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 b-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.

Both OsRecQ1 and OsRDR1 Are Required for the Production of Small RNA in Response to DNA-Damage in Rice

“…Nipponbare) and its knockout mutant lines were used in this study. OsRecQ1 (ND8004 and ND0059) and OsRDR1 (ND2001 and ND2059) mutant lines were reported previously [26], [27]. OsSGS3b (NE5050), OsCMT1 (NE7010) and OsCMT3 (NC4949) mutant lines were used in this study.…”

“…Total RNA was extracted from rice leaves and calli after DNA damage treatments as previously described [26], [27]. Low and high molecular weight RNAs were used to detect qiRNAs and aRNAs with 25 and 40 µg of total RNA, respectively.…”

“…Quantitative real-time PCR (RT-qPCR) was performed with a Light Cycler II system (Roche) using a previously described protocol [26], [27]. In brief, total RNA was isolated using an RNeasy plant mini kit (QIAGEN) and treated with RNase-free DNase I (Roche), and Poly (A) + mRNA was purified by an Oligotex-dT30 mRNA purification kit (TaKaRa).…”

“…In rice, previous studies suggest that OsRecQ1 , a QDE-3 homologue, is thought to participated in the process of allowing inverted repeat (IR) DNA to be transcribed into dsRNA that can trigger RNA silencing [26]. OsRDR1 has been reported to be involved in virus mediated RNA silencing [27], while rice chromomethyltransferase 3 ( OsCMT3 ) has been anticipated to be involved in the epigenetic process in affecting genome activity during abiotic stress [28]. Suppressor of gene silencing 3 ( SGS3 ) in Arabidopsis has been shown to be required for the biogenesis of trans -acting small interfering RNAs (ta-siRNAs) [24].…”

OsRDR6 plays role in host defense against double-stranded RNA virus, Rice Dwarf Phytoreovirus

“…Rice encodes five RDRs: OsRDR1, OsRDR2, OsRDR3a, OsRDR3b and OsRDR6 44 . OsRDR1 is required for the production of small RNA in response to DNA-damage in rice 45 and for the RNA silencing mediated by Brome mosaic bromovirus (BMV), but not by Wheat dwarf geminivirus (WDV) 46 . Recent report showed that OsRDR2 did not function in siR441 and siR446 production, which were previously annotated as microRNAs (miRNAs) 47 .…”