Exploiting An Intron-containing Tombusvirus-based Vector System

As a novel expression system, plant virus RNA-based vectors allow for the rapid, high level, transient expression of proteins in whole plants. Tomato bushy stunt virus (TBSV) has a wide host range and no biological vector, making TBSV a suitable and biosafe candidate for construction of plant virus RNA-based expression vector. Toward developing a novel plant virus RNA-based expression system, we have investigated the possibility of using TBSV as an expression vector. The TBSV-based vectors yield high levels of foreign gene expression in inoculated cells and leaves. However, results with the original TBSV-derived CP replacement gene vectors are inconsistent. Often the numbers of green foci were extremely low.To solve the above-mentioned problem, we optimaized TBSV-derived CP replacement gene vectors and the relative constructs were generated. The different TBSV-based vectors were delivered into Nicotiana benthamiana plants by means of an agrobacterium-mediated transformation assay (agroinfiltration) and the accumulation level of GFP in agroinfiltrated tobacco leaves has been analyzed. The results and conclusions were showed as follows.①The viral vector carrying modified insertion site, pTBSV-WY2, was genetically engineered based on pTBSV-WY1 vector by subcloning technology. Those two vectors were agroinfiltrated on Nicotiana benthamiana plants and then analyzed for their expression levels of green fluorescence protein by Western blotting and ELISA. The results showed that the relative GFP accumulation lelvel of pTBSV-WY2 was higher than pTBSV-WYl and the results form pTBSV-WY2 were more consistent than the results form pTBSV-WY1. The results implicate the insertion site in viral genome either have effect on the productivity or stability.②2To compare the effect of promoter on TBSV-based expression vector, the ACT2 promoter (AB026654,57962-58748) from arabidopsis thaliana was used to construct the viral vector pTBSV-WY3. The results showd that the expressed GFP have indistinguishable expression levels in the presence of 35S promoter or ACT2 promotor. The changing of GFP expression levels across various time points was different from each other and we speculated that the inherent characteristics of the promoter are the possibility of the variation in the GFP expression pattern. ③To test the effect of the presence of introns within TBSV genome, introns were added at up to 11 positions within the RdRp and the P19/P22 sequences. Three intron-containing Tombusvirus-based Vectors, pTBSV-WY3-2intr, pTBSV-WY3-9intr and pTBSV-WY3-11intr, were constructed. Northern blot results showed that the inserted introns can be recognized and removed by the RNA processing machinery properly. But addition of introns had no significant effect on production of heterologous proteins in plants. This may be related to intron positions, numbers and sizes within viral genome and it still need further research.These results will be beneficial for the further research and application of TBSV-based plant virus expression vectors and for guiding the development of other plant virus RNA-based expression systems.