DNA conformational dynamics in the context-dependent non-CG CHH methylation by plant methyltransferase DRM2.

DNA methylation provides an important epigenetic mechanism that critically regulates gene expression, genome imprinting, and retrotransposon silencing. In plants, DNA methylation is prevalent not only in a CG dinucleotide context but also in non-CG contexts, namely CHG and CHH (H = C, T, or A) methylation. It has been established that plant non-CG DNA methylation is highly context dependent, with the +1- and +2-flanking sequences enriched with A/T nucleotides. How DNA sequence, conformation, and dynamics influence non-CG methylation remains elusive. Here, we report structural and biochemical characterizations of the intrinsic substrate preference of DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2), a plant DNA methyltransferase responsible for establishing all cytosine methylation and maintaining CHH methylation. Among nine CHH motifs, the DRM2 methyltransferase (MTase) domain shows marked substrate preference toward CWW (W = A or T) motifs, correlating well with their relative abundance in planta. Furthermore, we report the crystal structure of DRM2 MTase in complex with a DNA duplex containing a flexible TpA base step at the +1/+2-flanking sites of the target nucleotide. Comparative structural analysis of the DRM2-DNA complexes provides a mechanism by which flanking nucleotide composition impacts DRM2-mediated DNA methylation. Furthermore, the flexibility of the TpA step gives rise to two alternative DNA conformations, resulting in different interactions with DRM2 and consequently temperature-dependent shift of the substrate preference of DRM2. Together, this study provides insights into how the interplay between the conformational dynamics of DNA and temperature as an environmental factor contributes to the context-dependent CHH methylation by DRM2.

Structure and Mechanism of Plant DNA Methyltransferases.

DNA methylation is an important epigenetic mark conserved in eukaryotes from fungi to animals and plants, where it plays a crucial role in regulating gene expression and transposon silencing. Once the methylation mark is established by de novo DNA methyltransferases, specific regulatory mechanisms are required to maintain the methylation state during chromatin replication, both during meiosis and mitosis. Plant DNA methylation is found in three contexts; CG, CHG, and CHH (H = A, T, C), which are established and maintained by a unique set of DNA methyltransferases and are regulated by plant-specific pathways. DNA methylation in plants is often associated with other epigenetic modifications, such as noncoding RNA and histone modifications. This chapter focuses on the structure, function, and regulatory mechanism of plant DNA methyltransferases and their crosstalk with other epigenetic pathways.

Turnip mosaic virus manipulates DRM2 expression to regulate host CHH and CHG methylation for robust infection.

DNA methylation is an important epigenetic marker for the suppression of transposable elements (TEs) and the regulation of plant immunity. However, little is known how RNA viruses counter defense such antiviral machinery. In this study, the change of DNA methylation in turnip mosaic virus (TuMV)-infected cells was analyzed by whole genome bisulfite sequencing. Results showed that the total number of methylated sites of CHH and CHG increased in TuMV-infected cells, the majority of differentially methylated regions (DMRs) in the CHH and CHG contexts were associated with hypermethylation. Gene expression analysis showed that the expression of two methylases (DRM2 and CMT3) and three demethylases (ROS3, DML2, DML3) was significantly increased and decreased in TuMV-infected cells, respectively. Pathogenicity tests showed that the enhanced resistance to TuMV of the loss-of-function mutant of DRM2 is associated with unregulated expression of several defense-related genes. Finally, we found TuMV-encoded NIb, the viral RNA-dependent RNA polymerase, was able to induce the expression of DRM2. In conclusion, this study discovered that TuMV can modulate host DNA methylation by regulating the expression of DRM2 to promote virus infection.

DORMANCY/AUXIN ASSOCIATED FAMILY PROTEIN 2 of Arabidopsis thaliana is a negative regulator of local and systemic acquired resistance.

To fine tune defense response output, plants recruit both positive and negative regulators. Here we report Arabidopsis DORMANCY/AUXIN ASSOCIATED FAMILY PROTEIN 2(DAP2) gene as a negative regulator of basal defense against virulent bacterial pathogens. Expression of DAP2 enhances upon pathogen inoculation. Our experiments show that DAP2 suppressed resistance against virulent strains of bacterial pathogens, pathogen-induced callose deposition, and ROS accumulation; however, it did not influence effector-triggered immunity. In addition, DAP2 negatively regulated systemic acquired resistance (SAR). DAP2 expression was enhanced in the pathogen-free systemic tissues of SAR-induced plants. Previously, Arabidopsis Flowering locus D (FLD) gene has been shown to be essential for SAR but not for local resistance. We show here that FLD function is necessary for SAR-induced expression of DAP2, suggesting DAP2 as a target of FLD for activation of SAR in Arabidopsis.

Hairy Root Transformation: A Useful Tool to Explore Gene Function and Expression in Salix spp. Recalcitrant to Transformation.

Willow (Salix spp. L.) species are fast-growing trees and shrubs that have attracted emergent attention for their potential as feedstocks for bioenergy and biofuel production, as well as for pharmaceutical and phytoremediation applications. This economic and environmental potential has propelled the creation of several genetic and genomic resources for Salix spp. Furthermore, the recent availability of an annotated genome for Salix purpurea has pinpointed novel candidate genes underlying economically relevant traits. However, functional studies have been stalled by the lack of rapid and efficient coupled regeneration-transformation systems for Salix purpurea and Salix spp. in general. In this report, we describe a fast and highly efficient hairy root transformation protocol for S. purpurea. It was effective for different explant sources and S. purpurea genotypes, with efficiencies between 63.4% and 98.7%, and the screening of the transformed hairy roots was easily carried out using the fluorescent marker DsRed. To test the applicability of this hairy root transformation system for gene functional analysis, we transformed hairy roots with the vector pGWAY-SpDRM2, where the gene SpDRM2 encoding a putative Domain Rearranged Methyltransferase (DRM) was placed under the control of the CaMV 35S constitutive promoter. Indeed, the transgenic hairy roots obtained exhibited significantly increased expression of SpDRM2 as compared to controls, demonstrating that this protocol is suitable for the medium/high-throughput functional characterization of candidate genes in S. purpurea and other recalcitrant Salix spp.

UAP56 associates with DRM2 and is localized to chromatin in Arabidopsis.

Repeated sequence expression and transposable element mobilization are tightly controlled by multilayer processes, which include DNA 5'-cytosine methylation. The RNA-directed DNA methylation (RdDM) pathway, which uses siRNAs to guide sequence-specific directed DNA methylation, emerged specifically in plants. RdDM ensures DNA methylation maintenance on asymmetric CHH sites and specifically initiates de novo methylation in all cytosine sequence contexts through the action of DRM DNA methyltransferases, of which DRM2 is the most prominent. The RdDM pathway has been well described, but how DRM2 is recruited onto DNA targets and associates with other RdDM factors remains unknown. To address these questions, we developed biochemical approaches to allow the identification of factors that may escape genetic screens, such as proteins encoded by multigenic families. Through both conventional and affinity purification of DRM2, we identified DEAD box RNA helicases U2AF56 Associated Protein 56 (UAP56a/b), which are widespread among eukaryotes, as new DRM2 partners. We have shown that, similar to DRM2 and other RdDM actors, UAP56 has chromatin-associated protein properties. We confirmed this association both in vitro and in vivo in reproductive tissues. In addition, our experiments also suggest that UAP56 may exhibit differential distribution in cells depending on plant organ. While originally identified for its role in splicing, our study suggests that UAP56 may also have other roles, and our findings allow us to initiate discussion about its potential role in the RdDM pathway.

DNA methylation maintenance consolidates RNA-directed DNA methylation and transcriptional gene silencing over generations in Arabidopsis thaliana.

In plants, 24 nucleotide short interfering RNAs serve as a signal to direct cytosine methylation at homologous DNA regions in the nucleus. If the targeted DNA has promoter function, this RNA-directed DNA methylation may result in transcriptional gene silencing. In a genetic screen for factors involved in RNA-directed transcriptional silencing of a ProNOS-NPTII reporter transgene in Arabidopsis thaliana, we captured alleles of DOMAINS REARRANGED METHYLTRANSFERASE 2, the gene encoding the DNA methyltransferase that is mainly responsible for de novo DNA methylation in the context of RNA-directed DNA methylation. Interestingly, methylation of the reporter gene ProNOS was not completely erased in these mutants, but persisted in the symmetric CG context, indicating that RNA-directed DNA methylation had been consolidated by DNA methylation maintenance. Taking advantage of the segregation of the transgenes giving rise to ProNOS short interfering RNAs and carrying the ProNOS-NPTII reporter in our experimental system, we found that ProNOS DNA methylation maintenance was first evident after two generations of ongoing RNA-directed DNA methylation, and then increased in extent with further generations. As ProNOS DNA methylation had already reached its final level in the first generation of RNA-directed DNA methylation, our findings suggest that establishment of DNA methylation at a particular region may be divided into distinct stages. An initial phase of efficient, but still fully reversible, de novo DNA methylation and transcriptional gene silencing is followed by transition to efficient maintenance of cytosine methylation in a symmetric sequence context accompanied by persistence of gene silencing.