Soybean RNA interference lines silenced for eIF4E show broad potyvirus resistance

Abstract

Soybean mosaic virus (SMV), a potyvirus, is the most prevalent and destructive viral pathogen in soybean-planting regions of China. Moreover, other potyviruses, including bean common mosaic virus (BCMV) and watermelon mosaic virus (WMV), also threaten soybean farming. The eukaryotic translation initiation factor 4E (eIF4E) plays a critical role in controlling resistance/susceptibility to potyviruses in plants. In the present study, much higher SMV-induced eIF4E1 expression levels were detected in a susceptible soybean cultivar when compared with a resistant cultivar, suggesting the involvement of eIF4E1 in the response to SMV by the susceptible cultivar. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that soybean eIF4E1 interacted with SMV VPg in the nucleus and with SMV NIa-Pro/NIb in the cytoplasm, revealing the involvement of VPg, NIa-Pro, and NIb in SMV infection and multiplication. Furthermore, transgenic soybeans silenced for eIF4E were produced using an RNA interference approach. Through monitoring for viral symptoms and viral titers, robust and broad-spectrum resistance was confirmed against five SMV strains (SC3/7/15/18 and SMV-R), BCMV, and WMV in the transgenic plants. Our findings represent fresh insights for investigating the mechanism underlying eIF4E-mediated resistance in soybean and also suggest an effective alternative for breeding soybean with broad-spectrum viral resistance.

Keywords: Potyvirus; Soybean mosaic virus; Agrobacterium-mediated transformation; RNA interference; broad-spectrum resistance; eIF4E; soybean.

Figure 1

Spatiotemporal expression analysis of eIF4E1 in soybean cultivars Tianlong 1 and Kefeng 1 using RT‐qPCR. (a) Temporal expression profiles of eIF4E1 in the inoculated leaves after challenge with soybean mosaic virus (SMV) strain SC3 at different time points. Data were calibrated using phosphate‐buffered saline (PBS)‐inoculated controls. (b) Spatial expression profiles of eIF4E1 in various healthy tissues. Error bars indicate SD (n = 3). Asterisks indicate significant difference between susceptible and resistant plants at the corresponding time points and tissues, t test, p < .001. Results are representative of three independent experiments

Figure 2

Subcellular localization of soybean eIF4E1 and analysis of protein–protein interaction with soybean mosaic virus (SMV). (a) Subcellular localization in Nicotiana benthamiana leaf cells. Soybean eIF4E1 fused with green fluorescent protein (GFP) was agroinfiltrated into leaves of 4‐week‐old N. benthamiana. Scale bars = 20 μm. (b) Yeast two‐hybrid screen system. Yeast co‐transformants were identified on selective quadruple dropout medium SD/−Leu/−Trp/−Ade/−His/+X‐α‐Gal with blue color staining. Yeast containing pBT3‐STE + pPR3, pBT3‐STE‐eIF4E1 + pPR3, or pBT3‐STE + pPR3‐SMV served as negative controls. Yeast cells co‐transformed with pPR3‐P3N‐PIPO + pBT3‐STE‐GOS12 were used as positive control. (c) Bimolecular fluorescence complementation assay. eIF4E1‐YN and SMV‐YC were co‐agroinfiltrated into leaves of 4‐week‐old N. benthamiana. Interactions between YN and YC, YN and SMV‐YC, and eIF4E1‐YN and YC were used as negative controls. Scale bars = 20 μm

Figure 3

RT‐qPCR and Southern blot analyses of transgenic soybean plants. (a) RT‐qPCR detection of relative expression levels of eIF4E1 in positive T₀ plants. The y axis indicates eIF4E1 transcript levels. The x axis indicates T₀ and nontransformed (NT) plants. Results are representative of three independent experiments with error bars indicating SD (n = 3). (b) Southern blot hybridization analysis in T₁ generation derived from T₀ line 1. Total genomic DNA (c.30 μg) was digested with HindIII and hybridized with a bar probe (Figure S1) labelled with DIG. M, DNA molecular size; +, pB7GWIWG2(II)‐eIF4E1i vector used as positive control; −, genomic DNA of nontransformed soybean plants used as negative control; 1–10 represent transgene‐positive T₁ plants

Figure 4

Soybean mosaic virus (SMV) resistance assessments in T₁/T₂ generations. (a) Appearance of symptoms in T₁ soybean plants after challenge with SMV strain SC3. Mock‐inoculated and SMV‐inoculated nontransformed plants were used as controls. (b) RT‐qPCR detection of systemic virus accumulation in leaves of T₂ plants derived from T₀ line 1 after challenge with SMV strain SC3. The y axis indicates SMV transcript levels at 15 and 30 days post‐inoculation (dpi). The x axis indicates T₂ and nontransformed (NT) plants. Results are representative of three independent experiments with error bars indicating SD (n = 3)

Figure 5

Broad‐spectrum resistance assessments in homozygous T₃/T₄ generations derived from T₀ line 1. (a) Appearance of systemic symptoms on leaves of T₃ soybean plants after challenge with different viruses. Virus‐inoculated nontransformed plants were used as controls. SMV, soybean mosaic virus; BCMV, bean common mosaic virus; WMV, watermelon mosaic virus; BPMV, bean pod mottle virus. (b) RT‐qPCR detection of systemic virus accumulation in leaves of T₄ plants after challenge with different viruses. The y axes indicate virus transcript levels at 15 and 30 days post‐inoculation (dpi). The x axes indicate T₄ and nontransformed (NT) plants. Results are representative of three independent experiments with error bars indicating SD (n = 3)