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mycological research 111 (2007) 14311436

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Ostreolysin enhances fruiting initiation in the oyster mushroom (Pleurotus ostreatus)

Sabina BERNEa, Jure POHLEVENb,c, Iztok VIDICd, Katja REBOLJe, Franc POHLEVENd, e,* EKe, Anton SONNENBERGf, Kristina SEPC IC Tom TURKe, Peter MAC
a

Medical centre for molecular biology, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia Department of Pharmaceutical Biology, Faculty of Pharmacy, University of Ljubljana, As kerc eva 7, 1000 Ljubljana, Slovenia c ef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia Department of Biotechnology, Joz d na dolina, Cesta VIII/34, Department of Wood Science and Technology, Biotechnical Faculty, University of Ljubljana, Roz 1000 Ljubljana, Slovenia e Department of Biology, Biotechnical Faculty, University of Ljubljana, Vec na pot 111, 1000 Ljubljana, Slovenia f Department of Plant Breeding, Wageningen University and Research Centre, P.O. Box 16, 6700 AA Wageningen, The Netherlands
b

article info
Article history: Received 4 May 2007 Received in revised form 17 June 2007 Accepted 10 September 2007 Published online 19 September 2007 Corresponding Editor: Daniel C. Eastwood Keywords: Fruiting initiation Ostreolysin Pleurotus ostreatus

abstract
Fruiting initiation in mushrooms can be triggered by a variety of environmental and biochemical stimuli, including substances of natural or synthetic origin. In this work ostreolysin, a cytolytic protein specically expressed during the formation of primordia and fruit bodies of Pleurotus ostreatus, was applied to nutrient media inoculated with mycelium of P. ostreatus, and its effects on mycelial growth and fructication of the mushroom studied. The addition of ostreolysin slightly inhibited the growth of mycelium, but strongly induced the formation of primordia, which appeared 10 d earlier than in control plates supplemented with bovine serum albumin or with the dissolving buffer alone. Moreover, ostreolysin stimulated the subsequent development of primordia into fruit bodies. However, direct involvement of this protein in the sporulation of the mushroom is unlikely, as it was also detected in large amounts in the non-sporulating strain of P. ostreatus. 2007 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction
The oyster mushroom (Pleurotus ostreatus) is an edible ligninolytic basidiomycete with medicinal, biotechnological, and environmental applications (Ku es & Liu 2000; Cohen et al. 2002). Commercially, it is the most cultivated mushroom worldwide after Agaricus bisporus (Ku es & Liu 2000). Knowledge of the cellular processes leading to the initiation of fruit body development is lacking in several edible mushrooms, including P. ostreatus. Therefore, the identication of

the genes and proteins involved in fruiting, as well as studying the effects of environmental and biochemical treatments on fruit body formation are extremely important biotechnologically and commercially. These ndings can be used to control fruit body initiation, which is the pivotal step in the production of mushrooms. In 2002, cDNA libraries were constructed from mRNA isolated either from the mycelium or fruit bodies of P. ostreatus. The libraries were sequenced, and expressed-sequence tags (ESTs) from both developmental stages were compared and

* Corresponding author. E-mail address: kristina.sepcic@bf.uni-lj.si 0953-7562/$ see front matter 2007 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2007.09.005

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1432 S. Berne et al.

analysed for changes in gene expression (Lee et al. 2002). Among the 1069 ESTs identied in fruit bodies, one set of unigene sequences, with a redundancy number of 29, was differentially expressed and found only in the fruit bodies. These sequences were similar to those of ostreolysin and aegerolysin, cytolytic proteins that were simultaneously isolated from the fruit bodies of P. ostreatus and Agrocybe aegerita, respectively (Berne et al. 2002). Both proteins were assigned to a novel aegerolysin protein family (Berne et al. 2005) that comprises identical acidic proteins from fungi, bacteria, and plants. Many of them have haemolytic and cytolytic properties et al. 2003; Tomita et al. (Ebina et al. 1985; Berne et al. 2002; Sepc ic 2004) which can cause toxic effects and/or death after intravenous administration (Sakaguchi et al. 1975; Ebina et al. 1982;  z ek et al. 2006; Rebolj et al. 2007). Aegerolysins are heatZu and pH-inactivated (Berne et al. 2002, 2005), and, therefore, are not harmful to humans on ingestion. The exact biological function of aegerolysin family members has not been resolved. Ostreolysin and aegerolysin are specically expressed during the formation of primordia and fruit bodies of the correspond` re 1997; Berne ing mushrooms (Fernandez Espinar & Labare et al. 2002; Vidic et al. 2005), and Clostridium bifermentans haemolysin-like proteins appear to be related to bacterial sporulation (Barloy et al. 1998), indicating a putative role in the developmental cycle of the organisms that produce them. Ostreolysin, a distinctive representative of the aegerolysin protein family, is absent in the vegetative mycelium of P. ostreatus. The expression of this acidic 15 kDa protein is initiated at the stage of rapidly growing primordia, and continues during the maturation of young fruit bodies, where it is preferentially found in growing regions of the mushrooms, especially in the basidia and basidiospores (Berne et al. 2002; Vidic et al. 2005). The spatial and temporal expression of ostreolysin suggests its involvement in the initiation of fructication and/or sporulation. Ostreolysin is also lytic at submicromolar concentra tions to erythrocytes and cell lines (Berne et al. 2002; Sepc ic et al. 2003, 2004), where the presence of cholesterol-enriched membrane domains existing in the liquid-ordered state is et al. 2004; Rebolj et al. a prerequisite for its activity (Sepc ic 2006). However, it is not lytic to vesicles reconstituted from to et al. 2003), and is poorly tal lipid extract of P. ostreatus (Sepc ic lytic to lipid vesicles enriched in ergosterol (Rebolj et al. 2006). In this work, ostreolysin was applied to solid nutrient medium inoculated with P. ostreatus mycelium, and its effects on mushroom growth were studied.

Wageningen University and Research Centre, Wageningen, the Netherlands. The strain is also present in the collection of Somycel SA, Langeais, France where it is used to produce commercial spawn. The strain is available for research on request but is protected for commercial spawn preparation by European Breeders Right and Plant Patent Right (for the USA).

Proteins
Ostreolysin was isolated from the fresh fruit bodies of Pleurotus ostreatus, strain Plo5, as described previously (Berne et al. 2002). Its purity was checked using polyacrylamide gel electrophoresis (PAGE) and reverse-phase HPLC. The protein concentration was determined spectrophotometrically using the BCA Protein Assay Reagent (Pierce, Rockford, IL). After isolation, the protein was desalted and kept frozen (20  C) in aliquots in deionised water. Bovine serum albumin (BSA) was obtained from SigmaAldrich (St. Louis, MO).

Effects of externally applied ostreolysin on fructication

Malt extract agar (MEA; Merck, Whitehouse Station, NJ) was poured into polystyrene Petri dishes (20 ml/plate). After solidication, 200 ml of ostreolysin dissolved in 50 mM TrisHCl (pH 8) were spread on the plates, applying 250, 100, 50, 10, 5, or 1 ng protein/mm2 of the agar surface. The control plates were supplemented with the same amounts of buffer-dissolved BSA, or with 200 ml dissolving buffer only. All the dishes were then inoculated centrally with Pleurotus ostreatus (strain Plo5) mycelial discs (V 5 mm) obtained from the periphery of overgrown 7-d-old potato dextrose agar (PDA, Difco, Franklin Lakes, NJ) inoculum plates. Each experiment was repeated three to ve times. The plates were kept in a growth chamber in the dark at 25  C, and growth of the mycelium was regularly monitored until the surface of the growth media was homogeneously overgrown. After 18 d, plates were transferred to the dark at 810  C for 4 d, in order to induce the formation of primordia. Subsequent fructication proceeded in a growth chamber with a constant temperature of 16  C, 90 % air humidity, less than 1000 ppm carbon dioxide, and a 12:12 h photoperiod (29.4 W/m2). The plates were regularly monitored for the formation of primordia over 70 d. To control the possible loss of ostreolysin activity, 1 ml of the protein (0.5 mg/ ml1) was kept in the growth chamber throughout the cultivation procedure, and its haemolytic activity was assayed weekly, using 10 ml of the protein solution from the test tube.

Materials and methods

Mushroom strains
Mycelium of Pleurotus ostreatus (strain Plo5) was taken from the ZIM collection of the Biotechnical Faculty, University of Ljubljana, Slovenia (Raspor et al. 1995). A non-sporulating strain of P. ostreatus (H-195) was obtained from the culture collection of the Department of Plant Breeding, Wageningen University and Research Centre. The sporeless oyster mushroom strain H-195 is stored in the collection of the Mushroom Research Group of Plant Research International,

Detection of ostreolysin in sporulating and non-sporulating strains

Pleurotus ostreatus, strain Plo5, was cultivated on 90 % pinewood sawdust and 10 % wheat bran as previously described (Berne et al. 2002). Young (1015 mm) mushrooms were collected and immediately homogenized, extracted with 50 mM TrisHCl (pH 8), and centrifuged for 20 min at 13 000 g and 4  C. The non-sporulating P. ostreatus strain H-195 was fruited as described previously by Baars et al. (2004). Initials and fruit bodies were lyophilized, homogenized in 50 mM TrisHCl (pH 8), and centrifuged as described above. After adjusting

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Ostreolysin induces fruiting in P. ostreatus 1433

the total protein concentrations of both fungal extracts to the same level (25 mg/ml1) with 50 mM TrisHCl (pH 8), the extracts were analysed using sodium dodecyl sulphate (SDS)-PAGE, and their protein contents and haemolytic activities determined. The presence of ostreolysin in both mushrooms was detected by performing Western blot analysis.

Results
Effects of externally applied ostreolysin on mycelial growth
The addition of ostreolysin to the nutrient plates inoculated with mycelium of Pleurotus ostreatus, strain Plo5, inhibited mycelial growth in a concentration-dependent manner (Fig 1). Seven days after inoculation, the mycelial growth was inhibited by approximately 13, 60, and 85 % on plates supplemented with 10, 100, and 250 ng/mm2 of ostreolysin, respectively. Nevertheless, this effect appeared to be transient, as all the plates were homogeneously overgrown at day 12 after the inoculation. BSA did not affect the mycelial growth, even at the highest concentration tested (250 ng/m2, Fig 1).

SDS-PAGE
Fungal extracts were dissolved in one volume of 1 M DTT (dithiothreitol) and two volumes of electrophoresis buffer [200 mM TrisHCl (pH 6.8), 10 % b-mercaptoethanol, 4 % SDS, 20 % glycerol and 0.1 % bromophenol blue]. The samples were analysed using SDS-PAGE on an electrophoresis apparatus (Bio-Rad, Hercules, CA), using a 12 % acrylamide gel. Proteins were stained with Coomassie Brilliant Blue.

Effects of externally applied ostreolysin on fructication

In control plates treated with various concentrations of BSA or TrisHCl buffer alone, and inoculated with Pleurotus ostreatus (strain Plo5), the formation of primordia was initiated at the 24th day after the induction. Only one primordium (on a control plate supplemented with 250 ng/mm2 of BSA) developed further into a fruit body. In contrast to the control, hyphal aggregation and formation of primordia on plates treated with ostreolysin was observed 14 d after induction, and young fruit bodies appeared on the 22nd day. The average size and number of young fruit bodies on the 25th day after the induction are shown in Fig 2. All ostreolysin concentrations tested

Western blot analysis

For Western blotting, mushroom protein extracts were separated using SDS-PAGE under reducing conditions as described above. Proteins were blotted for 2 h to a 0.45 mm pore size polyvinylidene diuoride Immobilon-P membrane (IPVH15150) (Millipore, Billerica, MA) previously soaked in methanol for 10 min. The blotted membrane was xed overnight in a 4 % BSA solution in WASH buffer, composed of 50 mM NaCl, 1 mM EDTA and 10 mM TrisHCl (pH 7.4). The membrane was soaked and gently stirred for 2 h in 20 ml 4 % BSA in WASH buffer combined with 200 ml rabbit immune serum, prepared as described in Berne et al. (2002), in order to allow binding of primary antibodies. After washing with WASH buffer, the membrane was incubated for 2 h with horse radish peroxidise (HRP)-labelled goat anti-rabbit IgG secondary antibodies (Southern Biotechnology Associates Birmingham, Alabama), previously diluted in 20 ml WASH buffer containing 4 % BSA. After unbound compounds had been washed out, the membrane was soaked in a peroxidase substrate (a mixture of 10 mg ethylcarbazole, 2.5 ml dimethylformamide, 47.5 ml 50 mM sodium acetate (pH 5), and 1.2 ml 0.6 % hydrogen peroxide).

Estimation of haemolytic activity

Haemolytic activity against bovine erythrocytes was measured by a turbidimetric test as described previously (Berne et al. 2002). Typically, 1050 ml of either protein extracts (from sporulating and non-sporulating P. ostreatus strains), or pure ostreolysin, were added into a cuvette containing 3.0 ml bovine erythrocyte suspension in 0.13 M NaCl, 20 mM TrisHCl (pH 7.4), at 25  C. The suspension had an apparent absorbance of 0.5 at a wavelength of 700 nm. Decrease of the apparent absorbance was recorded at 700 nm using a Shimadzu 2100 UV/visible light spectrophotometer to obtain the time course of haemolysis. The nal protein concentration of both fungal extracts used for the haemolytic assay was 16.7 mg/ml1, while monitoring of the ostreolysin activity during the cultivation of the mushrooms was performed at a nal concentration of 1.67 mg/ml1.

Fig 1 Effect of externally applied ostreolysin on Pleurotus ostreatus mycelial growth. Petri dishes containing malt extract agar (MEA) were supplemented with 200 ml of 50 mM TrisHCl (pH 8; control, -), with 200 ml BSA (250 ng/mmL2, 7), or with 200 ml ostreolysin in nal concentrations of 10 (,), 100 (B), or 250 ng/mm2 (C). Dishes were inoculated centrally with one P. ostreatus (strain Plo5) mycelial disc, respectively, and kept in the dark at 25  C for 18 d. All data represent the average radius of the grown mycelium standard error of three to ve independent experiments. The results showing the effects of lower concentrations of ostreolysin (5 and 1 ng/mm2) and BSA (up to 100 ng/mm2) are not shown as they did not differ from the control obtained with 200 ml dissolving buffer alone. BSA, bovine serum albumin; Oly, ostreolysin.

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1434 S. Berne et al.

Fig 2 Effect of externally applied ostreolysin on the fructication of Pleurotus ostreatus. P. ostreatus (strain Plo5) mycelium was centrally inoculated onto Petri dishes containing MEA supplemented with 50 mM TrisHCl (pH 8) or with various concentrations (1250 ng/mm2) of BSA or ostreolysin. The cultivation of mushrooms was carried out as described in the Materials and Methods section. The plates were regularly checked for the formation of primordia and young fruit bodies. (A) Representative control (BSA, 100 ng/mm2) and ostreolysin-treated plates photographed on the 25th day after induction. (B) The average number standard error of three to ve independent experiments, of young fruit bodies grown on plates supplemented with various concentrations of ostreolysin, Oly (C), or BSA (B), on the 25th day after the induction. Numbers near the data points denote the average dimensions (in millimetres) of young fruit bodies on the 25th day standard error.

markedly enhanced the yield and the size of young fruit bodies, reaching a peak at 10ng/mm2. The haemolytic activity of ostreolysin dropped by only 20 % during the 70-d cultivation process (data not shown).

Discussion
Stimulation of fruiting in mushrooms can be achieved by a variety of external stimuli, including reduced temperature (Eger 1976), light (Zadrazil 1978), and nutrient depletion (Eger 1968). Moreover, the fruiting process can be triggered by some fungal or plant secondary metabolites, or certain synthetic compounds. In particular, the growth and mushroom yield of Pleurotus ostreatus on nutrient medium plates were shown to be stimulated by the addition of veratryl alcohol (Sugimoto et al. 2001), b-adenosine (Domondon et al. 2004), phenol (Upadhyay & Hofrichter 1993), or saponin (Magae 1999). The signal by which surface-active compounds, such as saponins, trigger fungal fruiting was proposed to be related to cell membrane disruption. However, this hypothesis was later abandoned due to the nding that surfactants act as

Detection of ostreolysin in sporulating (Plo5) and non-sporulating (H-195) P. ostreatus strains

Fungal extracts of the sporulating and non-sporulating strain exerted nearly identical patterns and intensities of haemolytic activity (Fig 3), corroborating the presence of haemolytic compound(s) in both strains of Pleurotus ostreatus. Analysis of these extracts by SDS-PAGE (Fig 3A) also showed similar protein patterns. Evidence of the presence of ostreolysin in both extracts was further proved by Western blot analysis, showing a positive result in both cases, and suggesting that ostreolysin was present in its active form in both mushroom strains (Fig 3B).

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Ostreolysin induces fruiting in P. ostreatus 1435

Fig 3 Expression of ostreolysin in sporulating (Plo5) and non-sporulating (H-195) strains of Pleurotus ostreatus. The time course of haemolysis induced by extracts of P. ostreatus strains Plo5 (solid line) and H-195 (dashed line), both 16.7 mg/ml1. For details, see the Materials and Methods section. A700 [ apparent absorbance at a w.l. of 700 nm. Inset: SDS-PAGE (a), and western blot analysis (b) of mushroom extracts in 50 mM TrisHCl (pH 8). For SDS-PAGE, the proteins were separated on a 12 % acrylamide gel. Left lane: high molecular weight (MW) standards (Pharmacia, Uppsala) with corresponding MWs of 170, 130, 100, 72, 55, 40, 33, 24, 17, and 11 kDa (from the top). Western blot hybridization was performed using primary rabbit, and secondary HRP-conjugated goat anti-rabbit antibodies, as described in the Materials and Methods section. Oly, pure ostreolysin isolated from the Plo5 strain (control); Plo5, crude extract of the P. ostreatus sporulating strain; H-195, crude extract of the P. ostreatus non-sporulating strain.

fruiting stimulators only when bearing a sugar moiety. Pure commercial surfactants like SDS, CHAPS, CHAPSO, MEGA 8, MEGA 9, MEGA 10, or Triton were ineffective (Magae 1999; Magae et al. 2005; Magae & Ohara 2006), whereas the addition of some synthetic amphipathic derivatives, like 3-O-octyl- or 3-O-decyl-D-glucose, markedly enhanced hyphal aggregation, formation of primordia and consequent fructication of P. ostreatus (Magae et al. 2005). Finally, some reports indicate that fruiting in mushrooms can be enhanced by the addition of a nitrogen source (Eger 1976), but this appears not to be true in the present case, as the fructication of P. ostreatus was enhanced only on addition of ostreolysin, and not another protein, BSA (Fig 2). Recently, Cho et al. (2003) found a large increase of the mycelia-attached population of uorescent pseudomonads during the formation of P. ostreatus fruit bodies. Inoculation of the mycelium with isolated Pseudomonas strains promoted the formation of primordia and enhanced the development of the mushroom fruit bodies, suggesting that the compounds inducing the fruiting signals might be produced by the bacteria. Haemolytically active pseudomonads are often associated with cultivable mushrooms, including P. ostreatus, and are responsible for brown blotch disease of Agaricus bisporus or the

yellowing of Pleurotus eryngii. The haemolytic activity was shown to correlate with pathogenicity on mushrooms, and seems to be attributed to similar factors, extracellular lipodepsipeptides (Munsch & Alatossava 2002). The most investigated among them is tolaasin I from the Pseudomonas tolaasii (Nutkins et al. 1991), which was shown to be lytic to erythrocytes and model lipid membranes (Coraiola et al. 2006). In contrast to aegerolysin-like cytolysins that are mainly cholesteroldependent (Rebolj et al. 2006), the membrane activity of tolaasin I decreases with increasing the sterol membrane content, and is higher on ergosterol-containing membranes that are its main targets in nature (Coraiola et al. 2006). Ostreolysin (and probably other aegerolysin-like proteins) cannot permeabilize lipid vesicles composed of total lipids extracted from P. ostrea et al. 2003), has a weak permeabilizing activity on tus (Sepc ic et al. 2004; Rebolj ergosterol-containing membranes (Sepc ic et al. 2006), and, being pH- and heat-sensitive, is toxic only if  z ek et al. 2006). These data administered intravenously (Zu suggest that aegerolysins, although haemolytic, probably do not act as toxins in their natural environment. Their specic expression during the initiation of fungal fruiting (Fernandez ` re 1997; Berne et al. 2002; Vidic et al. 2005), Espinar & Labare or bacterial sporulation (Barloy et al. 1998), indicates that they are involved in the developmental cycle of the mushrooms and bacteria. Further, it seems that the lytic activity is not a prerequisite for the above-described function of aegerolysins. In fact, aegerolysin-like proteins isolated from Clostridium bifermentans, which were suggested to be involved in sporulation, are not haemolytic (Barloy et al. 1998). In this regard, it is interesting that an aegerolysin-like protein PA0122 (NP_248812.1), bearing 31 % identity and 52 % similarity to ostreolysin, was predicted from the genomic sequence of Pseudomonas aeruginosa (Stover et al. 2000). Hence, both ostreolysin and PA0122 could have the same function and serve as compounds for promoting the growth of mushrooms. The external application of ostreolysin, which could mimic similar putative pseudomonad-associated proteins involved in fruiting initiation, could hence be one of the reasons for the observed enhancement of P. ostreatus fruiting in our study. The present results indicate that ostreolysin, besides being strongly expressed during the process of fruiting initiation (Berne et al. 2002; Vidic et al. 2005), can induce fructication of P. ostreatus, and possibly of other mushrooms, when applied externally, which makes this protein and its genetically engineered recombinant forms commercially and biotechnologically interesting. Its proposed involvement in sporulation of the mushrooms (Vidic et al. 2005) appears to be less likely, as considerable amounts of ostreolysin were also detected in the non-sporulating strain of P. ostreatus. Therefore, it could be concluded that ostreolysin is associated with the appearance of primordia and mushroom growth, but is probably not involved directly in sporulation.

Acknowledgements
This work was supported by the Slovenian Research Agency (grant L4-6420). The authors thank Professor Roger Pain for reading and commenting on the manuscript.

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