Constraints to virus infection in Nicotiana benthamiana plants transformed with a potyvirus amplicon

Background: Plant genomes have been transformed with full-length cDNA copies of viral genomes, giving rise to what has been called 'amplicon' systems, trying to combine the genetic stability of transgenic plants with the elevated replication rate of plant viruses. However, amplicons' performance has been very variable regardless of the virus on which they are based. This has boosted further interest in understanding the underlying mechanisms that cause this behavior differences, and in developing strategies to control amplicon expression. - Results: Nicotiana benthamiana plants were transformed with an amplicon consisting of a full-length cDNA of the potyvirus Plum pox virus (PPV) genome modified to include a GFP reporter gene. Amplicon expression exhibited a great variability among different transgenic lines and even among different plants of the same line. Plants of the line 10.6 initially developed without signs of amplicon expression, but at different times some of them started to display sporadic infection foci in leaves approaching maturity. The infection progressed systemically, but at later times the infected plants recovered and returned to an amplicon-inactive state. The failure to detect virus-specific siRNAs in 10.6 plants before amplicon induction and after recovery suggested that a strong amplicon-specific RNA silencing is not established in these plants. However, the coexpression of extra viral silencing suppressors caused some amplicon activation, suggesting that a low level of RNA silencing could be contributing to maintain amplicon repression in the 10.6 plants. The resistance mechanisms that prevent amplicon-derived virus infection were also active against exogenous PPV introduced by mechanical inoculation or grafting, but did not affect other viruses. Amplicon-derived PPV was able to spread into wild type scions grafted in 10.6 rootstocks that did not display signs of amplicon expression, suggesting that resistance has little effect on virus movement. - Conclusions: Our results suggest that amplicon-derived virus infection is limited in this particular transgenic line by a combination of factors, including the presumed low efficiency of the conversion from the transgene transcript to replicable viral RNA, and also by the activation of RNA silencing and other defensive responses of the plant, which are not completely neutralized by viral suppressors