Arabidopsis RNA Polymerase V Mediates Enhanced Compaction and Silencing of Geminivirus and Transposon Chromatin during Host Recovery from Infection

Abstract

Plants employ RNA-directed DNA methylation (RdDM) and dimethylation of histone 3 lysine 9 (H3K9me2) to silence geminiviruses and transposable elements (TEs). We previously showed that canonical RdDM (Pol IV-RdDM) involving RNA polymerases IV and V (Pol IV and Pol V) is required for to recover from infection with lacking a suppressor protein that inhibits methylation (BCTV ). Recovery, which is characterized by reduced viral DNA levels and symptom remission, allows normal floral development. Here, we used formaldehyde-assisted isolation of regulatory elements (FAIRE) to confirm that>90% of BCTV chromatin is highly compacted during recovery, and a micrococcal nuclease-chromatin immunoprecipitation assay showed that this is largely due to increased nucleosome occupancy. Physical compaction correlated with augmented cytosine and H3K9 methylation and with reduced viral gene expression. We additionally demonstrated that these phenomena are dependent on Pol V and by extension the Pol IV-RdDM pathway. BCTV was also used to evaluate the impact of viral infection on host loci, including repressed retrotransposons and Remarkably, an unexpected Pol V-dependent hypersuppression of these TEs was observed, resulting in transcript levels even lower than those detected in uninfected plants. Hypersuppression is likely to be especially important for natural recovery from wild-type geminiviruses, as viral L2 and AL2 proteins cause ectopic TE expression. Thus, Pol IV-RdDM targets both viral and TE chromatin during recovery, simultaneously silencing the majority of viral genomes and maintaining host genome integrity by enforcing tighter control of TEs in future reproductive tissues. In plants, RdDM pathways use small RNAs to target cytosine and H3K9 methylation, thereby silencing DNA virus genomes and transposable elements (TEs). Further, Pol IV-RdDM involving Pol IV and Pol V is a key aspect of host defense that can lead to recovery from geminivirus infection. Recovery is characterized by reduced viral DNA levels and symptom remission and thus allows normal floral development. Studies described here demonstrate that the Pol V-dependent enhanced viral DNA and histone methylation observed during recovery result in increased chromatin compaction and suppressed gene expression. In addition, we show that TE-associated chromatin is also targeted for hypersuppression during recovery, such that TE transcripts are reduced below the already low levels seen in uninfected plants. Thus, Pol IV-RdDM at once silences the majority of viral genomes and enforces a tight control over TEs which might otherwise jeopardize genome integrity in future reproductive tissue.

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

“…Total RNA was extracted from uninfected silique tissue using PureLink RNA reagent (ThermoFisher Scientific) according to the manufacturer's instructions. 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).…”

“…Viral transcript levels were normalized to viral DNA levels determined using qPCR. DNA gel blotting and analysis were carried out essentially as previously reported (36).…”

“…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).…”

The NIa-Protease Protein Encoded by the Pepper Mottle Virus Is a Pathogenicity Determinant and Releases DNA Methylation of Nicotiana benthamiana

“…Cytosine DNA methylation, one of the fundamental aspects of stable epigenetic modification, modulates coding genes that involve various cellular biological processes and molecular functions to counter virus infection (Wang et al, 2019). The cytosines in the genome of DNA viruses could be directly methylated through plant DNA methylation; for example, geminivirus transcription is usually disrupted by host DNA methylation, which occurs in the viral genome through RNAdirected DNA methylation (RdDM) (Coursey et al, 2018). Although RNA viruses cannot be directly methylated by plant DNA methylation owing to the absence of DNA in the replication process, the 2b protein encoded by cucumber mosaic virus (CMV) could bind the AGO4-related small RNAs and impairs AGO4 activities to release the DNA methylation of Nicotiana benthamiana, suggesting the modulation of RNA virus replication by plant DNA methylation (Hamera et al, 2012).…”

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

“…The in vitro DNA methylation of TGMV reduces viral replication in tobacco protoplasts [32]. 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].…”