The EMBO Journal vol.13 no.24 pp.5848-5854, 1994

Interfacial self-assembly of a hydrophobin into anamphipathic protein membrane mediates fungalattachment to hydrophobic surfaces

Han A.B.Wosten, Frank H.J.Schuren andJoseph G.H.Wessels1Department of Plant Biology, Biological Centre, University ofGroningen, Kerklaan 30, 9751 NN Haren, The Netherlands

'Corresponding author

Communicated by R.Kahmann

The SC3p hydrophobin of Schizophyllum commune isa small hydrophobic protein (100-101 amino acids witheight cysteine residues) that self-assembles at a water/air interface and coats aerial hyphae with an SDS-insoluble protein membrane, at the outer side highlyhydrophobic and with a typical rodlet pattern. SC3pmonomers in water also self-assemble at the interfacesbetween water and oils or hydrophobic solids. Thesematerials are then coated with a 10 nm thick SDS-insoluble assemblage of SC3p making their surfaceshydrophilic. Hyphae ofS.commune growing on a Teflonsurface became firmly attached and SC3p was shownto be present between the fungal cell wall and theTeflon. Decreased attachment of hyphae to Teflon wasobserved in strains not expressing SC3, i.e. a straincontaining a targeted mutation in this gene and aregulatory mutant thn. These findings indicate thathydrophobins, in addition to forming hydrophobic wallcoatings, play a role in adherence of fungal hyphae tohydrophobic surfaces.Key words: adhesion/hydrophobin/interfacial self-assembly/pathogenicity/rodlet layer

IntroductionHydrophobins were discovered as the putative productsof members of a gene family highly expressed duringformation of aerial hyphae and fruit bodies in the homo-basidiomycete Schizophyllum commune (Schuren andWessels, 1990) and now appear to be of ubiquitousoccurrence among fungi (de Vries et al., 1993; Wessels,1994). They code for small secreted proteins (-100 aminoacids) that contain eight cysteine residues at conservedpositions and display a characteristic hydropathy pattern.In S.commune at least four divergent hydrophobin genes,SC], SC3, SC4 and SC6 have been identified (Schurenand Wessels, 1990; unpublished data). Of these, thehydrophobins encoded by the SC3 and SC4 genes havebeen identified as proteins that form SDS-insoluble com-plexes in the cell walls of aerial hyphae and fruit-body hyphae, respectively (Wessels et al., 1991a,b). Theinsolubility of these hydrophobin complexes, which canonly be dissociated by agents like formic acid and tri-fluoroacetic acid, explains why these abundant proteins

(in S.commune up to 10% of the total protein mass) havenot been detected earlier.When the purified SC3p hydrophobin monomer is

confronted with a water/gas interface it assembles spon-taneously into a 10 nm thick SDS-insoluble amphipathicmembrane, with a gas-exposed surface of high hydro-phobicity and a typical ultrastructure composed of 10 nmdiameter rodlets (Wosten et al., 1993). Such an SC3pmembrane with the hydrophobic rodlet surface facing theair is present on aerial hyphae and we have suggestedinterfacial self-assembly of SC3p as a mechanism inducinghyphae to leave the aqueous substrate and to grow intothe air (Wosten et al., 1994). Hydrophobic rodlets similarto those on aerial hyphae of S.commune have been seenon many hydrophobic fungal spores and disruption of ahydrophobin gene in Aspergillus nidulans (Stringer et al.,1991) and Neurospora crassa (Bell-Pedersen et al., 1992;Lauter et al., 1992) resulted in the disappearance of therodlet layer and the formation of easily wettable sporesthat can no longer be air-dispersed.

Here we report that the SC3p hydrophobin ofS.commune self-assembles at a variety of hydrophilic/hydrophobic interfaces. The results indicate that assemblyof hydrophobins may play an important role in adhesion offungi to hydrophobic surfaces, as in pathogenic interactionswith plant and animals.

ResultsAssembly of SC3p at a water/oil interfaceSonication of SC3p in water caused precipitation of theprotein, probably because the protein assembled aroundgas bubbles which subsequently disintegrated (Wostenet al., 1993). However, when a solution of SC3p (100 ,ug/ml) was sonicated in the presence of 2% oil (v/v) SC3pcould not be pelleted, neither was it found in the waterphase (Figure 1, lane 2); all SC3p was present in theupper phase oil emulsion. When SC3p was not sonicatedtogether with the oil but added to an oil suspension, SC3pdisappeared from the aqueous phase during overnightincubation, the amount disappearing decreasing as theamount of oil was lowered (Figure 1, lanes 3, 4 and 5).Apparently, sonication of SC3p and oil together is mosteffective in assembling SC3p on the oil. After treatmentof the stabilized oil emulsion with chloroform/methanol(2:1) all the assembled SC3p could be pelleted bycentrifugation (results not shown).When viewed in the light microscope, the SC3p-treated

oil droplets often appeared budding or otherwise deviatedfrom purely spherical, suggesting lowering of the surfacetension (Figure 2a).

After OS04 fixation, uranyl-acetate-stained thinsections of the SC3p-stabilized oil suspension showed adark-stained layer at the surface of the oil droplets, -10 nm

58 Oxford University Press5848

Self-assembly of a fungal hydrophobin

12 3 4 5

-100

-71-43

-28

-18

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Fig. 1. Decrease of SC3p (100 ig/ml) in the aqueous phase due to thepresence of an olive oil emulsion. This is shown after 12.5%SDS-PAGE and staining with silver. SC3p before introducing the oil(lane I), SC3p remaining after sonicating 2% (v/v) oil together withSC3p (lane 2) and SC3p remaining after overnight incubation with an8% (lane 3), 1.6% (lane 4) and 0.16% oil emulsion (lane 5). Arrowsindicate monomeric and dimeric forms of SC3p. Molecular weightmarkers in kDa.

thick (Figure 2b), sometimes disconnected from the oildroplet (Figure 2c). This layer was absent on the surfaceof water-emulsified oil droplets; only a diffuse staining atthe periphery of the droplet was observed (Figure 2d).These observations indicate the presence of assembledSC3p as a coating on the oil droplets, probably as aprotein monolayer.When oil droplets coated with assembled SC3p were

freeze-fractured, a mosaic of parallel rodlets was observedfacing the oil (Figure 2e). No indications were found forrodlets in etched surfaces facing the water (data notshown). In agreement, when glutaraldehyde-fixed orunfixed oil droplets coated with assembled SC3p weredirectly shadowed no rodlets were observed (Figure 2f).The rodlets seen on the oil-facing side of the SC3pmembrane are similar to those seen after shadowing the air-facing hydrophobic side of a SC3p membrane generated bydrying down a solution of SC3p on a solid surface (Wostenet al., 1993, 1994). This strongly suggests that the oildroplets in the stabilized oil emulsion are coated with anamphipathic SC3p membrane with the hydrophobic sidefacing the oil, the hydrophilic side facing the water.

Assembly of SC3p at the interface between waterand solidsThe property of SC3p to assemble on air bubbles and oildroplets and to coat these hydrophobic phases with anamphipathic protein membrane suggested that the samemight occur when an aqueous solution of SC3p is con-fronted with a hydrophobic solid surface. Therefore, sheets

of various materials were submerged in an aqueous solu-tion of 35S-labelled or non-labelled SC3p. Hydrophobicityof the surfaces was determined by measuring water contactangles (van der Mei et al., 1991); Teflon (PTFE) showingthe highest hydrophobicity, glass the lowest (Table I).The time course of adsorption of 35S-labelled SC3p on

Teflon shows that at 2 gg/ml SC3p (this is a concentrationnormally found in the culture medium) saturation wasreached after 16 h, while at 20 ,ug/ml SC3p saturationwas already reached after 2 min of incubation (Figure 3).As shown in Table I, after 16 h incubation in 4 ,ug/mlSC3p, 5.9X 10- 12 mol/cm2 had attached to the Teflon, i.e.6% of the SC3p present in the aqueous solution. Atthe same time, contact angles showed a decrease inhydrophobicity of the surface from 108 ± 2° to 48 ±10°. After extraction with hot 2% SDS, contact angles onSC3p-coated Teflon somewhat increased while 87% ofthe radioactivity remained attached. This insolubility inhot SDS indicates that assembled SC3p strongly interactswith the plastic. Subsequent extraction with trifluoroaceticacid (TFA), which dissociates assembled SC3p, releasedall the SC3p and restored normal hydrophobicity. Similarresults were obtained with parafilm which resemblesTeflon in hydrophobicity, but adsorbed somewhat moreSC3p. The amount of SC3p attaching to other materialsdecreased with decreasing hydrophobicity of the surfaces.Of these, only polyethylene (PE) became clearly morehydrophilic after immersion in SC3p, while contact angleson polymethylmeta-acrylate (PMMA), nylon (nylon 6)and glass were only slightly affected. It thus appears thatfor the formation of a stable hydrophilic coating ofassembled SC3p the surface must have a hydrophobicitycorresponding to water contact angles exceeding 760.Results (not shown) similar to those given in Table I wereobtained when materials were exposed for 16 h to aqueoussolutions of 100 ,ug/ml SC3p instead of 4 ,tg/ml.As a control, the same materials as shown in Table I

were incubated for 16 h in a solution of 12 gg/ml bovineserum albumin (BSA) (similar molarity to 4 gg SC3p)followed by three washes of water. Changes in hydro-phobicity were slight. Without washings, Teflon becameeasy wettable when incubated in 1 mg/ml BSA but, incontrast to coating with SC3p, hydrophobicity was largelyrestored by three washes in water and completely restoredby extraction with hot SDS (results not shown).

Immuno-localization of SC3p at the cell wall!Teflon interfaceSC3p is excreted as monomers at tips of hyphae growingin aqueous medium but assembles into a rodlet layer atthe surface of aerial hyphae (Wosten et al., 1994). SinceSC3p also assembles in vitro at the interface betweenwater and a hydrophobic solid, and becomes stronglyattached to the hydrophobic surface, it is plausible thathyphae excreting monomeric SC3p and growing over ahydrophobic surface will attach themselves to this surfaceby self-assembly of SC3p. Indeed, when leading hyphaeof S.commune were forced to grow over Teflon, theyfirmly attached, as indicated by the flattening of hyphaeat the site of attachment (Figure 4). Using a specificpolyclonal antiserum, the SC3p hydrophobin was localizedat the wall surface contacting the air, as previouslyobserved (Wosten et al., 1994) but, also, between the wall

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Fig. 2. Morphological appearance of SC3p assembled on a water/oil interface. (a) Light microscopy of mineral oil droplets coated with assembledSC3p. (b) Thin section of SC3p-coated oil droplets after OS04 fixation. (c) As (b) but showing the layer of assembled SC3p disconnected from anoil droplet. (d) Oil droplet without assembled SC3p on its surface. (e) Freeze-fractured preparation of SC3p-coated oil droplets showing rodlets atthe right and oil covering the rodlet layer at the left. (f) Shadowing of SC3p-coated oil droplets reveals the hydrophilic side of assembled SC3pwithout rodlets. The thin bar represents 50 jim, the thick bars represent 100 nm. Thin arrows at right top corners of photographs (e and f) indicatedirection of shadowing.

surface and the Teflon (Figure 4). This is consistent witha role of SC3p in attachment of these hyphae to thehydrophobic surface.

Disruption of the SC3 gene affects attachment ofhyphae to a Teflon surfaceTransformation was used to replace the active SC3 geneby the disrupted-gene construct illustrated in Figure 5a

(see Materials and methods). Gene replacement was con-firmed by Southern analysis (Figure Sb) for two trans-formants that no longer secreted SC3p. Where the wild-type gene was still present, this was detected on two BglIIfragments of 2.5 and 7.0 kb. Gene replacement led todetection of a single 10.4 kb fragment. One of thesetransformants was subjected to another round of proto-plasting and regeneration to ensure the removal of any

H.A.B.W6sten et al.

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Fig. 3. Time course of adsorption of SC3p to Teflon sheets duringincubation in 2 gg/ml (A) and 20 tg/ml SC3p (B). Amounts of SC3padsorbed before (0) and after hot SDS extraction (0).

nuclei containing the wild-type SC3 gene, and used fortesting its capacity to adhere to Teflon. In addition, wealso tested a co-isogenic thn mutant that does not expressthe SC3 gene (Wessels et al., 1991b). Strains were forcedto grow from an agarose base onto a Teflon surface andthe number of hyphae attached to the Teflon was recordedbefore and after embedding them in molten agarose which,after solidification, was separated from the Teflon sheets(see Materials and methods). When the sheets wereseparated from 2% solidified agarose, all hyphae of thewild-type strain and most hyphae of the thn mutant andSC3 disruptant remained attached. However, when a 3%agarose solution was used, >95% of the wild-type hyphaeremained attached to the Teflon surface while only 3%(11 out of 350) of the hyphae of the SC3 disruptant and11% (78 out of 700) of the hyphae of the thn mutant werestill found on the Teflon sheets in their original position.

DiscussionIn the present and previous papers (Wosten et al., 1993,1994) we show that monomers of the hydrophobin SC3pof S.commune have the unique property to self-assembleinto a stable amphipathic membrane when confrontedwith a hydrophilic/hydrophobic interface. Self-assemblyof SC3p at the wall/air interface (Figure 6b) is solelyresponsible for the high hydrophobicity (water contactangles 115° ± 10°) of aerial hyphae of S.commune andthe typical rodlet pattern seen on the surface of thesehyphae (Wosten et al., 1993, 1994). The results presentedin this paper show that monomers of SC3p self-assemble

Fig. 4. Immuno-localization of SC3p on a section of a hypha grownover a Teflon surface. The convex side contacts the air, the flattenedside contacts the Teflon. Note the bad preservation of the cytoplasmpossibly due to degradation of cytoplasm after growth on Teflon stops.Gold particles, indicating the presence of assembled SC3p, are seen atthe hyphal periphery where the wall contacts the air or the Teflon. Barindicates 100 nm.

into an insoluble amphipathic membrane not only at awater/air interface but also at interfaces of water and anymaterial of minimal hydrophobicity. Experiments to bereported elsewhere (Wosten et al., 1995), using a con-tinuous gradient in hydrophobicity by coupling dimethyl-dichlorosilane to glass, have shown that it is indeed thedegree of hydrophobicity that primarily determines thedifferences in degree of assembly. We assume that theexposure to the interface induces a conformational changein the hydrophobin monomers resulting in their assemblyinto a stable membrane with polar groups concentratedat one surface and apolar groups at the other surface(Figure 6).

After coating with SC3p, 5.9X 10- 12 mol/cm2 adsorbedto the Teflon. Assuming an uninterrupted monolayer, thiswould predict the availability of 28 nm2 for every SC3pmolecule. Similarly, a value of 44 nm2 can be derivedfrom the amount which assembled at a water/air interface(Wosten et al., 1994). SC3p, a glycoprotein with amolecular mass of 14 500 according to mass spectroscopy(O.M.H.de Vries, unpublished) would occupy only -7 nm2if assumed to have a spherical form after assembly, anassumption apparently not justified.

Contact angles of water droplets on the surface of solidmaterials decreased from >100° to as low as 36° aftercoating with SC3p (Table I). These low contact anglesare similar to those obtained when water droplets wereplaced on hyphal surfaces devoid of SC3p (Wosten et al.,1993). This shows the compatibility of the hydrophilicside of the SC3p membrane with the hydrophilic cell wall.In addition, the strong adhesion of the assembled SC3pmembrane to hydrophobic surfaces, resisting boiling in asolution of SDS, suggested to us that assembly of thishydrophobin could mediate the attachment of hyphae tohydrophobic surfaces (Figure 6c). Indeed, hyphae ofS.commune strongly attached while growing over Teflon,and assembled SC3p could be localized at the wall/Tefloninterface. A targeted mutation of the SC3 gene resultedin hyphae which showed decreased attachment to Teflon.Hyphae of a regulatory mutant of S.commune (thn) thatdoes not express any of the known hydrophobin genes(Wessels et al., 1991b) likewise showed reduced attach-ment to Teflon. Residual attachment can be explained byassuming that this is due to low activities of the otherhydrophobin genes in the monokaryotic strain carryingthe SC3 mutation (Schuren and Wessels, 1990; Wessels

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Table I. Relationships between the degree of surface hydrophobicity (00) of various materials, change in surface hydrophobicity after SC3p coatingbefore (01) and after hot-SDS extraction (02) and the amount of SC3p adsorbed

Material Contact angles Adsorption SC3p mol/cm2 X( 1012

00 0' 02 Total SDS resistant

Teflon 108±2 48 ±10 62±8 5.9 5.1Parafilm 105±2 36 ±3 - 6.9 6.2Polyethylene 95 ±1 43 ± 6 68 ±4 4.0 2.6Polymethylmeta-acrylate 76 ±1 70 ± 5 73 ±7 1.3 0.3Nylon 51±1 49 ±2 - 0.7 0.4Glass 15±2 23 ±2 28±3 0.1 <0.06

Materials were incubated in 4 gig/ml SC3p for 16 h.

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contains a 5 kb genomic EcoRI fragment of the SC3 gene interrupted

by a phleomycin resistance cassette (Schuren and Wessels, 1994),

cloned into the Bglll and BclI sites of the SC3 gene thereby removing

these restriction sites (indicated in parenthesis). (b) Southern analysis of

Bgfll digested genomic DNA of the wild-type strain 4-39 (lane 1) and

four p3ABBph transformants (lanes 2, 3, 4 and 5), all probed with a

SC3 cDNA insert probe (Schuren and Wessels, 1990). The position of

the endogenous SC3 signals are indicated at the right. Lanes 2 and 3

show ectopic integration of the plasmid, lanes 4 and 5 show

homologous replacement of the SC3 gene.

et al., 199 a) and a low activity of the SC3 gene in the

thn mutant (Wessels et al., 1991ib).The biological role of attachment of S.commune hyphae

to a hydrophobic surface is not known. Possibly, adhesion

of these hyphae to hydrophobic surfaces, mediated by

Fig. 6. Proposed model for self-assembly of hydrophobins on fungal

hyphae. (a) Hydrophobin monomers excreted into the aqueous medium

remain monomeric due to the absence of a hydrophilic/hydrophobic

interface. (b) Hydrophobin monomers excreted by aerial hyphae self-

assemble into an amphipathic membrane at the cell wall (hydrophilic)/

air (hydrophobic) interface making the surface hydrophobic.

(c) Excreted hydrophobin monomers self-assemble at the site where

the hypha grows over a hydrophobic surface and then act as an

adhesive. We assume that the self-assembly is accompanied by a

stable conformational change in the protein resulting in a separation of

apolar (black) and polar (light grey) groups in the protein. The non-

assembled monomers are indicated as grey spheres.

assembly of SC3p, plays a role in this wood-rotting fungus

during invasive growth in wood which is rich in lignin as

a hydrophobic component. However, in pathogenic fungi,

hydrophobicity of the fungal surface has long been sus-

pected to be an important factor in fungal disease initiation,

both in plants and animals (Hazen, 1990; Cole and Hoch,

1991), but no adhesins have been chemically identified.

T'he discovery that the SC3p hydrophobin may mediate

adhesion to hydrophobic surfaces suggests a general

mechanism by which pathogenic fungi attach to the mostly

hydrophobic surfaces of hosts, prior to penetration. In

many pathogens this involves formation of a strongly

attached appressorium from which an infection hypha

grows that penetrates the host, mechanically and/or

enzymically. With the phytopathogen Uromyces

appendiculatus a correlation has been found between the

strength of adhesion, number of appressoria formed and

the hydrophobicity of surface bearing the sporelings

(Terhune and Hoch, 1993). In this organism evidence was

also obtained suggesting that appressorium formation is

5852

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Self-assembly of a fungal hydrophobin

related to the strength of adhesion of the hyphae to thehydrophobic surface, possibly via activation of mechano-sensitive ion channels in the plasma membrane at thegrowing tips (Zhou et al., 1991). With regard to a possiblerole of hydrophobins in this process, St Leger et al. (1992)found strong expression of the hydrophobin gene ssgAduring in vitro induction of appressoria in the insectpathogen Metarhizium anisopliae while Talbot et al. (1993)found high expression of the hydrophobin gene MPG] inplanta during appressorium formation by the rice blastfungus Magnaporthe grisea. Significantly, disruption ofthe MPGJ gene strongly decreased both formation ofappressoria and pathogenicity, indicating that thishydrophobin is a real pathogenicity factor (Talbot et al.,1993).

Material and methodsAssembly of SC3pSC3p, labelled or not with 35S, was purified from the culture mediumas described (Wosten et al., 1993). The amount of radioactive SC3p wascalculated from the known sulfur content of SC3p (Schuren and Wessels,1990) and the specific radioactivity of the sulfate in the medium.Amounts of non-radioactive SC3p were estimated using the bicinchoninicacid reagent (Smith et al., 1985) with radioactive purified SC3p asa reference.

For assembly of SC3p at a water/oil interface, kitchen grade olive oilor mineral oil (diffusion pump oil, Lion Fat and Oil Co. Ltd) were used.Ten gl of oil was emulsified in 300 pl water by sonication after which300 g1 of an aqueous solution of SC3p (200 ,ug/ml) was added.Alternatively, the oil was directly emulsified in the SC3p solution and300 gl water added. After overnight incubation at room temperature inthe presence of 0.02% NaN3, the emulsions were centrifuged at 10 000 gfor 30 min and the oil droplets, which easily dispersed, washed fourtimes with water by centrifugation to remove monomeric SC3p. Oildroplets coated with assembled SC3p were then resuspended in a smallvolume of water and processed for light or electron microscopy.

For assembly of SC3p on solid materials, cleaned rectangular sheets(20X8 mm) of the material (0.1-0.3 mm thick) were submerged in900 p1 water in a chromic-acid-cleaned glass cuvette and degassed byevacuation. This was followed by addition of 900 pl of a degassedaqueous solution of SC3p containing at least 4.0 gg/ml of SC3p Thecuvette was closed with a glass cover slip avoiding entrapment of air.After overnight incubation at 25°C in the presence of 0.02% NaN3, thesheets were washed three times with water for 15 min each and dried.Extractions with 2% SDS were done at 100°C for 10 min. This wasfollowed by three washes with water for 15 min each. The amount ofadsorbed [%5S]SC3p was determined directly by counting the sheets byliquid scintillation or by extracting SC3p from the sheets with trifluoro-acetic acid (TFA) and counting the extract after evaporation of the acid.

Surface hydrophobicities were determined by measuring water contactangles according to van der Mei et al. (1991).

Electron microscopyFreeze fracturing and freeze etching were done in a Bioetch 2005(Leybold-Hereaus) after the material was frozen in a mixture of solidand liquid nitrogen. Replicas of Pt/C were cleaned in K2Cr2O7-saturatedH2SO4 for 90 min at room temperature.

Surface shadowing was done with Pt/C at an angle of 300 after dryingthe material on formvar-coated nickel grids.

For thin sectioning of oil droplets coated with assembled SC3p, thesewere taken up in molten low melting point agarose and fixed for 60 minin 3% glutaraldehyde, 0.1 M cacodylate buffer pH 7.2 on ice. The fixedmaterial was washed in buffer and post-fixed with 1% OS04 in 0.1 Mcacodylate buffer at room temperature for 2 h. After three washes ofwater, this was followed by overnight incubation in 1% uranyl acetateat room temperature. The material was subsequently dehydrated in an

ethanol/propylene oxide series and embedded in Epon 812.For immuno-localization of SC3p on hyphae growing over Teflon

sheets, the sheets with adhering hyphae were immediately fixed in 0.5%glutaraldehyde, 2.5% paraformaldehyde in 0.1 M cacodylate bufferpH 7.2 for 60 min on ice. The sheets were then thoroughly washed

under running tap water to remove any aerial hyphae which grew fromthe agar slab containing the colony and secondarily adhered duringremoval of the Teflon sheet. The samples were then dehydrated in anethanol series and embedded in Unicryl (Biocell Research Lab., Cardiff,UK) under UV illumination. Thin sections were cut perpendicular to theembedded Teflon sheets and treated with purified antibodies againstSC3p as described (Wosten et al., 1994).

Materials were examined in a Philips CM 10 electron microscope.Photographs were made on FGP Kodak film.

ElectrophoresisSDS-PAGE was done in 12.5% (w/v) polyacrylamide gels (Laemmli,1970) in a Phast System (Pharmacia, Bromma, Sweden). After separation,proteins were stained with silver in the development unit according toinstructions of the manufacturer. Samples taken up in SDS sample bufferafter treatment with TFA were adjusted to pH 6.8 with I M Tris,if necessary.

Disruption of the SC3 gene in a wild-type strainClone pSg3E containing a 5 kb genomic EcoRI fragment encompassingthe SC3 gene was digested with BgllI and BclI, cutting unique sites inthe SC3 gene. The excised 200 bp fragment was replaced with a cassettecontaining the gene for the Streptoalloteichus hindustanus phleomycinbinding protein fused to the Scommune GPD promotor and terminatorsequences (Schuren and Wessels, 1994) resulting in plasmid p3ABBphin which the internal BgllI and BclI sites are no longer present (seeFigure 5). Schizophyllum commune strain 4-39 was protoplasted andtransformed to phleomycin resistance as described (Schuren and Wessels,1994). Phleomycin resistant transformants (163) were tested for excretionof SC3p on PVDF membranes underlying the colonies (Wosten et al.,1994). Nineteen colonies showing reduced, or no, SC3p excretionwere used for genomic DNA analysis. DNA was digested with Bglll,electrophoresed, blotted and hybridized to an SC3 cDNA insert asdescribed (Schuren et al., 1993). One of two transformants containing adisruption in the SC3 gene was protoplasted and phleomycin resistantcolonies resulting from single protoplasts obtained to ensure removal ofany non-transformed nuclei.

Attachment of hyphae to TeflonWild-type Scommune strain 4-40, a derived thn mutant (Wessels et al.,1991b) and the co-isogenic strain 4-39 with a targeted mutation in SC3were grown from small amounts of mycelial homogenate placed centrallyon 12 ml minimal medium (Dons et al., 1979) in 9 cm Petri disheswhich were closed with Parafilm. After 5 days of growth at 24°C in thelight, the agar medium in front of the colony was removed and chromic-acid-cleaned Teflon sheets were placed in such a way that hyphae wereforced to grow from the agarose slab over the Teflon. After 2 days, thecolony slab was removed, the Teflon was washed in running water toremove any aerial hyphae, and the pattern of hyphae attached recordedphotographically. The side of the Teflon with adhering hyphae was thenfloated on a low melting point agarose solution (2 or 3%) maintained at37°C. After solidifying for 15 min at room temperature, the Teflon sheetswere separated from the agarose and again photographed. Differences inthe number of hyphal tips before and after lifting the sheets from theagarose were taken as a measure of the strength of adhesion.

AcknowledgementsWe are indebted to M.A.van Wetter for participation in the work thatled to the targeted gene mutation and to J.van den Dolder, J.M.J.Scheer,K.Sjollema and J.Zagers for excellent technical assistance. With respectto hydrophobicity measurements we acknowledge the advice given byDrs H.C.van der Mei and H.J.Busscher.

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Received on July 29, 1994; revised on September 12, 1994

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