Abstract
Transient gene expression approaches are valuable tools for rapid introduction of genes of interest and characterization of their functions in plants. Although agroinfiltration is the most effectively and routinely used method for transient expression of multiple genes in various plant species, this approach has been largely unsuccessful in soybean. In this study, we engineered soybean mosaic virus (SMV) as a dual-gene delivery vector to simultaneously deliver and express two genes in soybean cells. We further show the application of the SMV-based dual vector for a bimolecular fluorescence complementation assay to visualize in vivo protein–protein interactions in soybean and for a co-immunoprecipitation assay to identify cellular proteins interacting with SMV helper component protease. This approach provides a rapid and cost-effective tool for transient introduction of multiple traits into soybean and for in vivo characterization of the soybean cellular protein interaction network.
Highlights
Cleavage site between SMV P1 and helper component protease (HC-Pro) cistrons and successfully expressed single recombinant proteins in soybean[7]
We showed that simple rub-inoculation of plasmid DNAs of the SMV-based viral vectors was successful to cause infection and systemically express recombinant proteins in soybean plants[7]
We described the detailed procedure for a co-immunoprecipitation assay in combination with the SMV-based dual vector to identify cellular proteins interacting with SMV HC-Pro
Summary
Engineering of SMV as a dual-gene delivery vector. We previously developed a promising gene delivery system by engineering the full-length infectious cDNA clone of SMV strain G7H (pSMV-G7H)[7]. The desired genes can be stably and systemically delivered into soybean by simple rub-inoculation with intact plasmid DNA of this recombinant SMV-based vector. We modified pSMV-MCS by engineering an additional gene insertion cassette between nuclear inclusion b (NIb) and coat protein (CP) cistrons. We showed that DNA-mediated rub-inoculation of the SMV infectious cDNA construct yielded highly efficient infection on soybean plants[7]. In the present study, we sought to examine whether the additional insertion of the gene cassette between the NIb and CP cistrons affects the infectivity of the pSMV-Dual plasmid. RT-PCR analysis further confirmed that all the inoculated plants were systemically infected (Table 1)
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