Evidence for sense RNA-mediated protection to PVYN in tobacco plants transformed with the viral coat protein cistron

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Abstract
The coat protein (CP) cistron of the tobacco veinal necrosis strain of potato virus Y (PVYN), supplemented with translational start signals, was cloned into an Agrobacterium tumefaciens Ti transformation vector. Transformation of tobacco leaf discs resulted in 99 transgenic lines which were subsequently analysed for the presence and expression, at both the transcriptional and translational level, of the CP-gene. Although CP-specific RNA transcripts were produced in all plants no CP could be detected by several sensitive immunological techniques. Upon mechanical inoculation of progeny lines of selfpollinated original transformants (S1) with PVYN, protection levels of 20 and 95%, respectively, could be observed in two out of ten lines tested. This level of protection increased to 100% in the S2 progeny obtained from self-pollination of virus-protected S1 plants. Transformation of tobacco leaf discs with a PVYN CP construct from which the ATG start codon had been removed by site-directed mutagenesis resulted in 57 transgenic lines that all produced CP-specific transcripts. Mechanical inoculation with PVYN of S1 progeny plants of several of these lines resulted in resistance to a similar level and extent as in the S1 progeny of plants transformed with the intact CP cistron. The results obtained strongly suggest that the resistance observed in the transgenic plants is principally based on the presence of PVYN CP RNA sequences rather than on the accumulation of viral coat protein.