Neither the hydrophobin triple knock-out mutants nor the wild type conidia were covered with rodlet-shaped structures, and no differences were observed between the strains (Figure 4A-C). When wild type conidia were treated with hexane, only small changes in their surface structures were observed. Similarly, spores washed for several times with water left the conidial surface structures rather intact. In contrast, chloroform treatment

had a drastic effect on the appearance of the conidial surface, leading VX-689 manufacturer to almost complete abrasion of the spinose surface (Figure 4D-G). Figure 4 Scanning electron microscopy of B. cinerea conidia. A: Overview showing the jagged spore surface (scale bar: 1 μm). B, C: Higher magnifications, showing irregular jags of wild type (B) and triple mutant (C) spores. C59 wnt concentration D: After treatment of wild type conidia with chloroform, the jags appeared abraded. E: Treatment of wild type conidia with hexane does not cause obvious changes in surface topography. F, G:

Repeated washing with water caused minor abrasions of the spiny surface of wild type (F) and triple mutant (G) conidia. Scale bar for higher BIBF 1120 manufacturer magnifications in B-G: 250 nm. Discussion The genomes of filamentous basidiomycetes and ascomycetes generally contain multiple hydrophobin genes [2]. 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 acetylcholine 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.