Utilizing ppGalNAc-T-knockout T. gondii strains, we demonstrate that hierarchical O-GalNAc glycosylation occurs with the initiator ppGalNAc-T2 and filling-in ppGalNAc-T3 glycosylating the mucin-like domain of CST1 and that this hierarchical glycosylation is critical for the ability of CST1 to generate a rigid cyst wall. This evidence concerns the gene CST1 and cyst.