The heat shock factor BdHsf1 is important for stress adaptation, asexual reproduction, and intracellular peroxide homeostasis in Botryosphaeria dothidea

Abstract Botryosphaeria dothidea , the primary causal agent of trunk cankers in Chinese hickory ( Carya cathayensis ), threatens a multimillion-dollar industry in China. Understanding the fundamental biological mechanisms underlying B. dothidea ’s stress adaption and pathogenesis is critical for developing novel control strategies against canker disease. Heat shock factors (Hsfs) are evolutionarily conserved regulators of stress responses and pathogen virulence, yet their roles in woody plant pathogenic fungi remain largely unexplored. This study investigated the functional contributions of Hsfs in B. dothidea , focusing on their involvement in stress adaptation, reproduction, and pathogenesis. A total of two Hsf orthologs, BdHsf1 and BdHsf2, were identified in B. dothidea , and BdHsf2 was found to be indispensable for fungal viability. Deletion of BdHSF1 accelerated hyphal growth, abolished asexual fruiting body formation, and increased sensitivity to oxidative stress, extreme pH, osmotic pressure, cell wall disruptors, and quinone outside inhibitors (QoIs) and dicarboximide fungicides, but not to thermal stress or benzimidazole and DMI fungicides. Unlike in other fungi, BdHsf1 is dispensable for thermal stress response and virulence though BdHsf1 was upregulated during the infection. Additionally, BdHsf1 critically governs oxidative defense through nuclear translocation and domain-specific mechanisms. Further transcriptomic analysis underscores the broad influence of BdHsf1 on antioxidant, cellular metabolism, signaling, and chaperone pathways, which correlates well with phenotypic deficiency observed in BdHSF1 deletion mutant. Collectively, these results provide the first comprehensive functional characterization of Hsfs in B. dothidea , which advances our understanding of Hsf evolution and function in phytopathogens and provides potential targets for managing canker diseases.