Dissection of Sclerotinia sclerotiorum biology using genetic and cell biology approaches

Fungal pathogens constitute a major threat to agriculture and human health. Plant pathogenic fungi cause widespread and often devastating diseases across diverse crops, yet many remain insufficiently studied. Recent advances in molecular and genetic technologies provide powerful tools to dissect their developmental and pathogenic mechanisms, supporting development of improved disease-management strategies. In my PhD research, I aimed to identify molecular components essential for the development and virulence of Sclerotinia sclerotiorum, a destructive soilborne fungus responsible for severe crop yield and quality losses worldwide. Using both forward and reverse genetic approaches, I identified four Ras guanine nucleotide exchange factors (RasGEFs) in S. sclerotiorum and demonstrated that they regulate fungal growth, sclerotia development, compound appressoria functionality and virulence. In parallel, four homologous RasGEFs were identified in Botrytis cinerea, an airborne plant pathogen closely related to S. sclerotiorum. These RasGEFs similarly influenced growth, conidiation， sclerotia formation, and virulence, underscoring their conserved importance. Given the promise of host-induced gene silencing (HIGS) for engineering plants that suppress essential pathogen genes, I further assessed whether RasGEFs could serve as effective HIGS targets. Tobacco leaves expressing RNAi constructs against SsRasGEFa and SsRasGEFc showed reduced development of lesions following S. sclerotiorum inoculation, indicating that silencing these genes attenuates fungal virulence. Additionally, through reverse genetic analysis, three hydrophobins (HPs) were identified in S. sclerotiorum, representing two classes, which are involved in surface hydrophobicity, cell wall integrity and resistance to environmental stresses. Analyses of single- and double- deletion mutants revealed that the HPs influence fungal development and virulence to varying degrees, highlighting the important roles these proteins play during the life cycle of this soilborne pathogen. Furthermore, during forward genetic studies, we uncovered an unexpected phenomenon of chromosomal distribution. In both S. sclerotiorum and B. cinerea, haploid chromosomes are partitioned across multiple nuclei, challenging the “one nucleus, one genome” paradigm. A comparable pattern in the non-pathogenic ascomycete Neurospora crassa suggests that this unconventional phenomenon may be more widespread than previously appreciated. In summary, my PhD research advances our understanding of S. sclerotiorum biology and provides new insights to support innovative, effective approaches for managing fungal diseases in crops.