Analysis of transgenic grapevine ( Vitis rupestris) and Nicotiana benthamiana plants expressing an Arabis mosaic virus coat protein gene

Abstract

A disarmed LBA4404 strain of Agrobacterium tumefaciens harboring a binary vector which contained chimeric genes encoding the neomycin phosphotransferase ( npt II) and the coat protein (CP) of Arabis mosaic nepovirus (ArMV) was used in co-cultivation experiments with leaf discs of Nicotiana benthamiana and somatic embryos of the grapevine rootstock cultivar Vitis rupestris. Transgenic N. benthamiana expressing the ArMV CP gene were regenerated and six independent lines were characterized. Enzyme-linked immunosorbent assay (ELISA) performed on leaf tissue demonstrated the accumulation of the ArMV CP in five of the six lines analyzed. Immunosorbent electron microscopy (ISEM) studies revealed the presence of virion-like isometric particles (VLPs) reacting to a rabbit antiserum specific to ArMV virions. ArMV-CP expressing transgenic N. benthamiana lines showed protection against ArMV expressed as a delay in infection and a reduction of the percentage of infected plants. Four independent transgenic lines of V. rupestris transformed with the ArMV CP gene were regenerated and characterized. In contrast to N. benthamiana, transgenic V. rupestris did not accumulate the ArMV CP at levels detectable by ELISA and no VLPs could be observed by ISEM. Northern blot analysis showed that the ArMV CP mRNA was expressed at lower level in V. rupestris compared with N. benthamiana. The reason for this difference in transgene expression and/or mRNA stability between grapevine and N. benthamiana is unclear, but the genetic state of the transgene(s) (homozygous in N. benthamiana versus hemizygous in V. rupestris) may have an effect on gene expression.