Article Figures & Data
Figures
- FIG 1
MapMan overview of primary and secondary metabolic pathways perturbed during PMV infection in Brachypodium. The square boxes in green and red indicate reduced and induced level of expression (>2 log2 fold-change, FDR < 0.05), respectively. The fold change expression data are provided in Data Set 1 in the supplemental material. Mito, mitochondrial; TCA, tricarboxylic acid; OPP, oxidative pentose phosphate; CHO, carbohydrates.
- FIG 2
Quantification of salicylic acid (SA), cinnamic acid (CA), jasmonic acid (JA), and precursors in PMV- and PMV+SPMV-infected Brachypodium. Levels of SA (A), CA (B), trans- and cis-JA (C), and trans- and cis-12-OPDA (D) were determined using GC-MS. The fresh weight (FW) leaf samples were collected at three different stages of disease progression (early stage I, 7 dpi; mid-stage II, 14 dpi; late stage III, 21 dpi), as described previously (24). The y axes represent average contents of respective metabolites from five biological replicates. The error bars represent standard errors of means (n = 3 and n = 4 for early and for mid and late stages, respectively). *, P ≤ 0.05; **, P ≤ 0.01 between mock- and virus-infected samples as determined by two-sample t test (one-tailed). M, mock; P, PMV; PS, PMV+SPMV; OPDA, 12-oxo-phytodienoate.
- FIG 3
Quantification of 18-carbon unsaturated fatty acid levels in PMV- and PMV+SPMV-infected Brachypodium. Levels of free and total 18:1, 18:2, and 18:3 fatty acids in mock-, PMV-, and PMV+SPMV-infected plants at stages II (14 dpi) and III (21 dpi) of infection. The error bars represent standard errors of means (n = 3 and n = 4 for stages II and III, respectively). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 between mock- and virus-infected samples as determined by two-sample t test (one-tailed). M, mock; P, PMV; PS, PMV+SPMV.
- FIG 4
Functional analysis of wild-type (WT) and BdPAL1 RNAi plants during PMV and PMV+SPMV infection. Expression of BdPAL1 (A) and PAL activity (B) in mock-, PMV-, and PMV+SPMV-inoculated WT and BdPAL1 RNAi plants at stage II (14 dpi) of infection. Levels of soluble carbohydrates (sucrose and glucose) (C), structural carbohydrates (cellulose and hemicellulose) and total lignin (D), and salicylic acid (E) at stage II (14 dpi) of infection. M, mock; P, PMV; PS, PMV+SPMV. Relative expression of defense-related genes (SA and JA signaling components) in PMV-infected (F) and PMV+SPMV-infected (G) WT and BdPAL1 RNAi plants. The error bars represent standard errors of means (n = 3 for panels A to D, F, and G and n = 5 for panel E). Statistically significant differences in panel A were determined by two-sample t test (one-tailed) and, in panels B to G, were assessed using one or two-way ANOVA followed by the Tukey’s test. Unlike lowercase letters represent significant differences among the group means (P ≤ 0.05). F and t test statistics of ANOVA and two-sample t tests are indicated.
- FIG 5
BdPAL1 RNAi plants showed enhanced susceptibility to PMV+SPMV infection. (A) Mock- and PMV+SPMV-infected WT (left) and BdPAL1 RNAi (right) plants at stage III (21 dpi). (B) Closeup of mock- and PMV+SPMV-infected leaves of WT (left) and BdPAL1 RNAi (right) plants at stage III (21 dpi). (C) Percentages of leaves with necrosis of wild-type (WT) and BdPAL1 RNAi plants, mock- and virus-infected plants at stage III (21 dpi). Statistically significant differences were assessed using one-way ANOVA followed by the Tukey’s multiple-comparison test. Unlike lowercase letters represent significant differences among the group means (P ≤ 0.05). F statistics of ANOVA are indicated. (D) RT-qPCR detection of mRNA encoding PMV CP (PCP) and SPMV CP (SPCP) in noninoculated leaves at 14 dpi. M, mock; PS, PMV+SPMV. Statistically significant differences were assessed between mock- and virus-inoculated samples using two-sample t test (one-tailed). *, P ≤ 0.05; ***, P ≤ 0.001. The error bars represent standard errors of the means (n = 3).
- FIG 6
Exogenous application of salicylic acid (SA) attenuates PMV and SPMV disease symptoms. Representative chlorosis and necrosis at 21 dpi (A) and overall stunting symptoms at 42 dpi (B) of PMV- and PMV+SPMV-infected plants treated with water or salicylic acid (SA; 100 ppm). Quantification of percent chlorotic (C) and necrotic (D) leaves in wild-type (WT) and BdPAL1 RNAi plants treated either with water or SA (100 ppm) at 21 dpi. Statistically significant differences were assessed using one-way ANOVA followed by the Tukey’s test. Unlike lowercase letters represent significant differences among the group means (P ≤ 0.05). F test statistics of ANOVA are indicated. The error bars represent standard errors of means (n = 3). M, mock; P, PMV; PS, PMV+SPMV.
Supplemental Material
DATA SET S1
Gene expression data (>2 log2 fold change, FDR < 0.05) in PMV-infected Brachypodium leaves at stage II. Download Data Set S1, XLSX file, 0.1 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
TABLE S1
Primers used for SA and JA defense marker gene expression analysis. Download Table S1, DOCX file, 0.1 MB.
This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
