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

Asparagine (Asn, N) -linked glycosylation within the glycosylation motif (Nglyco-X-S/T; X=P) is a ubiquitously distributed post-translational modification that participates in diverse eukaryotic cellular processes. However, little is known about the characteristic features and roles of N-glycosylation in oomycetes. In this work, it found that 2.5 g/ml tunicamycin (N-glycosylation inhibitor) completely inhibited Phytophthora sojae growth, suggesting that N-glycosylation is necessary for oomycete development. We conducted a glycoproteomic analysis of P. sojae to identify and map all N-glycosylated proteins and to quantify differentially expressed glycoproteins associated with mycelia, asexual cysts, and sexual oospores. A total of 355 N-glycosylated proteins were found, containing 496 glycosites that likely participate in glycan degradation, carbon metabolism, glycolysis, or other central metabolic pathways. To verify the glycoproteomic results and further examine the function of N-glycosylation in P. sojae, two proteins were selected for PNGase F deglycosylation assays and CRISPR/Cas9-mediated site-directed mutagenesis, including a GPI transamidase protein (GPI16) up-regulated in cysts, with the consensus Nglyco-X-S/T motif at Asn 94, and a heat shock protein 70 (HSP70) up-regulated in cysts and oospores with a previously unknown Nglyco-N motif at Asn 270. We demonstrated that the GPI16 and HSP70 are both N-glycosylated proteins, confirming that the Nglyco-N motif is a target site for asparagine - oligosaccharide N-glycosidic linkage. Glycosite mutations of Asn 94 in the GPI16 led to impaired cyst germination and pathogenicity, while HSP70 mutants exhibited decreased cyst germination and oospore production. This work describes an integrated map of oomycete N-glycoproteomes and advances our understanding of N-glycosylation in oomycetes. Moreover, we confirm that the consensus Nglyco-X-S/T and the Nglyco-N -linked glycosites are both essential for the growth of Phytophthora sojae, indicating that there are multiple N-glycosylation motifs in oomycetes.