A Signaling Cascade from miR444 to RDR1 in Rice Antiviral RNA Silencing Pathway^{<a href="#fn1" rid="fn1" class=" fn">1</a>}

Abstract

RNA silencing mediated by regulatory small RNAs (microRNAs [miRNAs] and small interfering RNAs [siRNAs]) negatively regulates gene expression at the posttranscriptional level or at the transcriptional level in eukaryotic organisms. Besides small RNAs, plant RNA-silencing pathways incorporate several kinds of core protein components, such as DICER-LIKE (DCL) RNase III endonucleases, which process long double-stranded RNA (dsRNA) into small RNA duplexes; ARGONAUTEs (AGOs), the major effector of the RNA-induced silencing complexes, which bind to small RNAs for silencing target RNAs; and RNA-dependent RNA polymerases (RDRs), which are required for copying single-stranded RNAs into dsRNAs for downstream processing by DCLs. Multiple DCLs, AGOs, and RDRs have evolved in plants and thus form an array of RNA-silencing pathways (Axtell, 2013; Martínez de Alba et al., 2013; Bologna and Voinnet, 2014). Among them, the antiviral RNA-silencing pathway is the earliest described and most extensively studied. It is well known that the antiviral silencing pathway directly targets viral RNAs. Briefly, as in Arabidopsis (Arabidopsis thaliana), the stem-loop structures and dsRNA replication intermediates of viral RNAs are recognized and cleaved by DCLs (DCL4 and DCL2) to produce primary virus-derived small interfering RNAs (vsiRNAs). Furthermore, abundant secondary vsiRNAs are produced from the dsRNAs amplified by host-encoded RDRs (RDR1 or RDR6). Both primary and secondary vsiRNAs are loaded into AGO proteins (AGO1 and AGO2) to direct the degradation of the viral RNAs. The amplified vsiRNAs are believed also to trigger the systemic silencing of the viral RNAs in distant tissues (Ding and Voinnet, 2007; Ding, 2010; Pumplin and Voinnet, 2013).

On the other hand, recent studies have shown that antiviral RNA silencing also is attributed to alterations in the expression of host genes, including those directly involved in the antiviral RNA-silencing pathway. For example, DCL1, a target of microRNA162 (miR162), negatively regulated the expression of DCL4 and DCL3, two important DCLs for slicing viral RNAs and producing vsiRNAs (Qu et al., 2008; Azevedo et al., 2010). Also, the activation of AGO1 expression has been observed in Arabidopsis infected by different viruses (Zhang et al., 2006; Azevedo et al., 2010; Várallyay et al., 2010), and AGO2 expression increased in Turnip crinkle virus- and Cucumber mosaic virus-infected plants (Harvey et al., 2011). The induction of these genes would certainly enhance the function of antiviral RNA-silencing pathways.

In addition, silencing of other defense-related host genes contributes to the antiviral response. For example, virus infection reduced the accumulation of miR482, a 22-nucleotide miRNA targeting the nucleotide-binding site-leucine-rich repeat (NBS-LRR) class R genes, and consequently increased the expression of two miR482-targeted NBS-LRR mRNAs in infected plants (Shivaprasad et al., 2012). And down-regulation of an R gene due to increased expression of miR6019, another 22-nucleotide miRNA, resulted in the attenuated R gene-mediated resistance to Tobacco mosaic virus in Nicotiana benthamiana (Li et al., 2012). Similarly, Turnip mosaic virus infection in Brassica spp. induced the production of miR1885, which targeted the TIR-NBS-LRR class R genes (He et al., 2008). In addition to miRNAs, siRNA-mediated RNA silencing also is involved in host defense against virus infection. For instance, a miniature inverted repeat transposable element inserted in the third intron of the tobacco mosaic virus resistance gene N generated 24-nucleotide siRNAs to regulate the expression of the N gene via an RNA-directed DNA methylation-mediated RNA-silencing mechanism (Kuang et al., 2009).

RDR1 orthologs have been reported to be virus or salicylic acid inducible in different plants, including Arabidopsis, Nicotiana spp., Medicago truncatula, maize (Zea mays), and rice (Oryza sativa; Yang et al., 2004; Alamillo et al., 2006; He et al., 2010; Satoh et al., 2010; Du et al., 2011), and to provide basal resistance to several viruses by participating in the biogenesis of virus-derived secondary siRNAs (Diaz-Pendon et al., 2007; Qi et al., 2009; Garcia-Ruiz et al., 2010; Wang et al., 2010). Recently, it was reported that RDR1 also is responsible for the production of a distinct class of virus-activated siRNAs, which direct widespread silencing of host genes to confer broad-spectrum antiviral activity in Arabidopsis (Cao et al., 2014), indicating that RDR1 plays a key role in antiviral resistance by silencing both the viral RNAs and the host immunity-related genes. Rice OsRDR1 might have similar roles in antiviral RNA silencing (Chen et al., 2010; Wang et al., 2014). However, the signaling pathway from virus infection to RDR1 expression is unclear.

miR444 is specific to monocots and plays roles in rice tillering and nitrate signaling (Guo et al., 2013; Yan et al., 2014). miR444 is a kind of natural antisense miRNA and targets four MIKC^{-type MADS box homologous genes (}OsMADS23, OsMADS27a, OsMADS27b, and OsMADS57) in rice (Sunkar et al., 2005; Lu et al., 2008; Wu et al., 2009; Li et al., 2010; Yan et al., 2014). Plant MADS box proteins regulate gene expression by binding to a highly conserved DNA motif known as the CArG box using the MADS box of the DNA-binding domain (de Folter and Angenent, 2006; Ito et al., 2008; Fujisawa et al., 2013). To bind the CArG motif, MADS box transcription factors need to form homodimeric or heterodimeric complexes (Riechmann et al., 1996). Here, we show that miR444 plays key roles in relaying the antiviral signal from virus infection to OsRDR1 expression in rice plants. Infection with Rice stripe virus (RSV) induces miR444 accumulation, and the activation of miR444 results in an increase in OsRDR1 expression, leading to rice resistance to RSV infection. We reveal a regulatory mechanism for the activation of OsRDR1 gene transcription in which the repressors formed by miR444 target proteins are released from the OsRDR1 promoter upon RSV infection. To our knowledge, such a derepression mechanism has not been described in the regulation of other RDR1 ortholog genes.

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