Reverse Genetic Analyses of Hydrophobins in Sclerotinia sclerotiorum Revealed Their Diverse Roles in Development, Environmental Survival, and Virulence

<i>Sclerotinia sclerotiorum</i> is a notorious soilborne fungal pathogen that causes white mold in a wide range of host plants, leading to globally significant yield loss in many crops. Hydrophobins (HPs) are small, secreted proteins unique to filamentous fungi, with diverse roles in fungal biology. However, their functions in <i>S. sclerotiorum</i> remain poorly understood. Here, we systematically investigated the roles of three HP genes, <i>SsHP1</i>, <i>SsHP2</i>, and <i>SsHP3</i>, through reverse genetic analyses. By analyzing their deletion mutant phenotypes, we demonstrate that class I HP (<i>SsHP1</i>) is specifically required for proper sclerotia development, whereas class II HPs (<i>SsHP2</i> and <i>SsHP3</i>) are essential for compound appressoria functionality. All three HPs contribute to fungal surface hydrophobicity, cell wall integrity, and stress tolerance. Using mycelial fusion, we generated double mutants lacking both class II HPs, which exhibited more severe defects in appressoria development, virulence, cell wall integrity, and stress adaptation, indicating their partially redundant roles. SsHP2 is required for both host penetration and post-penetration virulence, whereas SsHP3 mainly affects host penetration, revealing their overlapping yet distinct contributions to pathogenic development. Although all HP mutants formed normal apothecia and asci, they released significantly fewer ascospores, suggesting that HPs are dispensable for sexual morphogenesis but crucial for the biophysical process of ascospore dispersal. Furthermore, carbohydrate analyses uncovered that these HPs affect cell wall composition, more broadly influencing stress adaptation and virulence. Taken together, our study reveals both conserved and divergent roles of HPs across fungi and highlight their multifaceted contributions to <i>S. sclerotiorum</i> biology, offering new perspectives for disease management.