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...

Arabidopsis thaliana defense against distinct positive-strand RNA viruses requires production of virus-derived secondary small interfering RNAs (siRNAs) by multiple RNA-dependent RNA polymerases. However, little is known about the biogenesis pathway and effector mechanism of viral secondary siRNAs. Here, we describe a mutant of Cucumber mosaic virus (CMV-D2b) that is silenced predominantly by the RNA-DEPENDENT RNA POLYMERASE6 (RDR6)-dependent viral secondary siRNA pathway. We show that production of the viral secondary siRNAs targeting CMV-D2b requires SUP-PRESSOR OF GENE SILENCING3 and DICER-LIKE4 (DCL4) in addition to RDR6. Examination of 25 single, double, and triple mutants impaired in nine ARGONAUTE (AGO) genes combined with coimmunoprecipitation and deep sequencing identifies an essential function for AGO1 and AGO2 in defense against CMV-D2b, which act downstream the biogenesis of viral secondary siRNAs in a nonredundant and cooperative manner. Our findings also illustrate that dicing of the viral RNA precursors of primary and secondary siRNA is insufficient to confer virus resistance. Notably, although DCL2 is able to produce abundant viral secondary siRNAs in the absence of DCL4, the resultant 22-nucleotide viral siRNAs alone do not guide efficient silencing of CMV-D2b. Possible mechanisms for the observed qualitative difference in RNA silencing between 21-and 22-nucleotide secondary siRNAs are discussed.

Effectors are essential virulence proteins produced by a broad range of parasites, including viruses, bacteria, fungi, oomycetes, protozoa, insects and nematodes. Upon entry into host cells, pathogen effectors manipulate specific physiological processes or signaling pathways to subvert host immunity. Most effectors, especially those of eukaryotic pathogens, remain functionally uncharacterized. Here, we show that two effectors from the oomycete plant pathogen Phytophthora sojae suppress RNA silencing in plants by inhibiting the biogenesis of small RNAs. Ectopic expression of these Phytophthora suppressors of RNA silencing enhances plant susceptibility to both a virus and Phytophthora, showing that some eukaryotic pathogens have evolved virulence proteins that target host RNA silencing processes to promote infection. These findings identify RNA silencing suppression as a common strategy used by pathogens across kingdoms to cause disease and are consistent with RNA silencing having key roles in host defense.

Antiviral immunity controlled by RNA interference (RNAi) in plants and animals is thought to specifically target only viral RNAs by the virus-derived small interfering RNAs (siRNAs). Here we show that activation of antiviral RNAi in Arabidopsis plants is accompanied by the production of an abundant class of endogenous siRNAs mapped to the exon regions of more than 1,000 host genes and rRNA. These virus-activated siRNAs (vasiRNAs) are predominantly 21 nucleotides long with an approximately equal ratio of sense and antisense strands. Genetically, vasiRNAs are distinct from the known plant endogenous siRNAs characterized to date and instead resemble viral siRNAs by requiring Dicer-like 4 and RNA-dependent RNA polymerase 1 (RDR1) for biogenesis. However, loss of EXORIBONUCLEASE4/ THYLENE-INSENSITIVE5 enhances vasiRNA biogenesis and virus resistance without altering the biogenesis of viral siRNAs. We show that vasiRNAs are active in directing widespread silencing of the target host genes and that Argonaute-2 binds to and is essential for the silencing activity of vasiRNAs. Production of vasiRNAs is readily detectable in Arabidopsis after infection by viruses from two distinct supergroups of plant RNA virus families and is targeted for inhibition by the silencing suppressor protein 2b of Cucumber mosaic virus. These findings reveal RDR1 production of Arabidopsis endogenous siRNAs and identify production of vasiRNAs to direct widespread silencing of host genes as a conserved response of plants to infection by diverse viruses. A possible function for vasiRNAs to confer broad-spectrum antiviral activity distinct to the virus-specific antiviral RNAi by viral siRNAs is discussed.RNA silencing | microbiology | plant biology | viral suppressor of RNAi