Lineage-specific gene family innovations underpin the pathogenicity of woody-plant pathogens in Botryosphaeriaceae

Botryosphaeriaceae is an important fungal family that colonizes a wide range of woody hosts, including economically important crops, and causes serious diseases worldwide. This family has undergone substantial genomic innovations during its evolutionary history. However, the role of gene family innovations, particularly gene contractions, in shaping pathogenicity within Botryosphaeriaceae remains unclear. To address this, we generated five high-quality genomes from three species representing three genera of Botryosphaeriaceae, and combined them with four previously published genomes. These nine genomes were analyzed in a comparative genomic framework together with 47 publicly available fungal genomes. Multiple rounds of gene family expansions were identified in Botryosphaeriaceae, including ancient expansions that occurred prior to the divergence of genera and genus-specific expansions that arose afterward. These expansions were associated with genes involved in host infection and virulence. In contrast, gene family contractions were less frequent but were also associated with functions potentially related to pathogenicity when they occurred after the divergence of Botryosphaeriaceae into distinct genera. Transcriptomic analysis of Lasiodiplodia theobromae further supported the functional significance of lineage-specific gene family expansions and contractions in virulence evolution. Silencing a lineage-specific expanded gene encoding a plant cell wall-degrading enzymes from the GH11 family significantly reduced the virulence of L. theobromae, confirming its role in pathogenicity. Together, these findings reveal that lineage-specific gene family innovations, including both expansions and contractions, have driven virulence diversification and adaptation among Botryosphaeriaceae species during host-pathogen interactions.