Abscisic Acid Induces Resistance against Bamboo Mosaic Virus through Argonaute2 and 3

Abstract

Plant resistance to pathogens is tuned by defense-related hormones. Of these, abscisic acid (ABA) is well documented to moderate resistance against fungi and bacteria. However, ABA's contribution to resistance against viruses is pleiotropic. ABA affects callose deposition at plasmodesmata (therefore hindering the viral cell-to-cell movement), but here, we show that when callose synthase is down-regulated, ABA still induces resistance against infection with Bamboo mosaic virus (BaMV). By examining the potential connections between the ABA and RNA-silencing pathways in Arabidopsis (Arabidopsis thaliana), we showed that ABA regulates the expression of almost the whole ARGONAUTE (AGO) gene family, of which some are required for plant resistance against BaMV Our data show that BaMV infection and ABA treatment regulate the same set of AGOs, with positive effects on AGO1, AGO2, and AGO3, no effect on AGO7, and negative effects on AGO4 and AGO10 The BaMV-mediated regulation of AGO1, AGO2, and AGO3 is ABA dependent, because the accumulation of these AGOs in BaMV-infected ABA mutants did not reach the levels observed in infected wild-type plants. In addition, the AGO1-miR168a complex is dispensable for BaMV resistance, while AGO2 and AGO3 were important for ABA-mediated resistance. While most ago mutants showed increased susceptibility to BaMV infection (except ago10), ago1-27 showed reduced BaMV titers, which was attributed to the up-regulated levels of AGO2, AGO3, and AGO4 We have established that ABA regulates the expression of several members of the AGO family, and this regulation partially contributes to ABA-mediated resistance against BaMV These findings reveal another role for ABA in plants.

Figure 1.

Effects of ABA on plant resistance to BaMV in the gsl8i line. A, Plant responses to induction of the gsl8-RNAi line by treatment with dexamethasone. Wild-type (WT) and gsl8i plants treated with water showed similar phenotypes with or without ABA. At age ∼24 d, selected regions of gsl8i plants were watered with dexamethasone (25 µm in 1 L per watering) for 6 d, then all plants received double ABA treatments (100 µm): 1 d before BaMV infection and at 3 d postinfection (dpi). Leaves were inoculated with BaMV virions (1 µg in 5 µL of diethyl pyrocarbonate water per leaf) and collected at 4 dpi for further analysis. Photographs were taken when plants were 35 d old. B, BaMV RNA levels measured at 4 dpi by northern blotting in wild-type and gsl8i-induced plants with or without ABA. Average densities of the second subgenomic BaMV RNA were calculated from three different replicates, and the results were normalized to those in mock-treated lines. Statistical analysis was carried out using one-sided Student’s t test to determine the significance of regulation (**, P < 0.05). rRNA was used as an internal control. Reverse transcriptase (RT)-PCR was used to measure transcripts of Gsl8, HAI1/2 (an ABA-responsive gene), and eIF-1α (an internal control). Experiments were repeated three times with similar results. A, ABA-treated plants; M, mock-treated plants.

Figure 2.

Identification of ABREs in promoters of AGO genes, and the effect of ABA and BaMV infection on the expression of AGOs and ABA-related transcription factors (TFs). A, ABREs in AGO promoters. Promoter fragments located between −1,500 and +100 bp of the start codon were examined for the presence of ABREs, as described by Chang et al. (2008). Several ABREs were identified, with a confidence level of 100% and P value of ∼0.0002, in AGO1, AGO2, AGO3, AGO4, AGO5, AGO6, and AGO10. B and C, Relative expression of AGOs (B) and ABA-related TFs (C) upon BaMV infection with or without ABA treatment. Plants were first treated with ABA at 24 h before BaMV infection and then were given a second dose of ABA at 3 dpi. Leaves were collected at 4 dpi for RNA extraction and the determination of expression levels of members of the AGO family. Expression levels of AGO5, AGO6, and AGO9 were undetectable. RAB18 is an ABA-responsive gene. Actin was used as an internal control. Data are means ± sd from three biological replicates. Statistical analysis was carried as described in Figure 1: *, P < 0.05; **, P < 0.01; and ***, P < 0.005, by Student t test. Additional statistical analyses were carried out to compare levels between mock-sprayed and ABA-sprayed plants (both infected with BaMV).

Figure 3.

Progression of BaMV accumulation in Arabidopsis and its effect on AGO gene expression. Approximately 25-d-old Arabidopsis plants were inoculated with 1 µg of BaMV virions, while mock-inoculated plants were inoculated with water. A, Northern-blot analysis of BaMV RNA level in inoculated leaves at 3, 7, 10, and 14 d after BaMV infection. The rRNA level was examined as a loading control. M, Mock-treated plants; V, BaMV-infected plants. B to G, RTqPCR of the relative dynamics of AGO expression during BaMV progression: AGO1 (B), AGO2 (C), AGO3 (D), AGO4 (E), AGO7 (F), and AGO10 (G). Data are means ± sd of three biological replicates. Statistical analyses were carried out as described in Figure 1: *, P < 0.05 and **, P < 0.01, by Student t test.

Figure 4.

Effects of ABA on BaMV and AGO levels in the OsN3-O/E transgenic line. A, Northern-blot analysis (left) and quantitative analysis (right) of BaMV RNA level in wild-type (WT) and OsN3-O/E leaves at 10 dpi. M, Mock infected; V, BaMV infected. B to G, RTqPCR analysis of AGO gene levels in the OsN3-O/E line: AGO1 (B), AGO2 (C), AGO3 (D), AGO4 (E), AGO7 (F), and AGO10 (G). Data are means ± sd of three biological replicates. Statistical analysis was carried out as described in Figures 1 and 2: *, P < 0.05; **, P < 0.01; and ***, P < 0.005, by Student t test. Additional statistical analyses were performed to compare levels between wild-type infected and OsN3-O/E-infected lines.

Figure 5.

BaMV accumulation and AGO transcript expression in ABA-deficient mutants. RTqPCR analysis is shown for the levels of BaMV CP gene (A), AGO1 (B), AGO2 (C), AGO3 (D), AGO4 (E), AGO7 (F), and AGO10 (G) in mock and infected lines of aba2-1 and aao3 ABA-deficient mutants. Plants were infected with 1 µg of BaMV virions and then collected at 10 dpi for analysis as described previously (Alazem et al., 2014). Data are means ± sd of three biological replicates. Statistical analysis was carried out as described in Figure 1: *, P < 0.05; **, P < 0.01; and ***, P < 0.005, by Student t test. WT, Wild type.

Figure 6.

Effects of AGO mutants on the accumulation of BaMV in Arabidopsis. A and B, Northern-blot analyses of genomic and two subgenomic BaMV RNAs in ago1-27 and ago10-1 (A) and ago2-1, ago3-2, ago4-3, and ago7-2 (B) at 10 dpi. The relative accumulation of BaMV in the wild-type (WT) and mutants is shown below each blot. The northern blot in B was not exposed for as long as that in A to prevent saturation of the bands and allow for the measurement of BaMV density in ago2-1 and other backgrounds. M, Mock infected; V, BaMV infected. C, Protein blots for BaMV CP in the wild type and ago2-1 and ago3-2 mutants pretreated with 100 µm ABA 1 d before BaMV infection and then given another treatment at 3 dpi. The bottom gel represents Ponceau S staining, and data are means ± sd of three independent replicates. D, Protein blots of BaMV-GFP (top) and AGO-HA (middle). Commassie Blue staining (bottom) data are means ± sd of three independent replicates. Statistical analysis was carried out as described in Figure 3: **, P < 0.01.

Figure 7.

Effects of impaired AGO1 or miR168a on the accumulation of BaMV in Arabidopsis. A, Northern-blot analysis of miR168a levels in wild-type mock- and BaMV-infected lines. Relative accumulation of miR168a is shown at right. Data are means ± sd of three biological replicates. Statistical analysis was carried out as described in Figure 3: *, P < 0.05. B and C, Northern blots of BaMV RNAs in the transgenic line 4mAGO1 (B) and the mutant line mR168a-2. Plants were infected with 1 µg of BaMV virions, and leaves were collected at 10 dpi for further analysis. Col, Columbia-0; Ler, Landsberg erecta; M, mock infected; V, BaMV infected; WT, wild type.

Figure 8.

Effects of the ago1-27 mutant on the expression of AGO2, AGO3, AGO4, AGO7, and AGO10 and the susceptibility of ago2-1 and ago3-2 mutants. A to E, RTqPCR analysis of the levels of AGO2 (A), AGO3 (B), AGO4 (C), AGO7 (D), and AGO10 (E) in mock and infected lines of ago1-27 mutants at 10 dpi. Data are means ± sd of three biological replicates. Statistical analysis was carried out as described in Figure 1: *, P < 0.05 and **, P < 0.01. Additional statistical analysis was carried out to compare transcript levels in the infected wild-type (WT) and ago1-27 lines. F, Protein blot for BaMV-CP in ago1-27, ago2-1, and ago3-2 and the double mutants ago1-27;ago2-1 and ago1-27;ago3-2. Data are means ± sd of three replicates.

Figure 9.

Roles of ABA in modulating plant antiviral defenses. The schematic representation shows the central roles of ABA in modulating the plant response to BaMV infection. The ABA pathway is induced by BaMV infection (Alazem et al., 2014), which regulates several defense responses. However, BaMV benefits from this stimulation by inducing ABA2, which specifically supports the accumulation of BaMV (Alazem et al., 2014). In response to BaMV infection, AGO1, AGO2, and AGO3 are all up-regulated in an ABA-dependent manner, but only AGO2 and AGO3 have critical effects on resistance to BaMV (Figs. 3 and 5). AGO1 seems to play a different role in plant resistance to BaMV despite being up-regulated by ABA, as the mutant showed a resistance phenotype (Fig. 6A). The AGO1 regulator miR168a is also up-regulated in response to ABA or BaMV infection, and this up-regulation maintains mAGO1 RNA within certain levels (Vaucheret et al., 2006; Mallory and Vaucheret, 2009). When miR168a is impaired, AGO1 does not affect plant resistance to BaMV (Fig. 6). However, the mutant ago1-27 exhibits a resistance phenotype due to the increased levels of AGO2, AGO3, and AGO4 (Fig. 8). AGO4 is down-regulated by ABA and BaMV infection (Fig. 2). However, ABA overexpression and BaMV infection together increase AGO4 level (Fig. 4). Nevertheless, AGO4 still contributes to anti-BaMV defense (Fig. 5). ABA also was reported to be responsible for the enhanced callose deposition on plasmodesmata, which hinders viral cell-to-cell movement (Fraser and Whenham, 1989; Iriti and Faoro, 2008). However, induction of the dexamethasone-inducible RNAi line gsl8i, which inhibits callose synthase, did not affect the defensive role of ABA in plants (Fig. 1).