Characterization of an extracellular β-1,3-glucanase of Claviceps purpurea

Physiological and Molecular Plant Pathology (1992) 40, 191-201

191

Characterization of an extracellular f3-1,3-glucanase of Claviceps purpurea BARBARA BROCKMANN, RALF SMIT

and

PAUL TUDZYNSKI *

Allgemeine Botanik/Mikrobiologie, Institut fur Botanik der Westfdlischen Wilhelms-Universiuit Munster, Schlossgarten 3, W-4400 Munster, Germany (Accepted for publication February 1992)

The phytopathogenic fungus Claviceps purpurea produces an extracellular #-1,3-glucanase in axenic culture. The enzyme was purified and characterized . It is a glycoprotein with a pH optimum of 4 .5, a molecular weight of about 90 kDa, and an isoelectric point of 9 .0 . The latter two properties are unusual for a fungal f-1,3-glucanase . Analysis of substrate specificity confirmed that the enzyme is a specific endo-/3-1,3-glucanase . A comparable enzyme was also detected in honeydew of infected plants . A possible role for this enzyme in host-parasite interaction (degradation of callose protecting the host's vascular tissue) is discussed .

INTRODUCTION

The ascomycete Claviceps purpurea is a common parasite on grasses and cereal crops, especially wheat and rye . It is highly organ specific, since it infects only young flowers, causing a typical replacement disease of the ovary . Important for the strategy of the fungus in colonizing its host is a successful tapping of the vascular tissue : the assimilates are efficiently drawn from the phloem and used to produce honeydew, a conidia containing sugar-rich fluid serving for dispersal of the fungus by insects . Details of the plant-fungus interaction, especially the nature of the defense reaction of the plant and the strategy of the fungus to overcome it are unknown . It was postulated by Dickerson et al . [9] that callose, a f-1,3-glucan involved in several plant defense reactions also plays an important role in the protection of the vascular tissue . They found that the developing sclerotium and the honeydew contain f-1,3-glucanase activity, and postulated that this enzyme is an important tool in the colonization process functioning to degrade induced callose . Since f-1,3-glucanases are plant defense enzymes [13, 15, 20, 22] and have recently been detected in rye [I], the activity reported by Dickerson et al . [9] could be of plant origin . Dickerson & Pollard [10] proved by immunological methods the fungal nature of the enzyme in planta . In this paper we confirm that C. purpurea produces an extracellular fl- 1,3-glucanase in axenic culture, identical to the * To whom correspondence should be addressed . Abbreviations used in text : FPLC, fast performance liquid chromatography ; IEF, isoelectric focusing ; IEP, isoelectric point ; PAGE, polyacrylamide gel electrophoresis ; UV-LDI-MS, ultraviolet laser desorption ionization mass spectrometry . 0885-5765/92/030191 + 11 $03 .00/0

© 1992 Academic Press Limited



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enzyme detected in honeydew of infected plants . In order to allow a molecular study of the role of this enzyme in pathogenesis, it was purified and characterized . MATERIALS AND METHODS

Strains and culture conditions C. purpurea strain T5a, isolated from rye [11] was grown for 5 days in liquid MAmedium [19], without yeast extract, with 1 glucose on a rotary shaker at 30 ° C, and 170 rpm . Protein extracts Mycelial pellets were separated from the culture fluid by filtration . Extracellular proteins were precipitated from the culture fluid by ammonium sulphate (90 saturation), desalted by gel filtration (Sephadex G-25) in sodium-succinate buffer (0 . 05 M, pH 5 . 2) and concentrated with Centricon microconcentrators . Cellular proteins were extracted from mycelial pellets (ground in liquid nitrogen) with 0 . 1 M Tris-HCl pH 7 . 0 and precipitated with ammonium sulphate (90 % saturation) . Protein content was determined according to Lowry et al . [18] . /3-1,3-glucanase activity was determined by a modification of the method described by Kombrink et al . [16] . The reaction mixture contained 2 . 5 mg ml - ' laminarin (Sigma) in sodium-succinate buffer (50 mm, pH 5 . 2) . Reactions were stopped at various intervals by adding samples to a solution ofp-hydroxybenzoic acid hydrazine (0 . 5 % in 0 . 5 N NaOH) . After incubation at 100 °C for 5 min, absorption was determined at 410 nm . For estimation of pH optimum Davies universal buffer was used at various pH values . Ion exchange chromatography (a) Protein extracts were applied to small columns of anion exchange (QAE-sephadex A 25, Pharmacia) and cation exchange resin (CM-cellulose C-50, Pharmacia), equilibrated with Tris-HC1 buffer (20 mm, pH 5 . 0, 6 . 0, 7 .0) and potassium-phosphate buffer (20 mm, pH 7 . 5, 8 . 0) or glycine-NaOH buffer (20 mm, pH 8 . 5, 9 . 5) . Binding of f-1,3-glucanase activity was determined under the different conditions . (b) FPLC (LKB) : protein extracts were applied to a cation exchange column (TSK SP-5PW, LKB) in potassium-phosphate buffer (20 mM, pH 6 . 5) with a flow rate of 1 ml min' . Bound proteins were eluted in a NaCl gradient (elution buffer : 1 M NaCl in potassium-phosphate buffer 20 mm pH 6 . 5) . Gel-electrophoretic techniques SDS-polyacrylamide gel electrophoresis . (SDS-PAGE) was performed as described by Laemmli [17] for 15-17 h at 50-60 V (10 % PAA) . Proteins were stained with Coomassie brilliant blue . Native PAGE was performed according to Maurer [21 ] in 7 . 5 % PAA-gels ; the buffers used were KOH/TEMED pH 6 . 7/pH 4 . 3 (cationic system ; electrophoresis buffer : 35 mm fl-Alanine in 0 . 08 % acetic acid) and Tris/phosphate pH 7 . 3, Tris/TEMED pH 7 . 2 (anionic system, electrophoresis buffer : 38 . 5 mm glycine, pH 8 . 5) . /3-1,3-



193 fl-1,3-Glucanase of Claviceps purpurea glucanase activity staining of native PAA gels was performed as described by Pan et al . [26] with laminarin as substrate (in 20 mm sodium-succinate, pH 4. 5) and 2,3,5triphenyl-tetrazolium-chloride as detection reagent (incubation time with substrate was reduced to 16-15 min, with tetrazolium-chloride to 2-3 min) . Ultra thin layer isoelectric focusing

IEF was performed in rehydrated horizontal PAA gels (0 . 3 mm ; 7 . 5 °jo) on a solid surface . After polymerization the gel was washed with water (2 x 20 min) and water +2 % glycerol (1 x 20 min) and dried over night (50 ° C) . For rehydration the gel was incubated in a solution containing 10 % sorbitol and 2 . 5 % Servalyt (pH 3-10) for 2 h . The gel was run at 10 mA/ 1500 V for 30 min and the 14 mA/2000 V for 3 h . The samples were loaded in a solution containing 60 % glycerol and 4 % Servalyt (pH 3-10) . Protein staining with Coomassie brilliant blue was performed after fixation with 20% trichloroacetic acid . Activity staining was according to Pan et al. [26] . Glycoprotein detection

Proteins separated in a SDS-PAGE (10%) were blotted on nitrocellulose membrane (0 . 2 µm ; 2 h, 55 V), and incubated with Concanavalin A and peroxidase according to Conrads [6] . Positive controls were transferrin, calf intestinal phosphatase, and laminarinase from Penicillium sp ., trypsin was used as a negative control . Determination of molecular weight

In addition to SDS-PAGE (see above) ultraviolet laser desorption ionization massspectrometry (UV-LDI-MS) was performed according to Karas and Hillenkamp [14] . Cytochrome c was used as standard . RESULTS Detection of f-1,3-glucanase activity in axenic culture

Preliminary experiments showed that C. purpurea strain T5a produces an extracellular f-1,3-glucanase in media with low glucose concentrations . A time course experiment (see Fig . 1) using 1 % glucose as starting concentration showed that f-1,3-glucanase activity in the culture fluid was detectable after 4 days of incubation, when the glucose was exhausted [8] . The total and specific /3-1,3-glucanase activity reached their maximum after 6 days, when the culture had reached the stationary phase . Addition of laminarin, a fl-1,3-glucan, to the culture medium (0 . 1 %) resulted in no significant increase of /f-1,3-glucanase activity, indicating that synthesis of the enzyme is not induced by this substrate, but is probably only subject to glucose repression . Mycelial extracts also contained /3-1,3-glucanase activity . After 5 days of incubation, about 25 % of the activity was detectable in the mycelium . Preliminary comparative analysis of mycelial and culture fluid extracts using native PAGE with substrate staining (data not shown) indicated that C. purpurea secretes only one extracellular enzyme detectable in a cationic gel system, which is present in the mycelial extract in only very low amounts . There seem to be at least two additional intracellular /3-1,3glucanases, one detectable in the cationic gel system, the other one in an anionic gel system . Both showed rather low activity in native PAGE gels with substrate staining (data now shown) .



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0

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20

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n rn °

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Time (days) FIG . 1 Growth parameters and /l-1,3-glucanase activity in the medium of C . purpurea in axenic culture (C-source : 1 ;o glucose . •, Glucose concentration ; p, dry weight ; Q, protein content ;

0, specific f-1,3-glucanase activity ; [1, total f-1,3-glucanase activity .

Since only the secreted enzyme is likely to play a role in the host-parasite interaction, it was purified and araysed in detail . Purification of an extracellular /'-1,3-glucanase SDS-PAGE of culture fluid extracts showed the presence of more than 40 proteins . Fractionation by ammonium sulphate precipitation did not lead to a significant purification of the /-1,3-glucanase, therefore proteins were quantitatively precipitated by 900/0 ammonium sulphate . Binding of /j-1,3-glucanase activity was tested on different ion-exchange resins at various pH values . The anion exchange column was not effective (pH 5 . 0, pH 6 . 0, pH 7 . 0), whereas the cation exchange resin (CMcellulose) bound the activity completely up to pH 8 . 0, indicating a rather basic IEP of the protein(s) . This cation exchange chromatography was already a highly effective purification step, because it reduced the number of proteins in the eluate considerably to three major and seven minor bands on a SDS-PAGE, (data not shown) . As a final purification step a separation on a FPLC-cation-exchange column was performed with a different ion-exchange material (G 5000PW ; sulphopropyl-groups) . This resin was most effective at pH 6 . 5 . As shown in Fig . 2, application of a shallow NaCl-gradient led to quantitative elution of /3-1,3-glucanase activity in a single peak . One of the two active fractions (no . 17, see Fig . 2) contained a single major protein, and only l-3 minor proteins (Fig . 3) . fl- 1,3-glucanase-activity staining after separation of proteins on cationic native PAGE proved that this major protein is a f-1,3-glucanase [Fig . 3(b)] . No other active protein was detected in any other fraction of the gradient, supporting



fl-1,3-Glucanase of Claviceps purpurea

195

-absorbance at 280 nm 100%

C N

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3 5 7 9 II 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48

Fraction Fin . 2 Elution profile of culture filtrate of C . purpurea after cation exchange chromatography (fraction size 1 ml ; f-1,3-glucanase activity (- -) of each fraction was determined in 100 p1 aliquots), , absorbance at 280 nm ; •, NaCl gradient . (a) I

(b) 2

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2

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Fir- 3 PAGE of fractions containing fl-1,3-glucanase activity after cation exchange chromatography (fraction numbers according to Fig . 2) . (a) SDS-PAGE of fraction 17 .1, active fraction ; 2, marker proteins . (b) Native, cationic PAGE of fraction 17 .1, Coomassie staining ; 2, activity staining ; 3, control (without substrate) .



B . Brockmann et al.

196

the initial assumption (based on preliminary data obtained with mycelial and medium extracts using SDS and native gel electrophoresis, as outlined above) that C . purpurea strain T5a produces only one extracellular f-1,3-glucanase . The resulting enzyme preparation was considered to be sufficiently pure to be used for characterization experiments . Characterization of the extracellular /3-1,3-glucanase The molecular weight of the extracellular a-1,3-glucanase was determined by two methods . SDS-PAGE (see Fig . 3) in comparison to standard marker proteins gave a value of about 93000 Da . UV-LD I-MS-analysis showed a typical multiple-signal spectrum (Fig . 4) . The signals at 89055 and 44503 Da most probably correspond to 4 M 0 OD

m

r=

mN

I

I

3

r

20 000

50 000 M, /Z

FIG . 4 UV-laser desorption ionization mass spectrometry of purified extracellular fl-1,3glucanase of C . purpurea . Matrix : nicotinic acid ; wavelength : 266 nm ; diagram represents addition of 10 spectra ; reference : cytochrome C ([M+4] = 12360, [2+H] = 24716, [3M+H] = 37078) ; the arrow indicates the localization of the putative /3-1,3-glucanase monomer .

the mono- and double-charged f-1,3-glucanase monomer, respectively . The signals at 81767 and 8018 Da, which together add up to the molecular weight of the monocharged monomer, are possibly cleavage products . The signal at 40933 Da probably represents the double-charged large cleavage product, the one at 48969 Da may correspond to one of the minor contaminating proteins present in this fraction . Taken together, these results point to a molecular weight of about 90 kDa with no indications for a subunit structure . Determination of enzymatic activity over a pH range of 2 . 5 to 8 . 0 indicated a pH optimum of the extracellular fl- 1,3-glucanase of pH 4 . 5 and a rapid decline of activity at pH values beyond 5 . 0 (see Fig . 5) .



197

f-1,3-Glucanase of Claviceps purpurea

pH

FIG . 5 Influence of pH on enzyme activity of the extracellular

fl- 1,3-glucanase

of C. purpurea .

Since many fungal exoenzymes are highly glycosylated, the extracellular /3-1,3glucanase was analysed with the Concanavalin A/peroxidase coupling reaction . The protein showed a positive reaction in this system (data not shown), indicating that it is a glycoprotein, containing glucose and/or mannose units . The IEP of the protein was determined using isoelectric focusing. In good accordance with the results obtained with various cation exchange systems the IEP was determined at about pH 9 . 0 (see Fig . 6) . This result was confirmed by chromatofocusing (Mono P, Pharmacia) . The f-1,3-glucanase activity eluted immediately from the column, when a pH gradient of 9-6 was applied (data not shown) . An important point in the characterization of such an extracellular enzyme is its substrate specificity . The data in Table 1 clearly demonstrate that the C . purpurea enzyme is strictly specific for /3-1,3-linkages characteristic for callose . Thin-layer chromatography of laminarin digests showed that initially only oligosaccharides were formed, no free glucose was observed (data not shown) ; together with the negative results obtained with the substrate p-nitrophenyl-f-o-glucoside these data strongly suggest that the enzyme represents an endo-fl-1,3-glucanase . /3-1,3-glucanase activity in honeydew

Since honeydew of infected rye plants was shown to contain rather high levels of /3-1,3glucanase activity [9], a protein extract of conidia-free honeydew was analysed by ultra thin layer IEF with activity staining . As shown in Fig . 6, there are several active proteins detectable with this system . The protein with the highest activity has the same IEP as the extracellular enzyme of C . purpurea . Since also in native PAGE and SDSPAGE both proteins have identical properties, these data strongly indicate that the C. purpurea enzyme accounts for a major portion of the f-1,3-glucanase activity found in honeydew .



1 98

B . Brockmann et at. A

B

pH

FIG . 6 /3-1,3-glucanase activity-stained ultra thin layer IEF gel of (a) purified extracellular /3-1,3-glucanase of C. purpurea and (b) honeydew of rye plants infected with C . purpurea .

TABLE I

Substrate specificity of the extracellular /3-1,3-glucanase Substrate

Linkage

Laminarin p-Nitrophenyl-f-n-glucoside Carboxymethyl cellulose Lichenan Pustulan Nigerian Starch

/3-1,3 exo-/3-L3 /3-1,4 alt . fl-I, 4/fl-1,3 /3-1,6 a-1,3, a-1,4 a-1,4

'substrate had only limited solubility .

Hydrolysis +



/3-1,3-Glucanase of C/aviceps purpurea

199

DISCUSSION The data presented in this paper confirm earlier reports [9, 10] that C . purpurea produces an extracellular /3-1,3-glucanase . This is not an unexpected result, since most fungi analysed so far seem to synthesize such enzymes [5, 28] . They are probably involved in morphogenetic processes like elongation of hyphal tips and branching [3, 23, 27] by hydrolysing /3-1,3-glucans, which are normal components of fungal cell walls . The extracellular /3-1,3-glucanase of C . purpurea shares some properties such as an acidic pH-optimum, and glycosylation (both properties are rather common for fungal exoenzymes) with similar enzymes of other fungi, as well as constitutive synthesis which may be subject to catabolite repression . Other features, however, distinguish the C . purpurea enzyme from common /3-1,3-glucanases of other fungi . The molecular weight of 90 kDa is considerably higher than those reported for other fungal /3-1,3glucanases (20 to 70 kDa, [2, 25, 29]) . Another interesting feature is the extreme basic IEP, since fungal /3-1,3-glucanases are normally acidic enzymes [2, 4, 7, 12, 25, 29] . This and the high molecular weight may indicate a special function for this exoenzyme, in contrast to the intracellular /3-1,3-glucanases, which might represent the classical, `morphogenetic' /3-1,3-glucanases of C . purpurea . Dickerson et al . [9] reported /3-1,3-glucanase activity at the base of developing sclerotia and in honeydew of infected rye plants . They postulated that this enzyme could play a part in host-pathogen interaction . The present data prove the existence of one extracellular /3-1,3-glucanase in C . purpurea and indicate that this enzyme is responsible for a major portion of the /3-1,3glucanase activity in honeydew, supporting the model of Dickerson . C. purpurea uses the plant tissue as a nutritional source only during the first days of infection, when it necrotrophically colonizes the ovary . For its further development (conidia production, formation of sclerotium) C. purpurea is nutritionally dependent on assimilates of the host . Obviously the production of honeydew is an essential driving force for an efficient influx of plant assimilates into the infected tissue [24] . Secretion of /3-1,3-glucanases at the interface between mycelium and plant tissue could be important for maintaining this assimilate flux by degrading callose, a /3-1,3-glucan normally produced by the plant to protect the vascular tissue . In addition, preliminary analysis of the infection process indicates that callose is also deposited in several other areas of the rye flower, for example at the top of the style and in the outer cell walls of stigma trichome cells (E . Peveling et al ., unpublished) . The /3-1,3-glucanase could also, therefore, be important at very early stages of infection . To analyse in detail the potential functions of the extra-cellular /3-1,3-glucanase in C . purpurea during pathogenesis, molecular methods will be helpful . Efficient transformation systems for C . purpurea were developed recently [30, 31 ] . With the /3-1,3glucanase protein sufficiently purified for sequencing, it should be possible to isolate the corresponding gene and to perform gene disruption experiments which, in contrast to classical unspecific mutagenesis, will allow a detailed functional analysis .

The authors wish to thank H . Conrad for technical assistance, M . Steup, R . Tenhagen, M . Freiburg for technical advice, H . Nordhoff for performance of UV-



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