Summary The RNA-directed DNA methylation (RdDM) pathway in plants controls gene expression via cytosine DNA methylation. The ability to manipulate RdDM would shed light on the mechanisms and applications of DNA methylation to control gene expression. Here, we identified diverse RdDM proteins that are capable of targeting methylation and silencing in Arabidopsis when tethered to an artificial zinc finger (ZF-RdDM). We studied their order of action within the RdDM pathway by testing their ability to target methylation in different mutants. We also evaluated ectopic siRNA biogenesis, RNA polymerase V (Pol V) recruitment, targeted DNA methylation, and gene-expression changes at thousands of ZF-RdDM targets. We found that co-targeting both arms of the RdDM pathway, siRNA biogenesis and Pol V recruitment, dramatically enhanced targeted methylation. This work defines how RdDM components establish DNA methylation and enables new strategies for epigenetic gene regulation via targeted DNA methylation.

SignificanceDNA methylation is an epigenetic modification involved in gene silencing. Studies of this modification usually rely on the use of mutants or chemicals that affect methylation maintenance. Those approaches cause global changes in methylation and make difficult the study of the impact of methylation on gene expression or chromatin at specific loci. In this study, we develop tools to target DNA demethylation in plants. We report efficient on-target demethylation and minimal effects on global methylation patterns, and show that in one case, targeted demethylation is heritable. These tools can be used to approach basic questions about DNA methylation biology, as well as to develop new biotechnology strategies to modify gene expression and create new plant trait epialleles.

The natural outcome of some plant-virus interactions is symptom recovery, which is characterized by the emergence of asymptomatic leaves following a systemic symptomatic infection. Symptom recovery is generally accompanied with reduced virus titers and sequence-specific resistance to secondary infection and has been linked with the induction of antiviral RNA silencing. Recent studies have revealed an unsuspected diversity of silencing mechanisms associated with symptom recovery in various host-virus interactions, including degradation or translation repression of viral RNAs and in the case of DNA viruses, transcriptional arrest of viral minichromosomes. RNA silencing may also contribute to symptom alleviation by regulating plant gene expression. In this review, we discuss the evidence supporting the role of various RNA silencing mechanisms in symptom recovery. We also discuss how a delicate equilibrium between RNA silencing and virus counter-defense responses in recovered leaves may help maintain virus titers at levels below the threshold required for symptom induction.

Symptom recovery in nepovirus-infected plants has been attributed to the induction of RNA silencing. However, recovery is not always accompanied with viral RNA clearance. In this study, we show that recovery of Nicotiana benthamiana plants infected with the tomato ringspot virus (ToRSV) is associated with a reduction of the steady-state levels of RNA2-encoded coat protein (CP) and movement protein but not of RNA2. In vivo labeling experiments revealed efficient synthesis of the CP early in infection, but reduced RNA2 translation later in infection. Silencing of Argonaute1-like (Ago1) genes prevented both symptom recovery and RNA2 translation repression. Similarly, growing the plants at lower temperature (21 °C rather than 27 °C) alleviated the recovery and the translation repression. Taken together, our results suggest that recovery of ToRSV-infected plants is associated with an Ago1-dependent mechanism that represses the translation of viral RNA2.

CRISPR-based targeted modification of epigenetic marks such as DNA cytosine methylation is an important strategy to regulate the expression of genes and their associated phenotypes. Although plants have DNA methylation in all sequence contexts (CG, CHG, CHH, where H = A, T, C), methylation in the symmetric CG context is particularly important for gene silencing and is very efficiently maintained through mitotic and meiotic cell divisions. Tools that can directly add CG methylation to specific loci are therefore highly desirable but are currently lacking in plants. Here we have developed two CRISPR-based CG-specific targeted DNA methylation systems for plants using a variant of the bacterial CG-specific DNA methyltransferase MQ1 with reduced activity but high specificity. We demonstrate that the methylation added by MQ1 is highly target specific and can be heritably maintained in the absence of the effector. These tools should be valuable both in crop engineering and in plant genetic research.