arachidonic acid \hyperref[b5]{(Bostock et al., 1981)}. A family of extracellular proteins produced by Phytophthora species, termed elicitins, also induces defense responses in plant cells \hyperref[b37]{(Ricci et al., 1989)}. Elicitins are highly conserved, 10-kDa proteins that are produced by several Phytophthora and Pythium spp. \hyperref[b26]{(Kamoun et al., 1994}\hyperref[b36]{, Pernollet et al., 1993}\hyperref[b37]{, Ricci et al., 1989)}. However, a 13-mer oligopeptide within a 42 kDa glycoprotein secreted by Phytophthora megasperma, also caused ion fluxes across the plasma membrane, the oxidative burst and phytoalexin biosynthesis in parsley cells \hyperref[b32]{(Nurnberger et al., 1994}\hyperref[b39]{, Sacks et al., 1995)}. From the findings, it was suggested in the present report that we isolated PiPE elicitor from the fungal cell wall fractions in the Phytophthora. We described previously the elicitor activity of a hyphal cell wall preparation (HW), from Pi  {\ref (Furuichi and Suzuki, 1989)}. This HW has a protein content of about 22\% \hyperref[b22]{(Ikeda and Furuichi, 1993)}. Treatment of the HW with pronase or trichloroacetic acid prior to its addition to potato tuber tissues reduced elicitor activity in a dose-dependent manner \hyperref[b22]{(Ikeda and Furuichi, 1993)}. Purification of the activity that induced HR and phytoalexin accumulation in the potato cells, correlated with proteinaceous materials. Response to a monoclonal Abs (Abs), selected from mice hybridoma immunized with the HW of Pi, was retained by the purified protein \hyperref[b22]{(Ikeda and Furuichi, 1993)}.Naotaka Furuichi ? , Kazutoshi Yokokawa ? , Hisakazu Okamura ? \& Masahiro Ohta ?\par
The reported nucleotide sequence appears in the DDBJ/EMBL/GenBank databases under the accession number AB051573. 
\section[{II.}]{II.} 
\section[{Results}]{Results} 
\section[{a) Purification of antigens that recognize the-PiPE monoclonal Abs}]{a) Purification of antigens that recognize the-PiPE monoclonal Abs}\par
Elicitor activity and the level of extractable proteins from mycelia (race 0 of Pi) grown in liquid culture increased for three weeks. We used homogenates of 15-days cultures as the initial PiPE source. Fractionation of elicitor activity by anion exchange chromatography at pH 8.1 (Fig.  {\ref 1A}) with elution of a linear gradient of NaCl (0-0.5 M) resulted in seven protein peaks. The results of assaying each fraction for Abs-binding activity are illustrated in Figure  {\ref 1B}. The maximum Abs-binding activity was detected in fractions F17 to F20, eluted at 0.35 M NaCl. 
\section[{b) Elicitor activity of the fractions recognized by anti-PiPE Abs}]{b) Elicitor activity of the fractions recognized by anti-PiPE Abs}\par
Treatment of tubers from the resistant cultivar Eniwa (R 1 ) with the elicitor preparation (containing proteins and carbohydrates) showed that it was more active than on tubers from the susceptible cv. Irish Cobbler (r-gene) (Fig.  {\ref 3B}). Thus the initial preparation possesses the same specificity as the Pi isolate from which the cell wall elicitor was derived. The elicitor activity in the concentrated samples of fractions F15 to F21 from anion exchange chromatography showed similar response being more active on cv. Eniwa than on cv. Irish Cobbler. The intensity of the symptoms of browning and cell death characteristic of HR was much higher in fractions F19 and F20 than in the other fractions. Fractions F17-F20 were pooled and the proteins separated by SDS-PAGE and silver-stained (Fig. \hyperref[fig_6]{2B}). Several peptide bands were detected in each fraction. The anti-PiPE Abs recognized one clear band of protein (47 kDa) in fractions F17 and F18, and three protein bands (47, 38, and 34 kDa) in fractions F19 and F20 (Fig. \hyperref[fig_6]{2C}). Because no elicitor activity was detected in F17 and F18, containing 47 kDa peptides, we concluded that elicitor activity resided with either or both of the 38 kDa and 34 kDa protein bands. 
\section[{c) Elicitor activity of affinity-purified PiPE}]{c) Elicitor activity of affinity-purified PiPE}\par
Affinity-purified PiPE peptides by using the anti-PiPE-Abs from fractions F19 and F20 had higher elicitor activity on tuber tissue from the resistant potato cv. Eniwa than on the susceptible cv. Irish Cobbler. The affinity-purified proteins transiently enhanced the production of AOS in suspension cultures of both potato cultivars after 30 min returning to the control treatment level after 150 min. AOS generation was higher in cv. Rishiri than in cv. Mayqueen. The cv. Eniwa showed similar enhanced production of AOS in potato tuber disks, whilst cv. Irish Cobbler showed lesser enhancement of the AOS production.\par
d) The effect of His-Strboh1 for the activation of AOS in potato plasma membrane\par
To examine the effect of His-Strboh1 to AOS generation in potato cells, entire sequence of Strboh1 was expressed with the (BL21pLysS) containing a C-terminal 6His-tag.\par
Affinity purification yielded approximately 110 kDa translation product and His-Strboh1 was confirmed by SDS-PAGE and immunoblot analysis by using His-Abs. The effect of His-Strboh1 on the activity of AOS generation was measured by using luciferase subustrate (CLA) in potato microsomal fraction (cv Eniwa). Because it was reported that activated NADPH oxidase was localized in plasma membrane in mammalian cells, AOS measurements were performed with microsomal fraction prepared from potato tuber tissue.\par
expressed His-Strboh1 (1.2?M) was treated to potato microsomal fraction and measured its chemiluminescence. In potato microsomal fraction treated His-Strboh1, AOS generation was transiently increased 5 to 10 min after the treatment, whereas treatment of Tiron (1, 2-dihydroxy-3, 5benzenedisulfonic acid disodium salt) which was a scavenger of AOS inhibited the generation. Ten min after AOS generation was declined to the basal level in the microsomal fraction. These results suggested that His-Strboh1 expressed in E.coli was activated after the treatment to microsomal fraction.\par
Expression vector containing a N-terminal 6Histag construct was employed for the production of His-Strboh1. Affinity purification yielded approximately 120 kDa product and the production of His-Strboh1 was confirmed by immunoblot analysis by using His-Abs. The effect of insect cells expressed His-Strboh1 on the generation of AOS was performed. The AOS generation was transiently increased up to 10 min after treatment. However, AOS generation was increased until 40 min, and the peak of AOS generation was 7 times higher than with the fusion from These results suggested that glycosylation of His-Strboh1 had effect on the generation of AOS in membrane fraction of potato. 
\section[{e) Immunochemical analysis of germination fluid using anti-PiPE Abs}]{e) Immunochemical analysis of germination fluid using anti-PiPE Abs}\par
Immunoblotting of the germination fluid from zoospores of Pi with anti-PiPE Abs, detected proteins of 47 and 38 kDa (Fig.  {\ref 3D, E}). We have reported that the germination fluid from race 0 caused a typical HR response and browning in the potato tuber disks of cv. Rishiri  {\ref (Furuichi et al. 1979}). Thus one protein of a size associated with elicitor activity from mycelial extracts, the 38 kDa, was detected in the germination fluids. We have tested the localization of PiPE antigens by using PiPE Abs and by using electron microscope of germinating zoospores (Fig.  {\ref 3D}). Figure  {\ref 3D} shows the germinative zoospores at five hour after the 2 We had examined if the PiPE protein is a glycoprotein or not, by using SDS-PAGE and the stain by periodic acid-Shiff's reagent. Digestion of the purified PiPE with Staphylococcus aureus V8 protease, to generate peptides for amino acid sequencing, generated two major bands. A sequence of 20 amino acids determined from the N-terminus of the native peptide was identical to the sequence of one of the V8 protease-digested fragments (Table  {\ref 1}).\par
We designed oligonucleotides based on the Nterminus amino acid sequence (Table1) as PCR primers. We derived cDNA from RNA extracted from a freshly harvested mycelium (for 10 days) of Pi race 0 and used this material as template in PCR. A 674-bp PCR product was obtained, cloned and sequenced (Fig.  {\ref 4A}).\par
Searches using the Blast program \hyperref[b45]{(Stephen et al. 1997}) revealed significant similarity to fructose 1, 6 bisphosphate aldolase (FBA) genes, with the highest similarity to yeast FBA (Fig.  {\ref 4B}). A database survey of Blast searches against Pi ESTs in DDBJ revealed a full length EST of 674 bp to correspond to the cDNA sequence. Our finding that a protein with homology to a FBA produced by Pi has elicitor activity is novel because the PiPE is secreted by the germinating spores also and is detected in extracts from the mycelia. 
\section[{g) Southern blot analysis of the PiPE}]{g) Southern blot analysis of the PiPE}\par
Southern blot hybridization was used to determine the number of copies of the cloned PiPE gene sequences in the genomic DNA of Pi, races 0 and 1. Using a probe containing a 674-bp fragment of the PiPE open reading frame from Pi, four Pst I fragments and five Hind III fragments from the Pi race and two Pst I fragments and three Hind III fragments, four Pst I fragments and five Hind III fragments were detected in the race of Pi, and two Pst I fragments and three Hind III fragments were detected in Pi 831 (Fig. \hyperref[fig_8]{5}) were detected from Pi831 (Fig. \hyperref[fig_8]{5}). Because there is one Pst I site and no Hind III site in the cloned partial cDNA (674 bp) coding the PiPE (Fig.  {\ref 4B}), at least two copies of the 
\section[{PiPE gene occur in the Pi genome. h) Elicitor activity and generation of AOS in functional analysis of the tagged PiPE}]{PiPE gene occur in the Pi genome. h) Elicitor activity and generation of AOS in functional analysis of the tagged PiPE}\par
We generated a 6His-PiPE to demonstrate that the protein produced from the gene had elicitor activity. A His-PiPE band (27.5 kDa) was observed after SDS-PAGE and CBB-staining (Fig. \hyperref[fig_9]{6A}). A protein band of this size, was not obtained when the His-control from the plasmid vector without insert cDNA was analyzed (Fig. \hyperref[fig_9]{6A}, lane 1). The purified His-PiPE was recognized by both the anti-His Abs and anti-PiPE-monoclonal Abs (Fig. \hyperref[fig_9]{6B}).\par
The His-PiPE had stronger elicitor activity on Eniwa (R 1 -gene) tuber cells than on Irish Cobbler (r-gene) suspension cultured cells responded with a stronger oxidative burst than those of cv. Irish Cobbler after exposure to the His-PiPE. 
\section[{i) Presence of PiPE genes in different Phytophthora species}]{i) Presence of PiPE genes in different Phytophthora species}\par
To investigate whether sequences encoding PiPE are present in other Phytophthora isolates, RT-PCR was performed. The primers indicated in Table  {\ref 1} was used with cDNA derived from total RNA from freshly harvested mycelium of Pi, DN101 (race 0) and E003 (race 0), Pi 831 and St401 (race 1); P. megasperma; P. nicotianae; P. cryptogea; and P. capsici as templates.\par
PCR products of 674-bp were generated from all tested samples (Fig.  {\ref 8A}). These RT-PCR products all hybridized with the probe from race 0 (Fig.  {\ref 7B}).\par
III. 
\section[{Discussion a) Immunochemical analysis of the localization of PiPE Abs in potato cells}]{Discussion a) Immunochemical analysis of the localization of PiPE Abs in potato cells}\par
Based on this similarity, we assume that fructose 1,6-bisphosphate aldolase peptides (FBA) may exist in the cell wall, and may be an elicitor of Pi against potato cells. Though FBA may exist in the cytosol of Pi, it could also be expressed and translocated into the cell wall of the germinating tube during the infection process. Secretion peptide from FBA was produced after germination of Pi spoors. Aldolases are known to be glycoproteins.\par
We propose a model for the elicitation of HR mediated by recognization of the PiPE by the StCDPK (a Ca 2+ dependent protein kinase) in the potato cell plasma membrane \hyperref[b12]{(Furuichi et al., 1997)}. It was suggested that the PiPE binds to a 6H-StCDPK2 from a resistant cv. Rishiri (R 1 ), based on an ELISA assay using a monoclonal Abs of PiPE. It remains to be clarified what domain of the PiPE interacts with the CDPK of the host cells.\par
The isolated PiPE is showing the activity for AOS generation and tissue browning; it induced HR and AOS generation in cv. Rishiri, a resistant potato (R 1gene), but induced only AOS generation in cv. Irish Cobbler, a susceptible potato (r-gene).\par
During the infection process, the PiPE might be produced by the oomycete directly from the germ tubes and infection hyphae. It may be recognized specifically by CDPK of potato cells, causing some conformational change in the structure of the kinase domain, which would result in induction of CDPK activation within several minutes. It is possible that as a consequence of accessible to the substrates for the kinase in potato cells. However, the activation pattern of the kinase is 2 The glycoprotein has 65\% homology to FBA from yeast, which has a elicitor activity like as fungal cell the PiPE binding, the catalytic domain of CDPK is now wall elicitor of Pi. As shown by Figure  {\ref 3D}, 3E PiPE was secreted from fungal cell wall surfes and that PiPE was detective in the germination fluids by using immunochemical methods.©\par
It was reported that ectopic expression of a heterologous CDPK (AK1-6H, an Arabidopsis calcium dependent protein kinase) in tomato protoplasts enhanced plasma membrane-associated NADPH oxidase activity \hyperref[b46]{(Tena et al., 2011)}. We examined the effect of CDPK Abs , recognizing kinase domain-III, to AOS generation in potato microsomal fraction. It was observed that treatment of CDPK Abs to the microsomal fraction of potato, which was added with expressed His-Strboh1 protein from insect cells, inhibited approximately 50 \% of AOS generation. It was suggested that CDPK kinase play an important role in the NADPH oxydase activation in potato microsome.\par
In the Cf9-Avr9 gene-for-gene interaction, a 68 to 70 kDa CDPK is activated in the plasma membrane fraction of tobacco cell cultures. The reported inhibitor studies were consistent with the evidence that CDPK was located upstream in the signal pathway that leads to the induction of AOS generation \hyperref[b38]{(Romeis et al., 2000)}, and it is in accordance with the results of quantitative RT-PCR in this study. Furthermore, arachidonic acid, an elicitor of Pi, induces activation of 78 kDa protein kinase C-like enzyme in potato tubers in calcium-dependent manner \hyperref[b46]{(Tena et al., 2011)}. These lines of evidence suggest that the NADPH oxidase is activated by the regulation of CDPK. Despite the importance of Phytophthora species as devastating plant pathogens, the basis of it specificity on potato cultivars is not resolved although elicitor active fractions have been isolated \hyperref[b13]{, Furuichi and Suzuki, 1990}\hyperref[b25]{, Kamoun et al., 1998}\hyperref[b23]{, Joosten et al., 1999)} The studies in the present results suggest that the PiPE with a sequence resembling that of fructose-1, 6bisphosphate aldolase could be functioning as an avirulence factor. This protein incited browning and AOS production to a greater extent on the resistant cultivars than the susceptible cultivars.\par
However all four (two race 0, avirulent and two race 1, virulent) of the Phytophthora species tested, possessed sequences that hybridized to the PiPE gene.\par
Since the PiPE was recognized by anti-HW Abs, this PiPE was considered to exist in the cell wall of Pi cells. We observed that FITC-labeled Abs bound to the surfes of germinated spores of Pi by using fluorescent microscopy (×800) (unpublished data).\par
It was reported that ectopic expression of a heterologous CDPK in tomato protoplasts enhanced plasma membrane-associated NADPH oxidase activity  {\ref (Tena et al., 2011, Furuichi and}\hyperref[b17]{Yokokawa, 2010)}. We examined the effect of CDPK Abs, recognizing kinase domain-III, to AOS generation in potato microsomal fraction . It was observed that treatment of CDPK Abs to the microsomal fraction of potato, which was added with expressed His-Enrboh1 protein from insect cells, inhibited approximately 50 \% of AOS generation. It was suggested that CDPK kinase play an important role in the NADPH oxidase activation in potato plasma membrane. In the Cf9-Avr9 gene-forgene interaction, a 68 to 70 kDa CDPK is activated in the plasma membrane fraction of tobacco cell cultures. The reported inhibitor studies were consistent with the evidence that CDPK was located upstream in the signal pathway that leads to the induction of AOS generation \hyperref[b46]{(Tena et al., 2011)}, and it is in accordance with the results of quantitative RT-PCR in this study. Furthermore, arachidonic acid, an elicitor of Pi, induces activation of 78 kDa protein kinase C-like enzyme in potato tubers in calcium-dependent manner (Subramaniam et al. 1997). These lines of evidence suggest that the NADPH oxidase is activated by the regulation of CDPK \hyperref[b19]{(Furuichi et al., 2012)}.\par
IV. Tooley, Cornell University) were maintained on rye agar medium supplemented with 2\% sucrose and 0.2\% bacto yeast extract at 18? in the dark. Other Phytophthora species were maintained in the dark. For liquid culture of the oomycete. The mycelia were grown in the dark at 18? for 2-3 weeks on the synthetic medium as described by \hyperref[b13]{Furuichi and Suzuki (1990)}. The mycelial mat was harvested by gentle filtration, washed, and frozen at -20?. Zoospores from the mycelial mat of PI and the germination fluid were prepared using the methods reported previously \hyperref[b9]{(Doke and Tomiyama, 1977)}. on a column (4.6 mm-100 mm, 1.7 ml, PoRos QE/M; PerSeptive Biosystems, Tokyo) equilibrated in the same buffer. The flow rate was 5 ml/min. Proteins were eluted with a linear gradient of 0 to 0.5 M NaCl in 25 mM Tris-HCl, pH 8.1. The column eluate was monitored at 280 nm and recovered in fractions of 1 ml. 
\section[{Materials and Methods}]{Materials and Methods}\par
HW were isolated and purified after the homogenization and FPLC-anion exchange chromatography. 
\section[{c) Protein Measurement d) Monoclonal Abs of the PiPE e) Biological Assay}]{c) Protein Measurement d) Monoclonal Abs of the PiPE e) Biological Assay}\par
Induction of HR by the elicitor was assayed by using microscopic observation. 1) The cessation of cytoplasmic streaming, 2) Loss of stain ability by neutral red, and 3) Loss of ability for plasmolysis, of potato tuber tissue at 12 h after treatment.\par
The parenchymatous tissues of potato tuber from cvs. Eniwa (R 1 ) and Irish Cobbler (r) were aged for 16 h at 18? prior to being treated with 30 µl of the PiPE (1 mg ml -1 distilled water), which were isolated from fungal mat, and fractionated by FPLC then various FPLC fractions were used. The materials in the FLPC fractions were concentrated with a Centricon-30 micro concentrator (Amicon, Tokyo) (600 µg ml -1 ). State replicates of studies here.\par
The cystospores were germinated by shaking in a flask with CaCl 2 (10 -4 M). 
\section[{f) Measurement of AOS generation from suspension of potato cells}]{f) Measurement of AOS generation from suspension of potato cells}\par
A luciferase substrate, (CLA) was used to measure the concentration of AOS produced by suspension cultured potato cells. .Suspension-cultured cells (5 ml of 3-to 4-day-old cells of potato cv. Eniwa (R1-gene) and cv. Irish Cobbler (r-gene) were treated with His-tagged PiPE (500 µl of 600 µg ml 1 ). At each ml sample tube containing 426 µl of 39 mM HEPES (pH 7.0), 5 µl of 10 mM MgCl2 or of 10 mM CaCl2, 5 µl of 10 mM EGTA, 1.5 µl of 10 mM guanosine 5´ triphosphate (GTP)--S and 15 µl of 500 µl CLA(Cypridina Luciferin Analog;\par
2 Methyl-6-phenyl-3,7-dihydroimidazo[1,2a]pyrazin-3-one). CLA was added to the tube last. The sample was then incubated at 37ºC for 3 min. Radiated light was measured for 15 sec just after adding 23 µl of 3.3 mM NADPH (Luminescence Reader, Atto, Tokyo). The Abs used were monoclonal anti-PiPE Abs generated by mouse hybridomas as described in \hyperref[b22]{Ikeda and Furuichi (1993)}. PiPE was isolated from the Pi homogenate as reported previously \hyperref[b13]{(Furuichi and Suzuki, 1990)}.\par
Enzyme-linked immunosorbent assay (ELISA) was performed following the procedure described by \hyperref[b31]{McLaughlin et al. (1989)}. The wells of microtiter plates (Dynatech, Tokyo) were coated with the Abs diluted at 1/1,000 with 1\% bovine serum albumin in phosphatebuffered saline (PBS). Immunoglobulin-alkaline phosphatase conjugates from rabbit were used as secondary Abs at 1/2,000 dilution. Absorbance due to alkaline phosphatase was measured with a Microplate Reader (Bio-Rad, Tokyo) at 595 nm. 
\section[{?}]{?} 
\section[{Proteins}]{Proteins}\par
were separated by SDSpolyacrylamide gel electrophoresis as described previously (Laemmli, 1970) with a 12.5\% acrylamide separation gel and 4.5\% acrylamide stacking gel. Following electrophoresis, gels were silver-stained following the previously reported method or the proteins were transferred as described by Towbin et al.  {\ref (1979)} onto a polyvinylidene difluoride (PVDF) membrane (Immobilon-P, pore size 0.45 µM, Millipore, Tokyo) using a MilliBlot TM -SDS System (Millipore) (Stephen et al.) at 2 mA per cm 3 for 30 min. The PVDF membrane was incubated for 1 h at 25? in the blocking buffer (10 mM Tris-HCl, 150 mM NaCl (pH 7.5), and 5\% skimmed milk). Membranes were washed twice in TBS-Tween 20 for 5 min, incubated for 1 h at room temperature in the primary HW-Abs diluted at 1/1,000 with TBS. After washing a third time in TBS-Tween 20 for 10 min, membranes were incubated for 1 h at room temperature in rabbit anti-mouse IgG conjugates with alkaline phosphatase (Bio-Rad, Tokyo, Japan) diluted at 1/2,000 with TBS. After three washes in TBS-Tween-20, each for 10 min detection of the antigen-Abs complexes was carried out with alkaline phosphatase color reagent (Bio-Rad, Tokyo, Japan).The reactive membranes were majored by using typhoon (GE-Science,Tokyo,Japan). Controls were recorded by using without anti-PiPE-Abs and with control antisera. The experiments were determined by using 3 times measurements. The membrane with antigen-Abs complexes were recording by using the typhoon gel scanner. \hyperref[b6]{Cleveland et al. (1977)}. Briefly, protein bands from an SDS gel, stained after SDS-PAGE with Coomassie brilliant blue, were digested by the V8 protease without prior elution, by placing gel slices containing these bands in the sample wells of a second SDS gel, then overlaying each slice with the V8 protease. Digestion proceeded directly in the stacking gel during the subsequent electrophoresis. time point, 500 µl of the treated suspension cells were centrifuged for 15 sec at room temperature to collect the supernatant. The supernatant (25 µl) was added to a 15 i) N-terminal amino acid sequencing For N-terminal amino acid sequence determination, PiPE peptides were concentrated with a Centricon-30 micro concentrator in a final concentration of 100 pmol and transferred to a PVDF membrane as described previously \hyperref[b44]{(Southerton et al., 1993)}. Automated Edman degradation of the PiPE peptides was performed with a Shimadzu PPSQ-21 sequencer (Shimadzu, Kyoto) using the reagents and method of the manufacturer. j) Fungal RNA preparation and RT-PCR Total RNA from freshly harvested Pi mycelia was isolated using the guanidine hydrochloride extraction method reported by \hyperref[b29]{(Logemann et al., 1987)}. Amplification of cDNA with the degenerate primers shown in Table  {\ref 1} was carried out using Ready-To-Go TM RT-PCR beads (Amersham-Pharmacia, Tokyo) according to the methods of the manufacturer. Reverse transcription was carried out by adding 20 ng to 2 µg of total RNA and a final concentration of 1 pM oligo d (T)18 primer to dissolved beads in DEPC-treated water. The incubation conditions were as follows: 30 min, 72?; 5 min, 95?. After that, the degenerate primer indicated in Table  {\ref 1} was added for PCR. The incubation conditions were as follows: 4 min, 94?; 35 cycles (40 min, 94?; 1 min, 50?; 1.5 min, 72?); 7 min, 72?. µg of digested DNA was electrophoresed on a 1\% agarose gel. Alkaline DNA was transferred to a nylon membrane (Hybond N + , Amersham-Pharmacia, Tokyo), and Southern blot hybridizations were performed at 55ºC as reported  {\ref (Ausubel et al., 1987)}. Probes for hybridization were synthesized by PCR using the primers described in Table  {\ref 1} and comprised the nucleotide sequences (674-bp fragment -(Fig.  {\ref 9}). Probes were labeled using a AlkPhos Direct labeling and detection system (Amersham, Tokyo) according to the supplier's instructions. Membranes were washed twice at 65ºC for 10 min in the primary wash buffer, then washed in secondary wash buffer (50 mM Tris base, 100 mM NaCl and 2 mM MgCl 2 ). Positive cDNA clones were detected using the CDP-Star chemiluminescent detection reagent according to the manufacturer's instructions. 
\section[{PiPE protein was digested by Staphylococcus aureus V8 protease as described by}]{PiPE protein was digested by Staphylococcus aureus V8 protease as described by} 
\section[{l) Expression of recombinant PiPE}]{l) Expression of recombinant PiPE}\par
insert cDNA the 674 base pairs. His tag added to C terminus. They were added to 10 ml LB medium. The for 24 h. The protein was harvested. Cells were harvested by centrifugation at 4000 rpm for 10 min at 4?, and the pellet was resuspended in guanidinium lysis buffer (pH 7.8) and slowly shaken for 7 min before sonicated at ice-water temperature. The insoluble debris was removed by centrifugation at 6500 rpm for 15 min at 4?. The supernatant was collected and stored at 4? for subsequent His-tag purification by following the Xpress TM System protocol as described by Invitrogen. The polyhistidine-tagged fusion protein was loaded onto a ProBond TM histidine-bind resin column equilibrated with lysate buffer. The column was washed with denaturing binding buffer (8 M urea, 20 mM sodium phosphate, 500 mM sodium chloride, pH 7.8). Then, the column was washed with denaturing wash buffer, ranging at pH 6.0 and pH 5.3. Finally, the protein was eluted with denaturing elution buffer (8 M urea, 20 mM sodium phosphate, 500 mM sodium chloride, pH 4.0). The elute was dialyzed against 10 mM Tris-HCl, pH 8.0, and 0.1 \% Triton X-100 overnight at 4? to remove urea. Each samples were stocked at -30?What about the germination fluid also contains those PiPE. Affinity purified PiPE fraction were contained in a germination flud. 
\section[{m) Purification of recombinant protein}]{m) Purification of recombinant protein}\par
For purification of His-fusion protein, ProBond TM Protein Purification kit (Invitrogen) was used. The polyhistidine-tagged fusion protein was loaded onto a ProBond TM histidine-bind resin column equilibrated with lysate 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). Then, the column was washed with 8 ml of denaturing buffer (8 M urea, 20 mM sodium phosphate, 500 mM sodium chloride) pH 6.0 and pH 5.3 successively. Finally, the protein was eluted with 5 ml of denaturing elution buffer (8 M urea, 20 mM sodium phosphate, 500 mM sodium chloride, pH 4.0). the elute was dialyzed against 10 mM Tris-HCl, pH 8.0, 0.1 \% Triton X-100 overnight at 4 ? to remove urea. During this time, the dialysis buffer was replaced 4 times. The purified protein concentration was determined using the BCA protein assay kit (Pierce) with bovine serum albumin (BSA) as standard. 
\section[{V.}]{V.}\par
Volume XIII Issue V Version I Year 013 2 ( ) 
\section[{K}]{K}\par
The E. coli, BL21 pLysS cells, harboring apCR T7/CT TOPO plasmid vector (Invitrogen) containing the We thank N. Hatsugai , R.Ikeda (Niigata Univ.) and T.Oikawa (Taane Ltd, Sendai Ltd., Yamagata) for the preparation of monoclonal Abs, M.Kato (Hokkaido Agricultural Exp. Sta.) for providing the strains of Pi, and .A.Fujiwara (Denka Seiken Ltd.) for the preparation of the mice monoclonal Abs. We also thank Anne J. 10 ml of LB medium was inoculated with 2 ml of the E. coli cultured, and then cultured for 4 h at 37? with shaking.\par
Isopropyl--D-thiogalactopyranoside was added to a final concentration of 0.6 mM and cultured ? Anderson (USU, USA) and A. Shirata (Sendai) for advice throughout the project. This work was supported in a part by a grant from the Ministry of Education, Science and Culture of Japan?JST and by a grant from the     B, Western immunoblot analysis of fusion proteins with anti-His Abs (lane a) and anti-HW-Abs (lane b). C, Elicitor assay of the affinity purified protein expressed from E. coli on tuber tissue of potato cultivars.\par
Control 1: water treatment. Control 2: Expressed fusion protein and purified from the plasmid vector alone, then applied onto the tuber disks. Fusion protein: His-PiPE. The photograph was taken 96 h after the treatment. D, Effect of the His-PiPE on the generation of active oxygen species in suspension cultured cells of potato cv. Rishiri (R 1 -gene) and Mayqueen (r-gene). The CLA index was measured by a luminometer. \begin{figure}[htbp]
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  			\footnote{© 2013 Global Journals Inc. (US)} 			\footnote{( )K} 		 		\backmatter   			  			  			  				\begin{bibitemlist}{1}
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\end{bibitemlist}
 			 		 	 
\end{document}
