The core technology of Seoul Women’s University is to provide a pPepMoV-Vb1/GFP system as a useful tool for researching plant virus replication, cell-to-cell and long-distance movements of virus and virus-host interaction, and for screening target genes (e.g., pathogenesis-related genes) faster, earlier and more efficient in transgenic or breeding plants.
Background and unmet needs: A plant virus-based vector is a useful tool for efficient expression of target foreign proteins in plants. Plant expression systems have a significant advantage compared to other methods of recombinant protein production since plants are much cheaper and easier in cultivation than cell cultures. This system provides rapid and transient expression of heterogonous genes systemically in plants. These virus-based vectors have been used to express genes of pharmaceutical, agronomic value, elicit genetically dominant, gene-silencing phenotypes in plants to determine the functions of unknown genes.
In cases like the full-length clone of other potyvirus, pPepMoV-Vb1 was also observed undesirable effects in bacteria cell. Although full-length cDNA clones were successfully obtained, these clones were unstable and produced very small colonies and low yield in E. coli. A multitue of approaches and researches have been made to obtain the full-length clone with high infectious abilities but have finally failed to.
Discovery and Achievements: The full-length cDNA clones of PepMoV-Vb1 of Seoul Women’s University are constructed, sequenced and modified downstreams from the bacteriophage SP6 promoter and 35S CaMV promoter for generating in vitro- or in vivo- transcripted viral RNAs, respectively. The biological activities are analyzed on systemic host N. benthamiana and C. annuum ECW by mechanical inoculation. p35SPepMoV-Vb1/GFP of Seoul Women’s University also demonstrate the construction of an infectious full-length cDNA clone of SP6 promoter-based PepMoV-Vb1 for generation of a novel plant viral vector.
This is the first demonstration of infectious full-length clone in PepMoV isolates, and suggests that pPepMoV-Vb1 can be used for understanding replication property of PepMoV and viral functional genomics. The pSP6PepMoV-Vb1-based system that permits both efficient systemic spread and high insertion capacity is suitable for the development of a viral vector for expressing foreign proteins in Solanaceae plant. The ability to generate infectious in vitro transcripts from cloned cDNA of potyvirus also opens the possibility to study their biological activities at the molecular level in the sense that the ability to generate biologically functional RNA transcripts from those full-length cDNA clones provides a powerful tool to study the gene functions, replication, and pathogenicity of PepMoV at the molecular level.
Fig. 1. The procedures schematically represent a screening method to isolate infectious full-length cDNA clones.
Fig. 2. pSP6PepMoV-Vb1 contains the full-length cDNA clone of PepMoV-Vb1 (a) and pSP6PepMoV-Vb1/GFP (b) is fused with turboGFP.
Fig. 3. The isolated full-length cDNAs of p35SPepMoV-Vb1 (a) and pSP6PepMoV-Vb1/GFP (b) is confirmed by restriction fragment length polymorphism (RFLP).
Fig. 4. The phenotypes between Wt-pepMoV-Vb and SP6PepMoV-Vb1 in the leaves of C. annuum (a) and N. benthamiana (b) exhibit no differences after infection.
Fig. 5. Green fluorescence is stably detected on the leaves of C. annuum (a) and N. benthamiana (b), and the flowers and roots of N. benthamiana (c) infected with the pSP6PepMoV-Vb1/GFP.
Fig. 6. Infection of N. benthamiana with pSP6PepMoV-Vb1/GFP and pSP6PepMoV-Vb1/GFP-NAT exhibits the systemic expression of the GFP gene.
Accordingly, the pPepMoV-Vb1/GFP system will be a useful and powerful tool to study plant virus replication, cell-to-cell and long-distance movements of virus and virus-host interaction at whole plant and cellular level. Additionally, GFP-tagged PepMoV-Vb1 should offer the possibility to screen faster, earlier and more efficient the response of resistance plants to PepMoV infection in transgenic or breeding plants.