The genomes of filamentous basidiomycetes and ascomycetes generally contain multiple hydrophobin genes . In contrast, hydrophobin genes have not been found in yeasts, for example Cryptococcus neoformans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Candida albicans. Despite their important role, hydrophobins are not the only proteins that confer hydrophobic properties to fungal cell walls. The basidiomycete Ustilago maydis encodes a single hydrophobin, Hum2, and a much larger protein called Rep1. While Hum2 plays only a minor role, the peptides released from Rep1 during secretion are mainly responsible for conferring surface hydrophobicity to aerial hyphae in this fungus [23, 24].
Our search in the annotated genome sequences of B. cinerea strains B05.10 and T4 has revealed the presence of three unambiguous hydrophobins, and a total of six hydrophobin-like proteins, according to the criteria defined in the results. For all except one of these genes, homologues in the closely related Sclerotinia sclerotiorum have been identified. In contrast, homologues in other fungi were only found for the three hydrophobins and for the hydrophobin-like protein BC1G_02483. BC1G_02483 was unusual because its size (234 amino acids), the dense spacing of the 8 consensus cysteines, and the presence of 4 additional N-terminal cysteines. The three hydrophobins share typical properties of class I (Bhp1) and class II (Bhp2, Bhp3) proteins. Expression of bhp1, bhp2 and bhp3 was found to be low in conidia and mycelium. This was confirmed by a qRT-PCR analysis that showed generally low expression levels of the three hydrophobin genes and the hydrophobin-like genes in conidia. However, Bhp1 was found to be strongly upregulated in fruiting bodies. This is supported by EST data from a cDNA library from apothecia of B. cinerea. Among 3189 ESTs, 15 (0.5%) were found to represent Bhp1 mRNA, while no ESTs of other hydrophobin sequences were identified in the apothecial library (J. Amselem and M.-H. Lebrun, personal communication). Our RT-PCR data did not provide evidence that deletion of the hydrophobin genes significantly changes the expression level of any other hydrophobin (-like) genes analysed in this study (Figure 2A; additional file 3 : Figure S2).
Several of the hydrophobin (-like) protein encoding genes showed their highest expression levels either in sclerotia (bhp2, BC1G_12747) or in fruiting bodies (bhp1, bhl1). While we did not find any effects of the Δbhp2 mutants on sclerotia formation, the role of BC1G_12747 for sclerotia remains to be determined. Since we have not yet been able to perform crosses with B. cinerea in our laboratory, the role of Bhp1 and Bhl1 in fruiting body development and function also remains to be clarified. The strong upregulation of bhp1 and the apparently exclusive expression of bhl1 in fruiting bodies suggest that these genes might play a role during sexual development.
Using three different resistance markers for selection, mutants that lacked one, two, and all three hydrophobin genes bhp1, bhp2 and bhp3 were generated. To our knowledge, this is the first triple knock-out mutant described for B. cinerea. It was difficult to isolate because phleomycin is less suited for transformant selection compared to the commonly used hygromycin and nourseothricin, because of the growth of many false transformants. In addition to the hydrophobins, the hydrophobin-like gene bhl1 was knocked out. The resulting mutants were analysed for a variety of parameters of growth, differentiation and plant infection. In no case, significant differences between the phenotypes of wild type and mutant strains were observed. Specifically, the mutants showed wild type-like surface hydrophobicity of conidia and hyphae, and normal conidial surface structures when viewed by scanning electron microscopy. In agreement with a previous study , there is no evidence for the presence of a rodlet-like surface layer on B. cinerea conidia. This finding is in contrast to a variety of other fungi which have hydrophobin-coated cell walls surrounding conidia, germ tubes or aerial hyphae . Interestingly, hydrophobin layers have been recently found to protect conidia from immune recognition . While airborne conidia of Botrytis are usually less prevalent compared to the major genera Cladosporium and Alternaria, they have significant allergenic potential . It is possible that this might be due to the absence of hydrophobin layers in B. cinerea conidia.
Our data indicate that B. cinerea hydrophobins do not play a major role in the hydrophobic coating of spores and hyphal wall, and thus are not important for attachment to hydrophobic surfaces or formation of aerial hyphae. Although we cannot completely exclude that any of the other five hydrophobin-like proteins listed in additional file 1 : Table S1 are relevant in this respect, they are more hydrophilic than Bhp1, Bhp2, Bhp3 and Bhl1 and therefore not very likely to represent hydrophobins. As mentioned before, we do not exclude the possibility that Bhp1 or Bhl1 are involved in sexual development. Hydrophobins are known to be important for the formation of fruiting bodies in basidiomycetous mushrooms such as Agaricus bisporus and Schizophyllum commune . In the chestnut blight fungus Cryphonectria parasitica, the class II hydrophobin cryparin has been shown to cover the walls of fruiting bodies and to be required for normal fruiting body development .
Because several hydrophobins are encoded in the genomes of filamentous fungi, it is difficult to fully assess their roles and to exclude complimentary functions. In the tomato pathogen Cladosporium fulvum, six hydrophobins have been identified. Using single mutations, one of them (Hcf1) was found to be required for spore surface hydrophobicity, another one (Hcf6) seems to be involved in adhesion of germinating spores to glass surfaces . An attempt to assess the function of all hydrophobins simultaneously by multiple RNAi silencing failed to result in complete knock-down of the genes . In Fusarium verticillioides, five hydrophobin genes (hyd1 - hyd5) have been identified up to now in the genome. Phenotypical analysis of single mutants in these genes and of a hyd1/hyd2 double mutant revealed that hyd1 and hyd2 are required for normal microconidia formation, but did not provide evidence for a role of these hydrophobins in growth, infection behaviour, and mycelium hydrophobicity . This indicates that in some fungi, including B. cinerea and F. verticillioides, hydrophobins play only a minor - if any - role in generating cell wall surface hydrophobicity. However, they might serve other, as yet unknown functions.
By far not all fungal spores contain superficial rodlet layers. For example, they are missing in the urediospores of rust fungi , and conidia of several powdery mildews . Rust urediospores have been shown to be covered with a layer of lipids that can be extracted with organic solvents, leading to a significantly decreased hydrophobicity, and increased attachment to hydrophilic surfaces [32, 33]. Surface bound lipids, containing hydrocarbon and fatty acid constituents, have been described for spores of several but not all fungal species analysed. The lack of visible effects of hexane treatment on the surface structure of B. cinerea conidia indicates that simple lipids are not a major surface component of these spores. Alternatively, proteins other than hydrophobins could play a role in conferring surface hydrophobicity. In Stagonospora nodorum, preformed surface glycoproteins have been proposed to play a role in the attachment of conidia to hydrophobic surfaces . In the yeasts S. cerevisiae and C. glabrata, cell wall surface proteins called flocculins and adhesins, are involved in adhesion to various surfaces and in biofilm formation; their expression has also been correlated with an increased hydrophobicity of the cell surfaces [35, 36]. Adhesin-like proteins are also encoded in the genomes of filamentous ascomycetes; however, their function remains to be analysed .