ABSTRACTGlycosylphosphatidylinositol (GPI) anchoring of proteins is a conserved posttranslational modification in eukaryotes. GPI-anchored proteins are widely distributed in fungal plant pathogens, but the specific roles of the GPI-anchored proteins in the pathogenicity ofSclerotinia sclerotiorum, a devastating necrotrophic plant pathogen with a worldwide distribution, remain largely unknown. This research addressesSsGSR1, which encodes anS. sclerotiorumglycine- and serine-rich protein named SsGsr1 with an N-terminal secretory signal and a C-terminal GPI-anchor signal. SsGsr1 is located at the cell wall of hyphae, and deletion ofSsGSR1leads to abnormal cell wall architecture and impaired cell wall integrity of hyphae. The transcription levels ofSsGSR1were maximal in the initial stage of infection, andSsGSR1-deletion strains showed impaired virulence in multiple hosts, indicating thatSsGSR1is critical for the pathogenicity. Interestingly, SsGsr1 targeted the apoplast of host plants to induce cell death that relies on the glycine-rich 11-amino-acid repeats arranged in tandem. The homologs of SsGsr1 inSclerotinia,Botrytis, andMoniliniaspecies contain fewer repeat units and have lost their cell death activity. Moreover, allelic variants ofSsGSR1exist in field isolates ofS. sclerotiorumfrom rapeseed, and one of the variants lacking one repeat unit results in a protein that exhibits loss of function relative to the cell death-inducing activity and the virulence ofS. sclerotiorum. Taken together, our results demonstrate that a variation in tandem repeats provides the functional diversity of GPI-anchored cell wall protein that, inS. sclerotiorumand other necrotrophic pathogens, allows successful colonization of the host plants.IMPORTANCESclerotinia sclerotiorumis an economically important necrotrophic plant pathogen and mainly applies cell wall-degrading enzymes and oxalic acid to kill plant cells before colonization. In this research, we characterized a glycosylphosphatidylinositol (GPI)-anchored cell wall protein named SsGsr1, which is critical for the cell wall architecture and the pathogenicity ofS. sclerotiorum. Additionally, SsGsr1 induces rapid cell death of host plants that is dependent on glycine-rich tandem repeats. Interestingly, the number of repeat units varies among homologs and alleles of SsGsr1, and such a variation creates alterations in the cell death-inducing activity and the role in pathogenicity. This work advances our understanding of the variation of tandem repeats in accelerating the evolution of a GPI-anchored cell wall protein associated with the pathogenicity of necrotrophic fungal pathogens and prepares the way toward a fuller understanding of the interaction betweenS. sclerotiorumand host plants.