Robust RNAi-Based Resistance to Mixed Infection of Three Viruses in Soybean Plants Expressing Separate Short Hairpins from a Single Transgene

Abstract

Transgenic plants expressing double-stranded RNA (dsRNA) of virus origin have been previously shown to confer resistance to virus infections through the highly conserved RNA-targeting process termed RNA silencing or RNA interference (RNAi). In this study we applied this strategy to soybean plants and achieved robust resistance to multiple viruses with a single dsRNA-expressing transgene. Unlike previous reports that relied on the expression of one long inverted repeat (IR) combining sequences of several viruses, our improved strategy utilized a transgene designed to express several shorter IRs. Each of these short IRs contains highly conserved sequences of one virus, forming dsRNA of less than 150 bp. These short dsRNA stems were interspersed with single-stranded sequences to prevent homologous recombination during the transgene assembly process. Three such short IRs with sequences of unrelated soybean-infecting viruses (Alfalfa mosaic virus, Bean pod mottle virus, and Soybean mosaic virus) were assembled into a single transgene under control of the 35S promoter and terminator of Cauliflower mosaic virus. Three independent transgenic lines were obtained and all of them exhibited strong systemic resistance to the simultaneous infection of the three viruses. These results demonstrate the effectiveness of this very straight forward strategy for engineering RNAi-based virus resistance in a major crop plant. More importantly, our strategy of construct assembly makes it easy to incorporate additional short IRs in the transgene, thus expanding the spectrum of virus resistance. Finally, this strategy could be easily adapted to control virus problems of other crop plants.

Generation of an Attenuated, Cross-Protective Pepino mosaic virus Variant Through Alignment-Guided Mutagenesis of the Viral Capsid Protein

“…For each PepMV variant (WT as well as mutants), four infected plants were chosen, and the youngest systemic leaves were collected, processed separately, and assessed for the presence of PepMV using a commercial ELISA kit (Agdia Inc., Elkhart, IN) at 2 and 4 weeks postinoculation (wpi). The results were recorded by measuring the absorbance at 405 nm with an ELISA plate reader (DTX 880, Multimode Detector; Beckman Coulter Inc., CA), and converted to relative values as described in a previous study (35).…”

“…One typical set of results was shown in Figure 2C. To permit comparisons between different sets of experiments, the optical readings of ELISA results were converted to relative values by dividing the readings of test wells with the average of readings of negative control wells, with a relative value of two or above counted as positive (35). Although the CP of most mutants accumulated to levels that were at least fivefold higher than negative controls, that of both KD and KD/VC mutants was consistently low, at approximately threeand twofold higher than the negative controls, respectively (Fig.…”

Genetic Engineering and Sustainable Crop Disease Management: Opportunities for Case-by-Case Decision-Making

“…While consumers may be hesitant to eat transgenic papaya containing a viral coat-protein gene, they may be surprised to know that they are eating complete virus particles in fruit harvested from non-transgenic, infected trees, including fruit from cross-protected trees. RNAi provides control of other destructive viruses of crops, including the viral complexes that attack cassava in East Africa [22,82], soybean [83], and summer squash [84], and others [47,85].…”

DNA Sequencing and Crop Protection

“…Different plant-virus systems have been investigated for inducing immunity by means of RNAi technology and they are well resumed by and . Very recently Zhang et al (2011) were able to achieved robust resistance to multiple viruses (Alfalfa mosaic virus, Bean pod mottle virus, and Soybean mosaic virus) with a single dsRNA-expressing transgene in soybean plants opening new interesting perspectives in practical viruses control. It has also been assessed that plant viruses have evolved mechanism to overcome the host antiviral silencing by encoding diverse viral suppressors of RNA silencing (VSRs).…”