Published online 25 June 2004
Nucleic Acids Research, Vol. 32 No. 11 © Oxford University Press 2004; all rights reserved
The preferred route for the degradation of silencing target RNAs in transgenic plants depends on pre-established silencing conditions
Unit of Molecular Signal Transduction in Inflammation, Department for Molecular Biomedical Research and 1 Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology (VIB), Ghent University, Technologiepark 927, B-9052 Gent (Zwijnaarde), Belgium and 2 Bayer Bioscience N.V., Technologiepark 38, B-9052 Gent, Belgium
* To whom correspondence should be addressed. Tel: +32 9 33 13775; Fax: +32 9 33 13609 Email: matthews{at}dmbr.ugent.be
Present addresses: Nausicaa Lannoo, Department of Molecular Biotechnology, Ghent University, Coupure Links 653, B-9000 Gent, Belgium
Wendy Maddelein, Devgen N.V., Technologiepark 30, B-9052 Gent (Zwijnaarde), Belgium
Received April 20, 2004; Revised and Accepted June 3, 2004
RNA silencing can be initiated upon dsRNA accumulation and results in homology-dependent degradation of target RNAs mediated by 21–23 nt small interfering RNAs (siRNAs). These small regulatory RNAs can direct RNA degradation via different routes such as the RdRP/Dicer- and the RNA-induced silencing complex (RISC)-catalysed pathways. The relative contribution of both pathways to degradation of target RNAs is not understood. To gain further insight in the process of target selection and degradation, we analysed production of siRNAs characteristic for Dicer-mediated RNA degradation during silencing of mRNAs and chimeric viral RNAs in protoplasts from plants of a transgenic tobacco silencing model line. We show that small RNA accumulation is limited to silencing target regions during steady-state mRNA silencing. For chimeric viral RNAs, siRNA production appears dependent on pre-established cellular silencing conditions. The observed siRNA accumulation profiles imply that silencing of viral target RNAs in pre-silenced protoplasts occurs mainly via a RISC-mediated pathway, guided by (pre-existing) siRNAs derived from cellular mRNAs. In cells that are not silenced at the time of infection, viral RNA degradation seems to involve Dicer action directly on the viral RNAs. This suggests that the silencing mechanism flexibly deploys different components of the RNA degradation machinery in function of the prevailing silencing status.