Figures Captions
Fig. 1 The key common differential metabolites of EI vs. EF and EICL vs. EFCL. Box-and-whisker plots for selected metabolites comparing normalized intensities. The data of six biological replicates were normalized by summing and Pareto scaling. The significant values were confirmed by Student’s t -tests. *A significant difference (0.001< P < 0.05); **A extremely significant difference (P < 0.001). EF, endophyte-free plants; EI, endophyte-infected plants; EFCL, EF plants inoculated withCurvularia lunata ; EICL, EI plants inoculated with C. lunata.
Fig. 2 Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways of differentially expressed genes and identified metabolites in EI vs. EF (a) and EICL vs. EFCL (b) . The statistical significance of pathway enrichments was identified using Fisher’s exact test, andP values from the KEGG enrichment analyses were corrected by Benjamini and Hochberg as well. The corrected P values (false discovery rate, FDR) ≤0.05 were considered significantly enriched. Bars across the dashed lines indicated the KEGG pathways were significantly enriched. EF, endophyte-free plants; EI, endophyte-infected plants; EFCL, EF plants inoculated with Curvularia lunata ; EICL, EI plants inoculated with C. lunata.
Fig. 3 Transcription and metabolism roadmap in plant signal molecular JA. ET and Pip synthesis pathways. Heatmaps show the values of relative expression of each treatment group compared to the EF (values = Log2 (fold change)). Fold change calculation was based on fragments per kilo base of exon per million fragments mapped (FPKM) values (the information of genes were shown in Table S1). Box-and-whisker plots for selected metabolites comparing normalized intensities. The metabolite data of six biological replicates were normalized by summing and Pareto scaling. The significant values were confirmed by Student’s t -tests. Ns means no significance; *A significant difference (0.001< P < 0.05); **A extremely significant difference (P < 0.001). The red font represents the upregulated gene expression or increased metabolite content in EI vs. EF or EICL vs. EFCL, and the black font represents no significant change. LOX, lipoxygenase; AOS, allene oxide synthase; OPR, 12-oxo-phytodienoate reductase; OPCL1, OPC-8:0 CoA ligase 1; ACOX, acyl-coenzyme A oxidase; ACAA, 3-ketoacyl-CoA thiolase; SAMS, S-adenosylmethionine synthase; ACS, 1-aminocyclopropane-1-carboxylate synthase; ACO, 1-aminocyclopropane-1-carboxylate oxidase; ALD1, aminotransferase; SARD4, systemic acquired resistance-deficient4; FMO1, flavin-dependent-monooxygenase 1. EF, endophyte-free plants; EI, endophyte-infected plants; EFCL, EF plants inoculated with Curvularia lunata ; EICL, EI plants inoculated with C. lunata.
Fig. 4 Endogenous levels and roles verification of plant signaling molecules. The concentration of jasmonic acid (a) , internal ethylene (b) , and pipecolic acid (c) were quantified in EF and EI Achnatherum sibiricum before and afterCurvularia lunata inoculation. The disease index (d) ofA. sibiricum treated with water (control), salicylic acid (SA, 0.2 mM), methyl jasmonate (MeJA, 0.2 mM), ethephon (ETH, 0.2 mM), or pipecolic acid (Pip, 1 mM) were measured at five days post pathogen inoculation. Data are represented as means ± SEM of five biological replicates. Different letters above the bars denote statistically significant differences between different treatments (P< 0.05, Two-way ANOVA and post hoc Duncan’s test). EF, endophyte-free plants; EI, endophyte-infected plants; EFCL, EF plants inoculated withCurvularia lunata ; EICL, EI plants inoculated with C. lunata.
Fig. 5 Transcription and metabolism roadmap of phenols in the phenylpropanoid synthetic pathway. Heatmaps show the values of relative expression of each treatment group compared to the EF (values = Log2 (fold change)). Fold change calculation was based on fragments per kilo base of exon per million fragments mapped (FPKM) values (the information of genes were shown in Table S1). Box-and-whisker plots for selected metabolites comparing normalized intensities. The metabolite data of six biological replicates were normalized by summing and Pareto scaling. The significant values were confirmed by Student’s t-tests. Ns means no significance; *A significant difference (0.001< P < 0.05). The red font represents the upregulated gene expression or increased metabolite content in EI vs. EF or EICL vs. EFCL; the green font represents the decreased gene expression or metabolites content, and the black font represents no significant change. Solid arrows represent the direct synthesis pathway, and dashed arrows represent the indirect synthesis pathway. PAL, phenylalanine ammonia-lyase; PTAL, phenylalanine/tyrosine ammonia-lyase; C4H, trans-cinnamate 4-monooxygenase, 4CL, 4-coumarate-CoA ligase; F5H, ferulate 5-hydroxylase. EF, endophyte-free plants; EI, endophyte-infected plants; EFCL, EF plants inoculated withCurvularia lunata ; EICL, EI plants inoculated with C. lunata.
Supplementary Material
Fig. S1 Hyphal growth ofCurvularia lunata on nutrient agar medium containing chemicals.
Fig. S2 Metabolite profiling of Achnatherum sibiricum .
Fig. S3 Enrichment analysis of KEGG pathway in EI vs. EF(a) and EICL vs. EFCL (b) .
Table S1 Identificated key differentially expressed genes involved in the biosynthesis of plant signaling molecules and phenylpropanoids.
Method S1 The method of screening optimum concentrations for verification experiment.
Method S2 LC-MS instrumentation, metabolite identification, and data analysis.
Method S3 RNA sequencing, de novo assembly, functional annotation of unigenes, and data analysis.