Identification of O-mannosylated Virulence Factors in Ustilago maydis

Abstract

The O-mannosyltransferase Pmt4 has emerged as crucial for fungal virulence in the animal pathogens Candida albicans or Cryptococcus neoformans as well as in the phytopathogenic fungus Ustilago maydis. Pmt4 O-mannosylates specific target proteins at the Endoplasmic Reticulum. Therefore a deficient O-mannosylation of these target proteins must be responsible for the loss of pathogenicity in pmt4 mutants. Taking advantage of the characteristics described for Pmt4 substrates in Saccharomyces cerevisiae, we performed a proteome-wide bioinformatic approach to identify putative Pmt4 targets in the corn smut fungus U. maydis and validated Pmt4-mediated glycosylation of candidate proteins by electrophoretic mobility shift assays. We found that the signalling mucin Msb2, which regulates appressorium differentiation upstream of the pathogenicity-related MAP kinase cascade, is O-mannosylated by Pmt4. The epistatic relationship of pmt4 and msb2 showed that both are likely to act in the same pathway. Furthermore, constitutive activation of the MAP kinase cascade restored appressorium development in pmt4 mutants, suggesting that during the initial phase of infection the failure to O-mannosylate Msb2 is responsible for the virulence defect of pmt4 mutants. On the other hand we demonstrate that during later stages of pathogenic development Pmt4 affects virulence independently of Msb2, probably by modifying secreted effector proteins. Pit1, a protein required for fungal spreading inside the infected leaf, was also identified as a Pmt4 target. Thus, O-mannosylation of different target proteins affects various stages of pathogenic development in U. maydis.

Unraveling the Novel Structure and Biosynthetic Pathway of O-Linked Glycans in the Golgi Apparatus of the Human Pathogenic Yeast Cryptococcus neoformans

“…Recent studies on the human pathogenic yeast C. albicans and the plant pathogenic fungus Ustilago maydis also demonstrated that O-mannosylation is required for adherence, invasion of host tissues, and virulence (45,46). A previous study on the function of the PMT family proteins in C. neoformans reported that pmt1⌬ and pmt4⌬ mutants displayed defects in cell morphology and severely attenuated virulence in a murine inhalation model of cryptococcosis (31).…”

Roles and delivery mechanisms of fungal effectors during infection development: common threads and new directions

“…An example of each comes again from U. maydis. Pmt4 encodes an o-mannosyl transferase, modifying proteins as they leave the endoplasmic reticulum; its targets include a mucin involved in appressorium formation encoded by the Msb2 gene, and the Pit1 gene, whose product is involved in later stages of virulence, allowing the fungus to spread throughout the leaf [40]. Pit1 is genetically linked to a secreted putative effector encoded by Pit2; the Pit2 protein is membrane bound at fungal tips, and the authors hypothesize that Pit2 shuttles defense signals to Pit1, helping disguise the fungus from being detected [41].…”

Spatiotemporal Monitoring of Pseudomonas syringae Effectors via Type III Secretion Using Split Fluorescent Protein Fragments

“…In plants, most of the localization studies of T3Es have been performed in Agrobacterium tumefaciens-mediated transient expression systems using constitutive promoters (Block and Alfano, 2011;Macho, 2016). However, several biochemical studies revealed bacterial effectors undergo posttranslocational modification for proper targeting in the host cells (Boucrot et al, 2003;Reinicke et al, 2005;Patel et al, 2009;Fernández-Álvarez et al, 2012). Therefore, Agrobacterium-mediated expressions may not reflect dynamic localization of respective effectors originally secreted by T3SS.…”