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Lichen-forming fungi obtain carbohydrates and in some cases fixed nitrogen through symbiotic association with photoautotrophic partners (i.e. cyanobacteria or green algae or both). Lichen-forming fungi have polyphyletic origins (Hawksworth & Hill, 1984; Gargas et al., 1995; Lutzoni et al., 2001) and occur primarily in the Ascomycotina of which 13 250 species (or 46%) are lichenized (Hawksworth, 1988). Only about 50 species (0.3%) of more than 16 000 basidiomycetes are lichenized. In contrast to most ascolichens, basidiolichens form fruiting bodies that are similar to those made by nonlichen-forming basidiomycetes.
The lichen-forming basidiomycete Dictyonema glabratum (Sprengel) D. Hawksw. (syn. Cora pavonia (Web.) E. Fries) in symbiosis with the cyanobacteria Scytonema sp. is the only known example of a basidiolichen in which the fruitbody itself is lichenized. It develops a large and internally stratified basidiocarp that grows from a pseudomeristematic marginal curl (Fig. 1a). The fruitbody produces a hymenium that eventually covers the lower surface of the older regions (Fig. 1b). Photosynthetic rates and annual carbon gain in Dictyonema glabratum are higher than in other lichens, indicating that this symbiotic association is very efficient (Lange et al., 1994). The fungal uptake of photosynthates takes place primarily across a highly developed fungal haustorial–cyanobacterial plasma membrane interface (Roskin, 1970; Slocum & Floyd, 1977; Oberwinkler, 2001). Typical of all lichens, there are no specialized structures in the fruitbody for water allocation and gas exchange, which are both essential for photosynthesis, nitrogen fixation and respiration. Fruitbodies of Dictyonema glabratum, which grows in the tropics, undergo daily desiccation and hydration cycles in which water content (% dry weight) may fluctuate from less than 10% to 1000% (Lange et al., 1994). This presents a major challenge in maintenance of the symbiotic phenotype.
Figure 1. Morphology of the lichenized fruitbody of Dictyonema glabratum. (a) Dorsal view: the fruitbody grows from the curled edge. (b) Ventral view: older regions of the fruitbody are covered by hymenia.
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Hydrophobins are secreted fungal proteins that function in several capacities, including fungal water relations. Hydrophobins were first characterized in the nonlichenized basidiomycete Schizophyllum commune (Schuren & Wessels, 1990; Wessels et al., 1991) and have since been isolated from numerous fungi. Class I hydrophobins have the inherent ability to self-assemble at interfaces into amphipathic films that have a rodlet pattern on the hydrophobic side (Wösten et al., 1993; Wösten et al., 1994a; Wessels, 1997). Once assembled, hydrophobin membranes are highly stable and insoluble in boiling sodium dodecyl sulphate (SDS) solutions but can be rendered into monomers with trifluoroacetic acid (TFA) (Wessels et al., 1991). Hydrophobin layers function in various aspects of fungal development such as differentiation of aerial hyphae, asexual sporulation and fruitbody formation (Wessels, 1994, 1997). Hydrophobin layers are also involved in mediating attachment to surfaces (St Leger et al. 1992; Wösten et al., 1994b; Kershaw & Talbot, 1998) and in early ectomycorrhizal development (Martin et al., 1999a). Hydrophobins are assumed to be involved in creating hydrophobic wall layers in lichenized fungi (Honegger, 1997). Hydrophobins have been characterized from Dictyonema glabratum (Trembley et al., 2002) as well as from two lichen-forming ascomycetes (Scherrer et al., 2000).
The three hydrophobins DGH1, DGH2 and DGH3 have been cloned from the fruitbodies of Dictyonema glabratum (Trembley et al., 2002). The proteins are small (93–108 amino acids) and are 54–66% identical. They each contain eight cysteines that are present in the conserved pattern typical of hydrophobins and the hydropathy profiles of the polypeptides are consistent with class I hydrophobins (Wessels, 1997). When aqueous solutions of the hydrophobin extract are air-dried, the proteins self-assemble into a rodlet-patterned layer. A similar rodlet mosaic is found on the surface of hyphae lining interhyphal gas-filled spaces in the photobiont layer of the fruitbody (Trembley et al., 2002) and is thought to be formed by hydrophobins.
The aims of the present study were to investigate the expression of hydrophobin genes DGH1, DGH2 and DGH3 in the fruitbody of Dictyonema glabratum and to immunolocalize DGH1, the most abundant of the hydrophobins, with antibodies raised against a recombinant DGH1 protein. The possible involvement of hydrophobin layers in the maintenance of gas-filled interhyphal spaces, in creating an apoplastic continuum into which free water is forced and in defining strata within the fruitbody is discussed.
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The present study describes the expression patterns of the hydrophobin genes DGH1, DGH2 and DGH3 in the lichenized fruitbody of Dictyonema glabratum. DGH1 was also expressed in E. coli as a fusion protein for antibody production. The anti-6xHisDGH1 antiserum was used to immunolocalize the DGH1 protein in Dictyonema glabratum.
DGH1, DGH2 and DGH3 hydrophobin genes are highly expressed throughout all differently aged regions of the Dictyonema glabratum fruitbody. In a similar manner, the hydrophobin gene XPH1 is highly expressed in the medullary layer in both young and old parts of the lichen-forming ascomycete Xanthoria parietina (Scherrer, 2000; Scherrer et al., 2002). Hydrophobins in lichens thus seem to be in demand in the initial and long-term development, differentiation and/or functioning of the symbiotic phenotype. All of the hydrophobin genes in Dictyonema glabratum have similar patterns of expression in the curl and younger regions of the basidiocarp but show differential expression in the older, stratified parts. It is possible that in young, developing parts the proteins work together synergistically to create hydrophobic surface layers, as proposed elsewhere (Stringer & Timberlake, 1995; Ásgeirsdóttir et al., 1998) or are functionally redundant.
In E. coli, DGH1 was expressed as a recombinant protein. The fusion protein failed to self-assemble into a rodlet mosaic. The reasons for this might be that the presence of the tag or the inability of bacteria to carry out the appropriate post-translational protein modifications (Stüber et al., 1990) interfered with polymerization. Self-assembly of a rodlet layer has not been reported for the two other histidine-tagged hydrophobins that were expressed in E. coli (Peñas et al., 1998; Tagu et al., 2001). It cannot be excluded that components such as polysaccharides (Martin et al., 1999b) may have an impact on the formation of rodlet layers. When the native DGH1 was isolated from the hydrophobin extract it also failed to form rodlets: thus, other components in the extract are also important.
The anti-6xHisDGH1 antiserum was used to immunolocalize DGH1 in Dictyonema glabratum. In the curl region, the detection of DGH1 is insignificant and thus lower than the levels expected from the in situ hybridization data. Reasons for this might be that fungal cell wall properties in the curl hinder accessibility, or protein accumulation is too low to allow detection. In the younger and older regions of Dictyonema glabratum, localization patterns of DGH1 are in agreement with the in situ hybridization results. DGH1 accumulates in fungal cell walls, which is in agreement with the presence of a signal peptide for secretion in the DGH1 gene (Trembley et al., 2002). DGH1 is concentrated in the electron-dense, outer cell wall layer of aerial hyphae and hyphae at hyphal–gas-filled interfaces in the photobiont layer and the lower stratum. In a similar manner, the hydrophobins SC4 and ABH1, produced in fruitbodies of the nonlichenized basidiomycetes S. commune and Agaricus bisporus, respectively, are localized at the surface of interior, gas-filled channels (Lugones et al., 1999). In Dictyonema glabratum, DGH1 is additionally localized at hyphal–hyphal interfaces in between two strata. Similarly, localization of the HydPt-1 hydrophobin at hyphal–hyphal interfaces was observed in early stages of the ectomycorrhizal Pisolithus tinctorius–Eucalyptus globulus interaction (Tagu et al., 2001). However, in contrast to DGH1, labelling was confined primarily to an electron-transparent cell wall layer. The DGH1 protein is localized in the photobiont layer of Dictyonema glabratum, where rodlet layers line air spaces. Hydrophobin extracts purified from Dictyonema glabratum self-assemble in vitro into similar rodlet layers. Similar to DGH1, SC4 and ABH1 are localized in wall surfaces where rodlet layers, which resemble the ones formed in vitro by extracts of the corresponding hydrophobins, are observed (Lugones et al., 1999). The rodlet mosaic in Dictyonema glabratum may be formed primarily by DGH1, but DGH2 may also be present.
The spatial distribution patterns of water and hydrophobins in the stratified region of Dictyonema glabratum were diagrammatically superimposed in order to elucidate the possible function(s) of hydrophobins (Fig. 5). The boundary layer, which is sealed by DGH3, is the switching point in water translocation within the fruitbody; free interhyphal water is forced to move into the interior underneath hydrophobic wall surfaces. A similar situation may occur on the lower side of the basidiocarp where DGH3 seals off the hymenial attachment strands. The photobiont layer is lined by DGH1 and DGH2 on the lower side and by the DGH3-lined boundary layer on the upper side. Thus, the apoplastic continuum of the photobiont layer is three-dimensionally sealed by hydrophobins. Hydrophobins in this capacity probably facilitate the passive, apoplastic translocation of water and nutrients from the hydrophilic periphery into and within the photobiont layer. Hydrophobin rodlet layers, that enclose the photobiont layer, probably function at the same time to keep interhyphal spaces water free during saturation, such that gas exchange, important for photosynthesis, nitrogen-fixation and respiration, can take place. DGH1 may also be involved in hyphal–hyphal attachment and/or fruitbody stratification.
In the fruitbody of S. commune, SC4 functions to keep internal channels gas filled. This was proven with SC4-deficient mutants whose air channels lack rodlets and fill up with water when the fruitbody is soaked (van Wetter et al., 2000). We speculate that the hydrophobins in Dictyonema glabratum fulfil similar roles. Ultimate proof of the functional role of hydrophobins in Dictyonema glabratum would require knock-out mutants. However, as is the case for all lichen-forming fungi, it is not possible to resynthesize routinely the symbiotic phenotype under laboratory conditions from aposymbiotic (separated) sterile cultures of Dictyonema glabratum and its cyanobacterial photobiont, which makes the testing of mutagenized, symbiosis-related genes impossible at this time.
Hydrophobins seem to be widespread or even ubiquitous in fungi. Assuming that polymerised hydrophobin layers function in Dictyonema glabratum like they do in fruitbodies of non-lichenized basidomycetes suggests interesting evolutionary aspects regarding its transition to lichanization. Is it possible that potential photoautotrophic partners simply colonized fruitbody primordia and developed in the sheltered environment within a fruitbody that, utilizing hydrophobin layers, had already been constructed in such a way that the challenges of water allocation and gas exchange had been resolved? Similarities in ultrastructure and the ways in which hydrophobins function in lichenized and nonlichenized fungi indicate that this may be true. Conversely, development of fungal structures such as haustoria and differentiation of unique strata suggest that morphological innovations in Dictyonema glabratum have taken place, even if the initial cohabitation may have required no modification at all.