Amorphous-to-rodlet structural transition governs the interfacial functions of Aspergillus oryzae hydrophobin RolA

Hydrophobins are low-molecular-weight biosurfactant proteins that coat the cell surface of filamentous fungi, making the surface hydrophobic and supporting morphogenesis. On conidia, hydrophobins self-assemble to form rod-shaped multimeric structures known as rodlets. Previously, we reported that hydrophobin RolA from the industrial fungus Aspergillus oryzae first forms an amorphous film at the air-water interface and then undergoes structural rearrangement to form a densely packed rodlet film. This raised the question of whether the amorphous film or the rodlet film is more important for the biological functions of RolA. In this study, to investigate the properties of amorphous films in comparison with those of rodlet films, we created RolA mutants with reduced hydrophobicity in the hydrophobic loop regions by substituting L137, L142, or both with the polar serine residues, thereby suppressing rodlet formation driven by hydrophobic interactions. Rodlet formation was suppressed in the mutants and was abolished in one of them. The amorphous state of RolA in the mutants enabled direct comparison of the properties of amorphous and rodlet film. We found that the rodlet film was more rigid than the amorphous film and had stronger surface activity and a greater capacity to change surface wettability. RolA altered the properties of A. oryzae conidia only when it was in the rodlet state. These findings highlight the functional versatility of RolA and show that its dynamic structural transitions directly modulate its function.