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

• Published in: Environmental microbiology Vol. 23; no. 9; pp. 5147 - 5163
• Main Authors: Zhang, Can, Cai, Meng, Chen, Shanshan, Zhang, Fan, Cui, Tongshan, Xue, Zhaolin, Wang, Weizhen, Zhang, Borui, Liu, Xili
• Format: Journal Article
• Language: English
• Published: Hoboken, USA John Wiley & Sons, Inc 01-09-2021; Wiley Subscription Services, Inc
• Subjects: Asparagine; Cysts; Deglycosylation; Developmental stages; Germination; Glycan; Glycolysis; Glycoproteins; Glycosylation; Growth; Heat shock; Heat shock proteins; Hsp70 protein; Metabolic pathways; Metabolism; Mutagenesis; Mutation; Mycelia; Oligosaccharides; Oospores; Pathogenicity; Pathogens; Proteins; Site-directed mutagenesis
• ISSN: 1462-2912; 1462-2920
• DOI: 10.1111/1462-2920.15468

Summary
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.