Primers used in this study.

Protein glycosylation, a co- and post-translational modification that enhances the functional diversity of the proteome, contributes to various molecular and cellular functions by transferring different polysaccharides onto proteins. During the last decade, the role of glycosylation in plant pathogenic fungi has received significant attention, and glycoproteins are expected to play essential roles in various biological processes including pathogenicity. However, the comprehensive functional genetic analyses for protein glycosylation pathways and glycan structures of phytopathogenic fungi are still largely unknown. Here, we investigated the role of protein glycosylation in Fusarium graminearum by identifying 65 putative genes involved in protein glycosylation and characterizing their functions. Through cell wall component profiling and HPLC analysis, we characterized the overall N- and O-glycan structures in F. graminearum and found that deletion of ALG3 and ALG12 led to truncated core N-glycan structures. Quantitative proteomics analysis revealed that the truncated core N-glycans, generated by the loss of two key enzymes in the initial core N-glycosylation pathway, Alg3 and Alg12, affected a wide range of glycoproteins—including transcription factors, phosphatases, kinases, peroxidases, and other proteins involved in various biological processes—ultimately impacting the virulence of F. graminearum. This study elucidates the complex roles of glycosylation, highlighting the connections among genes involved in the protein glycosylation pathway, glycans, and glycoproteins in regulating the general biology and pathogenicity of F. graminearum. It also would be the fungal glycobiology study initiative.