Functional Analysis Of The Secretory Protein SsCFEM1 In Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum?Lib.?de Bary is a broad host range necrotrophic plant pathogen and the sclerotinia rot causes lots of loss in the worldwide scale.In the process of infecting the host,S.sclerotiorum can form a special structure—comprounded appressorium to invade the host and establish a parasitic relationship.Meanwhile,S.sclerotiorum can be able to synthesize and screcte pathogenic factors,for example oxalic acid and cell wall degrading enzymes,in order to resist the host defense response.CFEM?common in several fungal extracellular membrane proteins?domain is unique in fungi and contains eight conserved cysteine residues.CFEM domain-containing proteins have been detected in the transcriptome of many fungi,especially in the secretome of broad-host-range necrotrophs S.sclerotiorum.The CFEM domain may function as a cell surface receptor or an adhesion molecule in host–pathogen interaction,and play an important role in the development of infection structures and pathogenicity.However,the function of CFEM domain-containing proteins in S.sclerotiorum has not been reported.In the interaction between S.sclerotiorum and soybean,the transcript level of the Sscfem1 gene increased highly at 24 hours post inoculation?hpi?and then maintained at a high level expression during late stages?24⁴8 hpi?,indicating that SsCFEM1may be involved in infection of S.sclerotiorum.In order to futher investigate the role of SsCFEM1 in S.Sclerotiorum,the knock-out mutant and complement mutant were obtained by PEG-CaCl₂ mediated protoplast transformation.Further research found that:?1?SsCFEM1 is localized on the cell membrane;?2?Sscfem1 gene knockout mutants showed no significant changes on mycelial growth,colony morphology sclerotial development and oxalic acid secretion compared with the wild type and complement mutant.?3?Sscfem1 gene knock-out mutants can form infectin cushion,but the number of it and virulence were significantly reduced;?4?the addition of exogenous cAMP suppressed the defects in the formation of infectin cushion.The results of this study indicated that the SsCFEM1 protein may be localized on the cell membrane,and be involved in the development of infection cushion and pathogenic process.The Sscfem1 gene may be located upstream of the cAMP signaling pathway.This study preliminarily analyzed the function of SsCFEM1,and laid the foundation for further research on the function of CFEM protein in S.sclerotiorum.