Stress responsive glycosylphosphatidylinositol-anchored protein SsGSP1 contributes to Sclerotinia sclerotiorum virulence.

Fungal cell wall acts as a defense barrier, shielding the cell from varying environmental stresses. Cell wall proteins, such as glycosylphosphatidylinositol (GPI)-anchored proteins, are involved in swift and appropriate responses to minor environmental changes in fungi. However, the roles of these proteins in the pathogenic Sclerotinia sclerotiorum remain largely unexplored. Here, we identified a novel GPI-anchored protein in S. sclerotiorum, SsGSP1, comprising a Kre9_KNH domain. SsGSP1 was upregulated during infection, and the loss-of-function mutants of SsGSP1 exhibited the compromised cell wall integrity and reduced β-glucan content. During inoculation on Arabidopsis thaliana, Nicotiana benthamiana, and Brassica napus, the SsGSP1-deletion strains demonstrated the decreased virulence. The transgenic A. thaliana line carrying the sRNA targeting SsGSP1 enhanced resistance to S. sclerotiorum via Host-Induced Gene Silencing (HIGS). The SsGSP1-deficient strains displayed the heightened sensitivity to various stresses, including osmotic pressure, oxidative stress, and heat shock. The yeast two-hybrid and BiFC assays confirmed that SsGSP1 interacted with the key stress-related proteins catalase SsCat2, heat shock protein Sshsp60, and ABC transporter SsBMR1. Accordingly, transcriptome analysis revealed that the disruption of SsGSP1 downregulated the expression of genes involved in oxidative stress response, heat shock response, and chemical agent resistance. These results collectively delineate the intricate role of GPI-anchored protein SsGSP1 in β-glucan, cell wall integrity, and virulence and may act as a potential surface sensor to elicit signal transduction in response to environmental stresses in S. sclerotiorum.