Comparative Proteomic Analysis of Plasma Membrane Proteins in Rice Leaves Reveals a Vesicle Trafficking Network in Plant Immunity That Is Provoked by Blast Fungi

Abstract

Rice blast, caused by Magnaporthe oryzae, is one of the most devastating diseases in rice and can affect rice production worldwide. Rice plasma membrane (PM) proteins are crucial for rapidly and precisely establishing a defense response in plant immunity when rice and blast fungi interact. However, the plant-immunity-associated vesicle trafficking network mediated by PM proteins is poorly understood. In this study, to explore changes in PM proteins during M. oryzae infection, the PM proteome was analyzed via iTRAQ in the resistant rice landrace Heikezijing. A total of 831 differentially expressed proteins (DEPs) were identified, including 434 upregulated and 397 downregulated DEPs. In functional analyses, DEPs associated with vesicle trafficking were significantly enriched, including the "transport" term in a Gene Ontology enrichment analysis, the endocytosis and phagosome pathways in a Encyclopedia of Genes and Genomes analysis, and vesicle-associated proteins identified via a protein-protein interaction network analysis. OsNPSN13, a novel plant-specific soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) 13 protein, was identified as an upregulated DEP, and transgenic plants overexpressing this gene showed enhanced blast resistance, while transgenic knockdown plants were more susceptible than wild-type plants. The changes in abundance and putative functions of 20 DEPs revealed a possible vesicle trafficking network in the M. oryzae-rice interaction. A comparative proteomic analysis of plasma membrane proteins in rice leaves revealed a plant-immunity-associated vesicle trafficking network that is provoked by blast fungi; these results provide new insights into rice resistance responses against rice blast fungi.

Keywords: Oryza sativa L.; iTRAQ; plasma membrane; proteomics; rice blast; vesicle trafficking.

FIGURE 1

Overview of the experimental scheme. Schematic representation of the proteomics workflow. Leaf samples were collected at 0 and 24 h after M. oryzae infection from the resistant rice landrace Hei. Total PM protein was extracted, digested and quantified via iTRAQ labeling and LC–MS/MS analysis. Bioinformatic analyses were used to explore the constitution and function of DEPs.

FIGURE 2

Quantitative identification of proteins from the iTRAQ analysis. (A) Distribution of the number of unique peptides for the identified proteins. The X-axis is the scope of the unique peptides, the left Y-axis is the number of proteins, and the right Y-axis is the cumulative percent. (B) Volcano plot of the proteins identified. The X-axis specifies the fold change between 24 h/0 h, and the Y-axis specifies the -log10 (FDR); upregulated, downregulated and non-significant proteins are shown as red, blue, and gray dots, respectively; gray vertical and horizontal lines reflect the filtering criteria (fold change > 1.1 or < 0.909 and FDR < 0.1). (C) The number of total proteins and DEPs (including upregulated and downregulated). (D) The distribution of DEP fold changes.

FIGURE 3

Gene ontology enrichment analysis. GO enrichment analysis of DEPs (FDR < 0.05). The enriched terms from three levels (biological process, molecular function, and cellular component) are shown in the figure. The X-axis is the rich factor, and the Y-axis is the description of GO term.

FIGURE 4

Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. KEGG pathway enrichment analysis of DEPs (p-value < 0.05). (A) KEGG pathway enrichment analysis of upregulated DEPs. (B) KEGG pathway analysis of downregulated DEPs. The X-axis is the protein ratio (the number of DEPs annotated with this pathway term/the total number of DEPs), the Y-axis is the KEGG pathway, the size of the bubble represents the protein count, and the color of the bubble represents –log10 (p-value).

FIGURE 5

Protein–protein interaction network of DEPs. A protein–protein interaction network of DEPs associated with vesicle trafficking (including the transport terms from the GO analysis and the phagosome and endocytosis terms from the KEGG analysis) was built using the STRING database. Identified interactions with confidence scores ≥ 0.4 were visualized using Cytoscape software. The red nodes are upregulated DEPs, and the green nodes are downregulated DEPs; the size of the nodes represents the number of related interactions in the network; and the colors of the edges indicate the strength of the interaction: orange represents a strong interaction, and blue represents a weak interaction.

FIGURE 6

The identification of transcriptional expression patterns of 10 DEPs after M. oryzae infection. The expression patterns of 10 selected DEPs in Heikezijing (Hei) inoculated with the M. oryzae strain Hoku1 were investigated via qRT–PCR. Hei seedlings were collected 0, 8, 24, 48, and 72 h after inoculation. The rice actin gene was used as an internal control. The bar chart shows the average ± standard deviation (n = 3). The information of Fold Change (FC) and Peptide Coverage (PC) in proteomic data was also shown in the figure. Red and green colored names represent upregulated and downregulated, respectively, in the proteomic analysis.

FIGURE 7

Resistance phenotype of representative transgenic plants expressing OsNPSN13 and the subcellular localization of OsNPSN13. (A) Lesion length in wild-type Su and transgenic plants expressing OsNPSN13 at 7 dpi with M. oryzae. (B) Lesion number per leaf in wild-type Su and transgenic plants expressing OsNPSN13 inoculated with M. oryzae at 7 dpi. S13, transgenic overexpression lines; A13, transgenic knockdown lines. The bar charts show the average, standard deviation (n = 12) and p-value. (C) Rice blast resistance phenotypes of transgenic plants and wild-type Su inoculated with M. oryzae strain Hoku1. The leaves with lesions are shown. Bar = 1 cm. (D) The subcellular localization of OsNPSN13 transiently expressed in rice protoplasts. OsAMT3.2-RFP was used as PM marker. Bar = 5 μm.

FIGURE 8

Overview of the possible vesicle trafficking network of DEPs. Possible model for the roles of DEPs in vesicle trafficking during the rice-blast fungus interaction. Upregulated DEPs are shown in red, downregulated DEPs are shown in blue, green arrows indicate exocytosis, orange arrows indicate endocytosis, and the translational patterns of DEPs are shown in the charts.