Different roles for RNA silencing and RNA processing components in virus recovery and virus-induced gene silencing in plants

Abstract

A major antiviral mechanism in plants is mediated by RNA silencing, which relies on the cleavage of viral dsRNA into virus-derived small interfering RNAs (vsiRNAs) by DICER-like enzymes. Members of the Argonaute (AGO) family of endonucleases then use these vsiRNA as guides to target viral RNA. This can result in a phenomenon known as recovery, whereby the plant silences viral gene expression and recovers from viral symptoms. Endogenous mRNAs can also be targeted by vsiRNAs in a phenomenon known as virus-induced gene silencing (VIGS). Although related to other RNA silencing mechanisms, it has not been established if recovery and VIGS are mediated by the same molecular mechanisms. We used tobacco rattle virus (TRV) carrying a fragment of the phytoene desaturase (PDS) gene (TRV-PDS) or expressing green fluorescent protein (TRV-GFP) as readouts for VIGS and recovery, respectively, in Arabidopsis ago mutants. Our results demonstrated roles for AGO2 and AGO4 in susceptibility to TRV, whereas VIGS of endogenous genes appeared to be largely mediated by AGO1. However, recovery appeared to be mediated by different components, as all the aforementioned mutants were able to recover from TRV-GFP inoculation. TRV RNAs from recovered plants associated less with ribosomes, suggesting that recovery involves translational repression of viral transcripts. Translationally repressed RNAs often accumulate in RNA processing bodies (PBs), where they are eventually processed by decapping enzymes. Consistent with this, we found that viral recovery induced increased PB formation and that a decapping mutant (DCP2) showed increased VIGS and virus RNA accumulation, indicating an important role for PBs in eliminating viral RNA.

Functional and Genetic Analysis Identify a Role for Arabidopsis ARGONAUTE5 in Antiviral RNA Silencing

“…None of these mutants was more susceptible to PVX than the single ago2 mutant at either the local or systemic level and ago1 ago2 mutants even appeared somewhat less susceptible to PVX (Figures 5C and 5D; Supplemental Figures 8C and 8D). These results confirm that AGO1 and AGO7 do not play important roles in defense against PVX in Arabidopsis and are consistent with ago1 mutants being more resistant to TRV (Ma et al, 2015).…”

“…However, these two proteins appear to be dispensable for defense against TRV (Ma et al, 2015), and AGO2 is more important than AGO1 in defense against wildtype TCV (Zhang et al, 2012). Likewise, even though AGO1 and AGO7 are destabilized by the PVX VSR, our genetic and functional analyses suggest that they are not major determinants of PVX infection.…”

“…Hypomorphic ago1 mutants have increased susceptibility to Brome mosaic virus and Cucumber mosaic virus (CMV) and to VSR-defective CMV and Turnip crinkle virus (TCV) (Morel et al, 2002;Qu et al, 2008;Azevedo et al, 2010;Dzianott et al, 2012). However, an ago1 mutant is less susceptible than the wild type to Tobacco rattle virus (TRV), while AGO1 is required for virus-induced gene silencing (Ma et al, 2015). In addition, an ago7 mutant has increased susceptibility to specific derivatives of VSR-defective TCV (Qu et al, 2008), and ago4 mutants are more susceptible to TRV (Ma et al, 2015).…”

“…However, an ago1 mutant is less susceptible than the wild type to Tobacco rattle virus (TRV), while AGO1 is required for virus-induced gene silencing (Ma et al, 2015). In addition, an ago7 mutant has increased susceptibility to specific derivatives of VSR-defective TCV (Qu et al, 2008), and ago4 mutants are more susceptible to TRV (Ma et al, 2015). By contrast, AGO2 appears to be broadly required for antiviral defenses, having been shown to be involved in defense against a wide range of viruses, including CMV, TCV, TRV, PVX, Turnip mosaic virus (TuMV), and Tomato bushy stunt virus (Harvey et al, 2011;Jaubert et al, 2011;Scholthof et al, 2011;Wang et al, 2011;Carbonell et al, 2012;Zhang et al, 2012;Ma et al, 2015).…”

“…In addition, an ago7 mutant has increased susceptibility to specific derivatives of VSR-defective TCV (Qu et al, 2008), and ago4 mutants are more susceptible to TRV (Ma et al, 2015). By contrast, AGO2 appears to be broadly required for antiviral defenses, having been shown to be involved in defense against a wide range of viruses, including CMV, TCV, TRV, PVX, Turnip mosaic virus (TuMV), and Tomato bushy stunt virus (Harvey et al, 2011;Jaubert et al, 2011;Scholthof et al, 2011;Wang et al, 2011;Carbonell et al, 2012;Zhang et al, 2012;Ma et al, 2015). RISC complexes containing AGO1, 2, 3, or 5 act on viruses in in vitro Tombuvirus replication assays, and AGO1, 2, 3, 4, 5, and 9 can all bind to sRNAs derived from viruses or viroids (Takeda et al, 2008;Wang et al, 2011;Schuck et al, 2013;Minoia et al, 2014).…”