Sense and antisense coat protein gene constructs confer high levels of resistance to tomato ringspot nepovirus in transgenic Nicotiana species

The coat protein (cp) gene and the 3' end untranslated region of a peach isolate of tomato ringspot virus (TomRSV) were cloned from purified total viral RNA and sequenced. Reverse transcription and polymerase chain reaction (RT-PCR) were used to engineer the TomRSV cp gene so that it could be cloned into plasmid vectors designed for either in vitro transcription or plant expression. The cloned TomRSV cp gene was used to transform Nicotiana benthamiana and N. tabacum plants, a systemic and a local lesion host, respectively. After challenge inoculation with the TomRSV peach isolate, several R(0), R(1), and R(2) resistant transgenic lines containing sense and antisense cp constructs exhibited different levels of protection ranging from complete resistance to delay in symptom appearance or reduction in symptom severity. Interestingly, cp gene expression levels were undetectable by enzyme-linked immunosorbent assay (ELISA) in the resistant lines containing cp sense constructs, and levels of cp transcripts were low or undetectable by Northern blot on resistant sense and antisense lines. The high level of resistance obtained in Nicotiana spp. offers important prospects for the engineering of TomRSV resistance into several economically important fruit and berry crops susceptible to this nepovirus.