The consensus N glyco ‐X‐S /T motif and a previously unknown N glyco ‐N ‐linked glycosylation are necessary for growth and pathogenicity of Phytophthora

Summary Asparagine (Asn, N )‐linked glycosylation within N glyco ‐X‐S/T; X ≠ P motif is a ubiquitously distributed post‐translational modification that participates in diverse cellular processes. In this work, N ‐glycosylation inhibitor was shown to prevent Phytophthora sojae growth, suggesting that N ‐glycosylation is necessary for oomycete development. We conducted a glycoproteomic analysis of P. sojae to identify and map N ‐glycosylated proteins and to quantify differentially expressed glycoproteins associated with mycelia, asexual cyst, and sexual oospore developmental stages. A total of 355 N ‐glycosylated proteins was found, containing 496 glycosites, potentially involved in glycan degradation, carbon metabolism, glycolysis, or other metabolic pathways. Through PNGase F deglycosylation assays and site‐directed mutagenesis of a GPI transamidase protein (GPI16) upregulated in cysts and a heat shock protein 70 (HSP70) upregulated in oospores, we demonstrated that both proteins were N ‐glycosylated and that the N glyco ‐N motif is a target site for asparagine – oligosaccharide linkage. Glycosite mutations of Asn 94 N glyco ‐X‐S/T in the GPI16 led to impaired cyst germination and pathogenicity, while mutation of the previously unknown Asn 270 N glyco ‐N motif in HSP70 led to decreased oospore production. In addition to providing a map of the oomycete N ‐glycoproteome, this work confirms that P. sojae has evolved multiple N ‐glycosylation motifs essential for growth.