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Expression of exo-polygalacturonases in Botrytis cinerea
FEMS Microbiology Letters 201 (2001) 105^109
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Expression of exo-polygalacturonases in Botrytis cinerea Eugene Rha, Hong Jai Park, Myeong Ok Kim, Young Ryun Chung, Chang-Won Lee, Jae Won Kim * Division of Life Science and Research Institute of Natural Science, College of Natural Science, Gyeongsang National University, Jinju 660-701, South Korea Received 24 May 2001; accepted 29 May 2001
Abstract The pathogenic fungus, Botrytis cinerea, causing gray mold disease in a variety of plant species, secretes at least four polygalacturonases (PGs), cell wall degrading enzymes. Among them, we prepared polyclonal antibody against purified 66-kDa exo-PG in rabbit. Immunoblot analysis revealed that the antibody recognized two exo-PGs, 66 kDa and 70 kDa in molecular mass, secreted from B. cinerea cultured in the medium containing citrus pectin as a carbon source. By immunohistochemical analysis, the expression of exo-PGs was identified in cucumber leaves inoculated with spores of B. cinerea. The exo-PGs were observed 9 h after inoculation, and the amount of exo-PGs increased with time in the leaves. The exo-PGs were induced by polygalacturonic acid as well as its monomer, galacturonic acid, in vitro. The expression of 66-kDa exo-PG (exo-PG I) increased with time of culture, while 70-kDa exo-PG (exo-PG II) was transiently expressed soon after the start of culture. Therefore, exo-PGs might play an important role in pathogenesis at an early stage of infection as well as in tissue maceration of host plant. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Polygalacturonase; Exo-polygalacturonase; Botrytis cinerea
1. Introduction Decomposition of plant cell walls is a characteristic feature of numerous plant diseases caused by a variety of plant pathogenic fungi. They secrete a number of extracellular enzymes capable of degrading plant cell wall components. Amongst them, pectic enzymes such as polygalacturonases (PGs), pectin-methyl esterase and pectin lyase play an important role in pathogenesis [1,2]. Botrytis cinerea Pers.: Fr. (telemorph: Botryotinia fuckeliana (de Bary) Whetzel) produces several PG isozymes [3,4]. There are two classes of PGs according to their mode of action on the polygalacturonic acid chain: endo-PGs and exo-PGs. Biochemical studies on PGs have been carried out in several laboratories [4,5]. However, it is unclear how many PG isozymes are produced by B. cinerea because the numbers of PG isozymes separated by isoelectric focusing (IEF)^polyacrylamide gel electrophoresis (PAGE) vary with isolates and age of culture. Recently, at least six endo-PG genes were cloned and sequenced [6,7]. Among them, Bcpg1 was eliminated by partial gene replacement
and the resulting mutants were still pathogenic and displayed similar primary infections, although a signi¢cant decrease in secondary infection was observed on host tissue. These studies also showed that two of the genes, Bcpg1 and Bcpg2, were expressed constitutively, and others were induced by galacturonic acid and ambient pH [7,8]. In contrast to endo-PGs, the studies on exo-PGs have not been performed extensively. According to Tobias et al. [9,10], there were four exo-PGs and one endo-PG in an apple tissue decayed by B. cinerea indicating the possibility of a crucial role of exo-PGs in pathogenesis. Although the role of exo-PGs during penetration is not clear yet, it is important to know the expression pattern of exo-PGs to elucidate pathogenicity of B. cinerea. However, the induction of exo-PG has not been studied in detail. Here we examine the in vitro induction of exo-PGs of B. cinerea by immunological techniques. 2. Materials and methods 2.1. Fungal strains and growth condition
* Corresponding author. Tel. : +82 (55) 751-5944; Fax: +82 (55) 759-0187; E-mail: [email protected]
B. cinerea T91-1 strain, isolated from a lesion of a
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 2 5 2 - X
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tomato leaf in Daejeon area of South Korea, was obtained from National Institute of Chemistry, South Korea, and was maintained on potato dextrose agar (Difco Co.) at 20³C. The fungus was grown in a synthetic medium containing 10 g NaNO3 , 5 g KH2 PO4 , 2.5 g MgSO4 W7H2 O, 1 g CaCl2 , and 0.5 g yeast extract in 1 l of distilled water. Cultures were supplemented with citrus pectin, polygalacturonic acid, galacturonic acid, or glucose (Sigma Chemical Co., St. Louis, MO, USA) at the concentration of 0.5% as carbon source. At daily intervals, samples of culture were withdrawn. The culture £uids separated from the mycelium by ¢ltration were used for analyses. 2.2. Enzyme assays and protein determination PG was assayed as described previously with sodium polygalacturonate as substrate [11]. Protein concentrations were determined by the method of Bradford [12]. 2.3. Puri¢cation of exo-PG I Exo-PG I which has a molecular mass of 66 kDa was puri¢ed as described previously [11]. Puri¢ed enzyme was used for preparation of a polyclonal antibody and used as the standard protein for Western blot analysis. 2.4. Preparation of polyclonal antiserum against exo-PG I Puri¢ed enzyme (100 Wg) emulsi¢ed with 1 ml of Freund's complete adjuvant was intramuscularly injected to rabbit (New Zealand White, male) three times with a time interval of 2 weeks. One month after the third immunization, 200 Wg of the enzyme was ¢nally boosted without adjuvant. The serum was collected from the rabbit 10 days after ¢nal boost as described elsewhere [13]. 2.5. Determination of exo-PG by electrophoresis and immunoblotting Sodium dodecyl sulfate (SDS)^PAGE of extracellular proteins was carried out using a slab gel composed of 5% stacking gel and 10% resolving gel as described by Laemmli et al. [14]. The molecular mass of the proteins was estimated against pre-stained molecular mass markers (Bio-Rad, Richmond, CA, USA). The pI value of puri¢ed enzyme was determined by IEF^PAGE employing 5% ampholytes (pH range, 3.0^ 10.0). The separated protein band was visualized by Coomassie blue staining or activity staining using the polygalacturonic acid agarose overlay method [15]. Gel overlays contained 2 mg ml31 polygalacturonic acid in 0.1 M sodium acetate bu¡er (pH 4.5). Overlays remained in contact with gels for 30 min at 45³C, after which they were removed and stained for 30 min in 0.02% ruthenium red. For immunoblotting analysis following SDS^PAGE, the proteins were electrophoretically transferred to nitrocellu-
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lose. Goat anti-rabbit Q-globulin alkaline phosphatase conjugate (KPL) diluted at 1:2000 was used as the secondary antibody. 2.6. Immunohistochemistry of exo-PGs in cucumber leaves inoculated with B. cinerea Conidia of B. cinerea (1U106 ml31 ) were inoculated on to cucumber leaves. The leaves were collected 0, 9, 12, 24 and 48 h after inoculation. The cucumber leaves were dissected, ¢xed by immersion in 4% paraformaldehyde in 0.1 M sodium phosphate bu¡er for 24 h, and cryoprotected by immersion in phosphate bu¡er containing 20% sucrose for 24 h. Tissues were frozen by OCT compound (AO, 380³C). Sections (10 Wm) were cut in cross section on a cryomicrotome (Leica, CM 3050, Germany). Sections were thaw-mounted on the probe-on plus charged slides (Fischer) at room temperature, placed in the cryostat, and then stored at 370³C until use. For the localization of immunoreactive exo-PG I, the avidin^biotin complex (ABC) method was used [16]. Tissue sections on slides were air-dried, dipped twice in 0.02 M phosphate-bu¡ered saline (PBS, pH 7.4) for 5 min. Before the primary antibody application, the tissue sections were incubated for 30 min with 40 Wl of normal goat serum diluted 1:20 to eliminate non-speci¢c binding. Slides were then applied with 50 Wl of the primary antibody, rabbit-derived anti-exo-PG I with a ¢nal dilution of 1:400 for overnight at 4³C. The sections were washed with PBS for 10 min twice, incubated with 0.5% periodic acid to exclude the endogenous peroxidase, and then applied with 50 Wl of biotinylated goat anti-rabbit IgG (Vector, 1:200) for 30 min at room temperature. Then, the sections were applied with 50 Wl of ABC peroxidase labeled (Vector, 1:50) for 30 min at room temperature. After washing with PBS, the sections were incubated with 0.5% 3,3P-diaminobenzidine in 0.01% hydrogen peroxide in PBS for 5 min, washed, dehydrated, mounted in a synthetic mounting medium, and then observed under a light microscope. 3. Results and discussion 3.1. Puri¢cation of exo-PG I from B. cinerea T91-1 The exo-PG I was puri¢ed to electrophoretic homogeneity by a series of procedures including acetone precipitation, ion exchange, heparin a¤nity, and reverse phase column chromatography from 14-day-old culture ¢ltrates as described [11]. The molecular mass of the enzyme was estimated to be 66 kDa by denaturing PAGE as shown in Fig. 1A. Similarly, the molecular mass of the native enzyme was determined by gel permeation column chromatography to be 64 kDa, indicating the enzyme to be a single polypeptide chain (data not shown). The pI value of the enzyme was determined to be 4.85 by IEF and
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Fig. 1. Electrophoretic analysis of puri¢ed exo-PG I from B. cinerea. (A) Puri¢ed enzyme from 14-day-old culture ¢ltrates was analyzed by 10% SDS^PAGE (lane 1: 1 Wg, lane 2: 3 Wg, respectively). Proteins were visualized by Coomassie staining. (B) IEF and activity staining of the puri¢ed enzyme (lane 1: IEF of 2 Wg protein, lane 2: activity staining of puri¢ed enzyme)
Fig. 2. Western blot analysis of exo-PG I using polyclonal antibody. Puri¢ed enzyme (lane 1: 50 ng, lane 2: 500 ng) and 14-day-old culture ¢ltrates (lanes 3 and 5: 20 Wg, lanes 4 and 6: 200 Wg protein, respectively) were separated by 10% SDS^PAGE, and then triggered by polyclonal antibody against puri¢ed exo-PG I (lanes 3 and 4) and pre-immune serum (lanes 5 and 6).
activity staining as shown in Fig. 1B. The mode of cleavage of polygalacturonic acid by the puri¢ed enzyme was studied by comparison of the rate of reduction in viscosity with the corresponding increase in appearance of reducing end groups in solution, con¢rming this enzyme functioned as an exo-enzyme (data not shown). The enzyme puri¢ed in this study was similar to the enzyme denoted exo-PG I by Johnston and Williamson [3] in molecular mass, pI value and enzymatic properties such as the mode of cleavage, Km and Vmax values [11]. From the 60 l of culture ¢ltrates, 600 Wg of puri¢ed protein was obtained. The pro-
tein was used for the preparation of antibody against exoPG I from rabbit. 3.2. Preparation of polyclonal antiserum against exo-PG I The polyclonal antiserum against exo-PG I was prepared by injecting puri¢ed enzyme into a rabbit without deglycosylation. To determine the antibody speci¢city, puri¢ed enzyme and 14-day-old culture ¢ltrates were analyzed by Western blotting. As shown in Fig. 2, antiserum speci¢cally reacted with exo-PG I. When culture ¢ltrates
Fig. 3. Secretion of exo-PG I in the leaves of cucumber inoculated by conidia of B. cinerea. Leaves were immunoassayed after inoculation at 0 (A), 9 (B), 12 (C), and 24 h (D), respectively. Arrows indicate the hyphae of B. cinerea (U400).
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Fig. 4. Expression of exo-PG I by various carbon sources from B. cinerea. Puri¢ed enzyme (50 ng protein) and 5-day-old culture ¢ltrates (200 Wg protein) were analyzed by Western blotting. Lane 1: puri¢ed enzyme, lanes 2^7: secreted proteins in the medium containing glycerol, cellobiose, glucose, starch, sucrose, and pectin as a carbon source, respectively.
(14 days old) were analyzed, there was no other signi¢cant protein band except exo-PG I, showing that antibody did not react with endo-PGs. These results were consistent with those of Johnston and Williamson [5] showing that an antibody prepared against endo-PG did not bind to exo-PG I, while two endo-PGs immunocross-reacted. This suggests that exo-PGs are di¡erent from endo-PGs in molecular structure. To con¢rm the speci¢city of the antibody, enzyme activity was measured in supernatant and precipitates after immunoprecipitation. When immunoprecipitation was performed using pre-immune serum, enzyme activity was recovered in the supernatant fraction. There was no enzymatic activity in either the supernatant or the precipitates when the antiserum was used, indicating the formation of antigen^antibody complex. By Western blot analysis of immunoprecipitates, it was con¢rmed again that a single band appeared at the 66-kDa position corresponding to exo-PG I (data not shown).
biose, glucose, starch or sucrose for 5 days. Although the enzyme activity of PG was observed from the ¢rst day of culture, exo-PG I was only detected in a medium containing pectin, as shown in Fig. 4. Since the enzyme activity of the medium can be attributed to production of endo-PG, it was concluded that exo-PG I was induced by pectin. Interestingly, it was found that a protein band with a molecular mass of 70 kDa (Fig. 4, lane 7), in addition to exo-PG I, reacted with the antiserum in ¢ltrate of 5-day-old cultures, but was not detected in 14-day-old cultures as shown in Fig. 1A. The 70-kDa protein band corresponds with the molecular mass of the exo-PG II described by Johnston and Williamson [3]. This result suggests that two exo-PGs share common epitopes su¤cient to be recognized by the antibody raised against exoPG I. Together with these results, it suggests that the exoPGs are induced by pectin and the expression of exo-PG II di¡ers from exo-PG I. These results encouraged us to investigate the time course of expression of exo-PGs. 3.5. Induction of exo-PGs by galacturonic acid When the fungus was cultured over a 6-day period in the medium containing citrus pectin as a carbon source, exo-PG I was not detected until 2 days, and then increased up to 6 days (Fig. 5A). Meanwhile, the expression of exoPG II was only detected in 3^5-day-old culture ¢ltrates, and then disappeared after further mycelial growth. This
3.3. Immunohistochemistry of exo-PG in cucumber leaves inoculated with B. cinerea Immunostaining for exo-PG was carried out at 0, 9, 12, and 24 h after inoculation of conidia obtained from B. cinerea onto cucumber leaves. As shown in Fig. 3, secreted exo-PG from hyphae was detected on the surface of leaves at 9 h after inoculation. The secretion of exo-PG increased with time, and coincided with maceration of leaves observed at 12 and 24 h after inoculation. Considering the fact that germination of conidia in vitro usually takes 6^7 h in 0.1% PDB, the secretion of exo-PGs seems to occur at an early stage of infection. 3.4. Induction of exo-PGs by various carbon sources To investigate the e¡ect of various carbon sources on the expression pattern of exo-PGs, the fungus was cultured in six media containing citrus pectin, glycerol, cello-
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Fig. 5. Induction of exo-PGs from B. cinerea by pectin, polygalacturonic acid, and galacturonic acid. The secreted proteins (100 Wg) in the medium containing pectin (A), polygalacturonic acid (B), galacturonic acid (C), and glucose (D) were analyzed by Western blot. Numbers indicate the day of culture.
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result con¢rmed again that both enzymes are induced by pectin, and that the expression pattern of each exo-PG di¡ered (Fig. 5A). Under the same conditions, the fungus was cultured in a medium containing polygalacturonic acid as a carbon source and a similar pattern of the enzyme expression was observed (Fig. 5B). As shown in Fig. 5C, exo-PGs were also induced strongly and rapidly by galacturonic acid, especially the exo-PG II that was only produced until day 3. Our data are consistent with those of Johnston and Williamson [5]. When they resolved culture ¢ltrates of B. cinerea grown in the medium containing glucose or galacturonic acid for 48 h by IEF, novel PG activity peaks between pH 3.7 and 6.3 were induced by galacturonic acid, which failed to react with an antibody raised against endo-PGs. They suggested that this novel activity peak is exo-PG I and/or exo-PG II induced by galacturonic acid. To determine whether or not exo-PGs are produced in the medium containing glucose as a carbon source, the fungus was cultured over periods longer than 6 days. No exo-PGs were produced up to 5 days, as described by Johnston and Willamson [5]. However, the exo-PGs were detected in the ¢ltrates grown up to 6 days as shown in Fig. 5D. Therefore, it is likely that time of expression of exo-PGs varies depending on the carbon source and isolate. Both enzymes were induced rapidly by galacturonic acid, while the induction of exo-PGs was delayed by glucose. In contrast with endo-PGs [5], there has been no direct evidence for the induction of exo-PGs. As far as we know, this is the ¢rst report to describe the induction of exo-PGs in B. cinerea. Since the cloning and sequencing of the genes for exo-PGs have not yet been reported, we do not know whether these enzymes are the products of a single gene. Although it is likely that the two exo-PGs are not identical proteins glycosylated to varying degrees as described previously [3], the possibility of proteolytic degradation cannot be excluded. Considering that six genes for endo-PGs have been cloned [6,7], it is probable that there are also multi-genes for exo-PGs. The transient expression of exo-PG II might be explained by the existence of proteinases such as aspartate proteinase [17]. However, we cannot suggest how proteinases may specifically degrade exo-PG II, but not exo-PG I. Although the genes of several endo-PGs have been cloned [6,7], we still do not know how, and to what extent, PGs contribute to aggressiveness in B. cinerea. Gene deletion studies of endo- or exo- or both endo- and exo-PGs may explain the role of PGs in pathogenesis of this fungus. Acknowledgements
Institute, Invergowrie, Dundee, UK, for providing an antibody against endo-PG I. The Basic Science Research Institute Program, Ministry of Education, BSRI-98-4405, supported this work. References [1] Staples, R.C. and Mayer, A.M. (1995) Putative virulence factors of Botrytis cinerea acting as a wound pathogen. FEMS Microbiol. Lett. 134, 1^7. [2] Walton, J.D. (1994) Deconstructing the cell wall. Plant Physiol. 104, 1113^1118. [3] Johnston, D.J. and Williamson, B. (1992) Puri¢cation and characterization of four polygalacturonases from Botrytis cinerea. Mycol. Res. 96, 343^349. [4] Marcus, L. and Schejter, A. (1983) Single step chromatographic puri¢cation and characterization of the endopolygalacturonases and pectinesterases of the fungus, Botrytis cinerea Pers. Physiol. Plant Pathol. 23, 1^13. [5] Johnston, D.J. and Williamson, B. (1992) An immunological study of the induction of polygalacturonases in Botrytis cinerea. FEMS Microbiol. Lett. 97, 19^24. [6] Ten Have, A., Mulder, W., Visser, J. and Van Kan, J.A.L. (1998) The endo-polygalacturonase gene Bcpg1 is required for full virulence of Botrytis cinerea. Mol. Plant-Microbe Interact. 11, 1009^1016. [7] Wuben, J.P., Mulder, W., Ten Have, A., Van Kan, J.A.L. and Visser, J. (1999) Cloning and partial characterization of endopolygalacturonase genes Botrytis cinerea. Appl. Environ. Microbiol. 65, 1596^1602. [8] Wubben, J.P., Ten Have, A., Van Kan, J.A.L. and Visser, J. (2000) Regulation of endopolygalacturonase gene expression in Botrytis cinerea by galacturonic acid, ambient pH and carbon catabolite expression. Curr. Genet. 37, 152^155. [9] Tobias, R., Conway, W. and Sams, C. (1993) Polygalacturonase isozymes from Botrytis cinerea grown on apple pectin. Biochem. Mol. Biol. Int. 30, 829^837. [10] Tobias, R., Conway, W. and Sams, C. (1995) Polygalacturonase produced in apple tissue decayed by Botrytis cinerea. Biochem. Mol. Biol. Int. 35, 813^823. [11] Lee, T.H., Kim, B.Y., Chung, Y.R., Lee, S.Y., Lee, C.W. and Kim, J.W. (1997) Puri¢cation and characterization of an exo-polygalacturonase from Botrytis cinerea. J. Microbiol. 35, 134^140. [12] Bradford, M.M. (1976) A rapid and sensitive method for the determination of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248^254. [13] Harlow, E. and Lane, D. (1988) Antibodies. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [14] Laemmli, U.K. (1970) Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227, 680^685. [15] Ried, J.L. and Collmer, A. (1985) Activity stain for rapid characterization of pectic enzymes in isoelectric focusing and sodium dodecyl sulfate^polyacrylamide gels. Appl. Environ. Microbiol. 50, 615^622. [16] Hsu, S.M., Raine, L. and Franger, H. (1981) The use of avidin^biotin peroxidase complex (ABC) in immunoperoxidase technique: a comparison between ABC and unlabeled antibody (PAP) procedure. J. Histochem. Cytochem. 29, 577^580. [17] Movahedi, S. and Heale, J.B. (1990) The roles of aspartic proteinase and endo-pectin lyase enzymes in the primary stages of infection and pathogenesis of various host tissues by di¡erent isolates of Botrytis cinerea Pers ex. Pers. Physiol. Mol. Plant Pathol. 36, 303^324.
We thank Dr. B. Williamson, Scottish Crop Research
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Expression of exo-polygalacturonases in Botrytis cinerea Eugene Rha, Hong Jai Park, Myeong Ok Kim, Young Ryun Chung, Chang-Won Lee, Jae Won Kim * Division of Life Science and Research Institute of Natural Science, College of Natural Science, Gyeongsang National University, Jinju 660-701, South Korea Received 24 May 2001; accepted 29 May 2001
Abstract The pathogenic fungus, Botrytis cinerea, causing gray mold disease in a variety of plant species, secretes at least four polygalacturonases (PGs), cell wall degrading enzymes. Among them, we prepared polyclonal antibody against purified 66-kDa exo-PG in rabbit. Immunoblot analysis revealed that the antibody recognized two exo-PGs, 66 kDa and 70 kDa in molecular mass, secreted from B. cinerea cultured in the medium containing citrus pectin as a carbon source. By immunohistochemical analysis, the expression of exo-PGs was identified in cucumber leaves inoculated with spores of B. cinerea. The exo-PGs were observed 9 h after inoculation, and the amount of exo-PGs increased with time in the leaves. The exo-PGs were induced by polygalacturonic acid as well as its monomer, galacturonic acid, in vitro. The expression of 66-kDa exo-PG (exo-PG I) increased with time of culture, while 70-kDa exo-PG (exo-PG II) was transiently expressed soon after the start of culture. Therefore, exo-PGs might play an important role in pathogenesis at an early stage of infection as well as in tissue maceration of host plant. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Polygalacturonase; Exo-polygalacturonase; Botrytis cinerea
1. Introduction Decomposition of plant cell walls is a characteristic feature of numerous plant diseases caused by a variety of plant pathogenic fungi. They secrete a number of extracellular enzymes capable of degrading plant cell wall components. Amongst them, pectic enzymes such as polygalacturonases (PGs), pectin-methyl esterase and pectin lyase play an important role in pathogenesis [1,2]. Botrytis cinerea Pers.: Fr. (telemorph: Botryotinia fuckeliana (de Bary) Whetzel) produces several PG isozymes [3,4]. There are two classes of PGs according to their mode of action on the polygalacturonic acid chain: endo-PGs and exo-PGs. Biochemical studies on PGs have been carried out in several laboratories [4,5]. However, it is unclear how many PG isozymes are produced by B. cinerea because the numbers of PG isozymes separated by isoelectric focusing (IEF)^polyacrylamide gel electrophoresis (PAGE) vary with isolates and age of culture. Recently, at least six endo-PG genes were cloned and sequenced [6,7]. Among them, Bcpg1 was eliminated by partial gene replacement
and the resulting mutants were still pathogenic and displayed similar primary infections, although a signi¢cant decrease in secondary infection was observed on host tissue. These studies also showed that two of the genes, Bcpg1 and Bcpg2, were expressed constitutively, and others were induced by galacturonic acid and ambient pH [7,8]. In contrast to endo-PGs, the studies on exo-PGs have not been performed extensively. According to Tobias et al. [9,10], there were four exo-PGs and one endo-PG in an apple tissue decayed by B. cinerea indicating the possibility of a crucial role of exo-PGs in pathogenesis. Although the role of exo-PGs during penetration is not clear yet, it is important to know the expression pattern of exo-PGs to elucidate pathogenicity of B. cinerea. However, the induction of exo-PG has not been studied in detail. Here we examine the in vitro induction of exo-PGs of B. cinerea by immunological techniques. 2. Materials and methods 2.1. Fungal strains and growth condition
* Corresponding author. Tel. : +82 (55) 751-5944; Fax: +82 (55) 759-0187; E-mail: [email protected]
B. cinerea T91-1 strain, isolated from a lesion of a
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 2 5 2 - X
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tomato leaf in Daejeon area of South Korea, was obtained from National Institute of Chemistry, South Korea, and was maintained on potato dextrose agar (Difco Co.) at 20³C. The fungus was grown in a synthetic medium containing 10 g NaNO3 , 5 g KH2 PO4 , 2.5 g MgSO4 W7H2 O, 1 g CaCl2 , and 0.5 g yeast extract in 1 l of distilled water. Cultures were supplemented with citrus pectin, polygalacturonic acid, galacturonic acid, or glucose (Sigma Chemical Co., St. Louis, MO, USA) at the concentration of 0.5% as carbon source. At daily intervals, samples of culture were withdrawn. The culture £uids separated from the mycelium by ¢ltration were used for analyses. 2.2. Enzyme assays and protein determination PG was assayed as described previously with sodium polygalacturonate as substrate [11]. Protein concentrations were determined by the method of Bradford [12]. 2.3. Puri¢cation of exo-PG I Exo-PG I which has a molecular mass of 66 kDa was puri¢ed as described previously [11]. Puri¢ed enzyme was used for preparation of a polyclonal antibody and used as the standard protein for Western blot analysis. 2.4. Preparation of polyclonal antiserum against exo-PG I Puri¢ed enzyme (100 Wg) emulsi¢ed with 1 ml of Freund's complete adjuvant was intramuscularly injected to rabbit (New Zealand White, male) three times with a time interval of 2 weeks. One month after the third immunization, 200 Wg of the enzyme was ¢nally boosted without adjuvant. The serum was collected from the rabbit 10 days after ¢nal boost as described elsewhere [13]. 2.5. Determination of exo-PG by electrophoresis and immunoblotting Sodium dodecyl sulfate (SDS)^PAGE of extracellular proteins was carried out using a slab gel composed of 5% stacking gel and 10% resolving gel as described by Laemmli et al. [14]. The molecular mass of the proteins was estimated against pre-stained molecular mass markers (Bio-Rad, Richmond, CA, USA). The pI value of puri¢ed enzyme was determined by IEF^PAGE employing 5% ampholytes (pH range, 3.0^ 10.0). The separated protein band was visualized by Coomassie blue staining or activity staining using the polygalacturonic acid agarose overlay method [15]. Gel overlays contained 2 mg ml31 polygalacturonic acid in 0.1 M sodium acetate bu¡er (pH 4.5). Overlays remained in contact with gels for 30 min at 45³C, after which they were removed and stained for 30 min in 0.02% ruthenium red. For immunoblotting analysis following SDS^PAGE, the proteins were electrophoretically transferred to nitrocellu-
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lose. Goat anti-rabbit Q-globulin alkaline phosphatase conjugate (KPL) diluted at 1:2000 was used as the secondary antibody. 2.6. Immunohistochemistry of exo-PGs in cucumber leaves inoculated with B. cinerea Conidia of B. cinerea (1U106 ml31 ) were inoculated on to cucumber leaves. The leaves were collected 0, 9, 12, 24 and 48 h after inoculation. The cucumber leaves were dissected, ¢xed by immersion in 4% paraformaldehyde in 0.1 M sodium phosphate bu¡er for 24 h, and cryoprotected by immersion in phosphate bu¡er containing 20% sucrose for 24 h. Tissues were frozen by OCT compound (AO, 380³C). Sections (10 Wm) were cut in cross section on a cryomicrotome (Leica, CM 3050, Germany). Sections were thaw-mounted on the probe-on plus charged slides (Fischer) at room temperature, placed in the cryostat, and then stored at 370³C until use. For the localization of immunoreactive exo-PG I, the avidin^biotin complex (ABC) method was used [16]. Tissue sections on slides were air-dried, dipped twice in 0.02 M phosphate-bu¡ered saline (PBS, pH 7.4) for 5 min. Before the primary antibody application, the tissue sections were incubated for 30 min with 40 Wl of normal goat serum diluted 1:20 to eliminate non-speci¢c binding. Slides were then applied with 50 Wl of the primary antibody, rabbit-derived anti-exo-PG I with a ¢nal dilution of 1:400 for overnight at 4³C. The sections were washed with PBS for 10 min twice, incubated with 0.5% periodic acid to exclude the endogenous peroxidase, and then applied with 50 Wl of biotinylated goat anti-rabbit IgG (Vector, 1:200) for 30 min at room temperature. Then, the sections were applied with 50 Wl of ABC peroxidase labeled (Vector, 1:50) for 30 min at room temperature. After washing with PBS, the sections were incubated with 0.5% 3,3P-diaminobenzidine in 0.01% hydrogen peroxide in PBS for 5 min, washed, dehydrated, mounted in a synthetic mounting medium, and then observed under a light microscope. 3. Results and discussion 3.1. Puri¢cation of exo-PG I from B. cinerea T91-1 The exo-PG I was puri¢ed to electrophoretic homogeneity by a series of procedures including acetone precipitation, ion exchange, heparin a¤nity, and reverse phase column chromatography from 14-day-old culture ¢ltrates as described [11]. The molecular mass of the enzyme was estimated to be 66 kDa by denaturing PAGE as shown in Fig. 1A. Similarly, the molecular mass of the native enzyme was determined by gel permeation column chromatography to be 64 kDa, indicating the enzyme to be a single polypeptide chain (data not shown). The pI value of the enzyme was determined to be 4.85 by IEF and
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Fig. 1. Electrophoretic analysis of puri¢ed exo-PG I from B. cinerea. (A) Puri¢ed enzyme from 14-day-old culture ¢ltrates was analyzed by 10% SDS^PAGE (lane 1: 1 Wg, lane 2: 3 Wg, respectively). Proteins were visualized by Coomassie staining. (B) IEF and activity staining of the puri¢ed enzyme (lane 1: IEF of 2 Wg protein, lane 2: activity staining of puri¢ed enzyme)
Fig. 2. Western blot analysis of exo-PG I using polyclonal antibody. Puri¢ed enzyme (lane 1: 50 ng, lane 2: 500 ng) and 14-day-old culture ¢ltrates (lanes 3 and 5: 20 Wg, lanes 4 and 6: 200 Wg protein, respectively) were separated by 10% SDS^PAGE, and then triggered by polyclonal antibody against puri¢ed exo-PG I (lanes 3 and 4) and pre-immune serum (lanes 5 and 6).
activity staining as shown in Fig. 1B. The mode of cleavage of polygalacturonic acid by the puri¢ed enzyme was studied by comparison of the rate of reduction in viscosity with the corresponding increase in appearance of reducing end groups in solution, con¢rming this enzyme functioned as an exo-enzyme (data not shown). The enzyme puri¢ed in this study was similar to the enzyme denoted exo-PG I by Johnston and Williamson [3] in molecular mass, pI value and enzymatic properties such as the mode of cleavage, Km and Vmax values [11]. From the 60 l of culture ¢ltrates, 600 Wg of puri¢ed protein was obtained. The pro-
tein was used for the preparation of antibody against exoPG I from rabbit. 3.2. Preparation of polyclonal antiserum against exo-PG I The polyclonal antiserum against exo-PG I was prepared by injecting puri¢ed enzyme into a rabbit without deglycosylation. To determine the antibody speci¢city, puri¢ed enzyme and 14-day-old culture ¢ltrates were analyzed by Western blotting. As shown in Fig. 2, antiserum speci¢cally reacted with exo-PG I. When culture ¢ltrates
Fig. 3. Secretion of exo-PG I in the leaves of cucumber inoculated by conidia of B. cinerea. Leaves were immunoassayed after inoculation at 0 (A), 9 (B), 12 (C), and 24 h (D), respectively. Arrows indicate the hyphae of B. cinerea (U400).
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Fig. 4. Expression of exo-PG I by various carbon sources from B. cinerea. Puri¢ed enzyme (50 ng protein) and 5-day-old culture ¢ltrates (200 Wg protein) were analyzed by Western blotting. Lane 1: puri¢ed enzyme, lanes 2^7: secreted proteins in the medium containing glycerol, cellobiose, glucose, starch, sucrose, and pectin as a carbon source, respectively.
(14 days old) were analyzed, there was no other signi¢cant protein band except exo-PG I, showing that antibody did not react with endo-PGs. These results were consistent with those of Johnston and Williamson [5] showing that an antibody prepared against endo-PG did not bind to exo-PG I, while two endo-PGs immunocross-reacted. This suggests that exo-PGs are di¡erent from endo-PGs in molecular structure. To con¢rm the speci¢city of the antibody, enzyme activity was measured in supernatant and precipitates after immunoprecipitation. When immunoprecipitation was performed using pre-immune serum, enzyme activity was recovered in the supernatant fraction. There was no enzymatic activity in either the supernatant or the precipitates when the antiserum was used, indicating the formation of antigen^antibody complex. By Western blot analysis of immunoprecipitates, it was con¢rmed again that a single band appeared at the 66-kDa position corresponding to exo-PG I (data not shown).
biose, glucose, starch or sucrose for 5 days. Although the enzyme activity of PG was observed from the ¢rst day of culture, exo-PG I was only detected in a medium containing pectin, as shown in Fig. 4. Since the enzyme activity of the medium can be attributed to production of endo-PG, it was concluded that exo-PG I was induced by pectin. Interestingly, it was found that a protein band with a molecular mass of 70 kDa (Fig. 4, lane 7), in addition to exo-PG I, reacted with the antiserum in ¢ltrate of 5-day-old cultures, but was not detected in 14-day-old cultures as shown in Fig. 1A. The 70-kDa protein band corresponds with the molecular mass of the exo-PG II described by Johnston and Williamson [3]. This result suggests that two exo-PGs share common epitopes su¤cient to be recognized by the antibody raised against exoPG I. Together with these results, it suggests that the exoPGs are induced by pectin and the expression of exo-PG II di¡ers from exo-PG I. These results encouraged us to investigate the time course of expression of exo-PGs. 3.5. Induction of exo-PGs by galacturonic acid When the fungus was cultured over a 6-day period in the medium containing citrus pectin as a carbon source, exo-PG I was not detected until 2 days, and then increased up to 6 days (Fig. 5A). Meanwhile, the expression of exoPG II was only detected in 3^5-day-old culture ¢ltrates, and then disappeared after further mycelial growth. This
3.3. Immunohistochemistry of exo-PG in cucumber leaves inoculated with B. cinerea Immunostaining for exo-PG was carried out at 0, 9, 12, and 24 h after inoculation of conidia obtained from B. cinerea onto cucumber leaves. As shown in Fig. 3, secreted exo-PG from hyphae was detected on the surface of leaves at 9 h after inoculation. The secretion of exo-PG increased with time, and coincided with maceration of leaves observed at 12 and 24 h after inoculation. Considering the fact that germination of conidia in vitro usually takes 6^7 h in 0.1% PDB, the secretion of exo-PGs seems to occur at an early stage of infection. 3.4. Induction of exo-PGs by various carbon sources To investigate the e¡ect of various carbon sources on the expression pattern of exo-PGs, the fungus was cultured in six media containing citrus pectin, glycerol, cello-
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Fig. 5. Induction of exo-PGs from B. cinerea by pectin, polygalacturonic acid, and galacturonic acid. The secreted proteins (100 Wg) in the medium containing pectin (A), polygalacturonic acid (B), galacturonic acid (C), and glucose (D) were analyzed by Western blot. Numbers indicate the day of culture.
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result con¢rmed again that both enzymes are induced by pectin, and that the expression pattern of each exo-PG di¡ered (Fig. 5A). Under the same conditions, the fungus was cultured in a medium containing polygalacturonic acid as a carbon source and a similar pattern of the enzyme expression was observed (Fig. 5B). As shown in Fig. 5C, exo-PGs were also induced strongly and rapidly by galacturonic acid, especially the exo-PG II that was only produced until day 3. Our data are consistent with those of Johnston and Williamson [5]. When they resolved culture ¢ltrates of B. cinerea grown in the medium containing glucose or galacturonic acid for 48 h by IEF, novel PG activity peaks between pH 3.7 and 6.3 were induced by galacturonic acid, which failed to react with an antibody raised against endo-PGs. They suggested that this novel activity peak is exo-PG I and/or exo-PG II induced by galacturonic acid. To determine whether or not exo-PGs are produced in the medium containing glucose as a carbon source, the fungus was cultured over periods longer than 6 days. No exo-PGs were produced up to 5 days, as described by Johnston and Willamson [5]. However, the exo-PGs were detected in the ¢ltrates grown up to 6 days as shown in Fig. 5D. Therefore, it is likely that time of expression of exo-PGs varies depending on the carbon source and isolate. Both enzymes were induced rapidly by galacturonic acid, while the induction of exo-PGs was delayed by glucose. In contrast with endo-PGs [5], there has been no direct evidence for the induction of exo-PGs. As far as we know, this is the ¢rst report to describe the induction of exo-PGs in B. cinerea. Since the cloning and sequencing of the genes for exo-PGs have not yet been reported, we do not know whether these enzymes are the products of a single gene. Although it is likely that the two exo-PGs are not identical proteins glycosylated to varying degrees as described previously [3], the possibility of proteolytic degradation cannot be excluded. Considering that six genes for endo-PGs have been cloned [6,7], it is probable that there are also multi-genes for exo-PGs. The transient expression of exo-PG II might be explained by the existence of proteinases such as aspartate proteinase [17]. However, we cannot suggest how proteinases may specifically degrade exo-PG II, but not exo-PG I. Although the genes of several endo-PGs have been cloned [6,7], we still do not know how, and to what extent, PGs contribute to aggressiveness in B. cinerea. Gene deletion studies of endo- or exo- or both endo- and exo-PGs may explain the role of PGs in pathogenesis of this fungus. Acknowledgements
Institute, Invergowrie, Dundee, UK, for providing an antibody against endo-PG I. The Basic Science Research Institute Program, Ministry of Education, BSRI-98-4405, supported this work. References [1] Staples, R.C. and Mayer, A.M. (1995) Putative virulence factors of Botrytis cinerea acting as a wound pathogen. FEMS Microbiol. Lett. 134, 1^7. [2] Walton, J.D. (1994) Deconstructing the cell wall. Plant Physiol. 104, 1113^1118. [3] Johnston, D.J. and Williamson, B. (1992) Puri¢cation and characterization of four polygalacturonases from Botrytis cinerea. Mycol. Res. 96, 343^349. [4] Marcus, L. and Schejter, A. (1983) Single step chromatographic puri¢cation and characterization of the endopolygalacturonases and pectinesterases of the fungus, Botrytis cinerea Pers. Physiol. Plant Pathol. 23, 1^13. [5] Johnston, D.J. and Williamson, B. (1992) An immunological study of the induction of polygalacturonases in Botrytis cinerea. FEMS Microbiol. Lett. 97, 19^24. [6] Ten Have, A., Mulder, W., Visser, J. and Van Kan, J.A.L. (1998) The endo-polygalacturonase gene Bcpg1 is required for full virulence of Botrytis cinerea. Mol. Plant-Microbe Interact. 11, 1009^1016. [7] Wuben, J.P., Mulder, W., Ten Have, A., Van Kan, J.A.L. and Visser, J. (1999) Cloning and partial characterization of endopolygalacturonase genes Botrytis cinerea. Appl. Environ. Microbiol. 65, 1596^1602. [8] Wubben, J.P., Ten Have, A., Van Kan, J.A.L. and Visser, J. (2000) Regulation of endopolygalacturonase gene expression in Botrytis cinerea by galacturonic acid, ambient pH and carbon catabolite expression. Curr. Genet. 37, 152^155. [9] Tobias, R., Conway, W. and Sams, C. (1993) Polygalacturonase isozymes from Botrytis cinerea grown on apple pectin. Biochem. Mol. Biol. Int. 30, 829^837. [10] Tobias, R., Conway, W. and Sams, C. (1995) Polygalacturonase produced in apple tissue decayed by Botrytis cinerea. Biochem. Mol. Biol. Int. 35, 813^823. [11] Lee, T.H., Kim, B.Y., Chung, Y.R., Lee, S.Y., Lee, C.W. and Kim, J.W. (1997) Puri¢cation and characterization of an exo-polygalacturonase from Botrytis cinerea. J. Microbiol. 35, 134^140. [12] Bradford, M.M. (1976) A rapid and sensitive method for the determination of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248^254. [13] Harlow, E. and Lane, D. (1988) Antibodies. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [14] Laemmli, U.K. (1970) Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227, 680^685. [15] Ried, J.L. and Collmer, A. (1985) Activity stain for rapid characterization of pectic enzymes in isoelectric focusing and sodium dodecyl sulfate^polyacrylamide gels. Appl. Environ. Microbiol. 50, 615^622. [16] Hsu, S.M., Raine, L. and Franger, H. (1981) The use of avidin^biotin peroxidase complex (ABC) in immunoperoxidase technique: a comparison between ABC and unlabeled antibody (PAP) procedure. J. Histochem. Cytochem. 29, 577^580. [17] Movahedi, S. and Heale, J.B. (1990) The roles of aspartic proteinase and endo-pectin lyase enzymes in the primary stages of infection and pathogenesis of various host tissues by di¡erent isolates of Botrytis cinerea Pers ex. Pers. Physiol. Mol. Plant Pathol. 36, 303^324.
We thank Dr. B. Williamson, Scottish Crop Research
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