Elements Involved in the Rsv3-Mediated Extreme Resistance against an Avirulent Strain of Soybean Mosaic Virus

Abstract

Extreme resistance (ER) is a type of R-gene-mediated resistance that rapidly induces a symptomless resistance phenotype, which is different from the phenotypical R-resistance manifested by the programmed cell death, accumulation of reactive oxygen species, and hypersensitive response. The Rsv3 gene in soybean cultivar L29 is responsible for ER against the avirulent strain G5H of soybean mosaic virus (SMV), but is ineffective against the virulent strain G7H. Rsv3-mediated ER is achieved through the rapid accumulation of callose, which arrests SMV-G5H at the point of infection. Callose accumulation, however, may not be the lone mechanism of this ER. Analyses of RNA-seq data obtained from infected soybean plants revealed a rapid induction of the abscisic acid pathway at 8 h post infection (hpi) in response to G5H but not to G7H, which resulted in the down-regulation of transcripts encoding β-1,3 glucanases that degrade callose in G5H-infected but not G7H-infected plants. In addition, parts of the autophagy and the small interfering (si) RNA pathways were temporally up-regulated at 24 hpi in response to G5H but not in response to G7H. The jasmonic acid (JA) pathway and many WRKY factors were clearly up-regulated only in G7H-infected plants. These results suggest that ER against SMV-G5H is achieved through the quick and temporary induction of ABA, autophagy, and the siRNA pathways, which rapidly eliminate G5H. The results also suggest that suppression of the JA pathway in the case of G5H is important for the Rsv3-mediated ER.

Keywords: Rsv3; extreme resistance; signal transduction pathways; soybean mosaic virus.

Figure 1

Venn diagrams illustrating the clustering of the differentially expressed genes (DEGs). The diagrams show up- and downregulated DEGs from soybean plants (cultivar L29 carrying the Rsv3 ER gene) that were mock inoculated, infected with SMV avirulent G5H, or infected with SMV virulent G7H. DEGs were subjected to calculation of intersection using Venn diagrams: Downregulated DEGs at 8 hpi (A), 24 hpi (B), and 54 hpi (C). Upregulated DEGs at 8 hpi (D), 24 hpi (E), and 54 hpi (F). DEG lists were obtained from RNA-seq data using EXPath Tool by comparing the expression levels of healthy plants to those in mock plants and G5H- and G7H-infected plants at different time points.

Figure 2

Relative distribution of enriched pathways in soybean plants (L29 Rsv3-cultivar) infected with SMV-G5H at different time points. Enriched pathways in the upregulated DEGs in G5H-infected plants at 8 hpi (A), 24 hpi (C), and 54 hpi (E). Enriched pathway in the downregulated DEGs in G5H-infected plants at 8 hpi (B), 24 hpi (D), and 54 hpi (F).

Figure 3

Heatmaps of genes involved in the ABA pathway, synthesis of β-1,3 glucanases and glucan in soybean plants (L29 Rsv3-cultivar) infected with SMV-G5H and SMV-G7H. Gene expressions from soybean plants infected with the avirulent SMV strain G5H and the virulent strain G7H at 8, 24, and 54 hpi were used to generate heatmaps for genes involved in the ABA pathway (A), genes that encode for β-1,3 glucanases (B), and glucan synthesis and plasmodesmata callose-binding proteins (PCBPs) (C). Red and blue indicate up- and down-regulation, respectively, in terms of the fold-change indicated by the scale above each heatmap.

Figure 4

Effect of exogenous application of ABA on the SMV-G7H-GFP and PP2C3a levels in L29 soybean cultivar. (A) Protein blot for SMV-G7H-GFP in response to exogenous application of ABA (100 µM) or Mock (0.1% MeOH). Upper panel is GFP level, and lower panel is ponceau-S as loading control. (B) RTqPCR of PP2C3a gene in response to SMV-G7H-GFP infection, ABA, or a mix of both. Actin11 was used as internal control. Data are means ± standard deviation from 3 biological replicates. Statistical analysis was carried out using one-sided student t test to determine the significance of the regulation compared to mock-treated plants (where * and ** indicates p < 0.05, and p < 0.01, respectively).

Figure 5

Heatmaps of genes involved in autophagy and siRNA pathways. Heatmaps for genes involved in authophagy (A) and the siRNA pathway (B) were generated as described in Figure 3 legend.

Figure 6

Heatmaps of genes involved in the JA pathway WRKY gene family. Heatmaps for genes involved in the JA pathway (A) and the WRKY gene family (B) were generated as described in Figure 3 legend.