The role of hydrophobins in sexual development of Botrytis cinerea

Hydrophobins are small secreted proteins that play a role in a broad range of developmental processes in filamentous fungi, e.g. in the formation of aerial structures. Hydrophobins allow fungi to escape their aqueous environment and confer hydrophobicity to fungal surfaces. In Botrytis cinerea (teleomorph Botryotinia fuckeliana), one class I and two class II hydrophobin genes have been identified, as well as a number of hydrophobin-like genes. Previous studies showed that hydrophobins are not required for conferring surface hydrophobicity to conidia and aerial hyphae. We investigated the role of hydrophobins in sclerotium and apothecium development. RNA seq analysis of gene expression during different stages of apothecium development revealed high expression of the Bhp1 (class I hydrophobin) gene and of the Bhl1 (hydrophobin-like) gene in certain stages, whereas Bhp2 and Bhp3 (class II hydrophobin) genes were always expressed at very low levels. We characterized different hydrophobin mutants: four single gene knockouts, three double knockouts as well as a triple knockout. Sclerotia of DBhp1/DBhp3 (double knock out) and DBhp1/DBhp2/DBhp3 (triple knock out) mutants showed easily wettable phenotypes. These results indicate that hydrophobins Bhp1 and Bhp3 are important for normal development of sclerotia of B. cinerea. For analyzing apothecial development, a reciprocal crossing scheme was set up. Morphological aberrations were observed in crosses with some hydrophobin mutants. When the DBhp1/DBhp2 (double knock out) and DBhp1/DBhp2/DBhp3 (triple knockout) mutants bearing a MAT1-1 mating type were used as maternal parents (sclerotia), and fertilized with microconidia of a wild type MAT1-2 strain, the resulting apothecia were swollen, dark brown in color and had a blotted surface. Instead of growing upwards, the apothecia in some cases fell down. This aberrant apothecial development was not observed in the reciprocal cross, when the same mutants were used as paternal parent (microconidia). These results indicate that the presence of hydrophobins Bhp1 and Bhp2 in maternal tissue is important for normal development of apothecia of B. cinerea.