In-depth site-specific glycoproteomic analysis reveals ER-resident protein PDI regulating wheat yellow mosaic virus infection

N-glycosylation is crucial in the process of wheat yellow mosaic virus (WYMV) infection, but changes in site-specific N-glycosylation of proteins during WYMV infection have not been well studied. In this study, we employed an intact glycopeptide approach to analyze mock- and WYMV-infected wheat plants. We found that most glycoproteins have N-glycans containing paucimannose or complex/hybrid chains. Notably, the H3N2F1X1 N-glycan was the most prevalent, comprising 40 % of the total glycan abundance. Six glycan types showed an increasing trend of glycosylation in WYMV-infected wheat. Overall, 1202 unique N-glycopeptides corresponding to 53 N-glycans at 562 N-glycosylation sites in 456 N-glycoproteins were identified, and 176 N-glycopeptides from 115 glycoproteins were significantly regulated in WYMV-infected wheat. Bioinformatics analysis of the hyperglycosylated and hypoglycosylated glycoproteins indicated that two N-glycoproteins with significant regulatory differences were specifically related to protein quality control, endoplasmic reticulum stress response, and protein folding. Furthermore, the protein disulfide isomerase TaPDI 1-4 and TaPDI regulate WYMV infection, and their N-glycosylation is involved in the regulatory process. To our knowledge, this is the first study to analyze the differences and roles of protein N-glycosylation in wheat virus infection at the level of intact glycopeptides.