Quantitative proteomics analysis reveals important roles of N-glycosylation on ER quality control system for development and pathogenesis in Magnaporthe oryzae

Abstract

Genetic studies have shown essential functions of N-glycosylation during infection of the plant pathogenic fungi, however, systematic roles of N-glycosylation in fungi is still largely unknown. Biological analysis demonstrated N-glycosylated proteins were widely present at different development stages of Magnaporthe oryzae and especially increased in the appressorium and invasive hyphae. A large-scale quantitative proteomics analysis was then performed to explore the roles of N-glycosylation in M. oryzae. A total of 559 N-glycosites from 355 proteins were identified and quantified at different developmental stages. Functional classification to the N-glycosylated proteins revealed N-glycosylation can coordinate different cellular processes for mycelial growth, conidium formation, and appressorium formation. N-glycosylation can also modify key components in N-glycosylation, O-glycosylation and GPI anchor pathways, indicating intimate crosstalk between these pathways. Interestingly, we found nearly all key components of the endoplasmic reticulum quality control (ERQC) system were highly N-glycosylated in conidium and appressorium. Phenotypic analyses to the gene deletion mutants revealed four ERQC components, Gls1, Gls2, GTB1 and Cnx1, are important for mycelial growth, conidiation, and invasive hyphal growth in host cells. Subsequently, we identified the Gls1 N-glycosite N497 was important for invasive hyphal growth and partially required for conidiation, but didn't affect colony growth. Mutation of N497 resulted in reduction of Gls1 in protein level, and localization from ER into the vacuole, suggesting N497 is important for protein stability of Gls1. Our study showed a snapshot of the N-glycosylation landscape in plant pathogenic fungi, indicating functions of this modification in cellular processes, developments and pathogenesis.

Comprehensive identification of lysine 2‐hydroxyisobutyrylated proteins in Ustilaginoidea virens reveals the involvement of lysine 2‐hydroxyisobutyrylation in fungal virulence

“…Precursor proteins are typically inactive and are converted into mature functional proteins through a series of PTMs, which modulate diverse protein properties and functions (Deribe et al, 2010). In phytopathogens, some protein modifications play important roles in development and pathogenesis, including sumoylation, N‐glycosylation, glycosylphosphatidylinositol (GPI), and acetylation (Liang et al, 2018a; Liu et al, 2020; Chen et al, 2020a).…”

“…Using a previously reported method (Chen et al, 2020c), approximately 1.5 kb of upstream and downstream flanking sequences of the target gene were ligated with pGKO to generate a gene deletion vector (Figure S3A). The vector was introduced into A. tumefaciens strain EHA105, and then transformed into conidia of the wild‐type strain using ATMT following the method of Chen et al (2020c). Hygromycin‐resistant transformants were isolated and subsequently screened and then confirmed by rigor polymerase chain reaction (PCR) strategy (Figure S3A; Table S5).…”

An Overview of Genomics, Phylogenomics and Proteomics Approaches in Ascomycota

“…A few studies explored the functions of post-translational modification (PTM) during infection. Chen et al [ 259 ], employed a label-free quantitative proteomics analysis to unravel the regulatory mechanisms of N-glycosylation—in which a glycan moiety is added to the amide group of an asparagine residue—in the rice blast fungus Magnaporthe oryzae . The regulated N-glycosylated proteins, nearly all key components of the endoplasmic reticulum quality control (ERQC) system in conidium and appressorium (i.e., Gls1, Gls2, GTB1 and Cnx1), were found to be involved in the coordination of different cellular processes for mycelial growth, conidiation and invasive hyphal growth.…”

“… [ 366 ] Knufia perforans Whole cell proteome 2D-E n.a. Characterization of protein patterns in response to sub-optimal temperature [ 338 ] Characterization of protein patterns under simulated Mars-like conditions [ 240 ] Characterization of protein patterns under drought [ 339 ] Lobaria pulmonaria Proteome of the symbiotic consortium 1D-E, LC-ESI-MS/MS 463 Profiling of the metaproteome [ 380 ] 4405 Comparative omics to explore the lichen-associated microbiome [ 380 ] 1D-E, GeLC-MS/MS 6590 Profiling of the metaproteome [ 381 ] Magnaporthe oryzae Mycelium and conidia, whole cell proteome Label-free LC-MS/MS 355 and 559 N-glycosylation sites Quantitative analysis of N-glycosylation regulation and elucidation of its role in development and pathogenesis of M. oryzae [ 259 ] LC-MS/MS 5498 Comparative proteomic analysis between nitrogen supplemented and starved conditions [ …”

Comparative secretome of Magnaporthe oryzae identified proteins involved in virulence and cell wall integrity

“…Protein N -glycosylation is a significant protein modification essential for fungal infection. Mutants of protein N -glycosylation showing defected in fungal virulence are reported not only for M. oryzae [45], but also for other plant pathogens such as Penicilium digitatum, Botrytis cinerea and Ustilago maydis [24, 25, 46-52]. Recent study on glycoprotein proteomics in Ustilago maydis using wildtype and mutants Δ gls1 , a mutant also involved in N -glycosylation process, had identified several protein with N -glycosylation modification [53].…”