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A monoclonal antibody and the lectin wheat germ agglutinin induce zoospore encystment in Pythium porphyrae, a marine microbial pathogen

     Graduate School of Science & Technology, Nagasaki University, 1–14 Bunkyo Machi Nagasaki 852-8521, Japan

Kinya Kanai

     Fish Pathology Laboratory, Faculty of Fisheries, Nagasaki University, 1–14 Bunkyo, Machi, Nagasaki 852-8521, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

Pythium porphyrae (Oomycota) is a microbial pathogen which causes red rot disease in the commercially cultivated red seaweed Porphyra. This disease is initiated by the motile zoospores of the fungus, which it has been suggested to recognize and process host specific signals by membrane bound receptors. Monoclonal antibodies (MAbs) were developed against the surface components of zoospores and cysts of this fungus in order to try and identify the putative receptor molecules involved in the zoospore encystment process. Screening of MAbs by immunofluorescence assays has revealed three different patterns of surface epitope binding, while labeling of zoospore and cysts components by FITC-conjugated lectins has identified different carbohydrate moieties. Of the MAbs and lectins tested, MAb 1A3 and wheat germ agglutinin have induced zoospore encystment under in vitro conditions. MAb 1A3 identified a 109 KDa band of a glycoprotein in western blot analysis which could be a putative receptor responsible for the induction of zoospore encystment.

Key words: encystment, enzyme-linked immunosorbent assay, flagella, immunofluorescence assay, monoclonal antibody, putative receptor, red rot disease

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Pythium porphyrae (Py. porphyrae) is a marine oomycetous pathogen that causes the devastating red rot disease in the commercially cultivated red seaweed Porphyra. Porphyra (popularly known as "Nori") is a commercially important edible red alga. The disease is initiated by the motile zoospores produced asexually from either vegetative hyphae or oospores under low nutrient conditions (Fujita 1990). Mechanisms of the disease initiation by zoospores are very similar to that of higher plant-infecting oomycetes (Kerwin et al 1992). The transition from motile wall-less zoospores to non-motile walled cysts is the critical event in the lifecycle of many oomycetes where the initial interaction between the fungal propagule and its potential host is established. The whole process of encystment-attachment leading to germ tube formation assumes a critical significance in the lifecycle of hemibiotrophic oomycetous fungi, which typically completes in about 30–40 min before entering to the biotrophic phase (Burr and Beakes 1994, Birgit et al 2000). However, it is difficult to differentiate temporally the process of encystment and attachment. Initiation of the zoospore encystment is regarded as a stimulus-mediated process where recognition of a stimulus may lead to calcium ion influence across the cell membrane leading to secretion of adhesive glycoproteins from peripheral vesicles (Burr and Beakes 1994, Warburton and Deacon 1998). Thus, the timing of the encystment process is an important step for the future successful infection of the host, since an early or late encystment may lead to inappropriate positioning of the cyst on the plant surface or failure to adhere to the host surface (Hardham and Suzaki 1986, Estrada-Garcia et al 1990). Understanding of the zoospore encystment mechanism paves the way to developing strategies for interfering with host-specific infection by the pathogen.

Nevertheless, little information is available about the biochemical mechanisms involved in the process of host-specific infection by zoospores of marine Pythium (Uppalapati and Fujita 2000). To understand the mechanisms of disease initiation and to identify the molecules involved in the process of host recognition and subsequent infection process, one needs to have appropriate probes for surface components of the disease causative agent. Monoclonal antibodies (MAbs) are such highly specific probes, which may facilitate the identification of putative receptor molecules involved in the host-parasite interaction (Hardham et al 1985). Earlier MAbs were developed to vegetative hyphae of Py. porphyrae (Amano et al 1995) and polyclonal antibodies were developed against zoospores and cysts by Addepalli and Fujita (2001). Since zoospore surface molecules are believed to be involved in host recognition and infection, the present study is aimed at developing MAbs against surface components of the zoospores and cysts of Py. porphyrae and identifying putative receptor molecules involved during the zoospore encystment process, employing the MAbs and lectins as probes.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Culture and induction of zoospores – Py. porphyrae Takahashi et Sasaki was collected from Chiba prefecture (Tokyo Bay, Japan, December 1998) designated as strain C-1 (Uppalapati and Fujita 2000), Kumamoto prefecture (Western Japan, January 1998) designated as strain K-1 and from Fukuoka prefecture (Western Japan, November 1999) designated as strains F1, F2, F3 and F4. All the strains of Py. porphyrae were maintained on Arasaki B medium (Arasaki et al 1968) at 20 C in the dark on agar slants. Py. apahanidermatum IFO 32440, Py. marsipium IFO 32579 and Phytophothora cryptogea IFO 32325 were obtained from Institute for Fermentation, Osaka (IFO), Japan. The growth conditions and maintenance media were as recommended by IFO.

The zoospores from Py. porphyrae were obtained as described by Addepalli and Fujita (2001). The zoospores of Py.aphanidermatum and Py.marsipium, and those of Phytophthora cryptogea were produced following the protocol of Donaldson and Deacon (1992) and Hardham and Suzaki (1986), respectively.

Production of hybridoma cell lines – Antigen preparation and immunization was carried out as described by Hardham et al (1985). A zoospore suspension of 1 x 10⁶ cells mL^-1 (containing 10% cysts; strain C-1) was used as antigen for raising MAbs. The serum antibody titer was estimated by enzyme-linked immunosorbent assay (ELISA). The final booster injection was given three days before the date of fusion.

Hybridoma cells were produced following the modified protocol of Goding (1996). Briefly, spleenocytes were isolated from an immunized mouse and fused with P3U1 cells in the presence of 45% polyethylene glycol, MW 1500. The fusion products were diluted in 100 mL of HAT medium and plated in two 48-well trays, 1 mL per well. Hybridoma cells were grown for 14 d in a 5% CO₂ incubator at 37 C, and the cultured supernatants were assayed by ELISA for the presence of desired antibodies with antigen coated micro-titer plates. The wells that were positive by ELISA were cloned by clonal dilution in 96-well flat-bottom plates. The MAb producing clones were distinguished based on their ability to recognize the cysts or zoospores by immunofluorescence assay (IFA).

Specificity of MAbs – The cross reactivity of MAbs has been tested by IFA against the respective cells (zoospores or cysts depending on the reactivity of MAbs as determined above) of various isolates of Py. porphyrae collected from different geographical regions of Japan and oomycetes infecting higher plants. The specificity of MAbs that were not reactive in IFA was determined by ELISA on antigen consisting of a mixture of zoospores and cysts at a 1:1 ratio. The cross reactivity of MAbs against the Olpidiopsis sp., an obligate parasite infecting Porphyra spp. was tested by IFA as described previously (Addepalli and Fujita 2001) and by ELISA using infected thallus homogenate. All assays were carried out on fixed antigens and with appropriate controls as detailed elsewhere.

Determination of immunoglobulin subclass – The isotype of the MAbs was determined by sandwich ELISA test using the Bio-Rad mouse isotyping antibody kit.

Purification of MAbs from hybridoma supernatants – MAbs 1A3, 2B6, and 2F6 were purified from the hybridoma supernatant using affinity chromatographic column Kaptiv M (Technogen) following the instructions of manufacturer. The activity and purity of the antibody were determined by ELISA and SDS-PAGE, respectively.

ELISA – ELISA was performed following the methodology of Liddle and Cryer (1991). Briefly, micro-titer plates were incubated with 0.1% (w/v) poly-L-lysine (Wako Pure Chemicals, Ind.) for 1 h at room temperature. After incubation, the plates were tapped over paper towel to remove the traces of poly-L-lysine. The poly-L-lysine coated plates were allowed to dry for 30 min at room temperature. A volume of 50 µL glutaraldehyde fixed antigen (zoospore suspension of 1 x 10⁶ cells mL^-1) was incubated for 1 h in the poly-L- lysine-coated wells of micro-titer plates. After incubation, the wells were washed three times with 200 µL of PBS containing 0.02% Tween 20 (TPBS), and the unbound sites were blocked with 5% skim milk for 30 min at room temperature. After three washes with TPBS, the wells were incubated with 50 µL of hybridoma supernatants for 2 h at room temperature. The wells were washed as earlier with TPBS and incubated further for 1 h with HRP-conjugated anti-mouse IgG + A + M (H + L) (Zymed laboratories). The substrate solution containing H₂O₂ (0.002%) and 2, 2¹-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (0.04%) was added to the wells after three washes with TPBS and incubated for 30 min before stopping the reaction with 0.01% (w/v) sodium azide in 0.1 M citric acid. The absorbance values were read at 405 nm using an ELISA reader.

All assays were performed in quadruplicate and the values were expressed as average of the quadruples. Unless otherwise stated all the assays included the following controls: 3% BSA as an irrelevant antigen, normal mouse IgM as irrelevant antibody, mouse immune serum as positive control, and medium spent for growth of P3U1 cell line as negative control.

Determination of spore developmental stage-specific MAbs by IFA – By using IFA, hybridoma clones secreting MAbs were tested for their ability to recognize the different developmental stages of spores, such as zoospores, cysts, germinating cysts, and vegetative hyphae. IFA was performed as detailed by Goding (1996). Briefly, zoospores were fixed in 0.2% glutaraldehyde + 4% formaldehyde mixture following the protocol of Hardham et al (1985). Cysts were obtained by vortexing the zoospore suspension in Eppendorf tubes for 70 s and allowing to encyst for 30 min at room temperature before fixation. Germinating cysts were obtained by incubating the cysts for 4 h at room temperature and fixing as above. Vegetative hyphae were obtained from the five-day-old liquid cultures. A 100 µL suspension (1 x 10⁵ cells mL^-1) of the fixed sample of each developmental stage was taken in an Eppendorf tube and IFA was performed as per the routine protocol. Fifty µL of neat undiluted hybridoma supernatant was used as primary antibody with an incubation period of 2 h at room temperature. The bound primary antibody was detected using fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse antibody (1:80 dilution). The cells were washed, suspended in 10 µL of glycerol-based mounting fluid, and mounted in multi-well slide glasses. Slides were examined with an Olympus fluorescent microscope equipped with B2 excitation filters. Photographs were taken with Kodak T max 400 film. All the assays include the following controls unless other wise stated: cells treated only with buffer to detect auto-fluorescence, cells treated with spent medium of P3U1 cell line, antigen treated with isotype matching normal mouse antibody as negative controls, antigen treated only with FITC-conjugated secondary antibody, and cells treated with mouse immune serum as a positive control.

Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting – A zoospore suspension of 1 x 10⁸ spores mL^-1 was solubilized in Triton X-114 (0.5% in PBS) and the membrane proteins were enriched following the protocol of Goding (1996). The membrane enriched fraction was precipitated by 10% TCA, solubilized in SDS sample buffer (125 mM Tris-HCl, pH 6.8, containing 2% SDS, 100 mM dithiothreitol, 0.1% bromophenol blue, and 10% glycerol) and subjected to 10% SDS-PAGE following the protocol of Sambrook et al (1989). SDS-PAGE resolved proteins were either stained by silver stain (Sambrook et al 1989), or blotted onto a polyvinylidene difluoride (PVDF) membrane for 90 min at 27 mA using semi dry blotter (ATTO corporation) following the instructions of manufacturer. The blotted PVDF membrane was stained with 0.2% Ponceau S and was cut into strips before incubating with 5% blotto for 1 h at room temperature to block the non-specific binding sites. At the end of incubation time, the strips were incubated in hybridoma culture supernatants (diluted 1:1 in ratio in 3% BSA) for 2 h at room temperature on a rocking platform. The stripes were rinsed three times in PBS containing 0.05% Tween 20 and once in Tris-NaCl (150 mM NaCl, 50 mM Tris-HCl, pH 7.5) for 10 min each with five gel volumes to remove unbound antibody, and incubated with secondary antibody [HRP-rabbit anti-mouse IgG + A + M (H + L) diluted 2000 times in 5% skim milk] for 2 h at room temperature. After incubation, the strips were washed three times in Tris-NaCl buffer for 10 min each and developed in 0.5% diaminobenzidine tetrahydrochloride (Wako Pure Chemicals, Ind.) + 0.002% H₂O₂ until the desired intensity of bands was obtained. During western blotting, the membrane that was treated only with the secondary antibody but not with the primary antibody was used as negative control to determine the non-specific staining.

Characterization of antigens by treating with heat, protease, and periodate – The heat stability of the respective antigens was tested by heating cell suspensions for 5 min at 65 C and 100 C or autoclaving for 15 min at 121 C and coating in the micro-titer wells for ELISA tests in the usual manner. Stability of antigens towards protease was tested following the protocol of Addepalli and Fujita (2001). The effect of periodate on antigens was investigated by modified protocol of Bossi and Dewey (1992). Sensitivity of antigens to periodate treatment was tested by incubating the cells with 25 mM sodium periodate in 50 mM acetate buffer at pH 4.5 for 4 h and 16 h at 4 C in the dark. At the end of incubation time, the cells were washed five times with TPBS before proceeding for blocking to do ELISA test in the usual manner. Controls have received only the acetate buffer.

Fluorescent labeling of zoospores, cysts and germinating cysts by FITC conjugated lectins – The zoospores, cysts and germinating cysts were fixed in a mixture of formaldehyde and glutaraldehyde as done for IFA, and fluorescent labeling was carried out as described for IFA by Addepalli and Fujita (2001) in multi well slides with the inclusion of glycine treatment for 45 min to reduce the aldehyde induced fluorescence. At the end of incubation, wells were incubated with FITC conjugated lectins, concanavalin A (Con A), wheat germ agglutinin (WGA), Bandeiraea simplicifolia agglutinin (BS II) and soybean agglutinin (SBA) at a final concentration of 100 µg protein mL^-1 for 45 min at room temperature. The slides were mounted in glycerol based anti-fading solution and examined with a fluorescent microscope as above. The photographs were taken using Kodak T max 400 film. The lectins treated with 50 mM of appropriate hapten sugars for 1 h were used as controls to determine the non-specific binding of lectins to the spore components. All the lectins and their substrates used in the present study were procured from Sigma.

Encystment assay by MAbs and lectins – Squares of 10 mm were marked at the bottom of the polystyrene Petri dishes (4 squares for each 9-cm dish). Either MAb or lectin of pre-determined quantity was diluted in 40 µL of either half-strength seawater or tenfold diluted seawater, respectively, and was put in each square. To the test solution (containing either MAb or lectin), 40 µL zoospore suspension of 1 x 10⁴ cells mL^-1 was added using a micropipette (The tip was widened to avoid friction-induced encystment during spore suspension manipulation). The Petri dishes were incubated undisturbed in a humidified chamber for 15 min at room temperature (22 C), and the assay was terminated by the addition of glutaraldehyde at a final concentration 2%. The Petri dishes were allowed to stand for 30 min, cover glass was laid and the number of cysts and zoospores were counted at random microscopic fields within each square using an inverted microscope. Each test concentration was carried out in quadruplicate, and in each at least 100 cells were counted. All the experiments were carried out twice. For control the nonspecific IgM mouse monoclonal antibody (Sigma) and lectins pre-incubated with respective substrates were used. The results were expressed as the percentage of zoospores encysted from the total number of spores counted.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Production of MAbs – Antigen preparation and the immunization regime of the mice have yielded the MAbs as determined by ELISA and IFA. From a single fusion of splenocytes from an antigen-boosted mouse, hybridomas were produced in 59 out of 96 wells plated. Of the supernatants tested, eight were found to be secreting antibodies that recognize the spores of Py. porphyrae by ELISA. From these hybridomas, eight MAb-producing cell lines were established. Of the eight cell lines, five cell lines (1A3, 2B6, 1H2, 6F4, and 2F6) produced MAbs that have reacted with the antigen by both ELISA and IFA, whereas the MAb of cell lines 2D1, 7E3, and 8H8 showed positive reaction only by ELISA. The five MAbs, which were positive both by ELISA and IFA, showed varying patterns of labeling of the spore components. MAbs 1A3 and 2B6 labeled the zoospores of Py. porphyrae whereas the cysts remained unlabelled (Fig. 1). MAbs 1H2, 6F4, and 2F6 labeled only the cysts but not the zoospores (Fig. 2). Of the two MAbs that reacted with the zoospores 1A3 labeled the flagella of the zoospores as well as the surface while MAb 2B6 labeled the zoospore membrane but not the flagella (Figs. 3 and 4). However, none of the five MAbs labeled the germinating cysts or vegetative hyphae of Py. porphyrae.

View larger version (75K): [in this window] [in a new window]    FIGS. 1–4. Zoospores and cysts of Strain C-1 stained by IFA.  Fig. 1a. MAb 2B6 bound to the surface of zoospores in the preparation but not to the cysts. Arrowhead indicates intensely stained zoospores. 1b. Light microscopic version of FIG. 1a showing presence of zoospores and cysts. Two round cells are cysts. Arrowhead indicates cyst. Zoospore flagella were not present in the plane of visibility. x1300.  Fig. 2a. MAbs (1H2, 6F4 & 2F6) bound to the surface of cyst in the preparation but not to the zoospore. Arrowhead indicates intensely stained cyst. 2b. Light microscopic version of Fig. 2a showing both zoospore and cyst. Arrowhead indicates zoospore. Zoospore flagella were not present in the plane of visibility. x1300.  Fig. 3a. MAb IA3 bound to the flagella as well as the surface of zoospores. Arrowhead indicates intensely stained flagellum by 1A3. 3b. Light microscopic version of Fig. 3a. x1300.  Fig. 4a. MAb 2B6 bound to the surface of zoospores but not to the flagella. Arrowhead indicates zoospore. 4b. Light microscopic version of Fig. 4a. Arrowhead indicates zoospore flagella. x 1300.

Characterization of MAbs and epitopes – The following antibodies were typed and all proved to be IgM antibodies with kappa as the light chain: 1A3, 2B6, 1H2, 6F4, and 2F6.

Epitope characterization – The epitopes of the respective antigens were heat resistant after treating the cells at 65 C for 5 min. However, heating at 100 C and autoclaving of the cells reduced the reactivity of the antigen with all the MAbs tested. Treatment of antigen with protease resulted in decrease in reactivity of MAbs with the antigen (Table II).

Purification of Mabs – The MAbs have been successfully purified from the hybridoma supernatants using Kaptiv M affinity column chromatography and the purity obtained was determined by SDS-PAGE, where only one single band of the expected molecular weight of 900 kDa was detected.

Fluorescent labeling of spore components by FITC-conjugated lectins – Of the four lectins tested, Con A and WGA labeled the zoospores with varying patterns while the cysts were labeled by both the lectins in similar pattern. Con A labeled the zoospores very intensely all throughout the surface and cytoplasm, while flagella remained unlabeled. However, WGA labeled the zoospores very intensely in a mosaic fashion at the posterior end, while flagella remained weakly stained. Germinating cysts also showed varying patterns of labeling by Con A and WGA (Fig. 5a–f). However, BS II bound very weakly to the zoospores and cysts (could not be photographed with the available equipment) while germinating cysts were labeled moderately (Fig. 5g). SBA did not label zoospores, cysts, or germinating cysts (Table III) (Detailed lectin binding patterns and their intensities will be published elsewhere). Incubation of lectins with respective hapten sugars has failed to label all the three stages, indicating the specific labeling of spore components by the lectins.

View larger version (33K): [in this window] [in a new window]    FIG. 5. FITC-conjugated lectin staining patterns of different developmental stages of Py.porphyrae (Strain C-1) spores where 5a–c were stained by Con A, 5d–f were stained by WGA and 5g was stained by BS II.  Fig. 5a. Con A has bound to the surface of the zoospore membrane as well as cytoplasm where as flagella remain not stained. Arrowhead indicates zoospore. x1300.  Fig. 5b. Con A has bound to the cyst wall as well as cytoplasm. Arrowhead indicates cyst. x1300.  Fig. 5c. Con A has bound only to the cytoplasm of the germinating cyst while the germ tube was weakly stained. Arrowhead indicates intensely stained cytoplasm of germinating cyst. x600.  Fig. 5d. WGA has bound intensely to the posterior region of the zoospore while the anterior region remains unbound. Flagella were weakly bound by WGA but not enough stained to be visible in the photomicrograph. Arrowhead indicates the posterior region of zoospore. x1300.  Fig. 5e. WGA has bound intensely both to the cyst wall and cytoplasm of cysts. Arrowhead indicates the cyst. x1300.  Fig. 5f. WGA has bound very intensely both to the germinating cyst and germ tube. Arrowhead indicates intensely stained germ tube. x600.  Fig. 5g. BS II has bound only to the cytoplasm of the germinating cyst and germ tube but not to the cyst wall. Arrowhead indicates the cytoplasm of germinating cyst. x600.

View larger version (16K): [in this window] [in a new window]    FIG. 6. The percentage of zoospores (strain C-1) encysted after incubation with MAbs 1A3, 2B6 and 2F6 for 15 min at 22 C (was plotted against the concentration of MAb in µg mL-1). All the experiments were carried out twice and each in quadruplicate. At least 100 spores were counted at random microscopic fields for each test sample and the standard deviations are represented

View larger version (34K): [in this window] [in a new window]    FIG. 7. Silver staining of zoospore (strain C-1) membrane proteins resolved by SDS-PAGE (Lane A) and western blotting probed with MAb 1A3 (Lane B). The molecular weight markers in kDa were indicated in the left

View larger version (17K): [in this window] [in a new window]    FIG. 8. The percentage of zoospores (strain C-1) encysted after incubation with lectins (WGA, WGA + N-acetyl glucosamine, Con A and BsII). WGA was incubated with 50 mM of N-acetyl glucosamine for 1 h before assaying for encystment induction

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Labeling of spore components by FITC conjugated lectins – Con A (specifically binds to mannosyl or glucosyl residues) bound to the surface of zoospores, cysts, and germinating cysts as in the case of other oomycetes, Phytophthora sp., Saprolegnia spp., and Py. aphanidermatum (Basic et al 1985, Burr and Beakes 1994).

Labeling of zoospores, cysts, and germinating cysts by FITC-WGA indicates the presence of N-acetyl-ß-d-glucosaminyl residues and N-acetyl-ß-d-glucosamine oligomers and/or sialic acid residues on their surface. In the case of saprophytic fish pathogen, Saprolegnia spp., the primary and secondary zoospores, cysts, and germinating cysts have been labeled by WGA with relatively different intensities and patterns (Lehnen and Powell 1993, Burr and Beakes 1994). However, WGA was found to fail in labeling the surface components of both zoospores and cysts of Phytophthora cinnamomi (Basic et al 1985). BS II also binds to the zoospores, cysts, and germinating cysts, indicating the presence of non-reducing terminals of oligosaccharides containing N-acetyl-d-glucosamine residues. However, BS II binds to zoospores, cysts, and germinating cysts very weakly in comparison with WGA even though both the lectins were conjugated with equivalent FITC molecules per mole of lectin. SBA failed to bind to the surface of zoospores, cysts, and germinating cysts, indicating the absence of N-acetyl-d-galactosamine residues in their cell membranes, in contrast to Phytophthora cinnamomi, where it binds to both zoospores and cysts (Basic et al 1985, Burr and Beakes 1994).

Induction of zoospore encystment by Mabs – MAb 1A3 induced 90% of zoospores to encyst in comparison with the controls at 90 µg mL^-1 within 15 min of its incubation. However, there was no induction of encystment either by MAb 2B6 or 2F6 even at 90 µg mL^-1 after incubation for 15 min. This shows that the epitope of specificity was different between the two MAbs (1A3 and 2B6) reacting with the zoospores. Further, the binding pattern of the MAbs 1A3 and 2B6 varied as determined by IFA. 1A3 binds to flagella as well as to the the zoospore surface, whereas 2B6 binds only to the surface of the zoospores but not to the flagella. In Py. aphanidermatum, the MAb PA1 binds to flagella and to the surface of the zoospore and induces encystment, whereas the MAb PA8 binds to the zoospore surface and to the cyst wall, and does not induce encystment (Estrada-Garcia et al 1990). In the case of Phytophthora, only MAb Zf-1 induced zoospore encystment, and binds only to the flagella but not to the surface of the zoospore (Hardham et al 1985). The results from the present and previous studies suggest that the induction of zoospore encystment by the MAbs is due to stimulation of membrane bound receptor rather than a physiological phenomenon. In addition, MAb 1A3 recognizes a glycoprotein of 109 kDa as determined by treatment with heat, protease, and periodate, and western blotting. MAb 2B6 also recognizes a glycoprotein but does not reacted at all with the SDS-PAGE-denatured proteins. However, in Py. aphanidermatum the MAb PA1 recognizes a surface glycoprotein of molecular weight 75 kDa (Estrada-Garcia et al 1990). The possibility of steric effect on encystment induction by MAb 1A3, due to its IgM nature, was disproved, as the MAb 2B6 also belongs to the IgM class and does not induce encystment of the zoospores.

Induction of zoospore encystment by lectins – Of the three lectins tested, WGA alone induced 80% of zoospores to encyst at 100 µg mL^-1, whereas WGA incubated with its hapten sugar did not cause encystment of the zoospores. This indicates that binding of WGA either to N-acetyl-ß-d-glucosaminyl residues or N-acetyl-ß-d-glucosamine oligomers/sialic acid residues was responsible for the induction of encystment of the zoospores. Even though BS II binds to non-reducing terminals of oligosaccharides containing N-acetyl-d-glucosamine residues, the encystment of the zoospores was not induced. Further, the metabolism of encysted spores remains unaffected even at 100 µg mL^-1 of lectin as judged by the development of germ tubes after incubating the lectin-treated spores at room temperature for 90 min. However, Kerwin et al (1992) have reported the absence of encystment induction in the case of Py. marinum upon incubation with WGA, Con A, and other lectins. In the case of Saprolegnia diclina there is a significant encystment of the zoospores upon incubation with WGA but not in the case of Saprolegina parasitica (Burr and Beakes 1994). These results suggest that differential behavior of different species within the same genus might have taxonomical significance. But, neither of the other two lectins does affect the zoospore encystment in agreement with the results of Kerwin et al (1993).

In the present study, MAbs were successfully developed against the surface components of zoospores and cysts. Using the MAbs a glycoprotein of molecular weight of 109-kDa, a putative receptor molecule involved in the zoospore encystment process, has been identified. In the future, the identification of the gene responsible for the receptor for encystment signaling using the MAbs will help in development of strategies to interfere with the host specific infection by the pathogen.

	ACKNOWLEDGMENTS

 
This work is partially supported by grants to the Marine plant science laboratory (No. 11660191), Nagasaki University and a scholarship to MK Addepalli from the Ministry of Education, Science, Sports and Culture, Japan.

	FOOTNOTES

 
¹ Corresponding author, Email: yfujita{at}net.nagasaki-u.ac.jp

Accepted for publication January 9, 2002.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
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