Suppression of Arabidopsis ARGONAUTE1-Mediated Slicing, Transgene-Induced RNA Silencing, and DNA Methylation by Distinct Domains of the Cucumber mosaic virus 2b Protein

Abstract

Unique among the known plant and animal viral suppressors of RNA silencing, the 2b protein interacts directly with both small interfering RNA (siRNA) and ARGONAUTE1 (AGO1) and AGO4 proteins and is targeted to the nucleolus. However, it is largely unknown which regions of the 111-residue 2b protein determine these biochemical properties and how they contribute to its diverse silencing suppressor activities. Here, we identified a functional nucleolar localization signal encoded within the 61-amino acid N-terminal double-stranded RNA (dsRNA) binding domain (dsRBD) that exhibited high affinity for short and long dsRNA. However, physical interaction of 2b with AGOs required an essential 33-residue region C-terminal to the dsRBD and was sufficient to inhibit the in vitro AGO1 Slicer activity independently of its dsRNA binding activities. Furthermore, the direct 2b-AGO interaction was not essential for the 2b suppression of posttranscriptional gene silencing (PTGS) and RNA-directed DNA methylation (RdDM) in vivo. Lastly, we found that the 2b-AGO interactions in vivo also required the nucleolar targeting of 2b and had the potential to redistribute both the 2b and AGO proteins in nucleus. These findings together suggest that 2b may suppress PTGS and RdDM in vivo by binding and sequestering siRNA and the long dsRNA precursor in a process that is facilitated by its interactions with AGOs in the nucleolus.

Salt Stress Reveals a New Role for ARGONAUTE1 in miRNA Biogenesis at the Transcriptional and Posttranscriptional Levels

“…This interaction may also affect RNAPII processivity and abolish splicing of selected alternative exons (Ameyar-Zazoua et al, 2012;Li et al, 2012b;Alló et al, 2014). There have also been some suggestions that, in Arabidopsis, AGO1 localization is not limited to the cytoplasm (Ye et al, 2012;Duan et al, 2012). Using immunolocalization and western-blot analysis, we showed that AGO1 is indeed localized in the nucleus of Arabidopsis cells (Fig.…”

“…It has been demonstrated that miR168 is stabilized by AGO1 (Vaucheret et al, 2006;Vaucheret, 2009). The main role of Arabidopsis AGO1 as a slicer in the RISC complex that cuts an mRNA target places AGO1 in the cytoplasm where the cleavage of miRNA targets occurs, yet there are experimental data suggesting that AGO1 can also be localized in the nucleus in plant cells (Ye et al, 2012;Duan et al, 2012). However, the role of AGO1 in this cell compartment remains elusive.…”

Antiviral Silencing and Suppression of Gene Silencing in Plants

“…2b of CMV (CM95R strain) and TAV exhibit high affinity for long dsRNAs and ds-sRNAs Duan et al 2012;Gonzalez et al 2012;Goto et al 2007). CMV 2b (Fny and SD strains) was also shown to interact with AGO1 through the PAZ-and partly PIWI domains and blocks RISC slicer activity (Duan et al 2012;Zhang et al 2006).…”

“…2b interacts both with AGO4-related siRNAs and with AGO4 protein through PAZ and PIWI domains. The interaction with 2b reduces AGO4 access to endogenous target loci and consequently modulates endogenous transcription to create a favorable niche for CMV infection (Duan et al 2012;Gonzalez et al 2010Gonzalez et al , 2012Hamera et al 2012). P38 of TCV may also suppress silencing at multiple levels.…”

Innate Barriers to Viral Infection

“…This viral protein has a dsRNA binding domain that is needed for RNAi suppression in vivo. Additionally, the CMV 2b protein appears to repress post-transcriptional gene silencing as well as RNA-directed DNA methylation by binding and sequestering siRNA and the long dsRNA precursor [29].…”