Host impact on the stability of a plant virus gene vector as measured by a new fluorescent local lesion passaging assay.
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abstract
Viruses can be used as vectors for transient expression of proteins in plants but frequently foreign gene inserts are not maintained stably over time due to recombination events. In this study the hypothesis was that the choice of plant host affects the foreign gene retention level by a Tomato bushy stunt virus (TBSV) vector expressing green fluorescent protein (GFP). To accomplish this, a novel virus vector integrity bioassay was developed based on an old concept, whereby RNA transcripts of the TBSV-GFP vector were rub-inoculated onto leaves of test plants, and at 3 days post inoculation (dpi), these leaves were used as inoculum for passage to cowpea (Vigna unguiculata), a local lesion host. Chlorotic lesions at points of virus infection were counted on cowpea at 4dpi and then the leaves were exposed to ultraviolet light to count green fluorescent foci. These tests with seven different plant species covering five families showed that the percentage of green fluorescent lesions varied on the cowpea indicator plants in a host-dependent manner. For instance, the vector was relatively unstable in Nicotiana benthamiana, tomato, bean, and spinach, but compared to those its stability in lettuce was significantly improved (~3-fold). This host-dependent effect suggests that some plants may present a more suitable environment than others to support or maintain optimum levels of virus vector-mediated foreign gene expression.
