Aberrant protein N‐glycosylation impacts upon infection‐related growth transitions of the haploid plant‐pathogenic fungus Mycosphaerella graminicola

Abstract

SummaryThe ascomycete fungus Mycosphaerella graminicola is the causal agent of Septoria Tritici Blotch disease of wheat and can grow as yeast-like cells or as hyphae depending on environmental conditions. Hyphal growth is however essential for successful leaf infection. A T-DNA mutagenesis screen performed on haploid spores identified a mutant, which can undergo yeast-like growth but cannot switch to hyphal growth. For this reason the mutant was non-pathogenic towards wheat leaves. The gene affected, MgAlg2, encoded a homologue of Saccharomyces cerevisiae ScAlg2, an alpha-1,2-mannosyltransferase, which functions in the early stages of asparagine-linked protein (N-) glycosylation. Targeted gene deletion and complementation experiments confirmed that loss of MgAlg2 function prevented the developmental growth switch. MgAlg2 was able to functionally complement the S. cerevisiae ScAlg2-1 temperature sensitive growth phenotype. Spores of DMgAlg2 mutants were hypersensitive to the cell wall disrupting agent Calcofluor white and produced abnormally hypo-Nglycosylated proteins. Gene expression, proteome and glycoproteome analysis revealed that DMgAlg2 mutant spores show responses typically associated with the accumulation of mis-folded proteins. The data presented highlight key roles for protein N-glycosylation in regulating the switch to hyphal growth, possibly as a consequence of maintaining correct folding and localization of key proteins involved in this process.

N-Glycosylation of Effector Proteins by an α-1,3-Mannosyltransferase Is Required for the Rice Blast Fungus to Evade Host Innate Immunity

“…Three earlier reports have shown that N-glycosylation plays an important role in the pathogenesis of plant pathogenic fungi. In U. maydis, the ER glucosidase I gene, Gls1, is required for the initial stages of infection following appressorium penetration (Fernández-Álvarez et al, 2013) and deletion of the ER glucosidase II gene, GAS1, resulted in arrested growth of IH in the epidermal cell layer of maize (Zea mays) (Schirawski et al, 2005), and in M. graminicola, the a-1,2-mannosyltransferase Alg2 was shown to be important for switching from the yeast-like phase to the hyphal form (Motteram et al, 2011). These studies suggested the importance of N-glycosylation of proteins in the pathogenesis of plant fungal pathogens but did not offer mechanistic insight into why N-glycosylation is important for pathogenesis in plant pathogens.…”

“…However, the role of N-glycosylation in the pathogenesis of filamentous fungi remains largely unexplored. To date, there are only three reports with glycosylation-related mutants in Ustilago maydis and Mycosphaerella graminicola that implicate N-glycosylation in the pathogenesis of plant pathogenic fungi (Schirawski et al, 2005;Motteram et al, 2011;Fernández-Álvarez et al, 2013). However, the underlying mechanism was not characterized in those studies; therefore, it is not clear why N-glycosylation is important for fungal pathogenesis.…”

A fungal avirulence factor encoded in a highly plastic genomic region triggers partial resistance to septoria tritici blotch

“…The three fragments were assembled into pES1 (E. H. Stukenbrock, unpublished) linearized with Kpn I and Pst I (New England Biolabs), resulting in pES1∆581 3D1 and pES1∆581 3D7 . To create the construct for ectopic integration of Avr3D1 3D1 , a fragment containing Avr3D1 3D1 including the 1.3‐kb sequence upstream of the start codon and the 1‐kb sequence downstream of the stop codon was amplified and cloned into pCGEN (Motteram et al ., 2011) that had been linearised with Kpn I, resulting in pCGEN‐581 3D1 ect. To exchange the coding sequence (CDS) in pCGEN‐581 3D1 ect, we first digested it with Xho I (New England Biolabs) to linearise it and remove the Avr3D1 3D1 CDS.…”

Fungal model systems and the elucidation of pathogenicity determinants

“…Deletion of MgALG2, involved in glycosylation of secreted proteins, led to a mutant unable to infect wheat. This mutant is also impaired in switching from yeast to filamentous growth and in protein secretion (Motteram et al, 2011). Among several ABC transporters studied, only MgATR4 had a quantitative effect on pathogenicity (Stergiopoulos et al, 2003).…”