N-glycosylation of the protein disulfide isomerase Pdi1 ensures full Ustilago maydis virulence.

Miriam Marín-Menguiano,Alfonso Fernández-Álvarez,José Ignacio Ibeas,Ismael Moreno-Sánchez,Ramón R Barrales,Jin-Rong Xu

doi:10.1371/journal.ppat.1007687

Abstract

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 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 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 important for normal pathogenic development in U. maydis. Our results provide new insights into the role of glycosylation in fungal pathogenesis.

Highlights

Protein glycosylation is a common eukaryotic post-translational mechanism required for the correct folding, activity and secretion of many proteins
In order to identify virulence-related glycosylation targets, we designed a selective glycoproteomic screen based on the hypothesis that glycoproteins whose expression is modified upon virulence program induction are likely to have important roles in the pathogenic phase of the U. maydis life cycle
For this screen we set out to analyze cell extracts corresponding to cytosolic, cell wall and secreted proteins using two-dimensional differential gel electrophoresis (2D-DIGE) to detect glycoproteins whose spot area or intensity were altered by the loss of Pmt4 or Gls1 and corresponding effect on O- or N- glycosylation activities, respectively

Summary

Introduction

Protein glycosylation is a common eukaryotic post-translational mechanism required for the correct folding, activity and secretion of many proteins. Glycosylation involves the synthesis and addition of different polysaccharide cores (sugars) to specific amino acids within a consensus sequence. Protein glycosylation is divided into different types based on the structure and composition of the oligosaccharide cores and the amino acids to which they are attached. N-glycosylation consists of the addition of an oligosaccharide core, composed of two N-acetylglucosamines (NAcGlc), nine mannoses (Man) and three glucose (Glc) molecules, NAcGlc2Man9Glc, to the nitrogen chain of an asparagine residue in the sequence Asn-x-Ser/Thr, where x can be any amino acid except proline [9,10]. In fungi O-mannosylation is the most common type of O-glycosylation and is characterized by the addition of Man residues to target proteins. N- and Olinked glycans are later processed during their transit across the ER and GA, and specific trimming of sugars is essential for the function and secretion of glycoproteins [5,12]

Methods

Results

Conclusion