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FEMS Microbiology Letters 36 (1986) 287-292 287 Published by Elsevier FEM 02545 Localisation of fl-glucosidase in Trichoderma reesei celI walls with immunoelectron microscopy (Trichoderma reesei; fl-glucosidase; immunoelectron microscopy; cell wall) B. Sprey Institut fftr Biotechnologieder KernforschungsanlageJiilich, Postfach 1913, D-5170J~lich, F.R.G. Received 24 April 1986 Revision received9 June 1986 Accepted 10 June 1986 1. SUMMARY Using ferritin-conjugated antibodies as an elec- tron microscopic marker, fl-glucosidase was local- ized within the cell walls of the imperfect fungus Trichoderma reesei QM9414. With different states of cell wall degradation obtained with a cell wall-lysing culture filtrate of Micromonospora chaicea, fl-glucosidase was mainly detected within the outer, fibrous exopolysaccharide layer and the outer face of the plasma membrane. 2. INTRODUCTION fl-Glucosidase (EC 3.2.2.21) of Trichoderma species is predominantly found in a cell wall-bound state and to a minor degree present in cell wall-free form in culture fluids [1,2,11]. Both the insertion and the integration of the enzyme within the cell walls of the imperfect fungus is not understood. Moreover, the mode of binding of fl-glucosidase to cell walls is unknown. Thus mild detergents (NP40, Triton X, Chaps, octylglucoside) effect no essential release of fl-glucosidase from cell wall fractions [2]. On the other hand the release of cell wall-bound fl-glucosidase in Trichoderma pseudo- koningii was found to be correlated to cell wall- bound fl-l,3-glucanase activities [3,4], thus giving evidence for the release of fl-glucosidase together with cell wall showing lytic events. The intention of this contribution was the lo- calization of fl-glucosidase of T. reesei in different layers of the cell wall. Cell wall digests were obtained with a crude enzyme system of M. chalcea, which mainly contains fl-l,3-glucanase activity [5]. Using ferritin-conjugated antibodies [7] to purified fl-glucosidase [2] as a visual marker, localization of fl-glucosidase was then examined by transmission electron microscopy. Spheroplasts and protoplasts from Trichoderma have been successfully prepared for regeneration experiments [7], heterokaryon formation [8], secre- tion studies [9] and interspecific fusion [10]. To date no experiments have been performed to dem- onstrate the localisation of fl-glucosidase in T. reesei by means of immunoelectron microscopy. 3. MATERIALS AND METHODS 3.1. Culture conditions T. reesei QM9414 was grown under detergent- free conditions given by [11] on 1% w/v cellobiose 0378-1097/86/$03.50 © 1986 Federationof European MicrobiologicalSocieties

Localisation of β-glucosidase in Trichoderma reesei cell walls with immunoelectron microscopy

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Page 1: Localisation of β-glucosidase in Trichoderma reesei cell walls with immunoelectron microscopy

FEMS Microbiology Letters 36 (1986) 287-292 287 Published by Elsevier

FEM 02545

Localisation of fl-glucosidase in Trichoderma reesei celI walls with immunoelectron microscopy

(Trichoderma reesei; fl-glucosidase; immunoelectron microscopy; cell wall)

B. Sprey

Institut fftr Biotechnologie der Kernforschungsanlage Jiilich, Postfach 1913, D-5170 J~lich, F.R.G.

Received 24 April 1986 Revision received 9 June 1986

Accepted 10 June 1986

1. SUMMARY

Using ferritin-conjugated antibodies as an elec- tron microscopic marker, fl-glucosidase was local- ized within the cell walls of the imperfect fungus Trichoderma reesei QM9414. With different states of cell wall degradation obtained with a cell wall-lysing culture filtrate of Micromonospora chaicea, fl-glucosidase was mainly detected within the outer, fibrous exopolysaccharide layer and the outer face of the plasma membrane.

2. INTRODUCTION

fl-Glucosidase (EC 3.2.2.21) of Trichoderma species is predominantly found in a cell wall-bound state and to a minor degree present in cell wall-free form in culture fluids [1,2,11]. Both the insertion and the integration of the enzyme within the cell walls of the imperfect fungus is not understood. Moreover, the mode of binding of fl-glucosidase to cell walls is unknown. Thus mild detergents (NP40, Triton X, Chaps, octylglucoside) effect no essential release of fl-glucosidase from cell wall fractions [2]. On the other hand the release of cell wall-bound fl-glucosidase in Trichoderma pseudo-

koningii was found to be correlated to cell wall- bound fl-l,3-glucanase activities [3,4], thus giving evidence for the release of fl-glucosidase together with cell wall showing lytic events.

The intention of this contribution was the lo- calization of fl-glucosidase of T. reesei in different layers of the cell wall. Cell wall digests were obtained with a crude enzyme system of M. chalcea, which mainly contains fl-l,3-glucanase activity [5]. Using ferritin-conjugated antibodies [7] to purified fl-glucosidase [2] as a visual marker, localization of fl-glucosidase was then examined by transmission electron microscopy.

Spheroplasts and protoplasts from Trichoderma have been successfully prepared for regeneration experiments [7], heterokaryon formation [8], secre- tion studies [9] and interspecific fusion [10]. To date no experiments have been performed to dem- onstrate the localisation of fl-glucosidase in T. reesei by means of immunoelectron microscopy.

3. MATERIALS AND METHODS

3.1. Culture conditions T. reesei QM9414 was grown under detergent-

free conditions given by [11] on 1% w / v cellobiose

0378-1097/86/$03.50 © 1986 Federation of European Microbiological Societies

Page 2: Localisation of β-glucosidase in Trichoderma reesei cell walls with immunoelectron microscopy

288

or Avicel cellulose (Serva) as C-source under the modified conditions previously reported [2].

3.2. Enzyme purification fl-Glucosidase was purified by preparative iso-

electric focusing as previously described [2]. Criteria for purity (after titration curves), molecu- lar weight determinations, isoelectric points and substrate specificities have been reported else- where [2].

3.3. General immunology techniques Antibodies to fl-glucosidase were raised in rab-

bits (New Zealand whites). 0.25-1.0 mg of puri- fied fl-glucosidase were subcutaneously injected (3 times) with weekly intervals. Sera were purified for IgG with differential ammonium sulfate pre- cipitation and ion-exchange chromatography [21]. The single purification steps for IgG were checked by agarose immunoelectrophoresis (ACI, Corning, CA) using a 0.1 M barbital buffer, pH 8.6 (15 V/cm for 20 min [14]). The specifity of the anti- body was further characterized by immunodiffu- sion [2] and 2-D electrophoresis together with Western blotting [2,22,23] using an enzyme-linked immunoassay (EIA) with Protein-horseradish per- oxidase conjugate (BioRad) and 4-chloro-l-naph- thol as colour development reagent.

3.4. lmmunoelectron microscopy Conjugation of fl-glucosidase antibodies to fer-

ritin was performed by the toluene diiso- thiocyanate procedure [13]. Ferritin-antibody con- jugates were fractionated on DEAE-cellulose and purified on Sepharose 4B columns [6]. Specificity of the antibody-ferritin conjugate was tested by immunoelectrophoresis in 1.5% agarose plates using a 0.1 M barbital buffer pH 8.6 [14] under conditions previously reported [15].

3.5. Transmission electron microscopy Specimens were glutaraldehyde-osmium-fixed

and Epon-embedded [16]. Thin sections were con- ventionally stained with lead citrate and observed in a Philips E.M.400. Some samples were exclu- sively glutaraldehyde-fixed to differentiate the fer- ritin molecules.

For immunolabeling of protoplasts and sphero-

plasts samples were prefixed in glutaraldehyde (6.25% (v/v) 4 h) in the presence of osmotic stabilizer (0.6 M (NH4)2SO4) and repeatedly washed in 0.15 M phosphate-buffered saline (PBS, pH 7.2). Ferritin-conjugated antibody (20-60 /~g total protein) were added to 0.1 ml of protoplast solution and incubated for 2 h by gentle agitation (4°C). Unbound conjugate was removed by dilu- tion with PBS followed by centrifugation (500 x g, 5 min). The sediments were glutaraldehyde-fixed (6.25%, v/v) and Epon-embedded [16].

For freeze-fracturing mycelia were glutaralde- hyde-fixed (6.25%, v/v) and kept in 30% glycerol (24 h). Cryofixation was performed by rapid freez- ing in Freon 22 cooled with liquid nitrogen [17]. Freeze-fracturing was carried out in a Balzers BA 350 apparatus.

3. 6. Protoplast formation Protoplasts from 2-5-day-old mycelia of Tri-

choderma were obtained after incubation with a cell wall lysing culture broth from Micromono- spora chalcea CETC3195 (Spanish Type Culture Collection, Salamanca) grown on a chitin and laminarin-containing medium [5]. Criteria for pro- toplast formation were the lack of Calcofluor staining, osmotic lability in the absence of the osmotic stabilizer 0.6 M (NH4)2SO 4 and trans- mission electron microscope (TEM) observations of the plasmalemma after freeze-fracturing (un- published results).

4. RESULTS AND DISCUSSION

The specificity of the ferritin-conjugated anti- body used for the further labeling studies is given in Fig. 1. After immunoelectrophoresis fl-gluco- sidase obtained by preparative IEF reacted with both the ferritin-conjugated antibody to fl-gluco- sidase and rabbit IgG (Fig. 1) indicating its mono- specificity.

A :cross-sectioned Trichoderma cell after con- ventional staining (OsO 4, lead citrate, uranyl acetate) is shown in Fig. 2. The compact cell wall portion is about 0.05 #m in diameter. Its outer- most fibrillar part is poorly preserved. Dehydra- tion in acetone or ethanol followed by Epon-em-

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289

Fig. 1. lmmunoelectrophoretic pattern of purified ferritin-/~- glucosidase conjugate (lower lane) and purified antibody (up- per lane). After electrophoresis the trough was filled with purified //-glucosidase (30 /~g/30 vl). Detection of precipitin after staining with Coomassie blue.

bedding partially altered and removed this ex- opolysaccharide layer. The presence of this fibril- lar cell wall portion (F), however, was clearly demonstrated after freeze-fracturing (Fig. 3). After cryosectioning or immunofixation its thickness was 0.1-0.3 #m (unpublished observation).

The effect of cell wall lysis of Trichoderma induced by addition of the lytic enzymes from M. chalcea is given in Figs. 4 and 5. Fig. 4 shows a cross-sectioned Trichoderma cell in a spheroplast- like state after 4 h of incubation. Remnants of the

L

Fig. 3. Freeze-fractured portion of a Trichoderrna cell showing the compact cell wall (cw) and the fibrous exopolysaccharide layer (F).

Fig. 2. Cross-sectioned Trichoderma cell of untreated controls. Fixation: glutaraldehyde-OsO4, staining: uranyl acetate, lead citrate (same as Figs. 4 and 5).

undigested cell wall (CW) were loosely attached to the cell. The outer plasma membrane completely surrounded the cytoplasm. A protoplast-like state of Trichoderma (incubation 12 h) exclusively bound by the outer plasma membrane is shown in Fig. 5.

Addition of ferritin-conjugated antibodies to intact cells of Trichoderma or to cells partially or totally digested by lytic Micromonospora enzymes resulted in a labelling pattern as shown in Figs. 6-11. Fig. 6 shows the laminar structure of an untreated cell wall composed of an outer ex- opolysaccharide layer (3), the electron transparent main portion (2) and the plasma membrane (1). Addition of ferritin-labeled antibody to controls demonstrated the occurrence of the label exclu- sively in the outermost exopolysaccharide layer ((3), Fig. 7).

When Trichoderma cells were subjected to cell wall digesting enzymes for 2-6 h followed by antibody-labeling the compact cell wall portion

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290

iii~ii!!iiiiiil

Fig. 4. Cross-sectioned spheroplast-like state of a Trichoderma cell with adhering undigested cell wall (cw) remnant. Cells were treated for 4 h with lytic enzymes from Micromonospora chalcea.

Fig. 5. Protoplast of Trichoderma obtained after lytic degrada- tion of the cell wall showing the peripheral outer plasma membrane (PM).

was partially digested (Fig. 8). Ferritin marker molecules were detected at 3 different sites. They occurred in the outer exopolysaccharide layer (3), on lightly electron dense carbohydrate structures (upon OsO 4 staining) of the cell-wall middle por- tion (2) and the outer face of the plasma mem- brane (1, Fig. 8). In oblique thin sections the plasma membranes were densely coated with the immunolabel (Fig. 8).

With further cell wall degradation (6-12 h) the cell wall middle portion (2) seemed to be com- pletely digested, since no further immunolabel was detected in this region (Fig. 9). Labelling was found to be restricted to the outer exopolysac- charide layer (3) and the plasma membrane (1, Fig. 9).

Longer incubation (> 12 h) resulted mainly in a complete digestion of the cell wall (Figs. 5 and 10). The protoplasts thus obtained showed a dou- ble-layered plasma membrane (Fig. 10), when con-

ventionally stained (OsO 4, lead citrate and uranyl acetate). Labeling of protoplasts with ferritin-con- jugated antibody to fl-glucosidase demonstrated the iron cores of the ferritin-immunolabel mainly present on the outer face of plasma membrane (Fig. 11).

Results obtained by immunolabelling together with TEM of cell walls of T. reesei showed the occurrence of fl-glucosidases in the outermost ex- opolysaccharide layer and in the plasma mem- brane. To a lesser degree carbohydrate portions of the compact cell wall also seem to beak fl-gluco- sidase sites. The mode and the time course of different ceUo-oligomer (G2-G9) degradation by fl-glucosidase of Trichoderma reesei has been re- ported [1,2,18,19], and fl-glucosidase action of Aspergillus aculeatus on water-insoluble cel- lodextrins (DP 20) has been described [20].

From these results it can be speculated that water-insoluble cellooligomers with higher degree

Page 5: Localisation of β-glucosidase in Trichoderma reesei cell walls with immunoelectron microscopy

of polymerization (DP) are attached and degraded in the outermost exopolysaccharide layer of the cell wall Further degradation to soluble lower

291

DP's might then be carried out by glucan-located fl-glucosidase within the cell wall, thus connecting enzyme activities in the exopolysaccharide layer

Fig. 6. Cross-sectioned portion of the cell wall region of Trichoderma showing the plasma membrane region (1), the compact cell wall (2), and the exopolysaccharide layer (3). Staining: OsO4, lead citrate and uranyl acetate.

Fig. 7. Immunolabeling of fl-glucosidase with ferritin-conjugated antibodies in cell walls of control cells. The immunolabel is exclusively present in the outermost exopolysaccharide layer (3). Fixation and staining: exclusively giutaraldehyde

Figs. 8 and 9. Distribution of antibody markers in digested cell wails of Trichoderma. Incubation time: 2 h (Fig. 8), 6 h (Fig. 9). Fixation: giutaraldehyde-OsO4, no further post-staining.

Fig. 10. Detail of a plasma membrane from a Trichoderma protoplast of controls

Fig. 11. Ferritin-immunolabel to fl-giucosidase present in the plasma membranes of Trichoderma protoplasts

Page 6: Localisation of β-glucosidase in Trichoderma reesei cell walls with immunoelectron microscopy

292

with that in plasma membrane. Plasma mem- brane-located fl-glucosidase could further remove glucose moieties from the non-reducing end of the cellooligomers with lower DP's. It can be specu- lated that a sequential degradation of cellooli- gomers with higher to lower DP's is performed by a possible, special arrangement of fl-glucosidases with different cell wall portions. This could be favourable for a uni-directional transport of the reaction products to the cytoplasm. Moreover, due to their localisation cell wall-bound and plasma membrane-bound fl-glucosidases could be pro- tected against proteolysis and thermal inactiva- tion.

ACKNOWLEDGEMENTS

The author is indebted to Camille Lambert and Hans-Peter Bochem for expert help. The obtain of Micromonospora chalcea was kindly given by Dr. Giorgio Canavascini, University of Fribourg, Switzerland.

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