RNAi-mediated viral immunity requires amplification of virus-derived siRNAs in Arabidopsis thaliana

Abstract

In diverse eukaryotic organisms, Dicer-processed, virus-derived small interfering RNAs direct antiviral immunity by RNA silencing or RNA interference. Here we show that in addition to core dicing and slicing components of RNAi, the RNAi-mediated viral immunity in Arabidopsis thaliana requires host RNA-directed RNA polymerase (RDR) 1 or RDR6 to produce viral secondary siRNAs following viral RNA replication-triggered biogenesis of primary siRNAs. We found that the two antiviral RDRs exhibited specificity in targeting the tripartite positive-strand RNA genome of cucumber mosaic virus (CMV). RDR1 preferentially amplified the 5′-terminal siRNAs of each of the three viral genomic RNAs, whereas an increased production of siRNAs targeting the 3′ half of RNA3 detected in rdr1 mutant plants appeared to be RDR6-dependent. However, siRNAs derived from a single-stranded 336-nucleotide satellite RNA of CMV were not amplified by either antiviral RDR, suggesting avoidance of the potent RDR-dependent silencing as a strategy for the molecular parasite of CMV to achieve preferential replication. Our work thus identifies a distinct mechanism for the amplification of immunity effectors, which together with the requirement for the biogenesis of endogenous siRNAs, may play a role in the emergence and expansion of eukaryotic RDRs.antiviral immunity | cucumber mosaic virus | RNA silencing | RNAdependent RNA polymerase | secondary small interfering RNA R NA silencing or RNA interference in fungi, nematodes and plants requires amplification of small interfering RNAs by eukaryotic RNA-directed RNA polymerases (RDRs) (1-3). Plant and fungal RDRs convert transcripts of target genes into dsRNA that is subsequently processed into secondary siRNAs by a Dicer or Dicer-like (DCL) nuclease. In contrast, Caenorhabditis elegans RDRs, such as RRF-1, may directly manufacture secondary siRNAs without dicing a dsRNA precursor (1-3). The genome of Arabidopsis thaliana encodes six RDRs that are grouped into four clusters (1, 2, 4), among which little is known about cluster III, consisting of RDRs 3a, 3b, and 3c. RDR2 and RDR6 are both required for the short-distance spread of transgene silencing and for the perception, but not the production, of the long-distance mobile silencing signal (1, 5-9). RDR2 is also essential for the biogenesis of the DCL3-dependent 24-nucleotide (nt) repeat-associated siRNAs (rasiRNAs) derived from transposons, retroelements, and other elements, which are the most abundant endogenous small RNAs in A. thaliana (3). Similarly, RDR6 coupled with DCL4 or DCL1 is responsible for the biogenesis of transacting siRNAs (tasiRNAs) and natural antisense siRNAs (nat-siRNAs), which silence expression of their target genes like microRNAs (miRNAs) (1-3).RNA silencing controls antiviral immunity in fungi, plants, and invertebrates by producing virus-derived siRNAs to be loaded in an Argonaute protein for antiviral silencing (10-12). In A. thaliana, DCL4 and DCL2 produce viral siRNAs against distinct positive (+)-strand RNA viruses in a hierarc...

The role of decapping proteins in the miRNA accumulation inArabidopsis thaliana

“…Mature siRNAs are incorporated into various AGO proteins as well as miRNAs, and have roles in processes such as anti-viral defense. [26][27][28] DCP1, DCP2 and Hedls/Ge-1 are involved in miRNA-mediated gene silencing at different levels in animals. 20,[29][30][31][32][33] Moreover, DCP1 co-localizes with AGO1 and GW182, which are essential for the function of miRNAs in mammals.…”

Diversity, evolution, and therapeutic applications of small RNAs in prokaryotic and eukaryotic immune systems

“…Arabidopsis encodes six different RDR proteins. RDR1 assists in the formation of CMV -and Tobacco mosaic virus (TMV) -derived siRNAs [38,111,130]. Tobacco rattle virus (TRV)-derived siRNAs are formed via the activity of RDR1, RDR2, and RDR6 in conjunction with one another [50].…”

“…Tobacco rattle virus (TRV)-derived siRNAs are formed via the activity of RDR1, RDR2, and RDR6 in conjunction with one another [50]. CMV accumulates in mutants lacking RDR6 [130]. Garcia-Ruiz and colleagues demonstrated that plants mutant for RDR1, RDR2, or RDR6 genes are more susceptible to primary infection.…”

Modulation of host plant immunity by Tobamovirus proteins

“…In Arabidopsis thaliana, six different RDRs have already been identified (RDR1-RDR6) (reviewed in Willmann et al, 2011). RDR1 and RDR6 are the two main antiviral RDRs (Wang et al, 2010). It was proposed that RDRs display specific sensitivities to different viruses, and small RNA deep sequencing analysis has shown that both RDR1 and RDR6 play a role in the biogenesis of TMV-Cg vsiRNAs (Qi et al, 2009).…”