Fig 1.
Biological importance of N-glycosylation.
(A) Different N-glycosylated protein levels tested by Western blot using the ConA-HRP antibody. MY, mycelia; AP, appressoria; CO, conidia. (B) ConA-FITC and FLAER staining assay in different tissues of M. oryzae. Mycelia, conidia, appressoria and invasive hyphae were stained with 10 μg/mL ConA-FITC or 50 nM FLAER. ConA-FITC, FITC fluorescence-fused lectin concanavalin A; FLAER, fluorochrome (Alexa 488)–labeled inactivated aerolysin. Bar, 10 μm. Effect of 5 μg/mL tunicamycin (Tu) or 10 mM dithiothreitol (DTT) on: (C) mycelia dry weight, (D) the length of mycelial cell, (E) conidiation, (F) conidiophore formation, (G) appressorium formation ratio, (H) invasive hypha formation. Bar, 10 μm.
Fig 2.
Schematic for quantitative analysis strategy of the N-glycoproteome in M. oryzae.
(A) Light micrographs of M. oryzae mycelia, conidia, and conidia germinated on a hydrophobic surface showing the development of appressoria at the time points used in the N-glycoproteomics study. (B) Flow chart of the integrated strategy for quantitative analysis of the N-glycoproteome. Samples were digested with trypsin after removal of high-abundance proteins. Glycopeptides were enriched with lectin chromatography from two different samples, desalted, and then catalyzed by immobilized trypsin and PNGase-F in 18O or 16O water, as indicated. Equal amounts of 16O and 18O -labeled glycopeptides were mixed, and the 6 Da mass shifts were generated between paired, labeled glycopeptides, which could be identified by subsequent LC-MS/MS. (C) Venn diagram showing the number of N-proteins identified in different stages. (D) Distribution of the number of glycosylated sites on proteins. (E) Relative frequency plots of the N-glycosylation sequon (N-X-S/T) in the entire population of N-glycopeptides. MY, mycelia; CO, conidia; AP, appressoria.
Fig 3.
Classification of identified and protein interactions of the N-glycoproteome.
(A) Functional classification of identified N-glycoproteins based on Gene Ontology analysis. (B) KEGG pathway enrichment of the N-glycosylated proteins. (C) Protein interaction networks of the N-glycoproteins. Protein interaction networks were generated with the complete list of N-glycosylated proteins using the STRING database and visualized using the Cytoscape program.
Fig 4.
Differentiation of the N-glycoproteome among different samples.
(A) The principal component analysis (PCA) was carried out to get an overview of the data to detect batch effects, and assess differences between replicates. After filtering the missing values, the retained proteins were identified in two replicates of at least one condition. (B) The heatmap is plotted to visualized the Pearson correlations between the different samples. Colors indicate no correlation (white) and strong correlation (red). (C) The heatmap shows a clustering analysis of replicates, and indicates 6 h, 12 h, and 24 h appressoria enrich a similar abundance of proteins. Significant expression differences can be compared between individual samples during the differentiation process. The rows represent different proteins and are clustered in six groups by k-means clustering. Rows and columns are hierarchically clustered on Euclidean distance. Colors represent enrichment in the linkage versus control (red: enriched; blue: depleted). MY, mycelia; CO, conidia; 6h, 12h, 24h, appressoria (AP) at 6, 12 and 24 hpi.
Fig 5.
Different cellular processes mediated by N-glycoproteins.
The heat map shows the N-glycosylation profile of various N-glycoproteins in different developmental stages, including (A) cell wall proteins, (B) protease and peptidase, and (C) glycoside hydrolyses. Data is the mean of two biological replicates. The colored bar represents the scale for the log10 fold change in expression from green (-2) to red (2), white is set as 0. MY, mycelia; CO, conidia; AP, appressoria.
Fig 6.
Glycosylation pathways are mediated by N-glycosylation.
(A) N-glycan biosynthesis and (B) GPI anchor biosynthesis. (C) Changes in N-glycosylation protein levels in three glycosylation pathways at different stages. N-glycosylated proteins were marked in red font; N-glycosite was indicated by red ring. The colored bar represents the scale for the log10 fold change in expression from green (-2) to red (2), white is set as 0. MY, mycelia; CO, conidia; AP, appressoria.
Fig 7.
The ERQC system is regulated by N-glycosylation.
(A) Schematic diagram of the ERQC pathway. N-glycosylated proteins were marked in red font; N-glycosites was indicated by red ring. (B) Changes in ERQC pathway N-glycosylation protein levels in three glycosylation pathways. The colored bar represents the scale for the log10 fold change in expression from green (-2) to red (2), white is set as 0. MY, mycelia; CO, conidia; AP, appressoria.
Fig 8.
Functional analysis of the N-glycosylated components of the ERQC pathway.
(A) Colony morphology of the wild-type strain P131, ERQC pathway gene deletion mutants and complementary strains of Gls1 on oatmeal agar (OTA) plates. (B) Colony diameters of different strains. (C) Conidiation of different strains. (D) Conidiophore morphologies formed by different strains. (E) Lesions formed on barley leaves by different strains at 5 d post-inoculation (dpi). (F) Appressorium formation by different strains at 12 dpi. (G) Invasive hyphae (IH) formed by the same set of strains in barley epidermal cells at 24 h post-inoculation (hpi). Bar, 20 μm. (H) Formed ratio of IH in barley epidermal cells at 24 hpi.
Fig 9.
Western blot analysis and subcellular localization of Gls1.
(A) Western blot of N-glycosylated ERQC proteins in mycelia and appressoria. Total proteins from extracts of the Δgls1 and the Δalg3 mutant expressing GFP-fused Gls1 were separated by SDS-PAGE and then subjected to western blot analysis with an anti-GFP antibody. Total proteins isolated from the transformants were treated with or without Endo H and detected with an anti-GFP antibody by immunoblot analysis. Anti-actin antibody was used to evaluate protein loading level. (B) Subcellular co-localization of GFP:Gls1/RFP:HDEL and GFP:Gls1N497Q/CMAC in conidium. Bar, 10 μm. (C) Subcellular co-localization of GFP:Gls1/RFP:HDEL and GFP:Gls1N497Q/CMAC in appressorium. Bar, 10 μm. (D) Subcellular localization of GFP:Gls1/RFP:HDEL and GFP:Gls1N497Q in invasive hyphae. Bar, 10 μm. For (B), (C) and (D), linescan graphs show fluorescence intensity in a transverse section of individual appressorium as the arrow indicated direction, line colors refer to corresponding fluorescence colors. (E) Proposed model of the N-glycosylation regulated ERQC system for development.