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1 Microbiology, Institute of Biomembranes, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands
2 MPI für Terrestrische Mikrobiologie, Karl-von-Frisch-Straße, 35043 Marburg, Germany
3 Institute für Genetik, Ludwig-Maximilians University, Maria-Ward-Straße 1a, 80638 Munich, Germany
Correspondence
Han A. B. Wösten
h.a.b.wosten{at}bio.uu.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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hum2 cross. However, the reduction in aerial hyphae formation was much more dramatic in the rep1 cross. Moreover, colonies of the rep1 cross were completely wettable, while surface hydrophobicity was unaffected and only slightly reduced in the hum2 and the hum2hum3 cross, respectively. It was also shown that the repellents and not the hydrophobins are involved in attachment of hyphae to hydrophobic Teflon. Deleting either or both hydrophobin genes in the rep1 strains did not further affect aerial hyphae formation, surface hydrophobicity and attachment. From these data it is concluded that hydrophobins of U. maydis have been functionally replaced, at least partially, by repellents.
These authors contributed equally to this work. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Banuett & Herskowitz, 1988; Christensen, 1963). These teliospores undergo meiosis, ultimately producing haploid sporidia.
Fusion of haploid cells and development of an infectious dikaryon only occur when the two partners carry different alleles in the a and b mating-type loci. The a locus controls cell fusion through a pheromone-based recognition system (Bölker et al., 1992), while the multiallelic b locus regulates post-fusion steps of pathogenic development, including hyphal growth. The b locus encodes two unrelated homeodomain proteins, bE and bW (Gillissen et al., 1992). These proteins form heterodimers when they are derived from different alleles (Kämper et al., 1995) and as such regulate a number of genes (Bohlmann, 1996; Brachmann et al., 2001; Romeis et al., 2000; Wösten et al., 1996). One of the genes that is strongly up-regulated by the bE/bW heterodimer is rep1 (Wösten et al., 1996). It encodes a pre-pro-protein, consisting of a signal sequence for secretion and twelve repeats. Each repeat, with the exception of the last two, ends with a Kex2-like protease recognition sequence. The pro-protein is cleaved in the endoplasmic reticulum, resulting in ten repeats of 34–55 aa and a larger peptide of 228 aa. The latter not only consists of the last two repeats of Rep1 but also contains a proline-rich region (Fig. 1). The 10 small repeats and the 228 aa polypeptide are collectively known as repellents. Eight of the repellents were identified in cell walls of aerial hyphae, where they reside as SDS-insoluble but TFA-extractable proteins (Wösten et al., 1996).
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). Moreover, outgrowth of hyphae from a water droplet onto a hydrophobic surface is affected. The phenotype of the rep1 strain is thus very similar to that of a Schizophyllum commune strain in which the SC3 hydrophobin gene is deleted (van Wetter et al., 1996; Wösten et al., 1994b). Moreover, solubility of the repellents in the cell wall is very similar to that of the class I hydrophobin SC3 (de Vries et al., 1993; Wessels et al., 1991; Wösten et al., 1996). We studied here the role of two hydrophobin genes of U. maydis. These genes, called hum2 and hum3, do not function in attachment of hyphae to hydrophobic Teflon. Moreover, they are not involved in surface hydrophobicity and pathogenicity and are only partly responsible for aerial growth. Repellents have thus functionally replaced the hydrophobins in formation of hydrophobic aerial hyphae and in attachment of hyphae to a hydrophobic surface. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. The repellent gene rep1 (Wösten et al., 1996) and the hydrophobin genes hum2 and hum3 were deleted in the haploid U. maydis strains FB1 (a1b1) and FB2 (a2b2) (Banuett & Herskowitz, 1989).
Growth conditions.
U. maydis was routinely grown at 25 or 28 °C in YepsL (0.4 % yeast extract, 0.4 % peptone, 2 % sucrose) at 200 r.p.m. or on solid potato dextrose agar (PDA, Sigma) either supplemented or not with 1 % charcoal.
Molecular techniques.
U. maydis chromosomal DNA was isolated as described by Hoffman & Winston (1987). DNA was blotted on Hybond-N+ (Amersham) and hybridized in 0.5 M sodium phosphate, 7 % SDS, 10 mM EDTA, pH 7.2, at 60 °C with [-32P]CTP-labelled probes. RNA was isolated from ground cells (Microdismembrator, B. Braun) with Trizol (Invitrogen) according to the manufacturer's protocol.
Deletion of hum2 and hum3.
A 1.3 kb SphI fragment spanning the ORF of hum2 (um05010; http://mips.gsf.de/genre/proj/ustilago/) was cloned in pUC19. The resulting plasmid pDik2S was used as a template in an inverse PCR reaction with primers hum2.5 and hum2.3, which contain NotI linkers at their 5' end (Table 1). The amplified fragment was digested with NotI and ligated to a hygromycin resistance cassette (Brachmann et al., 2001). In the resulting plasmid pHum2 the region of hum2 between the start and stop codons is replaced by the hygromycin resistance cassette.
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) were used to amplify the 1 kb upstream and downstream region, respectively, of the coding sequence of hum3 (um04433; http://mips.gsf.de/genre/proj/ustilago/). PCR products of both flanks were cloned in pGEM-T Easy (Promega). Internal HindIII and BamHI sites were removed from the upstream and downstream flank, respectively, by Quikchange mutagenesis (Stratagene) using the primer pairs mUF-Hf/mUF-Hr and mDF-Bf/mDF-Br (Table 1). The upstream flank was cloned as an EcoRI–HindIII fragment between the corresponding sites of pUC19. This was followed by introduction of the downstream flank as a BamHI–HindIII fragment. A NotI carboxin resistance cassette taken from pNEB-Cbx (Brachmann et al., 2001) was inserted in the NotI site at the 3' end of the upstream flank. This resulted in pHum3. The EcoRI–HindIII fragment of this vector, encompassing both flanks and the resistance cassette, was used to transform U. maydis.
The hum3 and/or the rep1 gene were deleted in compatible hum2 strains to yield strains hum2hum3, hum2rep1 and hum2hum3rep1. In this case pRep1 (Wösten et al., 1996) contained the nourseotricin (Brachmann et al., 2001) instead of the hygromycin resistance cassette. U. maydis was transformed according to Brachmann et al. (2004). Transformants were selected on PDA plates supplemented with hygromcyin, carboxin or nourseotricin at a final concentration of 200, 2 and 150 µg ml–1, respectively.
Assessment of surface hydrophobicity and hyphal attachment.
Surface hydrophobicity was assessed by determining the contact angle of water droplets of 1–5 µl with the Krüss Drop Shape Analysis System DSA10 Mk2 according to the instructions of the manufacturer. Attachment of hyphae was determined essentially as described previously (Wösten et al., 1994a). Strains were grown on squares of Teflon sheet (FEP; 0.25 mm thick; Norton Fluorplast, Raamsdonkveer, The Netherlands). To this end, 30 µl water was placed in the middle of the sheet. The droplet contained 50 000 cells of each mating type, isolated from a culture in exponential phase. Cells were grown under humid conditions. After 40 h, the water was removed with a pipette and the sheet was placed on top of a 200 µl droplet of low-melting-point agarose (40 °C; 0.5–3 %), which had been pipetted on a Petri dish. After solidification of the agarose at 20 °C for 30 min the Teflon sheet was stripped from the gel. Presence of hyphae was scored in the agarose slab and on the Teflon sheet.
Pathogenicity assay.
Overnight cultures (OD600 0.4–2) were centrifuged and cells were taken up in water to a final OD600 of 3. Mating partners were mixed 1 : 1 and 0.5 ml of the mixture was injected in the leaf whorl of 12–30 1-week-old Early Golden Bantam Z. mays plants. Injection was performed 1 cm above the potting soil. Plants were grown at 28 °C with 14 h of light with a minimum of 244 µE m–2 s–1.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). For instance, 34 hydrophobin genes have been identified in the genome of Coprinus cinereus (R. Velagapudi & U. Kües, unpublished data), while Schizophyllum commune contains at least five hydrophobin genes (Wessels et al., 1995; H. J. Deelstra & H. A. B. Wösten, unpublished results). In contrast, hydrophobin genes are absent in the genomes of the yeasts Cryptococcus neoformans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis and Yarrowia lipolytica, and the dimorphic fungus Candida albicans. These organisms have probably lost their hydrophobin genes during evolution. We show here that U. maydis does have hydrophobin genes but that the encoded proteins have been functionally replaced, at least partially, by the repellents. U. maydis thus seems to be in between the filamentous fungi and the yeasts with respect to the role of hydrophobins in the life cycle.
U. maydis contains two hydrophobin genes
Two class I hydrophobin genes were identified in the genome of U. maydis, which encompasses 6801 ORFs (MUMDB; http://mips.gsf.de/genre/proj/ustilago/). Since 99.7 % of the genome has been sequenced, it is likely that these are the only hydrophobin genes in this dimorphic fungus. The two hydrophobin genes were named hum2 (um05010) and hum3 (um04433). The hum2 gene encodes a typical class I hydrophobin of 117 aa with eight conserved cysteine residues (Fig. 1). The first cysteine residue is preceded by a signal sequence for secretion and 13 amino acids that are predicted to form the N-terminal part of the mature protein. The hum3 gene encodes an atypical hydrophobin. The encoded protein consists of a class I hydrophobin domain preceded by a signal sequence and 17 imperfect repeats of 41–46 aa. Fourteen repeats are separated by KEX2 recognition sites (Fig. 1). Two KEX2 processing sites are found in between the last repeat and the first cysteine of the hydrophobin domain. The protein is thus expected to be cleaved in the endoplasmic reticulum into 15 repeats, and a hydrophobin of 117 aa. This hydrophobin shows 53 % identity to Hum2. The repeats, which we have named questellents, have no homology with proteins in the database but their hydropathy pattern shows a remarkable similarity to that of the repellents (Fig. 1B).
Role of repellents and hydrophobins in formation of aerial hyphae
Both rep1 (Wösten et al., 1996) and hum2 were shown to be expressed during aerial hyphae formation when the wild-type strains FB1 and FB2 were crossed on PDA-charcoal medium (Fig. 2). In contrast, expression of hum2 and rep1 was absent and low, respectively, in the non-crossed parental strains. In no case could expression be shown for hum3, implying that this hydrophobin gene has no significant role in aerial hyphae formation. To investigate this, genes hum2 and/or hum3 were deleted in strains FB1 and FB2 either in combination or not with an inactivation of the rep1 gene (see Methods). Southern analysis confirmed deletion of the genes (results not shown). Aerial hyphae formation was almost completely abolished when the repellent gene rep1 was deleted (Wösten et al., 1996; Fig. 3, Fig. 4A). In contrast, formation of aerial hyphae was only partially reduced when compatible hum2 strains were crossed (Fig. 3, Fig. 4A) and was almost unaffected in the hum3 cross. A hum2hum3 cross formed fewer aerial hyphae than the hum2 cross. Inactivation of hum2 and/or hum3 in the rep1 background had little or no effect on aerial hyphae formation. The reduced formation of aerial hyphae in the hum2hum3 cross compared to the hum2 cross is not explained by an up-regulation of hum3 when hum2 is inactivated (data not shown) but seems to be due to a defect in fusion of compatible hum2hum3 partners. This is concluded from the fact that the cross of the hydrophobin double mutant also forms fewer hyphae in the aqueous environment. The reason for this is not yet known.
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hum2 and the hum3 cross were highly hydrophobic (Fig. 4A, B), showing water contact angles of 127±5, 125±4 and 127±4 degrees, respectively. Surface hydrophobicity was slightly reduced in the hum2hum3 cross (water contact angle 117±2 degrees). The colony surfaces were completely wettable when rep1 was inactivated (Fig. 4A, B; Wösten et al., 1996). Since water droplets were immediately absorbed by the colony, water contact angles could not be measured. Deletion of hum2 and/or hum3 had no additional effect in the rep1 background.
Taking these results together, we conclude that repellents have to a great extent replaced hydrophobins in formation of aerial hyphae in U. maydis. This contrasts with the suggestion that repellents would function by anchoring hydrophobins to the cell wall (Wösten et al., 1996). The fact that we have been unable to identify hydrophobins in cell wall extracts (R. Bohlmann & H. A. B. Wösten, unpublished data) supports the conclusion that hydrophobins are not the main structural proteins that mediate aerial hyphae formation.
Role of repellents and hydrophobins in attachment and pathogenicity
Mating partners of the wild-type or those of rep1, hum2, hum3, rep1hum2, rep1hum3, hum2hum3 and rep1hum2hum3 were injected into 7-day-old maize plants. In all cases 90–100 % of the plants showed disease symptoms after 1 week (anthocyanin formation, chlorosis and tumour formation), demonstrating that repellents and hydrophobins are not involved in pathogenicity. In contrast, deleting the class I hydrophobin gene MPG1 in Magnaporthe grisea did affect pathogenicity (Talbot et al., 1996). This phenotype was explained by the inability of the M. grisea mutant strain to attach to the hydrophobic surface of the plant. Hydrophobin-mediated attachment to hydrophobic surfaces was also observed in Schizophylllum commune (Wösten et al., 1994a). Attachment of U. maydis hyphae to a hydrophobic surface was studied by crossing compatible strains in a water droplet placed on a sheet of Teflon. Most hyphae were formed at the periphery of the water droplet and grew onto the dry hydrophobic Teflon. Outgrowth onto the dry Teflon surface was not affected in the hum2, hum3 and hum2hum3 crosses (data not shown) but was reduced in the rep1 cross (Wösten et al., 1996). No apparent further reduction of escape of hyphae was observed in rep1 strains in which either or both hydrophobins had been deleted. Hyphae of the wild-type cross that had escaped the aqueous environment and had grown onto the dry Teflon surface could not be removed by extensive washing with water (data not shown) or by stripping with 3 % agarose (Fig. 5). Similar results were obtained with the hum2, the hum3 and the hum2hum3 strains (data not shown). In contrast, 50 % of the hyphae of the rep1 cross were removed upon stripping with 3 % agarose (Fig. 5). Deleting either or both hydrophobin genes in the rep1 background did not further reduce attachment.
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rep1 cross is much less dramatic than that observed after inactivation of the SC3 gene of Schizophyllum commune. In this case, wild-type hyphae remained attached after stripping with 3 % agarose, whereas SC3 hyphae already detached at 1.5 % agarose. Apparently, other proteins are involved in attachment of U. maydis. The hydrophilic nature of the fungal cell wall and the hydrophobic surface of the host suggests the involvement of an amphipathic molecule. Interestingly, the U. maydis genome contains two ORFs (um05708 and um06112) with a repeat structure like rep1 and hum3. Both encode putative secreted proteins with 13 and 11 repeats, respectively, that are 21 aa in length. Most of these repeats end with KEX2 recognition sites. Future research should establish whether one of these genes mediates attachment.
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REFERENCES |
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Banuett, F. & Herskowitz, I. (1988). Ustilago maydis, smut of maize. In Genetics of Plant Pathogenic Fungi: Advances in Plant Pathology, pp. 427–455. Edited by G. S. Sidhu. London: Academic Press.
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Received 30 March 2006;
revised 3 August 2006;
accepted 20 September 2006.
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