Lack of evidence for a role of hydrophobins in conferring surface hydrophobicity to conidia and hyphae of Botrytis cinerea

Background: Hydrophobins are small, cysteine rich, surface active proteins secreted by filamentous fungi, forming hydrophobic layers on the walls of aerial mycelia and spores. Hydrophobin mutants in a variety of fungi have been described to show ‘easily wettable’ phenotypes, indicating that hydrophobins play a general role in conferring surface hydrophobicity to aerial hyphae and spores.Results: In the genome of the grey mould fungus Botrytis cinerea, genes encoding three hydrophobins and six hydrophobin-like proteins were identified. Expression analyses revealed low or no expression of these genes in conidia, while some of them showed increased or specific expression in other stages, such as sclerotia or fruitingbodies. Bhp1 belongs to the class I hydrophobins, whereas Bhp2 and Bhp3 are members of hydrophobin class II. Single, double and triple hydrophobin knock-out mutants were constructed by consecutively deleting bhp1, bhp2 and bhp3. In addition, a mutant in the hydrophobin-like gene bhl1 was generated. The mutants were tested forgermination and growth under different conditions, formation of sclerotia, ability to penetrate and infect host tissue, and for spore and mycelium surface properties. Surprisingly, none of the B. cinerea hydrophobin mutants showed obvious phenotypic defects in any of these characters. Scanning electron microscopy of the hydrophobicconidial surfaces did not reveal evidence for the presence of typical hydrophobin ‘rodlet’ layers.Conclusions: These data provide evidence that in B. cinerea, hydrophobins are not involved in conferring surface hydrophobicity to conidia and aerial hyphae, and challenge their universal role in filamentous fungi. The function of some of these proteins in sclerotia and fruiting bodies remains to be investigated