Silencing of a viral RNA silencing suppressor in transgenic plants

High expression levels of the helper component proteinase (HCpro), a known virus suppressor of RNA silencing, were attained in Nicotiana benthamiana transformed with the HCpro cistron of Potato virus A (PVA, genus Potyvirus). No spontaneous silencing of the HCpro transgene was observed, in contrast to the PVA coat protein (CP)-encoding transgene in other transgenic lines. HCpro- transgenic plants were initially susceptible to PVA and were systemically infected by 14 days post-inoculation (p.i.) but, I to 2 weeks later, the new expanding leaves at positions +6 and +7 above the inoculated leaf showed a peculiar recovery phenotype. Leaf tips (the oldest part of the leaf) were chlorotic and contained high titres of PVA, whereas the rest of the leaf was symptomless and contained greatly reduced or non-detectable levels of viral RNA, CP and transgene mRNA. The spatial recovery phenotype suggests that RNA silencing is initiated in close proximity to meristematic tissues. Leaves at position +8 and higher were symptomless and virus-free but not completely resistant to mechanical inoculation with PVA. However, they were not infected with the virus systemically transported from the lower infected leaves, suggesting a vascular tissue-based resistance mechanism. Recovery of the HCpro-transgenic plants from infection with different PVA isolates was dependent on the level of sequence homology with the transgene. Methylation of the HCpro transgene followed recovery. These data show that the transgene mRNA for a silencing suppressor can be silenced by a presumably 'strong' silencing inducer (replicating homologous virus).