RNA-dependent RNA polymerase 6 is required for efficient hpRNA-induced gene silencing in plants

In plants, transgenes with inverted repeats are used to induce efficient RNA silencing, which is also frequently induced by highly transcribed sense transgenes. RNA silencing induced by sense transgenes is dependent on RNA-dependent RNA polymerase 6 (RDR6), which converts single-stranded (ss) RNA into double-stranded (ds) RNA. By contrast, it has been proposed that RNA silencing induced by self-complementary hairpin RNA (hpRNA) does not require RDR6, because the hpRNA can directly fold back on itself to form dsRNA. However, it is unclear whether RDR6 plays a role in hpRNA-induced RNA silencing by amplifying dsRNA to spread RNA silencing within the plant. To address the efficiency of hpRNA-induced RNA silencing in the presence or absence of RDR6, Wild type (WT, Col-0) and rdr6-11 Arabidopsis thaliana lines expressing green fluorescent protein (GFP) were generated and transformed with a GFP-RNA interference (RNAi) construct. Whereas most GFP-RNAi-transformed WT lines exhibited almost complete silencing of GFP expression in the T1 generation, various levels of GFP expression remained among the GFP-RNAi-transformed rdr6-11 lines. Homozygous expression of GFP-RNAi in the T3 generation was not sufficient to induce complete GFP silencing in several rdr6-11 lines. Our results indicate that RDR6 is required for efficient hpRNA-induced RNA silencing in plants.     

Rice RNA-dependent RNA polymerase 6 acts in small RNA biogenesis and spikelet development

Higher plants have evolved multiple RNA-dependent RNA polymerases (RDRs), which work with Dicer-like (DCL) proteins to produce different classes of small RNAs with specialized molecular functions. Here we report that OsRDR6, the rice (Oryza sativa L.) homolog of Arabidopsis RDR6, acts in the biogenesis of various types and sizes of small RNAs. We isolated a rice osrdr6-1 mutant, which was temperature sensitive and showed spikelet defects. This mutant displays reduced accumulation of tasiR-ARFs, the conserved trans-acting siRNAs (tasiRNAs) derived from the TAS3 locus, and ectopic expression of tasiR-ARF target genes, the Auxin Response Factors (including ARF2 and ARF3/ETTIN). The loss of tasiR-mediated repression of ARFs in osrdr6-1 can explain its morphological defects, as expression of two non-targeted ARF3 gene constructs (ARF3muts) in a wild-type background mimics the osrdr6 and osdcl4-1 mutant phenotypes. Small RNA high-throughput sequencing also reveals that besides tasiRNAs, 21-nucleotide (nt) phased small RNAs are also largely dependent on OsRDR6. Unexpectedly, we found that osrdr6-1 has a strong impact on the accumulation of 24-nt phased small RNAs, but not on unphased ones. Our work uncovers the key roles of OsRDR6 in small RNA biogenesis and directly illustrates the crucial functions of tasiR-ARFs in rice development.     

Small RNAs from MITE-derived stem-loop precursors regulate abscisic acid signaling and abiotic stress responses in rice

Small silencing RNAs (sRNAs) are non-coding RNA regulators that negatively regulate gene expression by guiding mRNA degradation, translation repression or chromatin modification. Plant sRNAs play crucial roles in various developmental processes, hormone signaling, immune responses and adaptation to a variety of abiotic stresses. miR441 and miR446 were previously annotated as microRNAs (miRNAs) because their precursors can form typical stem-loop structures, but are not considered as real miRNAs because of inconsistency with some ancillary criteria of the recent guidelines for annotation of plant miRNAs. We tentatively rename them small interfering (si)R441 and siR446, respectively, in this study. It has recently been shown that the precursors of siR441 and siR446 might originate from the miniature inverted-repeat transposable element (MITE) Stowaway1. In this report, we show that, in contrast with Dicer-like (DCL)3- and RNA-dependent RNA polymerase (RDR)2-dependent MITE-derived ra-siRNAs, siR441 and siR446 are processed by OsDCL3a but independent of OsRDR2, indicating that siR441 and siR446 are generated from single-stranded stem-loop precursors. We also show that abscisic acid (ABA) and abiotic stresses downregulate the expression of siR441 and siR446 but, surprisingly, increase the accumulation of their precursors in rice plants, implying that processing of siRNA precursors is inhibited. We provide evidence to show that this defective processing is due to increased precursor accumulation per se, possibly by intermolecular self-pairing of the processing intermediate sequences, thus hindering their normal processing. Functional examinations indicate that siR441 and siR446 are positive regulators of rice ABA signaling and tolerance to abiotic stress, possibly by regulating MAIF1 expression.