Arabidopsis RNA Polymerases IV and V Are Required To Establish H3K9 Methylation, but Not Cytosine Methylation, on Geminivirus Chromatin

Abstract

In plants, RNA-directed DNA methylation (RdDM) employs small RNAs to target enzymes that methylate cytosine residues. Cytosine methylation and dimethylation of histone 3 lysine 9 (H3K9me2) are often linked. Together they condition an epigenetic defense that results in chromatin compaction and transcriptional silencing of transposons and viral chromatin. Canonical RdDM (Pol IV-RdDM), involving RNA polymerases IV and V (Pol IV and Pol V), was believed to be necessary to establish cytosine methylation, which in turn could recruit H3K9 methyltransferases. However, recent studies have revealed that a pathway involving Pol II and RNA-dependent RNA polymerase 6 (RDR6) (RDR6-RdDM) is likely responsible for establishing cytosine methylation at naive loci, while Pol IV-RdDM acts to reinforce and maintain it. We used the geminivirus Beet curly top virus (BCTV) as a model to examine the roles of Pol IV and Pol V in establishing repressive viral chromatin methylation. As geminivirus chromatin is formed de novo in infected cells, these viruses are unique models for processes involved in the establishment of epigenetic marks. We confirm that Pol IV and Pol V are not needed to establish viral DNA methylation but are essential for its amplification. Remarkably, however, both Pol IV and Pol V are required for deposition of H3K9me2 on viral chromatin. Our findings suggest that cytosine methylation alone is not sufficient to trigger de novo deposition of H3K9me2 and further that Pol IV-RdDM is responsible for recruiting H3K9 methyltransferases to viral chromatin. IMPORTANCEIn plants, RNA-directed DNA methylation (RdDM) uses small RNAs to target cytosine methylation, which is often linked to H3K9me2. These epigenetic marks silence transposable elements and DNA virus genomes, but how they are established is not well understood. Canonical RdDM, involving Pol IV and Pol V, was thought to establish cytosine methylation that in turn could recruit H3K9 methyltransferases, but recent studies compel a reevaluation of this view. We used BCTV to investigate the roles of Pol IV and Pol V in chromatin methylation. We found that both are needed to amplify, but not to establish, DNA methylation. However, both are required for deposition of H3K9me2. Our findings suggest that cytosine methylation is not sufficient to recruit H3K9 methyltransferases to naive viral chromatin and further that Pol IV-RdDM is responsible. R epressive chromatin methylation suppresses the expression of transposable elements and DNA viruses and leads to the establishment of transcriptional gene silencing (TGS). Plants employ RNA-directed DNA methylation (RdDM) to target methylation of cytosine residues and use cytosine methylation and associated histone 3 lysine 9 dimethylation (H3K9me2) to silence invasive DNAs, such as transposons and geminiviruses. As this study addresses the roles of RNA polymerases in repressive methylation of geminivirus chromatin, an overview of relevant pathways and their interrelationships is presented (1, 2).In the reference plant Arabido...

Arabidopsis Histone Reader EMSY-LIKE 1 Binds H3K36 and Suppresses Geminivirus Infection

“…Isolation of DNA and RNA from symptomatic floral heads and siliques of CaLCuVinfected plants was performed as described previously (36). RNA was reverse transcribed using Super-Script III with oligo(dT) primers, and cDNAs were used to perform quantitative reverse transcriptase PCR (RT-qPCR) with Bio-Rad iQ SYBR green supermix and the Bio-Rad CFX96 real-time PCR detection system (34). PP2A (At1G13320) was used as a reference gene, and comparisons of steady-state host and viral transcript levels were performed using the 2 ϪΔΔCT method (53).…”

“…The viral chromatin serves as the template for replication and transcription, which depends on host polymerases and associated machineries. In a classic example of host defense and pathogen counterdefense, multiple studies have shown that plants employ DNA and H3K9 methylation to compact and silence geminivirus chromatin, while viral pathogenicity factors inhibit repressive chromatin methylation (27)(28)(29)(30)(31)(32)(33)(34)(35)(36).…”

Exogenous Transposable Elements Circumvent Identity-Based Silencing, Permitting the Dissection of Expression-Dependent Silencing

“…To focus solely on the initiation and establishment phases of silencing, and separate these mechanisms from the maintenance of epigenetic marks, investigation has concentrated on transformation or infection of epigenetically naïve (chromatin modification-free) DNA into plants (Greenberg et al, 2011;Bond and Baulcombe, 2015;Jackel et al, 2016). Although transgenesis might be considered an artificial approach to investigate silencing, evidence suggests that transgenesis can occur naturally (Kyndt et al, 2015).…”

Superinfection by PHYVV Alters the Recovery Process in PepGMV-Infected Pepper Plants

“…Viral DNA methylation was demonstrated to be important for plant defense against geminiviruses. Hypermethylation of the viral genome has been associated with the recovery phenotype in different models of plant-geminivirus interaction [12,14,[34][35][36]. In addition, mutants of RdDM are more susceptible to infection and show an exacerbated symptomatology compared to wild type plants [34].…”

“…Low levels of viral DNA methylation are associated with symptomatic infection, whereas the host recovery infection phenotypes are related to hypermethylated viral genome, association to repressive histone marks and minichromosomes condensation [14,18,33]. Arabidopsis plants with mutations in components of RdDM pathway are unable to recover after infection with Beet curly top virus lacking a suppressor protein that inhibits methylation (BCTV L2 -), and viral DNA shows hypomethylation [12,[34][35][36]. In natural host-virus interaction, a recovery process also associated with gene silencing was observed in Cucurbit leaf crumple virus-infected watermelon [37].…”