Identification and characterization of glycoproteins as new virulence factors in "Ustilago maydis"

Resumo

Fungal 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 glycoproteins responsible for the loss of virulence observed in glycosylation-defective fungi have not been identified. Hence, we devise a proteomics approach to identify those proteins and use it to demonstrate the role for the highly conserved protein disulfide isomerase Pdi1, endo-β-1,4-xylanases Xyn1, Xyn2 and Xyn11A, and β-1,6-glucanase Glu1 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 fungus Ustilago 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 an important event for pathogenic development in U. maydis. Moreover, we have shown that xylanases Xyn1, Xyn2 and Xyn11A, and glucanase Glu1 are required for full virulence, being essential for fungal filamentation on the plant surface and progression inside the plant. Additionally, we have demonstrated that loss of fungal N-glycosylation affected maize plant gene expression during the establishment of the biotrophism, specially during the first stages. Our results provide new insights into the role of glycosylation in fungal pathogenesis.