Fate of hairpin transcript components during RNA silencing and its suppression in transgenic virus-resistant tobacco.
Transgenic tobacco plants, carrying a Potato virus Y (PVY)-NIa hairpin sequence separated by a unique unrelated spacer sequence were specifically silenced and highly resistant to PVY infection. In such plants neither PVY-NIa nor spacer transgene transcripts were detectable by specific quantitative real time reverse transcriptase PCR (RT-qPCR) assays of similar relative efficiencies developed for direct comparative analysis. However, small interfering RNAs (siRNAs) specific for the PVY sequence of the transgene and none specific for the LNYV spacer sequence were detected. Following infection with Cucumber mosaic virus (CMV), which suppresses dsRNA-induced RNA silencing, transcript levels of PVY-NIa as well as spacer sequence increased manifold with the same time course. The cellular abundance of the single-stranded (ss) spacer sequence was consistently higher than that of PVY dsRNA in all cases. The results show that during RNA silencing and its suppression of a hairpin transcript in transgenic tobacco, the ssRNA spacer sequence is affected differently than the dsRNA. In PVY-silenced plants, the spacer is efficiently degraded by a mechanism not involving the accumulation of siRNAs, while following suppression of RNA silencing by CMV, the spacer appears protected from degradation.
Mitter
Neena
N
Department of Primary Industries and Fisheries, Emerging Technologies, Queensland Agricultural Biotechnology Centre, Queensland Bioscience Precinct, The University of Queensland, St. Lucia, Qld. 4072, Australia.
Mitchell
Roger
R
Dietzgen
Ralf G
RG
D007113:Immunity, Innate / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics; D030821:Plants, Genetically Modified / Q000378:metabolism*; D017800:Potyvirus / Q000502:physiology*; D034622:RNA Interference / Q000502:physiology*; D011994:Recombinant Proteins / Q000235:genetics; D013489:Suppression, Genetic / Q000235:genetics; D014026:Nicotiana / Q000502:physiology* / Q000821:virology*; D014157:Transcription Factors / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics*
D010940:Plant Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D014764:Viral Proteins