AbstractFungal pathogenesis depends on accurate secretion and location of virulence factors which drive host colonization. Protein glycosylation is a common posttranslational modification of cell wall components and other secreted factors, typically required for correct protein localization, secretion and function. Thus, the absence of glycosylation is associated with animal and plant pathogen avirulence. While the relevance of protein glycosylation for pathogenesis has been well established, the main glycoproteins responsible for the loss of virulence observed in glycosylation-defective fungi have not been identified. Here, we devise a proteomics approach to identify such proteins and use it to demonstrate a role for the highly conserved protein disulfide isomerase Pdi1 in virulence. We show that efficient Pdi1 N-glycosylation, which promotes folding into the correct protein conformation, is required for full pathogenic development of the corn smut fungusUstilago maydis. Remarkably, the observed virulence defects are reminiscent of those seen in glycosylation-defective cells suggesting that the N-glycosylation of Pdi1 is necessary for the full secretion of virulence factors. All these observations, together with the fact that Pdi1 protein and RNA expression levels rise upon virulence program induction, suggest that Pdi1 glycosylation is a crucial event for pathogenic development inU. maydis. Our results provide new insights into the role of glycosylation in fungal pathogenesis.Author summaryFungal pathogens require virulence factors to be properly secreted and localized to guarantee complete infection. In common with many proteins, virulence factors must be post-translationally modified by glycosylation for normal localization, secretion and function. This is especially important for virulence factors, which are mainly comprised of cell wall and secreted proteins. Aberrant glycosylation leads to a loss of virulence in both animal and plant pathogenic fungi. We have previously demonstrated that glycosylation is important for virulence of the corn smut fungus,Ustilago maydis. However, the glycoproteins involved and their specific roles in the infection process have not yet been reported. Here, we describe a proteomic assay designed to identify glycoproteins involved in plant infection. Using this method, we define the role of Pdi1 protein disulfide isomerase in virulence. Interestingly, abolishing Pdi1 N-glycosylation mimics Δpdi1defects observed during infection, suggesting that Pdi1 N-glycosylation is required for the secretion of virulence factors. We hypothesize that Pdi1 N-glycosylation is crucial for maintaining proper effector protein folding during the infection process, especially in the harsh conditions found inside the maize plant.