Transcriptomic and Epigenomic Assessment Reveals Epigenetic Regulation of WRKY Genes in Response to Magnaporthe oryzae Infection in Rice

Abstract

Background: Histone acetylations acting as active hallmarks for gene transcription is involved in regulating numerous developmental and stress-responsive gene expression. Methods: The data from chromatin immunoprecipitation sequencing (ChIP-seq) was performed by using histone H3 lysine 9 acetylation (H3K9ac) antibody, and RNA sequencing (RNA-seq) utilizing rice seedlings inoculated by Magnaporthe oryzae (M. oryzae) were integrated. Results: RNA-seq data revealed that 422, 460 and 466 genes were up-regulated at 12h, 24h and 48h after inoculation. ChIP-seq data showed that 60%-80% of blast up-regulated genes at different time points were marked with H3K9ac, which was prone to be enriched in both TSS and gene body region. However, the H3K9ac level at a rather small proportion of the up-regulated genes was elevated after M. oryzae inoculation. We found that seven WRKY genes induced by rice blast fungus harbor H3K9ac. For different WRKY genes, blast fungus induction led to the increase of H3K9ac in distinct regions, including promoter, TSS or gene body, indicating that histone acetylation may play diverse roles in the activation of defense-related genes. By searching DNA-binding motifs of transcription factors in the promoter of genes with increased H3K9ac after M. oryzae infection, we found that ERF family protein-binding motifs were enriched with high -log P-value (>20), including ERF1, DEAR3, DREB2C, RAP2.6, RRTF1_3ARY, all of which contain GCC-box (GCCGCC). Conclusion: In this study, we revealed that the vast majority of genes induced by fungus M. oryzae were marked with H3K9ac preferring both TSS and gene body regions. However, H3K9ac enrichment was increased, responding to M. oryzae inoculation only at a low proportion of these genes, including several WRKY genes. Besides, for different genes, the increment of H3K9ac occurred in different regions. Finally, ERF proteins that have been proved to bind GCC-box might be one of the potential transcription factors for recruiting histone acetyltransferases to deposit histone acetylation at defense-related genes in rice.

Keywords: ERF; H3K9ac; Magnaporthe oryzae; Rice; WRKY; histone acetylation.

Fig. (1)

Differential gene expression after M. oryzae inoculation. (A) Volcano plot of differential gene expression at 12h, 24h and 48h after M. oryzae inoculation. Red dots indicate genes with unchanged expression, and blue dots indicate differentially expressed genes (DEGs). Overlapping of up-DEGs (B) and down-DEGs (C) at 12h, 24h and 48h after M. oryzae inoculation is presented by venn diagram. Heat maps show log2 (Fold change) of up-DEGs (D) and down-DEGs (E) at 12h, 24h and 48h respectively after M. oryzae inoculation.

Fig. (2)

Pathway enrichment of up-DEGs. (A) The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of up-DEGs at 12h, 24h and 48h after M. oryzae inoculation. (B) Pathway network diagram of up-regulated DEGs with P-value < 0.05 were visualized by Cytoscape.

Fig. (3)

H3K9ac alteration responding to M. oryzae infection. (A) Metaplots of genome-wide H3K9ac profile in genic regions before (H2O) and after (JC2) M. oryzae infection. (B) Correlation analysis of gene expression and H3K9ac enrichment responding to M. oryzae infection at 12h, 24h and 48h. (C) Metaplots of H3K9ac profiles of up-DEGs at 12h, 24 and 48h after inoculation.

Fig. (4)

Identification of transcription factor binding motifs related to H3K9ac. (A) Analysis of motif enrichment of the promoters with increased H3K9ac [log2 (Fold change)≥1]. B represents C or G or T, V represents A or C or G, W represents A or T, S represents C or G, R represents A or G, and M represents A or C. (B) The top five motifs in the promoters of genes with increased H3K9ac [log2 (Fold change) ≥1].

Fig. (5)

Changes of expression and H3K9ac levels of WRKY genes. (A) Heat map of log2(fold change) of WRKY genes at 12h, 24h and 48h after M. oryzae inoculation. The data were extracted from our RNA-seq data. (B) Genome browser screen shots of H3K9ac at seven WRKY genes before (H2O) and after (JC2) M. oryzae infection.