Characterization of Soybean mosaic virus resistance derived from inverted repeat-SMV-HC-Pro genes in multiple soybean cultivars

Abstract

Soybean mosaic virus resistance was significantly improved in multiple soybean cultivars through genetic transformation induced by inverted repeat-SMV- HC - Pro genes based on RNAi and post-transcriptional gene silencing. Here, we demonstrate Soybean mosaic virus (SMV) resistance in transgenic soybean plants. Transformation of five soybean genotypes with a construct containing inverted repeat-SMV-HC-Pro genes-induced high-level SMV resistance. Through leaf-painting assays, polymerase chain reaction (PCR) verification and LibertyLink(®) strip detection, 105 T0 and 1059 T1 plants were confirmed as transgene-positive. Southern blotting confirmed insertion of the T-DNA into the genomic DNA and revealed a low-copy integration pattern. Most T0 plants were fertile and transmitted the exogenous genes to their progenies (ratios of 3:1 or 15:1). In the T1 generation, virus resistance was evaluated visually after inoculation with SMV (strain SC3) and 441 plants were highly resistant (HR). SMV disease rating was classified on a scale with 0 = symptomless and 4 = mosaic symptoms with severe leaf curl. In the positive T1 plants, the disease rating on average was 1.42 (range 0.45-2.14) versus 3.2 (range 2-4) for the nontransformed plants. With the T2 generation, 75 transgene-positive plants were inoculated with SC3, and 57 HR plants were identified. Virus-induced seed coat mottling was eliminated in the resistant lines. Analysis of SMV levels in the plants was performed using quantitative real-time PCR and double-antibody sandwich enzyme-linked immunosorbent assays; the results revealed no virus or a gradual reduction over time in the viral content, thereby supporting the visual examination results. This is the first report demonstrating pathogen-derived resistance to SMV induced by inverted repeat-SMV-HC-Pro genes in multiple soybean cultivars. Our findings contribute positively to the study of transgenic SMV-resistance using RNA interference.

RNAi-mediated Soybean mosaic virus (SMV) resistance of a Korean Soybean cultivar

“…In this study, we used a modified half-seed method to transform a Korean cultivar, with a transformation efficiency of about 3 %. This is similar to the efficiency rate reported by Gao et al (2015), but lower than that of our previous experiment (Kim et al 2013). A transformation efficiency of about 3 % is not highly efficient, but is enough to produce more than 20 transgenic plants from 500 explant seeds.…”

“…Due to its limitation of SMV-CP RNAi for SMV resistance in soybean, HC-Pro was used to produce strong and complete resistance. Recently, Gao et al (2015) reported results similar to ours, obtained by producing SMV-resistant soybean plants of Chinese cultivars using an HC-Pro gene fragment from SMV SC3 strain via RNAi. One small difference is the presence of viral transcript after the SMV inoculation of transgenic plants; in our study, no accumulation of viral transcript was detected in resistant T 2 plants and T 3 seeds, via either northern blot analysis or reverse transcriptase PCR and quantitative real-time PCR analysis; however, a gradual decrease or low level of viral transcript accumulation was detected in T 1 plants via qRT-PCR during their study.…”

“…Using conventional breeding methods to produce SMV-resistant cultivars is a laborious and time-consuming procedure, and is often accompanied by the introgression of undesirable traits via the cross. To overcome the limitations of conventional breeding, genetic transformation can be a good method of improving virus resistance in soybean (Jagtap et al 2011;Furutani et al 2006;Kim et al 2013;Gao et al 2015).…”

The Current Status of the Soybean-Soybean Mosaic Virus (SMV) Pathosystem

“…The multiple soybean cultivars transformed with an RNA interference (RNAi) construct targeted for SMV HC-Pro displayed a significantly enhanced resistance against SMV (Gao et al, 2015). Soybean plants transformed with a single RNAi construct expressing separate short hairpins or inverted repeat (IR) (150 bp) derived from three different viruses (SMV, Alfalfa mosaic virus, and Bean pod mottle virus) confer robust systemic resistance to these viruses (Zhang et al, 2011).…”

Small RNA Based Genetic Engineering for Plant Viral Resistance: Application in Crop Protection

“…Stable transgenic plants for a variety of crops were generated expressing small RNAs in different ways and their reactions to targeted viruses were tested in both laboratory and field condition ( Supplementary Table S1 ). In some studies, the durability of resistance was tested for many generations (Wang et al, 2001, 2016; Liu et al, 2007; Cruz et al, 2014; Faria et al, 2014). According to the International Service for the Acquisition of Agri-Biotech Applications (ISAAA) website, dozens of transgenic crops resistance to virus generated with SRGE were approved for commercial release ( Supplementary Table S2 ).…”