Planta (1996)198:118-126

P l a n t ~ 9 Springer-Verlag1996

Rapid reactions of spruce cells to elicitors released from the ectomycorrhizal fungus Hebeloma crustuliniforme, and inactivation of these elicitors by extracellular spruce cell enzymes Peter Salzer, Gerhard Hebe, Andreas Reith, Barbara ZitterelI-Haid, Haraid Stransky, Katja Gaschler, Acbim Hager Botanisches Institut, AllgemeineBotanik and Pflanzenphysiologie,Universit~itTfibingen, Auf der Morgenstelle 1, D-72076 Tiibingen, Germany Received: 23 January 1995/Accepted: 17 May 1995

Abstract. Elicitors released from hyphae or cell walls of the ectomycorrhizal fungus Hebeloma crustuliniforme (Bull. ex Fries.) Qu~l. induced in suspension-cultured cells of Picea abies (L.) Karst. a set of fast reactions: (i) an immediate efflux of C1- into the medium, followed by a K § efflux; (ii) an influx of Ca 2 § (measured as accumulation of 45Ca2+ in the cells); (iii) a phosphorylation of a 63-kDa protein and dephosphorylation of a 65-kDa protein (detectable by 4 min after elicitor application); (iv) an alkalinization of the medium, and (v) a transient synthesis of H 2 0 2 . The removal of extracellular Ca 2 + by EGTA delayed the elicitor-induced alkalinization. A further reduction of this response could be achieved by TMB-8 an inhibitor of Ca 2 § release from intracellular stores. Moreover, the inhibition of protein kinase activity by staurosporine prevented the extracellular alkalinization completely. However, the effectiveness of the elicitors in inducing the extracellular alkalinization was strongly impaired by constitutively secreted enzymes of spruce cells which cleaved the elicitors to inactive fragments. It is suggested that in ectomycorrhizae the efficacy of elicitors released from fungal cell walls is controlled by apoplastic enzymes of the host; the plant itself is able to reduce the activity of fungal elicitors on their way through the plant cell wall. But those elicitors which finally reach the plasma membrane of host cells induce reactions that are similar to the early defense reactions in plant-pathogen interactions. Key words: Ectomycorrhiza - Elicitor inactivation - Elicitor-induced reaction - Hebeloma - Picea cells - Signal transduction

Abbreviations: DW = dry weight; FW = fresh weight; TMB-8 = 3,4,5 trimethoxybenzoic acid 8-(diethylamino)-octylester Correspondence to: A. Hager; FAX: 49 (7071)293287

Introduction Nearly all forest trees, except those in tropical regions, are in symbiotic association with ectomycorrhiza-forming fungi. Mycorrhiza formation is a dynamic process. Lateral roots of the host are completely wrapped up in hyphae which form the hyphal sheath and finally penetrate into the cell walls of the root cortex to form the Hartig net (Kottke and Oberwinkler 1986; Brunner and Scheidegger 1992; Scheidegger and Brunner 1993). In ectomycorrhizae of eucalypts (Hilbert and Martin 1988) and birch (Simoneau et al. 1993) the development of symbiotic structures is accompanied by the synthesis of new proteins. Interestingly, besides the differentiation of structures which serve the mutualistic interaction of ectomycorrhizal partners, induction of defense-related proteins occurs. In associations of Eucalyptus globulus and Pisolithus tinctorius, stimulation of chitinase and peroxidase activities have been demonstrated during formation of ectomycorrhizae (Albrecht et al. 1994a, b). Similar elicitor-induced responses have also been observed in suspension-cultured cells. In this context, Sauter and Hager (1989) have demonstrated that elicitors from the ectomycorrhizal fungus Amanita muscaria induce the formation of chitinase in spruce cells as well as in roots of spruce seedlings. In addition, elicitors from Hebeloma crustuliniforme (Bull. ex Fries.) Qu+l. induced peroxidase in suspension-cultured cells of their natural host, Picea abies (Salzer and Hager 1993). Furthermore, Schwacke and Hager (1992) reported that in spruce cells after elicitor application a very rapid and transient synthesis of H202 takes place. These results are an indication that spruce cells exhibit defense reactions similar to those of roots and cells in the naturally occurring ectomycorrhiza. On the other hand, varying concentrations of auxin were able to suppress the induction of defense-related enzymes, such as chitinase and peroxidase, in spruce cells, showing that hormones may act as modulators of defense reactions in the plant (Sauter and Hager 1989; Salzer and Hager 1993). Surprisingly, auxin could not influence the rapid elicitorinduced reactions in spruce cells, e.g. the synthesis of

P. Salzer et al.: Reactions of Picea cells to Hebeloma elicitors H 2 0 2 (Salzer a n d H a g e r 1993), o r the e x t r a c e l l u l a r alk a l i n i z a t i o n ( d a t a n o t shown). E m p l o y i n g s u s p e n s i o n - c u l t u r e d s p r u c e cells, we ext e n d e d t h e s t u d i e s o n t h e fast r e s p o n s e s of these cells to a p p l i c a t i o n of elicitors released f r o m the cell wall o r isolated f r o m the c u l t u r e m e d i u m of t h e e c t o m y c o r r h i z a l f u n g u s H e b e l o m a crustuliniforme. W e f o u n d a c o m p l e x p a t t e r n of r a p i d C I - , K § a n d C a 2§ fluxes, a p r o t e i n p h o s p h o r y l a t i o n a n d d e p h o s p h o r y l a t i o n , synthesis of active o x y g e n a n d a C a 2 + - d e p e n d e n t , p r o t e i n - k i n a s e m e d i a t e d e x t r a c e l l u l a r a l k a l i n i z a t i o n in these s p r u c e cells. O n the o t h e r h a n d , the effectiveness of the elicitors in i n d u c i n g r a p i d r e a c t i o n s was d r a s t i c a l l y r e d u c e d b y ext r a c e l l u l a r p l a n t e n z y m e s w h i c h were c o n s t i t u t i v e l y s y n thesized a n d secreted to the a p o p l a s t .

Materials and methods Chemicals. Staurosporine was purchased from Boehringer (Mannheim, Germany), N,N',N",N"' tetraacetylchitotetraose, purified crab shell chitin, ~-glucuronidase type H-2 crude solution from Helix pomatia, laminarin, N-acetylglucosamine and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester hydrochloride (TMB-8) were from Sigma (Deisenhofen, Germany). Marker proteins for electrophoresis were from Pharmacia (Uppsala, Sweden), tetramethylethylenediamine and ammonium peroxydisulfate from BIORAD (Richmond, Cal., USA) and Protogel (30% acrylamide, 0.8% bisacrylamide stock solution) from Hrlzel (Manville, N.J., USA). [32p]Orthophosphate, 45Ca2 § and Hyperfilm MP were purchased from Amersham Buchler (Braunschweig, Germany), Ultima Gold from Packard (Groningen, The Netherlands). The other chemicals used were p.a. grade from Merck (Darmstadt, Germany). All media were prepared with bidistilled water. Cultures. Suspension cultures of Picea abies (L.) Karst. were grown in 4 • Gamborg's medium (Gamborg et al. 1968) and subcultured weekly by transferring 20 ml of cell suspension into 60 ml of fresh medium. The cultures were shaken in 200-ml flasks on a rotary shaker at 100 rpm at 24 to 26 ~ in the dark. Hebeloma crustuliniforme (Bull. ex Fries) Qu61. (strain Tii 704) has been isolated from fruit bodies on Picea abies. Suspension cultures of the fungus were grown in Melin-Norkrans (MMN) medium according to Mason (1980) with the exception that 1.5 g KH2PO4"I- 1 and 0.05g FeCI3"1-1 were used. Further culture conditions were as previously described (Salzer and Hager 1991). Isolation of elicitors. Soluble wall fragments from Hebeloma crustuliniforme were prepared from three- to four-week-old cultures. One gram of hypliae was consecutively washed in 40 ml bidistilled water, in 40ml 0.1 M and in 40 ml 0.5 M potassium phosphate buffer (pH 7.2). Then the hyphae were homogenized in 0.5 M potassium phosphate buffer, using an Ultraturrax T25 (Janke and Kunkel, Staufen im Breisgau, Germany) 4 x 30 s (24000 rpm), and sonified 4 • 20 s (70 W; Sonifier B12, Branson, Danbury, Conn., USA). The homogenate was centrifuged (5 min, 4000-0) and the pellet was subsequently washed eight times in 0.5 M, then eight times in 0.1 M potassium phosphate buffer, eight times in bidistilled water, and was finally air-dried. To release soluble elicitors, 3 mg dry weight (DW) of the walls was shaken for 30 min at room temperature in a mineral solution containing 10 mM NaNO3, 1 mM KC1, 1 mM Na2SO4, 1 mM Mg(NO3)2, and 1 mM CaC12. The insoluble fragments were sedimented by centrifugation (5 min, 4000-9) and the elicitors in the supernatant (soluble elicitors) were used for experiments. The carbohydrate content of 1 mg (DW) of the insoluble fungal cell wall fragments was 4.8 Ixmol glucose equivalents (determined by the

119 method of Dubois et al. (1956) with glucose as standard). The content of alkali soluble protein (determined by the method of Ayers et al. (1976) with bovine serum albumin as standard) was 85.2 ~tg-(mg DW)- 1. The tiny amounts of carbohydrates and proteins in the supernatant were not detectable by the above-mentioned chemical methods. Soluble elicitors from walls of suspension cultured spruce cells were prepared following the same procedure. Colloidal chitin was prepared from crab shell chitin according to Berger and Reynolds (1958) and adjusted to a concentration of 16.6 mg-ml-1 and stored in bidistilled water at 4~ The supernatant of the colloidal chitin contained soluble chitin fragments which were used as elicitors. The NaOH-soluble fraction of the 13-glucan was isolated from commercially available bakers yeast according to Cabib and Bowers (1971). To release elicitor-active fragments 3 mg (DW) were agitated in 1 ml mineral solution (see above) for 30 min. Insoluble fragments were removed by centrifugation and the resulting supernatant was used as elicitor. Elicitors released to the culture medium were isolated from three- to four-week-old Hebeloma crustuliniforme cultures. The hyphae were separated from the culture medium by filtration with a nylon net (mesh 10 larn). The medium was adjusted to pH 6.2 by adding NaOH before it was used for induction of H202 release from spruce cells. When using the culture medium for the induction of an extracellular alkalinization by the spruce cells, the culture medium of Hebetoma crustuliniforme was dialysed against the mineral soiution and was adjusted to the actual pH value of the incubation medium of the spruce cells. Isolation and partial purification of extracellular spruce enzymes. Chitinase and glucanase were isolated from the medium of 7- to 10-d-old spruce suspension cultures. The cells were separated from the medium by filtration with a nylon net (mesh 10 lam). The filtrate was centrifuged (20 min, 38 000.g) and the proteins were precipitated with (NH4)2504 (saturation 70%) for 2 h at 4~ Protein was collected by centrifugation (20 min, 38000.9) and resuspended in 20 mM Na-phosphate-citrate buffer (pH 5.2) and partially purified by gel filtration on a Sephadex G75 column (bed 3.6 • 76 cm, flow 25 ml-h- t, eluent 20 mM Na-phosphate-citrate buffer, pH 5.2). The fractions with the highest chitinase and glucanase activities were pooled. Prior to activity assays, the enzymes were dialysed against the mineral solution (10 mM NaNO3, 1 mM KC1, 1 mM Na2SO4, I mM Mg(NO3)2, 1 mM CaC12). The specific activity of chitinase was determined with colloidal chitin as substrate in a 30 mM citrateNaOH buffer (pH 5.0) at 37 ~ according to Sauter and Hager (1989). Glucanase activity was determined as the amount of reducing sugars released from laminarin (5 mg.ml- 1). The sugars were measured by the method of Nelson (1944). Protein concentrations were determined by the method of Bradford (1976). Treatment of soluble elicitors with extracellular spruce enzymes. To study the time dependence of the elicitor inactivation by extracellular spruce enzymes, soluble elicitors obtained from Hebeloma crustuliniforme cell walls were incubated with partially purified chitinase and [3-1,3-glucanase from spruce cells (see above) with 46 pkat 13-1,3glucanase and 29 pkat chitinase activity at 37 ~ (other enzymatic activities were not determined). To check that the enzymatic inactivation of soluble elicitors was due to decomposition and not to generation of suppressors, part of the elicitor extract was incubated for 30 min at 37 ~ with extracellular enzymes as described above, and part was incubated without spruce enzymes. To inactivate the spruce enzymes, the samples containing elicitors plus enzymes were boiled for 5 min. In control samples the elicitors and an equivalent amount of spruce enzymes were also boited before application to the spruce cells. Elicitor-induced alkalinization, CI- and K + efflux. For experiments 7- to 9-d-old spruce cells were separated from the culture medium by filtration with a nylon net (mesh 10 ~tm) and washed with the mineral solution. For simultaneous measurement of pH, CI-

P. Salzer et al.: Reactions of Picea cells to Hebeloma elicitors

120 and K + concentrations, Picea abies cells from the same culture flask were used. Two batches of 5 g (wet weight) spruce cells were incubated in 20 ml of the solution and shaken for about 60 min at 120 rpm before the elicitors were added. In one batch the pH of the cell suspension was measured with half-micro pH electrodes (Ingold, Steinbach, Germany) which were connected to a Research Expandable Ion Analyzer EA 940 (Orion, Cambridge, Mass., USA). In the other batch the C1 and K + concentrations of the incubation medium were measured with ion-sensitive electrodes (model 94 17B chloride electrode and model 93-19 potassium electrode; Orion research, Boston, Mass., USA) with an Ag/AgC1 double-junction reference electrode (model 90 02; Orion). Instrument management and data acquisition were controlled by Quick Basic programs developed for IBM-compatible PCs.

8.0), 10% (w/v) glycerol and 2% (w/v) SDS by heating to 90~ for 10 min. The protein concentration of the samples was determined with the DC-protein assay (BIORAD, Richmond, Cal., USA) following the manufacturer's instructions. Twenty minutes before subjecting the samples to SDS-PAGE, dithiothreitol was added to a final concentration of 10 mM. The SDS-PAGE analysis was performed according to Laemmli (1970) using a 5% acrylamide stacking gel and a 10% separation gel with 20 p.g proteins per lane. The gels were dried and the phosphorylated proteins were detected by autoradiography with a Hyperfilm MP.

Synthesis of H202. To measure H202 induction, the spruce cells

Elicitor release from hyphae of Hebeloma crustuliniforme.

were washed with a buffer containing 24.7 m M KNO3, 1.1 mM NaHEPO4, 1 mM MgSO4, 1 m M (NH,02SO4, 1 mM CaC12 and 2% (w/v) sucrose adjusted to pH 6.6 with a 2 0 m M 2-(N-morpholino)ethanesulfonic acid-NaOH-buffer. Two grams fresh weight (FW) of the Picea abies cells was incubated in 20 ml of the buffer and 2 ml of the Hebeloma crustuliniforme culture medium was added. The concentration of H202 in the buffer was determined by the luminol method ~is described by Schwacke and Hager (1992).

S u s p e n s i o n - c u l t u r e d h y p h a e of the e c t o m y c o r r h i z a l fungus Hebeloma crustuliniforme are a b l e to release elicitors i n t o t h e c u l t u r e m e d i u m . F o r d e t e c t i o n of these elicitors cells o f Picea abies w e r e used. It was d e m o n s t r a t e d t h a t the t r a n s f e r o f f u n g a l c u l t u r e m e d i u m ( a d j u s t e d to p H 6.2) to t h e s p r u c e cell s u s p e n s i o n was sufficient to i n d u c e a r a p i d H 2 0 2 release f r o m Picea abies cells, i n d i c a t i n g the p r e s e n c e of elicitors in the c u l t u r e m e d i u m ( M M N c m e d i u m ) . L i k e w i s e , the e l i c i t o r - c o n t a i n i n g f u n g a l c u l t u r e m e d i u m i n d u c e d a r a p i d e x t r a c e l l u l a r a l k a l i n i z a t i o n of s u s p e n d e d s p r u c e cells (Fig. 1). M o r e o v e r , elicitors obt a i n e d f r o m Hebeloma crustuliniforme cell walls w i t h o u t a u t o c l a v i n g i n d u c e d a n e x t r a c e l l u l a r a l k a l i n i z a t i o n in s p r u c e cells w i t h a t i m e p a t t e r n s i m i l a r to t h a t i n d u c e d by elicitors f r o m the c u l t u r e m e d i u m (Fig. 1). T h e t i m e c o u r s e of t h e H 2 0 2 release i n d u c e d b y t h e s e wall elicitors ( d a t a n o t s h o w n ) was s i m i l a r to t h a t p r e v i o u s l y r e p o r t e d b y S c h w a c k e a n d H a g e r (1992).

Uptake of45Ca2+. Before incubation with 45Ca2 +, the spruce cells were washed in the same buffer as described for H202 synthesis (see above) with the exception that the pH was 5.5 and the concentration of CaCI2 was 80 ~tM. Then, 1.5 g (wet weight) cells was transferred into 15 ml of buffer containing 80 ~tM CaC12 and 55.3 kBq 4SCaC12 (specific activity 0.37 1.5 GBq-mg-z) until the equilibrium of extracellular and intracellular 45Ca2+ was reached. Then, about 30 min after application of 45Ca2 +, 300 lal soluble elicitors (or water) was added. Various times after elicitor addition 1.5 ml of the cell suspension was removed and washed to remove 45Ca2+ from the cell walls. The cells were washed three times for 20 min with 8 ml of an ice-cold solution of 25 m M CaC12 and 20 mM MgC12 and were finally dried by suction on a Biichner funnel. The amount of radioactivity taken up by the spruce cells was measured in Ultima Gold by liquid scintillation counting and was related to the fresh weight of the cells which was determined after suction.

Results

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Effect of EGTA and TMB-8. To remove extracellular Ca 2+, 4g spruce cells was washed three times for 20 min in 250 ml mineral solution, containing 1 mM E G T A and no Ca 2 +. Then the medium was separated from the cells by filtration on a nylon net (mesh 10 Ixm) and 0.5 g (wet weight) of the spruce cells was transferred to the mineral solution containing 1 p.M EGTA and no Ca 2 +. In samples containing extracellular Ca 2§ EGTA was removed by washing the cells in mineral solution without EGTA. Finally, Ca 2 + was supplemented by transferring the cells into mineral solution containing 1 mM Ca 2 § Release of Ca 2 + from intracellular stores was inhibited by application of TMB-8 to the cells after the last washing step. To 20 ml of cell suspension (either supplemented with Ca 2 + or without extracellular Ca 2 +) 50 ~tl TMB-8 (40 mM dissolved in ethanol) was added 1 h before elicitor application. The final concentration of TMB-8 was 100 p.M. To cells without TMB-8 an equivalent amount of ethanol was added.

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In-rive phosphorylation. To 1.5 ml of a 7- to 10-d-old Picea abies culture 780 kBq carrier-free [32p]Pi was added. Thirty minutes after application of [32p]Pi, 30 pl of the soluble elicitors (dissolved in water) was added. To control samples an equivalent amount of water was added. The phosphorylation reaction was stopped at indicated times, ranging from 0 to 20 min, by adding 300 lal of 30% (w/w) trichloroacetic acid and transferring the cells into liquid N2 according to the method described by Felix et al. (1991). After agitating for 30rain at room temperature and a 30-min storage at - 2 0 ~ the samples were centrifuged (15 000.g, 5 min). The pellet was washed three times with 80% acetone. Subsequently, the proteins were solubilized in a buffer containing 62.5 mM Tris-HC1 (pH

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Fig. l. Time course of elicitor-induced extracellular alkalinization and HzOz formation in Picea abies suspension-cultured cells. Elicitor-containing culture medium (2 ml) from Hebeloma crustuliniforme or soluble elicitors released from Hebeloma crustuliniforme cell walls (400 Ixl) were added to spruce cells. In control experiments fungal culture medium (MMNc) and mineral solution without elicitors were added

P. Salzer et al.: Reactions of Picea cells to Hebeloma elicitors I

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Fig. 2. Rapid alkalinization induced by hyphae of Hebeloma crustuliniforme added to suspension-cultured cells of Picea abies. Hyphae (0.4 g FW) suspended in 15 ml mineral solution were added to spruce cells (0.5gFW) suspended in 15ml mineral solution. In control samples, 15 ml of the mineral solution was added without hyphae. The diagram shows a typical result of three independent experiments

The permanent release of elicitors from hyphae of the living fungus could be demonstrated by the co-incubation of hyphae of Hebeloma crustuliniforme and spruce cells, which resulted in a rapid alkalinization of the surrounding mineral solution (Fig. 2). Before measuring the extracellular alkalinization, residual elicitors from the culture medium were removed from the hyphae by washing them six times with the mineral solution. Then, the extracellular p H of two Hebeloma crustuliniforme samples and two Picea abies samples were measured simultaneously over a period of 2 h. During this time the p H values of the fungal media were slowly adjusted to the p H values of the spruce media by a dropwise addition of 1 0 - 4 M N a O H . As soon as identical p H values were attained one of the hyphae-containing samples was completely transferred to a batch of spruce cells. After a lag of 2-3 min the p H of the medium started to increase (Fig. 2). These results show that elicitors are primary signals of the ectomycorrhizal fungus and that the fungal cell wall is a source of elicitors (Fig. 3). The fact that chitin fragments and 13-glucans are able to induce the same alkalinization response (Fig. 4) as elicitors from walls of the ectomycorrhizal fungus indicates that these wall constituents of Hebeloma crustuliniforme might also act as elicitors in vivo.

Elicitor-induced ionfluxes. Elicitors released from walls of Hebeloma crustuliniforme triggered an effiux of C1- from the spruce cells without a significant delay. The efflux of K § followed with a variable lag ranging from 0 to 2 min. The increase of the extracellular p H started 2 - 4 min after elicitor application (Fig. 5). The amounts of C1- and K § released from the spruce cells and the K + / C I - ratio varied to some extent in different experiments. Special care was

stourosporine + 600jut elicitors

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Fig. 3. Soluble elicitors (dissolved in mineral solution) released from walls of Hebelomacrustuliniformeincreased the extracellular alkalinization of spruce cells in a dose-dependent manner. Application of various amounts of mineral solution had no effect on the extracellular pH (data not shown). Staurosporine (100nM dissolved in dimethylsulfoxide), which was applied 20 min before elicitor addition, completely inhibited the alkalinization. Staurosporine alone induced a slight decrease of the extracellular pH. The diagram shows a typical result of three independent experiments

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(600 lal), fragments from colloidal chitin (600 lal), and N,N',N",N"' tetraacetylchitotetraose (final concentration was 1 IxM) were used as elicitors. Staurosporine (50 nM) was added 20 min prior to the elicitor application and suppressed the effectiveness of all tested elicitors. The diagram shows a typical result of three independent experiments taken to check the influence of ions added together with the soluble elicitors, because additional ions might cause artifacts due to ion-exchange processes of the plant cell wall. In control experiments, elicitors were dissolved in mineral solution which had been removed from the incubation medium of a parallel batch of spruce cells (removal

P. Salzer et al.: Reactions of Picea cells to Hebeloma elicitors

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Fig. 5. Time course of elicitor-induced C1- and K + effluxes and the extracellular alkalinization. The C1- and K + concentrations were continuously monitored with ion-selective electrodes in the bathing solution of the spruce cells; the pH was measured in parallel samples. A 600 ~tl aliquot of the soluble elicitors was added to 5 g (FW) spruce cells suspended in 20 ml mineral solution. The dotted lines show the time course of the C1- and K + concentrations and the pH in the incubation medium in comrol samples where 600 p,l incubation medium without elicitors was added. The diagram represents typical elicitorinduced ion fluxes as found at least in five independent experiments

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Fig. 7. Influx and mobilisation of Ca 2 + from intracellular stores influenced elicitor-induced extracellular alkalinization by spruce cells. Extracellular Ca 2 * was removed from spruce cells by repeated washing in mineral solution containing 1 m M EGTA ( + E G T A ) . Inhibition of opening of intracellular Ca 2 + stores by TMB-8 (added 1 h before elicitor application) had a small effect on the alkalinization in the presence of extracellular Ca 2 § ( + TMB-8) but a strong inhibitory effect without extracellular Ca 2§ ( + E G T A + TMB-8). In samples containing extracellular Ca 2 +, the cells were separated from the EGTA-containing solution and were transferred to mineral solution containing 1 m M Ca 2 § (control). The diagram shows a typical result of three independent experiments

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Fig. 6. Elicitors released from cell walls ofHebeloma crustuliniforme induce a rapid influx of r into spruce cells. The cells were incubated with 4SCa~+ for 30 rain prior to the addition of the elicitors. This time period was sufficient for the 4SCa2 § distribution between cells and medium to reach equilibrium. Then, 300 pl elicitors or 300 pl water was added. The elicitor-induced uptake ofr 2+ was saturated 10-15 rain after elicitor application. The diagram shows a typical time course of one of eight independent experiments

after 60 min of incubation). In this case the elicitorinduced effects were not different from those which were induced by elicitors dissolved in fresh mineral solution. In addition, application of mineral solution without elicitors had no effect on ion fluxes in spruce cells (Fig. 5).

The elicitor-dependent influx of 4 5 C a 2 + started very rapidly too (Fig. 6). A substantial increase of intracellular r + in spruce cells could be measured by 2 4 rain after addition of elicitors and was saturated 10-15 rain after elicitor application. Involvement of Ca z + and protein kinase in signal transduction leading to extracellular alkalinization. In the sequence of events from elicitor recognition to extracellular alkalinization at least two effects seemed to be involved: first the influx of extracellular Ca 2 § and, second, the action of protein kinases. The alkalinization was delayed for 12 min when the concentration of extracellular Ca 2 + was reduced by E G T A (Fig. 7). The drug TMB-8, known to prevent the release of Ca z+ from intracellular stores (RittenhouseSimmons and Deykin 1978) reduced the intensity of the elicitor-induced alkalinization (Fig. 7). A combination of E G T A plus TMB-8 not only prolonged the lag phase but also decreased the intensity of the elicitor-dependent alkalinization drastically. This might be an indication that the transient influx of Ca 2 § (Fig. 6) is a signal for opening intracellular Ca 2+ stores. The elevated cytosolic Ca 2+ level could then be a signal for the activation of protein kinases or protein phosphatases finally activating the mechanisms causing the alkalinization of the medium. The complete inhibition of this latter effect by 100 nM staurosporine, a potent protein-kinase inhibitor, favours this assumption (Fig. 3). As shown in Fig. 8 the phosphorylation of a 63-kDa protein was detectable by 4 min after application of

P. Salzer et al.: Reactions of Picea cells to Hebeloma elicitors

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0

~

15

Time after elicitor applicotion (mln) Fig. 8. Rapid elicitor-induced protein phosphorylation (63 kDa) and dephosphorylation (65 kDa). Elicitors (30 gl dissolved in water) released from the cell walls of Hebeloma crustuliniforme induced the phosphorylation and the dephosphorylation of proteins in spruce cells after a lag of 4-5 min. [32p]Orthophosphate was added to the suspension-cultured Picea abies cells 30 min before elicitor application. The phosphorylation was stopped 0, 4 and 15 min after elicitor addition and the extracted proteins were separated by SDS-PAGE and autoradiographed. Addition of an equivalent amount of water had no effect on protein phosphorylation. The protein phosphorylations which were found 0,4 and 15 min after addition of 30 pl water were similar to the phosphorylation pattern shown for 0 min after elicitor application. The diagram shows a typical result of five independent experiments

elicitors released from walls of Hebeloma crustuliniforme. The dephosphorylation of a 65-kDa protein started after a lag phase of 5 min and was completed after 15 min. Since the time course of protein phosphorylation correlates with that of the extracellular alkalinization and the H202 synthesis (Fig. 1) we assume that the phosphorylated proteins are important mediators in the signal transduction leading to the fast responses, such as extracellular alkalinization and H202 formation. The involvement of protein phosphorylations in signal transduction was also demonstrated by the fact that elicitor-effective compounds such as N,N',N",N"' tetraacetylchitotetraose and fragments released from colloidal chitin or 13-glucans could induce the same fast reactions as described above, and that these effects could also be inhibited by staurosporine (Fig. 4).

Enzymatic destruction of fungal elicitors by apoplasmic plant enzymes. On their way from the fungal cell wall to the presumptive receptor site at the outer surface of the plant plasma membrane, the elicitors must cross the plant cell wall. The question was whether hydrolytic enzymes occurring in the plant cell wall or in the culture medium of the spruce cells could modulate the effectiveness of the fungal elicitors. For this purpose proteins from the culture medium of 7- to 10-d-old Picea abies suspension cultures were collected by (NH4)2SO4 precipitation (70% satura-

I

I

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I

0

20

(,0

60

Time after elicitor oppIicution (rain)

Fig. 9. Inactivation of fungal elicitors by the action of extracellular spruce enzymes. Soluble elicitors obtained from Hebeloma crustuliniforme cell walls (400 pl) were incubated with partially purified 13-1,3-glucanase (46 pkat) and chitinase (29 pkat) at 37 ~ for various times. Three hundred microlitres of the elicitor-containing solution was applied to the spruce cells. In control experiments 300 I.tl of the mineral solution containing an equivalent amount of chitinase and glucanase was added. The diagram shows a typical result of three independent experiments

tion). The precipitated proteins were partially purified by gel filtration with Sephadex G 75 and the fractions with l~-glucanase and chitinase activity were pooled. Before incubation with the elicitors, the enzymes were dialysed against the mineral solution to remove the buffering eluent and to allow subsequent measurement of pH changes. A 300-gl aliquot of soluble elicitors released from cell walls of Hebeloma crustuliniforme was incubated with 100 pl enzyme solution, containing 29 pkat chitinase and 46 pkat [3-glucanase activity. As demonstrated in Fig. 9 the effectiveness of the fungal elicitors in inducing extracellular alkalinization decreased with increasing time of incubation with the apoplasmic spruce enzymes. This time-dependent decrease of the effectiveness of soluble elicitors was due to the degradation ~of these elicitors and not to the formation of suppressor molecules. This was demonstrated by a second addition of elicitors which had not been pretreated by apoplasmic enzymes. In this case a maximal alkalinization response in the spruce cell medium could be observed(Fig. 10).In the presence of suppressor molecules no effect would be expected. Discussion

Release of elicitors from hyphae of Hebeloma crustuliniforme. In this study we demonstrated that hyphae of the ectomycorrhizal fungus Hebeloma crustuliniforme released elicitors which induced a rapid extracellular

P. Saher et al.: Reactions of Picea cells to Hebeloma elicitors

124 I 5.7--

I

growth, chitin has not attained its mature structure, and a crosslinking of the [3-glucans is not completed (Wessels 1993). However, it remains to be proved that chitin and glucan fragments are the elicitors released in vivo.

I

untreQted

Z

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enz),maflcotly 5.5

-

prerreafed elicitors

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Fig. 10. Reduction of the effectiveness of wall-released elicitors from Hebeloma crustuliniforme by extracellular spruce enzymes is due to elicitor degradation and not to generation of suppressor molecules. When the elicitors had been pretreated with extracellular enzymes obtained from spruce cells the alkalinization response of the spruce cells was strongly decreased (300 ~1 soluble elicitors was incubated with 100 ~1 partially purified chitinase and [3-1,3-glucanase for 30 min at 37 ~ then boiled for 5 min). Enzymatically untreated elicitors (300 tal soluble elicitors and 100 lal enzymes were separately incubated for 30 min at 37 ~ then boiled for 5 min) were used as control. When added 35 min after the application of enzymatically treated elicitors, the untreated elicitors were able to induce a strong extracellular alkalinization by spruce cells in spite of the presence of degraded elicitors, showing that these elicitor fragments act not as suppressor molecules. The diagram shows a typical result of three independent experiments

alkalinization and the synthesis of H 2 0 2 in suspensioncultured cells of its natural host Picea abies. Previous work has demonstrated that elicitors which were prepared from walls of the ectomycorrhizal fungi Amanita muscaria and Hebeloma crustuliniforme induced chitinase and peroxidase in spruce cells (Sauter and Hager 1989; Salzer and Hager 1993). The finding that enzymes such as chitinase and peroxidase were also stimulated during formation of the eucalypt ectomycorrhiza (Albrecht et al. 1994a, b) indicates that the fungus must interact with the host cells during the colonisation of the plant root via elicitors. Colloidal chitin, N,N',N",N"' tetraacetylchitotetraose and ]3-glucans have been shown to induce an alkalinization response in tomato cells (Felix et al. 1993). Those compounds induced an alkalinization response in spruce cells similar to that of elicitors released from living hyphae and walls of Hebeloma crustuliniforme. In addition it was found that treatment of these wall-released elicitors by hydrolytic enzymes containing chitinase and glucanase activity diminished the effectiveness of these elicitors. This indirect evidence let us assume that the elicitors released from Hebeloma crustuliniforme might be chitin and [3glucan fragments of the fungal cell walls. Such chitin and [3-glucan elicitors would probably be released from the tips of growing hyphae, because in the regions of apical

Regulation of elicitor effectivity by extracellular plant enzymes and hormones. The intensity of the elicitor-induced extracellular alkalinization in Picea abies suspension-cultured cells depends on the concentration of the applicated elicitors (Fig. 3). Also in ectomycorrhizae of eucalypts, aggressive strains of Pisolithus induced higher levels of chitinase in the roots of their host than less-aggressive strains (Albrecht et al. 1994b). Therefore, it can be assumed that the amount of "active" elicitors that reach their presumptive receptors on the plant plasma membrane determines the intensity of the plant defense reactions in ectomycorrhizae. On their way from the site of release to the site of perception, the elicitors must cross the plant cell wall. But cell walls and the apoplasmic space of plant cells are rich in hydrolytic enzymes. Both chitinase and ~-l,3-glucanase activity were demonstrated in the extracellular fluid of spruce cells and presumably these enzymes are responsible for the reduction of the effectiveness of the elicitors of the mycorrhizal fungus Hebeloma crustuliniforme (Figs. 9, 10), probably by cleaving the elicitors into inactive fragments. A hint for such a mechanism is the finding by Felix et al. (1993) who showed that chitin fragments become inactive in inducing extracellular alkalinization in tomato cells when their degree of polymerisation falls below 4. In controlling the elicitor-induced synthesis of defense proteins, hormones, especially auxins that are synthesized by the mycorrhizal fungus (Rouillon et al. 1986; Gogala 1991), might be involved. In spruce cells, auxins specifically suppressed the induction of one peroxidase isoform, but did not influence the synthesis of HzO2 (Salzer and Hager 1993) and the extracellular alkalinization (data not shown). Elicitor-induced signal transduction in spruce cells. After crossing the plant cell wall, elicitors are suggested to bind to receptor proteins anchored in the plasma membrane (Cosio et al. 1988; Basse et al. 1993; Yoshikawa and Sugimoto 1993; Niirnberger et al. 1994). However, it cannot be excluded that the elicitors from Hebeloma crustuliniforme exert their effects by interaction with membrane lipids as shown for chitosan and polycations in inducing callose synthesis in plant cells (Kauss et al. 1989). Release of C1- was the most rapid elicitor-induced reaction in spruce cells, preceding K + efflux, Ca 2 + influx, extracellular alkalinization and H202 synthesis. Therefore, one possible mechanism involved :in the primary events at the plasma membrane might be a depolarisation of the membrane caused by the elicitor-triggered C1efflux. As a result depolarisation-activated K + and Ca 2 + channels might be opened, as was shown for Chara gymnophylla (Pottosin and Andjus 1994), Plantago media (Vogelzang and Prins 1994) and wheat roots (Huang et al. 1994). The influx of extracellular Ca 2+ into cells is necessary to trigger the rapid H202 formation and the extracellular alkalinization. Removal of extracelluar C a 2 + by EGTA

125

P. Salzer et al.: Reactions of Picea cells to Hebelorna elicitors

Fig. ll. Speculative model of early events in fungal plant interactions of ectomycorrhizal partners explaining the following steps: (i) release of the elicitors from the fungus without influence of the host cell; (ii) permeation of the cell wall of the host and partial destruction of the fungal elicitors by cell wall enzymes of the plant; (iii) binding of residual elicitors to presumptive receptor sites at the plant plasma membrane thereby triggering the efflux of CI- and K +, and (iv) triggering a Ca 2+ influx which is a precondition for the extracellular alkalinization and the H202 burst which both were inhibited by the protein-kinase inhibitor staurosporine; (v) suppression of the synthesis of defense-related proteins of the plant cell by hormones (e.g. auxins) of the fungus. For further information see Discussion

prolongs the lag-time of the elicitor-induced alkalinization from 3 to 12 min and, as previously shown by Schwacke and Hager (1992), completely inhibited the elicitor-induced H 2 0 2 synthesis. Moreover, the release of Ca 2 + from intracellular stores seems also to be involved in determining the intensity of the alkalinization response since TMB-8 an inhibitor of Ca 2 § release from intracellular stores (Rittenhouse-Simmons and Deykin 1978; Bourbouloux et al. 1992) decreased the elicitor-induced alkalinization in the absence of extracellular Ca 2 § (Fig. 7). The opening of Ca 2§ channels at intracellular stores might either depend on the elevated cytosolic Ca 2 § level or on inositol phosphates (Drobak 1993). The involvement of inositol-l,4-bisphosphate in elicitor-induced phytoalexin synthesis was recently demonstrated in tobacco suspension-cultured cells (Kamada and Muto 1994). The finding of elicitor-induced phosphorylation and dephosphorylation of proteins resembles the findings of Felix et al. (1991, 1993) for t o m a t o cells and supplies further evidence for the action of a protein kinase and the involvement of phosphorylated proteins in elicitor-induced signal transduction in spruce cells. The time course of the elicitor-induced phosphorylation of a 63-kDa protein and dephosphorylation of a 65-kDa protein correlates with reactions which can be inhibited by staurosporine, namely the elicitor-induced extracellular alkalinization (Fig. 3) and H 2 0 2 synthesis of spruce cells

(Schwacke and Hager 1992). Thus, the action of protein kinases seems to be involved in signal transduction in ectomycorrhizal as well as pathogenic interactions (Dietrich et al. 1990; Viard et al. 1994). The modulation of signal-dependent kinase activity might be mediated by an elevated cytosolic Ca 2 § level. The presence of Ca 2 +-dependent and Ca2§247 protein kinases in plant cells is well established ( H a r m o n et al. 1987; Roberts and H a r m o n 1992). The function of the proteins being phosphorylated or dephosphorylated after elicitor application is poorly understood. Interestingly, phosphorylations are involved in the regulation of H § ATPase and NAD(P)H oxidase activity. In Lycopersicon esculentum, activation of the plasmalemma H § by elicitor-induced dephosphorylation was demonstrated (Vera-Estrella et al. 1994), and in human neutrophils, activation of NAD(P)H oxidase depends on the phosphorylation of cytosolic 47-kDa and 67-kDa subunits of the plasma-membrane enzyme (Rotrosen and Leto 1990; Dusi and Rossi 1993). The H§ is probably involved in the cellular processes causing the extracellular alkalinization, and the NAD(P)H oxidase is assumed to play a role in elicitor-induced H 2 0 2 synthesis in spruce cells. The results of our studies with Picea abies cells and hyphae of the ectomycorrhizal fungus Hebeloma crustuliniforme cultured in suspension are summarized in a speculative model (Fig. 11). These reactions induced by elicitors of ectomycorrhizal fungi resemble those described for plant pathogen interactions (Niirnberger et al. 1994) and the initial events of a hypersensitive response. However, in ectomycorrhizae some reactions might attenuate the (aggressive) action of the fungal elicitors in vivo: (i) apoplasmic enzymes of the plant itself are able to destroy fungal glucan and chitin elicitors; (ii) plant hormones (e.g. auxins) produced by the mycorrhizal fungi are able to suppress elicitor-induced synthesis of defense-related proteins, such as chitinase and peroxidase. We thank Prof. M. Zenk (Universitiit Miinchen, Germany) for providing spruce cell cultures, and Dr. I. Kottke (Universit~it Tiibingen, Germany) for isolates of Hebeloma crustuliniforme TiJ 704. We are also thankful to Dr. W. Mayer (Universitiit Tiibingen) for valuble discussions. This work was supported by Deutsche Forschungsgemeinschaft. B. Zitterell-Haid was financed by "Graduiertenkolleg Interaktion in Waldrkosystemen" (supported by Deutsche Forschungsgemeinschaft) and G. Hebe by a scholarship of the Landesgraduiertenf'orderungsgesetz.

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