Abstract
Plant viral vectors enable the expression of proteins at high levels in a relatively short time. For many purposes (e.g., cell biological interaction studies) it may be desirable to express more than one protein in a single cell but that is often not feasible when using a single virus vector. Such a co-expression strategy requires the simultaneous delivery by two compatible and non-competitive viruses that can co-exist to each express a separate protein. Here, we report on the use of two agro-launchable coat-protein gene substitution GFP-expressing virus vector systems based on Tomato bushy stunt virus (TBSV) referred to as TG, and Tobacco mosaic virus (TMV) annotated as TRBO-G. TG expressed GFP in Nicotiana benthamiana, tomato, lettuce and cowpea, whereas expression from TRBO-G was detected only in the first two species. Upon co-infiltration of the two vectors co-expression was monitored by: molecular detection of the two slightly differently sized GFPs, suppressor-complementation assays, and using TG in combination with TRBO-RFP. All the results revealed that in N. benthamiana and tomato the TBSV and TMV vectors accumulated and expressed proteins in the same plants, the same leaves, and in the same cells. Therefore, co-expression by these two vectors provides a platform for fast and high level expression of proteins to study their cell biology or other properties.
Highlights
Expression of foreign proteins in plants is normally achieved via transformation with Agrobacterium tumefaciens or by biolistics, to generate stable transgenic plants within several months by incorporating foreign DNA into the plant chromosome
One technical disadvantage associated with virus-mediated protein expression is that due to size constraints with regards to the insertion of foreign material, most viruses only support the expression of one protein (Gleba et al, 2004), while many bioactive complexes are often composed of oligomers of different proteins
The present study addressed the question whether Tomato bushy stunt virus (TBSV) and Tobacco mosaic virus (TMV) virus vectors can express foreign proteins at high levels in the same cells of different plant species
Summary
Expression of foreign proteins in plants is normally achieved via transformation with Agrobacterium tumefaciens or by biolistics, to generate stable transgenic plants within several months by incorporating foreign DNA into the plant chromosome. Using the same viral vector construct as backbone for different proteins in coinfections often can lead to one construct interfering with or outcompeting the other (Gleba et al, 2007) This phenomenon is referred to as superinfection exclusion (Folimonova et al, 2010), which probably contributes at the molecular level to the control method of cross protection (Hull, 2002). Overcoming these limitations requires the use of at least two non-competitive plant viral vectors to achieve expression of more than one protein. Such a strategy was reported by co-expressing two polypeptides using vectors based on Tobacco mosaic virus (TMV) and Potato virus X (PVX) (Giritch et al, 2006) that is used to produce pharmaceutical vaccine
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