FEMS Microbiology
Letters 140 (1996) 265-269
Targeted mutation of the SC3 hydrophobin gene of Schizophyllum commune affects formation of aerial hyphae Marie-Anne
van Wetter *, Frank H.J. Schuren ‘, Theo A. Schuurs, Joseph G.H. Wessels
Department of Plant Biology, Groningen Biomolecular Sciences and Technology Institute (GBB), University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands Received 2 April 1996; accepted 29 April 1996
Abstract The SC3 hydrophobin gene of Schizophyllum commune was disrupted by homologous integration of an SC3 genomic fragment interrupted by a phleomycin resistance cassette. The phenotype of the mutant was particularly clear in sealed plates in which formation of aerial hyphae was blocked. In non-sealed plates aerial hyphae did form but these were hydrophilic and not hydrophobic as in wild-type strains. Complementation with a genomic SC3 clone restored formation of hydrophobic aerial hyphae in sealed plates. In a dikaryon homozygous for the SC3 mutation normal sporulating fruiting bodies were produced but aerial hyphae were hydrophilic. Keywords: Gene disruption in Schizophyllum; SC3 hydrophobin Schizophyllum commune
1. Introduction Hydrophobins are small proteins secreted by fungi that have eight characteristically spaced cysteine residues and common hydropathy patterns [l]. In Schizophyllum commune at least four distinct hydrophobin genes occur. The genes Xl, SC4 and SC6 are expressed in dikaryons whereas the SC3 hydrophobin gene is expressed in both monokaryons and dikaryons [ 1,2].
* Corresponding author. Tel.: +31 (50) 363 2325; Fax: +31 (50) 363 2273; E-mail:
[email protected] ’ Present address: Microbiological Institute, Swiss Federal Institute of Technology, Zirich, Switzerland. 0378.1097/96/$12.00 Copyright PII SO378-1097(96)00192-9
0 1996 Federation
of European
gene;
Aerial
hyphae;
Fruiting
body
formation
in Schizophylbm;
Good evidence was obtained for the involvement of SC3 in the formation of aerial hyphae. It was found that by assembling at a water-air interface the purified SC3 formed a highly insoluble amphipathic membrane with rodlet structures at the hydrophobic side [3]. SC3 was found as an SDS-insoluble assemblage in the cell walls of aerial hyphae [4,5], and by using antibodies raised against purified SC3 it was shown that these hyphae were covered with assembled SC3, showing rodlets at the hydrophobic surface [5]. To prove involvement of SC3 in the formation of aerial hyphae a strain containing a disruption of the endogenous SC3 gene was made and analyzed. Previously we showed that a strain of S. commune with a disrupted SC3 gene is unable to attach to a hydrophobic surface 161. Microbiological
Societies. Published
by Elsevier Science B.V.
2. Materials
and methods
agarose gel, blotted to Hybond-N+ (Amersham, UK) and hybridized with a SC3 cDNA probe as described
2. I. Strains and trunsfiwmution
[lOI.
The co-isogenic S. commune strains 4-39 (MATA MATB41. CBS 341.81) and 4-40 (MATA MATB43. CBS 340.81) were used as well as a derivative of 4-40 carrying the tlzrt mutation [4]. Disruption of the SC3 gene of strain 4-39 has been described previously [6]. The SC_? disruptant (strain 72-3) was obtained by homologous integration of a 5 kb genomic clone of the SC.3 gene (pSg3E) in which a small (0.2 kb) BglII/BcfI fragment was replaced by a bacterial phleomycin resistance gene driven by the S. commune GPD promoter (p3ABBph). To obtain a strain suitable for re-introduction of a functional SC3 gene. 72-3 was crossed with strain 12-43 (MATA MATB42 ural) and siblings selected for both phleomycin resistance and inability to grow on minimal medium. One of these showing the same morphology as 72-3 was then used to re-introduce a functional SC3 gene (on pSg3E) by co-transformation with the CJRAl gene located on
2.4. RNA analysis and protein una1~si.s
puBgB [71. The transformation protocol was as previously described [8] except that protoplasts were obtained from mycelia grown for 2 days in shaken culture. 2.2. Immunologicul
selection of’ transfiwmants
To select transformants for secretion of SC3. colonies were grown for 3 days on perforated polycarbonate membranes (Poretics Corporation, USA. pore size 0.1 p.m) overlying minimal medium containing 1.5% agar. Then a PVDF membrane (Millipore, USA) was placed underneath the polycarbonate membrane for 30 min after which immunodetection was performed on the PVDF membrane with an anti-SC3 antiserum purified against cell walls of the thn mutant of S. commune [9]. The antiserum was used in 1:2000 dilution and detection of the antibodies was as described [9]. 2.3. DNA analysis DNA was isolated using the CTAB procedure [IO]. The DNA samples (5 pg per transformant) were digested for 18 h with BglII, run on 1% TAE
One-tenth of a colony of 7 cm diameter was homogenized at maximum speed for I min in a Waring blender containing 100 ml minimal medium. 5 ml of this mixture was inoculated in 9 cm Petri dishes and incubated at 24°C for 3 days. Another 20 ml medium was then brought underneath the mycelial mat and growth allowed to continue for 2 days. RNA was isolated as described [IO]. Hydrophobins were isolated from the medium as described [3]. After 15% PAGE gels were semi-dry blotted onto a PVDF membrane for immunodetection of SC3 hydrophobin. 2.5. Meusurements
of‘hydrophobicity
A mycelial homogenate (see above) was spread onto perforated polycarbonate membranes overlying agar medium and grown at 24°C for 5 days. After washing with water (four times), pieces of the mycelial mats were dried overnight on chromicacid-cleaned glass cover slips. The dried mycelial mats were used for contact angle measurements using 1 ~1 water droplets placed on the surface of the samples [I I].
3. Results and discussion 3.1. Phenotvpe monokaryon
of
the
SC3
mutation
in
the
Transformation of strain 4-39 with p3ABBph to effect disruption of the SC3 gene [6] resulted in 163 phleomycin-resistant transformants which were then screened for the absence of SC3 secretion. After 3 days of growth, immunodetection of secreted SC3 caught on PVDF membranes showed 44 transformants secreting little or no SC3. Among these about 20 showed few or no aerial hyphae and these were further analyzed at the DNA level. In only two transformants the endogenous SC3 gene was replaced by the disrupted SC3 gene; in the other
M.-A. van Wetter et al./ FEMS Microbiolog! Letter.7 140 (19961 265-269
Fig. 1. Morphologies of strain 4-39 (a,c) and the same strain with a disrupted SC3 gene (b,d) cultured for 5 days. The lids of the Petri dishes were sealed in (c) and (d).
transformants p3ABBph had integrated ectopically [6]. The fact that many of the transformants with ectopically integrated copies of the p3ABBph construct showed low SC3 secretion and few aerial hyphae is noteworthy and may be explained by the fact that SC3 expression is often suppressed by integration of multiple copies of the SC3 gene (T.A. Schuurs and J.G.H. Wessels, unpublished). One of the two SC3 mutants (strain 72-3) was analyzed further. To ascertain that this strain had not acquired the thin mutation which frequently occurs spontaneously and down-regulates SC3 and other genes expressed during emergent growth [4], strain 72-3 was mated to a compatible thn tester strain. If the thn mutation were present in 72-3. the two strains should not complement each other. Since normal fruiting bodies were formed in the cross the thn mutation was absent from the SC3 disruptant. The SC3 disruptant could form some aerial hyphae (Fig. 1) although RNA and protein analysis showed that there was no formation of SC3 mRNA and SC3 protein (Fig. 2). It was only after growing the mycelium as a lawn from homogenized inoculum in sealed plates that the absence of aerial hyphae became clear (Fig. 1). Sealing the plates had only little effect on the phenotype of a monokaryon with
267
an intact SC3 gene. However, contact angles of 1 ~1 water droplets placed on the surface of the wild-type monokaryon were 115 + 10” whereas contact angles on the surface of the disruptant strain (grown in open plates) were not measurable; water droplets immediately soaked into the mycelial mat. There was thus a clear difference in the wettability of the surface of aerial hyphae produced by these two strains. It appeared that under conditions of poor aeration the activity of the SC3 gene was absolutely necessary for hyphae to leave the substrate and grow into the air. The factors involved in this environmental modulation of aerial growth are unknown. Apart from its influence on the formation and hydrophobicity of aerial hyphae, disruption of the SC3 gene also seemed to affect the radial growth rate of colonies which in the disruptant was 15-30% lower than in the wild type (Fig. 1). Also cell wall assembly appeared affected by the gene disruption because the SC3 disruptant formed protoplasts even after 5 days of growth whereas in the wild-type strain and in the strain with a re-introduced func-
(b) nt
Fig. 2. Aerial secreted SC3 disrupting the gene. Mycelia
1
2
3
1
2
3
kDa
growth (a), SC3 mRNA in the mycelium (b) and protein (c) in the wild-type strain 4-39 (I), after SC3 gene (2), and after re-introduction of the SC3 were grown as lawns in sealed plates for 5 days.
tional SC3 gene this ability dropped sharply after approximately 2 days of growth. At this time the SC3 gene normally becomes active [12] and the hydrophobin is secreted at hyphal tips [S]. How the absence of the SC3 hydrophobin can be responsible for these apparent changes in wall construction is unknown. 3.2. PhenoQpe
of SC3 mutation in the dikatyorl
A cross of the SC3 mutant carrying the phleomycin expression cassette (72-31 with a compatible wild-type strain yielded a normal dikaryon that fruited and sporulated with a I: 1 ratio of phleomycin-resistant and phleomycin-sensitive spore germlings. To investigate the effect of absence of SC3 expression in the dikaryon, strain 72-3 was crossed with strain 4-40. MATA MATB43 strains with an SC3 disruption were recovered and by mating such a strain to the original SC3 disruptant a dikaryon homozygous for the SC3 disruption was synthesized. This dikaryon formed normal sporulating fruiting bodies showing that the SC3 gene is dispensable for fruiting body formation. This agrees with the absence of SC3 expression within fruiting bodies except in the aerial hyphae covering the fruiting bodies [9] and the absence of hydrophobins on basidiospores (H.A.B. W&ten and J.G.H. Wessels, unpublished observation). However, the aerial hyphae formed by this dikaryon were hydrophilic, in accordance with a role of the SC3 hydrophobin in covering aerial hyphae of both monokaryon and dikaryon with a hydrophobic rodlet layer [5],[9]. In the secondary mycelium the hydrophobin genes SC4, SC1 [2] and SC6 [ 121 were active, in addition to the SC3 gene. Nevertheless, none of the hydrophobins encoded by these genes was present on the aerial hyphae formed on the secondary mycelium homozygous for the SC? mutation as indicated by their wettability. We have previously shown that the pattern of gene expression in the secondary mycelium was dominated by the dikaryotic hyphae but that aerial hyphae lacked the typical binucleate condition and had a monokaryotic type of gene expression, that is, they expressed SC3 but not the dikaryon-expressed hydrophobin genes [9]. On the basis of this observation, the absence of any hydrophobins on aerial hyphae of a secondary mycelium carrying the
SC3 mutation in both constituent genomes was expected. The observed hydrophilicity of aerial hyphae in the SC3-disrupted secondary mycelium did therefore not exclude the possibility that the dikaryonspecific hydrophobins were functionally able to substitute for the SC3 hydrophobin. Experiments to express the dikaryon-specific hydrophobins in monokaryons by placing them under control of the SC3 promoter are in progress. 3.3. Complementation
of the disrupted SC3 gene
To prove that the observed phenotype of the disruptant (i.e. absence of aerial hyphae in sealed plates, and hydrophilicity of aerial hyphae in open plates) was indeed caused by the disruption of the SC3 gene, the intact SC3 gene was re-introduced. After co-transformation of clone pSG3E with the cloned URA I gene (puBgB1, prototrophic transformants were tested in sealed plates for the formation of aerial hyphae. Under these conditions some transformants were able to form aerial hyphae. Fig. 2 shows recovery of SC3 expression on the RNA and protein levels in such a transformant that had regained the ability to form hydrophobic aerial hyphae under all conditions.
Acknowledgements We are indebted to J. van den Dolder for technical assistance. This research was supported by the Life Sciences Foundation (SLW) and the Technology Foundation (STW) which are subsidized by the Netherlands Organization for Scientific Research (NWO).
References [I] Wessels. J.G.H. (1994) Developmental
regulation of cell wall formation. Annu. Rev. Phytopathol. 32. 413-437. [2] Schuren, F.H.J. and Wessels J.G.H. (1990) Two genes specifically expressed in fruiting dikaryons of Schi:oph~?/wu c’ornmune: homologies with a gene not regulated by mating type genes. Gene 90. 199-205. [3] W&ten, H.A.B.. de Vries. O.M.H. and Wessels, J.G.H. (I 993) Interfacial self-assembly of a fungal hydrophobin into a hydrophobic rodlet layer. Plant Cell 5, I567- 1574.
M.-A. fan Wetter et al. / FEMS Microbiology [4] Wessels, J.G.H., de Vries, O.M.H., Asgeirsdbttir, S.A. and Schuren, F.H.J. (1991) Hydrophobin genes involved in formation of aerial hyphae and fruit bodies in Schizophyllum. Plant Cell 3, 793-799. [5] Wosten, H.A.B., Asgeirsd6ttir, S.A., Krook. J.H., Drenth, J.H.H. and Wessels, J.G.H. (1994) The fungal hydrophobin SC3p self-assembles at the surface of aerial hyphae as a protein membrane constituting the hydrophobic rodlet layer. Eur. J. Cell Biol. 63, 122-129. [6] W&ten, H.A.B., Schuren, F.H.J. and Wessels J.G.H. (1994) Interfacial self-assembly of a hydrophobin into an amphipathic protein membrane mediates fungal attachment to hydrophobic surfaces. EMBO J. 13, 584885854. [7] Froeliger, E.H., Ullrich, R.C. and Novotny, C.P. (1989) Sequence analysis of the CrRAl gene encoding orotidine-5’monophosphate decarboxylase of Schizophyllum commune. Gene 83, 387-393. [S] Schuren, F.H.J. and Wessels, J.G.H. (1994) Highly-efficient transformation of the homobasidiomycete Schizophyllum
[9]
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commune to phleomycin resistance. Curr. Genet. 26, 179183. Asgeirsdottir, S.A., van Wetter, M.A. and Wessels, J.G.H. (1995) Differential expression of genes under control of the mating-type genes in the secondary mycelium of Schizophyk lum commune. Microbiology 141, 1281-1288. Schuren, F.H.J., Harmsen, M.C. and Wessels, J.G.H. (1993) A homologous gene-reporter system for the basidiomycete Schizophyllum commune based on internally deleted genes. Mol. Gen. Genet. 238, 9 I-96. van der Mei, H.C., Rosenberg, M. and Busscher, H.J. (1991) Assessment of microbial cell surface hydrophobicity. In: Microbial Cell Surface Analysis (Mazes, N., Handly, P.S., Busscher, H.J. and Rouxhet, P.G.. Eds.), pp. 261-287. VCH Publishers, New York. Schuren, F.H.J., van der Lende, T.R. and Wessels, J.G.H. (1993) Fruiting genes of Schizophyllum commune are transcriptionally regulated. Mycol. Res. 97, 538-542.
