Arabidopsis Double-Stranded RNA Binding Protein DRB3 Participates in Methylation-Mediated Defense against Geminiviruses

Abstract

Arabidopsis encodes five double-stranded RNA binding (DRB) proteins. DRB1 and DRB2 are involved in microRNA (miRNA) biogenesis, while DRB4 functions in cytoplasmic posttranscriptional small interfering RNA (siRNA) pathways. DRB3 and DRB5 are not involved in double-stranded RNA (dsRNA) processing but assist in silencing transcripts targeted by DRB2-associated miRNAs. The goal of this study was to determine which, if any, of the DRB proteins might also participate in a nuclear siRNA pathway that leads to geminivirus genome methylation. Here, we demonstrate that DRB3 functions with Dicer-like 3 (DCL3) and Argonaute 4 (AGO4) in methylation-mediated antiviral defense. Plants employ repressive viral genome methylation as an epigenetic defense against geminiviruses, using an RNA-directed DNA methylation (RdDM) pathway similar to that used to suppress endogenous invasive DNAs such as transposons. Chromatin methylation inhibits virus replication and transcription, and methylation-deficient host plants are hypersusceptible to geminivirus infection. Using a panel of drb mutants, we found that drb3 plants uniquely exhibit a similar hypersensitivity and that viral genome methylation is substantially reduced in drb3 compared to wild-type plants. In addition, like dcl3 and ago4 mutants, drb3 plants fail to recover from infection and cannot accomplish the viral genome hypermethylation that is invariably observed in asymptomatic, recovered tissues. Small RNA analysis, bimolecular fluorescence complementation, and coimmunoprecipitation experiments show that DRB3 acts downstream of siRNA biogenesis and suggest that it associates with DCL3 and AGO4 in distinct subnuclear compartments. These studies reveal that in addition to its previously established role in the miRNA pathway, DRB3 also functions in antiviral RdDM. IMPORTANCEPlants use RNA-directed DNA methylation (RdDM) as an epigenetic defense against geminiviruses. RNA silencing pathways in Arabidopsis include five double-stranded RNA binding proteins (DRBs) related to Drosophila R2D2 and mammalian TRBP and PACT. While DRB proteins have defined roles in miRNA and cytoplasmic siRNA pathways, a role in nuclear RdDM was elusive. Here, we used the geminivirus system to show that DRB3 is involved in methylation-mediated antiviral defense. Beginning with a panel of Arabidopsis drb mutants, we demonstrated that drb3 plants uniquely show enhanced susceptibility to geminiviruses. Further, like dcl3 and ago4 mutants, drb3 plants fail to hypermethylate the viral genome, a requirement for host recovery. We also show that DRB3 physically interacts with the RdDM pathway components DCL3 and AGO4 in the nucleus. This work highlights the utility of geminiviruses as models for de novo RdDM and places DRB3 protein in this fundamental epigenetic pathway.

Antiviral Silencing and Suppression of Gene Silencing in Plants

“…Additional functional diversity between DCL4 and DCL2 has been reported: DCL2 stimulates transitivity and secondary siRNA production, while DCL4 is sufficient for silencing on its own (Parent et al 2015a). DCL3 has only a minor role against RNA viruses (Qu et al 2008;Raja et al 2014). The fact that silencing suppressors of RNA viruses interfere with DCL3 pathway suggests that DCL3 contributes to antiviral silencing (Azevedo et al 2010;Hamera et al 2012;Lacombe et al 2010).…”

“…The fact that silencing suppressors of RNA viruses interfere with DCL3 pathway suggests that DCL3 contributes to antiviral silencing (Azevedo et al 2010;Hamera et al 2012;Lacombe et al 2010). During antiviral silencing against DNA viruses DCL3 is essential and works presumably by inducing chromatin modifications (Akbergenov et al 2006;Blevins et al 2006;Raja et al 2014). DCL1 may act as a negative regulator limiting DCL4 and DCL3 through miRNA pathway (Azevedo et al 2010;Qu et al 2008).…”

“…DRB4, the cofactor of DCL4, takes part in antiviral defense against RNA viruses (Curtin et al 2008;Jakubiec et al 2012;Qu et al 2008 silencing suppressor of Cauliflower mosaic virus (CaMV) inhibits DRB4, strongly suggests that DRB4 is an antiviral factor against DNA viruses as well. DRB3, the cofactor of DCL3, contributes to antiviral defense through chromatin modification against DNA viruses (Raja et al 2014). HEN1-mediated vsiRNA methylation is critical for effective antiviral defense (Vogler et al 2007).…”

“…vsiRNA-binding VSRs do not compromise dicing, but efficiently inhibit antiviral silencing (Csorba et al 2015). The downstream AGO-dependent effector step is therefore necessary to restrict virus replication and spread of both RNA and DNA viruses (Azevedo et al 2010;Carbonell et al 2012;Harvey et al 2011;Pantaleo et al 2007;Qu et al 2008;Raja et al 2014;Raja et al 2008;Wang et al 2011). The properties of vsiRNAs like 5 0 -nucleotide, length, thermodynamical properties of sRNA duplex ends and sRNA's duplex structure define loading and sorting into AGO effectors (Khvorova et al 2003;Mi et al 2008;Schuck et al 2013;Schwarz et al 2003;Zhang et al 2014;Kontra et al unpublished).…”

“…Arabidopsis dcl3, drb3 and ago4 mutants fail to hypermethylate the viral genome that is required for host recovery (Raja et al 2014). Besides, AGO4 was proposed to be important in transcriptional regulation of host transcriptional response during CMV virus infection (Hamera et al 2012) or to be involved in PVX virus resistance induced by NB-LRR proteins involving AGO4-mediated translational control (Bhattacharjee et al 2009).…”