Analysis of rice RNA-dependent RNA polymerase 1 (OsRDR1) in virus-mediated RNA silencing after particle bombardment

Abstract

RNA-dependent RNA polymerases (RDRs) play a key role in various RNA silencing pathways in many organisms. Using the nucleotide sequence of SGS2/SDE1/RDR6 in Arabidopsis as the search query for sequences that flank the insertions of rice retrotransposon Tos17, we selected rice mutant lines (OsRDR1). RT-PCR analysis showed that OsRDR1 mRNA was undetectable in leaves and calli of the mutants, while it was detected in wild type. RNA silencing was induced by particle bombardment to investigate any effects of OsRDR1 on RNA silencing with β-glucuronidase or green fluorescent protein DNA/RNA in the mutant lines. The results showed that RNA silencing was impaired in these mutant lines by inverted repeat (IR) DNA or in vitro transcribed double-stranded RNA. Further, the mutant lines were bombarded with Brome mosaic bromovirus (BMV, a ssRNA virus) or Wheat dwarf geminivirus (WDV, a ssDNA virus), each carrying the IR sequence of a reporter gene. As a result, RNA silencing was impaired by BMV. Interestingly, however, it was not impaired by WDV. Thus we propose that OsRDR1 is required for RNA silencing mediated by Bromovirus, but not by Geminivirus in this system.