An Avirulent Strain of Soybean Mosaic Virus Reverses the Defensive Effect of Abscisic Acid in a Susceptible Soybean Cultivar

Abstract

In soybean cultivar L29, the Rsv3 gene is responsible for extreme resistance (ER) against the soybean mosaic virus avirulent strain G5H, but is ineffective against the virulent strain G7H. Part of this ER is attributed to the rapid increase in abscisic acid (ABA) and callose, and to the rapid induction of several genes in the RNA-silencing pathway. Whether these two defense mechanisms are correlated or separated in the ER is unknown. Here, we found that ABA treatment of L29 plants increased the expression of several antiviral RNA-silencing genes as well as the PP2C3a gene, which was previously shown to increase callose accumulation; as a consequence, ABA increased the resistance of L29 plants to G7H. The effect of ABA treatment on these genes was weaker in the rsv3-null cultivar (Somyungkong) than in L29. Besides, G5H-infection of Somyungkong plants subverted the effect of ABA leading to reduced callose accumulation and decreased expression of several RNA-silencing genes, which resulted in increased susceptibility to G5H infection. ABA treatment, however, still induced some resistance to G7H in Somyungkong, but only AGO7b was significantly induced. Our data suggest that Rsv3 modulates the effect of ABA on these two resistance mechanisms, i.e., callose accumulation and the antiviral RNA-silencing pathway, and that in the absence of Rsv3, some strains can reverse the effect of ABA and thereby facilitate their replication and spread.

Keywords: RNA-silencing pathway; Rsv3; abscisic acid; callose; extreme resistance; plant virus; plant–virus interactions; soybean mosaic virus.

Figure 1

Effect of abscisic acid (ABA) treatment on the accumulation of soybean mosaic virus (SMV) strains and on PP2C3a expression in L29 and Somyungkong (SMK) soybean cultivars. Protein blots of the soybean mosaic virus (SMV) in response to exogenous application of ABA (100 μM) or Mock (0.1 % MeOH) in: (A) L29 cultivar (carries the Rsv3 resistance gene) infected with the virulent strain G7H, or (B) SMK cultivar (rsv3-null) infected with G7H or the avirulent strain G5H (both strains express GFP). The upper panel shows the GFP level, and the lower panel shows Ponceau-S, which was used as a loading control. Relative expression levels of PP2C3a using RT-qPCR in response to G7H infection in L29 plants (C), G7H infection in SMK plants (D), and G5H plants (E). Actin11 was used as the internal control. Plants were sampled at 5 dpi. For (C–E), values are means standard deviation (SD) of three biological replicates. In each panel, values were compared to that of the mock-treated, uninfected plants (the bar on the left) with one-sided Student’s t-tests; * and ** indicate a significant difference at P < 0.05 and <0.01, respectively.

Figure 2

Levels of callose and reactive oxygen species (ROS) in L29 plants as affected by ABA treatment and G7H infection. L29 plants treated with Mock (0.1% MeOH) or with ABA (100 µM) were also treated with sap from healthy plants (HS) or sap from G7H-GFP-infected plants (G7H). Samples collected 5 dpi were subjected to aniline blue staining to reveal callose accumulation; scale bar = 20 µm (A). Callose fluorescence from aniline-blue-stained leaves was quantified using Image J software (B). ROS accumulation as indicated by DAB and as affected by ABA treatment, G7H-GFP infection, or both (C). The experiment was carried out in three independent replicates where values are means ± SD of three biological replicates, and statistical analysis was carried out as described in the legend of Figure 1, with additional comparison between Mock-G7H and ABA-G7H plants. * and ** indicate a significant difference at P < 0.05 and <0.01, respectively.

Figure 3

Levels of callose and ROS in SMK plants as affected by ABA treatment and G7H or G5H infection. SMK plants were treated with combinations of Mock (0.1% MeOH) or ABA (100 µM), and sap from healthy plants (HS), sap from G7H-GFP-infected plants (G7H), or sap from G5H-GFP-infected plants. Samples were collected 5 dpi and subjected to (A) aniline blue staining to detect callose accumulation levels, (B) quantification of callose fluorescence, and (C) DAB staining to reveal ROS accumulation. The experiment was carried out in three independent replicates, and values are means ± SD of three biological replicates. Statistical analysis was carried out as described in Figure legend 1, with additional comparison between Mock-G5H and ABA-G5H plants. Asterisks in red color indicate significant decrease at P < 0.05.

Figure 4

Expression levels of RNA-silencing genes in L29 plants infected with G7H. Expression levels of Argonaute (AGO) genes (A), and dicer like (DCL) and RNA polymerase (RDR) genes (B) in L29 plants in response to G7H infection, ABA treatment, or both. Plants were collected 5 dpi for RNA extraction and RT-qPCR. Values are means ± SD of three biological replicates. Statistical analysis was carried out for each gene using Student t-test; the means for plants treated with ABA alone, G7H alone, or the combination of ABA and G7H were individually compared with the mean for plants that were not treated with ABA or G7H: * and * indicate a significant increase or decrease, respectively, at P < 0.05. Additional analyses were carried for ABA-G7H in comparison with Mock-G7H where ns and * indicate non-significance and significance at P < 0.05, respectively.

Figure 5

Expression levels of RNA-silencing genes in SMK plants infected with G7H. Expression levels of AGO genes (A), and DCL and RDR genes (B) in SMK soybean cultivar (rsv3-null) in response to G7H infection, ABA treatment, or both. Plants were collected 5 dpi for RNA extraction and RT-qPCR. Values are means ± SD of three biological replicates. Statistical analysis was carried out for each gene using Student t-test; the means for plants treated with ABA alone, G7H alone, or the combination of ABA and G7H were individually compared with the mean for plants that were not treated with ABA or G7H: * and * indicate a significant increase or decrease, respectively, at P < 0.05. Additional analyses were carried for ABA-G5H in comparison with Mock-G7H where ns and * indicate non-significance and significance at P < 0.05, respectively.

Figure 6

Expression levels of RNA-silencing genes in SMK plants infected with G5H. Expression levels of AGO genes (A), and DCL and RDR genes (B) in SMK soybean cultivar (rsv3-null) in response to G5H infection, ABA treatment, or both. Plants were collected 5 dpi for RNA extraction and RT-qPCR. Data are means ± standard deviation from three biological replicates. Statistical analysis was carried out for each gene using Student t-test; the means for plants treated with ABA alone, G5H alone, or the combination of ABA and G5H were individually compared with the mean for plants that were not treated with ABA or G5H: * and * indicate a significant increase or decrease, respectively, at P < 0.05. Additional analyses were carried for ABA-G5H in comparison with Mock-G5H where ns and * indicate non-significance and significance at P < 0.05, respectively.