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.