Asparagine (Asn, N)-linked glycosylation within Nglyco -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 Nglyco -N motif is a target site for asparagine - oligosaccharide linkage. Glycosite mutations of Asn 94 Nglyco -X-S/T in the GPI16 led to impaired cyst germination and pathogenicity, while mutation of the previously unknown Asn 270 Nglyco -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.
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