Synthesis of complementary RNA by RNA-dependent RNA polymerases in plant extracts is independent of an RNA primer
Lei Wang A B , Neil A. Smith A , Lan Zhang B , Elizabeth S. Dennis A , Peter M. Waterhouse A , Peter J. Unrau C and Ming-Bo Wang A DA CSIRO Plant Industry, PO Box 1600, Canberra, ACT 2601, Australia.
B Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
C Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC V5A 1S6, Canada.
D Corresponding author. Email: ming-bo.wang@csiro.au
Functional Plant Biology 35(11) 1091-1099 https://doi.org/10.1071/FP08118
Submitted: 10 April 2008 Accepted: 15 August 2008 Published: 28 November 2008
Abstract
RNA-dependent RNA polymerase (RDR) activities were readily detected in extracts from cauliflower and broccoli florets, Arabidopsis thaliana (L.) Heynh callus tissue and broccoli nuclei. The synthesis of complementary RNA (cRNA) was independent of a RNA primer, whether or not the primer contained a 3′ terminal 2′-O-methyl group or was phosphorylated at the 5′ terminus. cRNA synthesis in plant extracts was not affected by loss-of-function mutations in the DICER-LIKE (DCL) proteins DCL2, DCL3, and DCL4, indicating that RDRs function independently of these DCL proteins. A loss-of-function mutation in RDR1, RDR2 or RDR6 did not significantly reduce the amount of cRNA synthesis. This indicates that these RDRs did not account for the bulk RDR activities in plant extracts, and suggest that either the individual RDRs each contribute a fraction of polymerase activity or another RDR(s) is predominant in the plant extract.
Additional keywords: Dicer-like protein, dsRNA, RNA-dependent RNA polymerase, RNA silencing.
Acknowledgements
We thank Limin Wu for technical assistance and Tony Ashton, John Watson and Andrew Eamens for critical reading of the manuscript.
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