Brachypodium Phenylalanine Ammonia Lyase (PAL) Promotes Antiviral Defenses against Panicum mosaic virus and Its Satellites

Abstract

Brachypodium distachyon has recently emerged as a premier model plant for monocot biology, akin to Arabidopsis thaliana We previously reported genome-wide transcriptomic and alternative splicing changes occurring in Brachypodium during compatible infections with Panicum mosaic virus (PMV) and its satellite virus (SPMV). Here, we dissected the role of Brachypodium phenylalanine ammonia lyase 1 (PAL1), a key enzyme for phenylpropanoid and salicylic acid (SA) biosynthesis and the induction of plant defenses. Targeted metabolomics profiling of PMV-infected and PMV- plus SPMV-infected (PMV/SPMV) Brachypodium plants revealed enhanced levels of multiple defense-related hormones and metabolites such as cinnamic acid, SA, and fatty acids and lignin precursors during disease progression. The virus-induced accumulation of SA and lignin was significantly suppressed upon knockdown of B. distachyonPAL1 (BdPAL1) using RNA interference (RNAi). The compromised SA accumulation in PMV/SPMV-infected BdPAL1 RNAi plants correlated with weaker induction of multiple SA-related defense gene markers (pathogenesis related 1 [PR-1], PR-3, PR-5, and WRKY75) and enhanced susceptibility to PMV/SPMV compared to that of wild-type (WT) plants. Furthermore, exogenous application of SA alleviated the PMV/SPMV necrotic disease phenotypes and delayed plant death caused by single and mixed infections. Together, our results support an antiviral role for BdPAL1 during compatible host-virus interaction, perhaps as a last resort attempt to rescue the infected plant.IMPORTANCE Although the role of plant defense mechanisms against viruses are relatively well studied in dicots and in incompatible plant-microbe interactions, studies of their roles in compatible interactions and in grasses are lagging behind. In this study, we leveraged the emerging grass model Brachypodium and genetic resources to dissect Panicum mosaic virus (PMV)- and its satellite virus (SPMV)-compatible grass-virus interactions. We found a significant role for PAL1 in the production of salicylic acid (SA) in response to PMV/SPMV infections and that SA is an essential component of the defense response preventing the plant from succumbing to viral infection. Our results suggest a convergent role for the SA defense pathway in both compatible and incompatible plant-virus interactions and underscore the utility of Brachypodium for grass-virus biology.

Keywords: bioenergy; defense hormones; grasses; metabolic pathways; plant-virus interactions.

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.