Fig 1.
Schematic overview of the integrative strategy for understanding aspects of response of barley transcriptome to biotic and hormonal stresses.
Table 1.
Primer sequences used in the qPCR.
Fig 2.
Venn diagrams illustrating the number of significant (P-value ≤ 0.05) down- and up-regulated DEGs between biotic and hormonal datasets.
(a) For the biotic datasets, 556 up- and 676 down-regulated DEGs were identified. For the hormonal datasets, 179 up- and 129 down-regulated DEGs were identified. (b) 61 Common DEGs between the biotic and hormonal studies were illustrated. Four-way Venn diagrams show common and up-/down-regulated DEGs in response to biotic and hormonal stresses. In total, 12 common down-regulated DEGs and 18 common up-regulated DEGs were identified. The red rectangle highlights the total up- and down-regulated DEGs (P-value ≤ 0.05). In fact, the completely biotic DEGs are common to the up-regulated hormonal DEGs in the four DEGs, but there is no commonality between the whole hormonal DEGs and the down-regulated biotic DEGs.
Fig 3.
Gene enrichment and pathway analyses of biotic DEGs.
(a) Gene ontology was performed using DAVID. The most significantly (P-value ≤ 0.05) enriched GO terms in Biological Process (BP), Molecular Function (MF), and Cellular Component (CC) categories related to the defense responses are presented. The horizontal axis shows the number of genes based on the P-value, while the vertical axis represents Go terms. (b) Shows KEGG pathway analysis of biotic DEGs. Y-axis shows the pathways and the X-axis shows the number of DEGs enriched in the pathways. The colors of each bubble are determined based on the P-value of the identified pathways. Size of the bubble represents number of DEGs enriched in the pathways. The figures were drawn using R and ggplot package.
Fig 4.
Gene enrichment and pathway analyses of hormonal DEGs.
(a) Shows gene ontology analysis using DAVID. The most significantly (P-value ≤ 0.05) enriched GO terms in biological process (BP), molecular function (MF) and cellular component (CC) categories related to signaling pathways are presented. The horizontal axis shows the number of genes based on the P-value, while the vertical axis represents Go terms. (b) Shows KEGG pathway analysis. Y-axis shows the pathways associated with hormonal signaling and the X-axis shows the number of DEGs enriched in the pathways. The colors of each bubble are determined based on the P-value of the identified pathways. Size of the bubble represents number of DEGs enriched in the pathways. The figures were drawn using R and ggplot package.
Table 2.
Modules of biotic stress studies.
Table 3.
Modules of hormonal treatment studies.
Fig 5.
Co-expression analysis and modules identification.
a, b) The module eigengene adjacency is showed by hierarchical clustering and heat map. In the heatmap, each row and column correspond to a specific module’s eigengene was denoted by module color. Within the heatmap, red indicates high adjacency (positive correlation) and blue shows low adjacency (negative correlation) as shown by the color legend. A module eigengene summarizes the gene expression profile of each module for (a) biotic and (b) hormonal studies. In the heatmap of module-module relationships, the progressively more saturated blue and red colors indicate the high co-expression interconnectedness. c-d) Multi-dimensional Scaling (MDS) map of each gene located in a module of the c) biotic or (d) hormonal studies. MDS plot demonstrating the similarity of gene expression patterns between different modules. Genes of different modules are marked in different colors. e-f) show network heatmap plots. Co-expression network modules of (e) biotic or (f) hormonal DEGs are presented. In the TOM plot, darker red color represents low overlap as progressively light color represents higher overlap among DEGs. Blocks of darker colors along the diagonal correspond to the modules. Gene dendrograms and module assignments are also displayed on the left side and the top.
Fig 6.
GO analysis of hub genes (P-value ≤ 0.05).
(a) Biotic hub genes. (b) Hormonal hub genes.
Fig 7.
TF families identified in DEGs.
(a) The number of up- and down-regulated biotic TF families. (b) The number of up- and down-regulated hormonal TF families. The Y-axis refers to the number of genes. The X-axis shows TF families.
Table 4.
PKs identified in biotic and hormonal treatment studies.
Fig 8.
Identification of miRNAs and their targets.
(a) Common miRNAs between biotic and hormonal studies are showed. The Y-axis refers to the number of miRNAs. The X-axis shows miRNA families. (b) Shows GO classification for the target genes of differentially expressed miRNAs. Categorization of miRNA target genes with WEGO for biotic and hormonal studies are presented. The horizontal axis is the GO classification; the vertical is the percentage of genes (left) and number of genes (right). The miRNAs are divided into GO categories. The genes targeted by the entire miRNAs: red column, the genes targeted by the differentially expressed miRNAs: gray column.
Table 5.
The conserved cis-acting elements found in the promoter of biotic DEGs by the MEME analysis.
Table 6.
The conserved cis-acting elements found in promoter of hormonal DEGs by the MEME analysis.
Fig 9.
The protein-protein interaction network of hub genes and DEGs using STRING database and Cytoscape software.
(a) The network of interactions among biotic hub genes and the DEGs is showed. (b) The network of interactions among hormonal hub genes and the DEGs is presented. The yellow nodes represent the selected key genes for qPCR validation, blue nodes represent the hub genes, and the purple nodes represent the most connected genes to the key genes in the network.
Table 7.
The most important molecular mechanisms related to the biotic and hormonal studies.
Fig 10.
The protein-protein interaction network of common DEGs using STRING database and Cytoscape software.
Interaction network of 61 common DEGs between hormonal and biotic datasets. The yellow colored nodes represent the key genes expressed in the pathways of resistance to the pathogens and signaling of hormones, and the pink colored nodes represent the genes with the most connectivity to major genes in the network.
Fig 11.
Relative expression analysis of hub genes.
The expression of hub genes at the time points of 3 h, 6 h, 24 h, and 48 h after 100 μM MeJA treatment. The different letters on the columns represent the significant differences given by Duncan’s multiple range statistical analysis. Vertical bars indicate ± SE of the mean (n = 3, P-value ≤ 0.05). The X-axis shows the times after MeJA treatment.