Abstract
The CFEM (Common in Fungal Extracellular Membrane) domain defines a family of cysteine-rich proteins unique to fungi, playing pivotal roles in host-pathogen interactions. However, the repertoire and functions of CFEM proteins in the broad-host-range necrotrophic pathogen Sclerotinia sclerotiorum remain largely unexplored. Through genome-wide bioinformatic analysis, we identified 13 CFEM-containing proteins (SsCFEM1-13) in S. sclerotiorum. Characterization revealed substantial diversity in their physicochemical properties, domain architecture, and predicted subcellular localization. Ten proteins possess a secretion signal, with six predicted to be GPI-anchored and three classified as high-confidence effectors. Members lacking transmembrane domains were predicted to adopt the conserved CFEM "helical-basket" fold. Phylogenetic analysis grouped SsCFEMs into two distinct clades and indicated a complex evolutionary history involving both conserved ancestry and lineage-specific expansion. Transcriptomic profiling showed that most genes were upregulated during early infection of various host plants, with SsCFEM8 exhibiting particularly strong and consistent induction. Crucially, transient expression assays in Nicotiana benthamiana revealed that several SsCFEM proteins, notably SsCFEM4 and SsCFEM9, function as cell death suppressors, validating their predicted effector roles and identifying key virulence candidates. This study provides the first comprehensive catalog and functional prediction of the CFEM protein family in S. sclerotiorum, establishing a foundation for future mechanistic studies on their roles in the pathogenesis of this devastating fungal pathogen.