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Parental epigenetic asymmetries, which contribute to the monoallelic expression of genes known as imprints, play a critical role in seed development in flowering plants. Primarily, differential DNA methylation patterns and histone modifications on parental alleles form the molecular basis of gene imprinting. Plants predominantly exhibit this non-Mendelian inheritance phenomenon in the endosperm and the early embryo of developing seeds. Imprinting is crucial for regulating nutrient allocation, maintaining seed development, resolving parental conflict, and facilitating evolutionary adaptation. Disruptions in imprinted gene expression, mediated by epigenetic regulators and parental ploidy levels, can lead to endosperm-based hybridization barriers and hybrid dysfunction, ultimately reducing genetic diversity in plant populations. Conversely, imprinting helps maintain genetic stability within plant populations. Imprinted genes likely influence seed development in various ways, including ensuring proper endosperm development, influencing seed dormancy, and regulating seed size. However, the functions of most imprinted genes, the evolutionary significance of imprinting, and the long-term consequences of imprinting disruptions on plant development and adaptation need further exploration. Thus, it is clear that research on imprinting has immense potential for improving our understanding of plant development and ultimately enhancing key agronomic traits. This review decodes the possible genetic and epigenetic regulatory factors underpinning genomic imprinting and their positive and negative consequences on seed development. This study also forecasts the potential implications of exploiting gene imprinting for crop improvement programs. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the relationship between breast cancer susceptibility gene-1 (BRCA1) gene methylation and the radiosensitivity of breast cancer. Materials and Methods: The authors studied three breast cancer cell lines: MDA-MB-435, MDA-MB-231, and MCF-7 cells. They constructed five short hairpin RNAs (shRNAs) and five small interfering RNAs to target selected promoter loci and initiate sequence-specific methylation in breast cancer cells. Pyrosequencing was used to analyze the state of DNA methylation. Quantitative real-time polymerase chain reaction was used to detect BRCA1 mRNA expression and RNA-directed DNA methylation (RdDM)-related gene expression. Western blotting was performed to analyze BRCA1 protein expression. Colony formation assays and γ-histone H2A foci formation assays were conducted to assess the surviving fraction (SF) and double-strand break (DSB) repair ability of cells after irradiation. Results: The authors constructed five strains of lentivirus vectors and five plasmid vectors targeting BRCA1 promoter region. In MDA-MB-435 cells, lentivirus-mediated RNA interference targeting Site 1 of BRCA1 increased the methylation levels of BRCA1 and reduced BRCA1 mRNA and protein expression. The SF and the ability to repair DNA DSBs were reduced in the combined LV-BRCA1RNAi-Site 1 infection and irradiation group. Conclusions: The authors' findings suggest that the shRNA suppressed the expression levels of the BRCA1 gene and reduced the SF and DNA repair ability of cells after irradiation through RdDM. In summary, the radiosensitivity of breast cancer cells may correlate with BRCA1 methylation. Advances in Knowledge: The authors first utilized a lentivirus-based shRNA-mediated specific-sequence DNA methylation of the BRCA1 gene mediated by RdDM. [ABSTRACT FROM AUTHOR]

RNA-directed DNA methylation (RdDM) is a plant-specific de novo methylation pathway that is responsible for maintenance of asymmetric methylation (CHH, H = A, T or G) in euchromatin. Loci with CHH methylation produce 24 nucleotide (nt) short interfering (si) RNAs. These siRNAs direct additional CHH methylation to the locus, maintaining methylation states through DNA replication. To understand the necessary conditions to produce stable methylation, we developed a stochastic mathematical model of RdDM. The model describes DNA target search by siRNAs derived from CHH methylated loci bound by an Argonaute. Methylation reinforcement occurs either throughout the cell cycle (steady) or immediately following replication (bursty). We compare initial and final methylation distributions to determine simulation conditions that produce stable methylation. We apply this method to the low CHH methylation case. The resulting model predicts that siRNA production must be linearly proportional to methylation levels, that bursty reinforcement is more stable and that slightly higher levels of siRNA production are required for searching DNA, compared to RNA. Unlike CG methylation, which typically exhibits bi-modality with loci having either 100% or 0% methylation, CHH methylation exists across a range. Our model predicts that careful tuning of the negative feedback in the system is required to enable stable maintenance.

In flowering plants, euchromatic transposons are transcriptionally silenced by RNA-directed DNA Methylation, a small RNA-guided de novo methylation pathway. RNA-directed DNA Methylation requires the activity of the RNA Polymerases IV and V, which produce small RNA precursors and noncoding targets of small RNAs, respectively. These polymerases are distinguished from Polymerase II by multiple plant-specific paralogous subunits. Most RNA-directed DNA Methylation components are present in all land plants, and some have been found in the charophytic green algae, a paraphyletic group that is sister to land plants. However, the evolutionary origin of key RNA-directed DNA Methylation components, including the two largest subunits of Polymerase IV and Polymerase V, remains unclear. Here, we show that multiple lineages of charophytic green algae encode a single-copy precursor of the largest subunits of Polymerase IV and Polymerase V, resolving the two presumed duplications in this gene family. We further demonstrate the presence of a Polymerase V-like C-terminal domain, suggesting that the earliest form of RNA-directed DNA Methylation utilized a single Polymerase V-like polymerase. Finally, we reveal that charophytic green algae encode a single CLSY/DRD1-type chromatin remodeling protein, further supporting the presence of a single specialized polymerase in charophytic green algae. [ABSTRACT FROM AUTHOR]

Key message: Barley AGO4 proteins complement expressional changes of epigenetically regulated genes in Arabidopsis ago4-3 mutant and show a distinct affinity for the 5′ terminal nucleotide of small RNAs, demonstrating functional conservation and divergence. The function of Argonaute 4 (AGO4) in Arabidopsis thaliana has been extensively characterized; however, its role in monocots, which have large genomes abundantly supplemented with transposable elements (TEs), remains elusive. The study of barley AGO4 proteins can provide insights into the conserved aspects of RNA-directed DNA methylation (RdDM) and could also have further applications in the field of epigenetics or crop improvement. Bioinformatic analysis of RNA sequencing data identified two active AGO4 genes in barley, HvAGO4a and HvAGO4b. These genes function similar to AtAGO4 in an Arabidopsis heterologous complementation system, primarily binding to 24-nucleotide long small RNAs (sRNAs) and triggering methylation at specific target loci. Like AtAGO4, HvAGO4B exhibits a preference for binding sRNAs with 5′ adenine residue, while also accepting 5′ guanine, uracil, and cytosine residues. In contrast, HvAGO4A selectively binds only sRNAs with a 5′ adenine residue. The diverse binding capacity of barley AGO4 proteins is reflected in TE-derived sRNAs and in their varying abundance. Both barley AGO4 proteins effectively restore the levels of extrachromosomal DNA and transcript abundancy of the heat-activated ONSEN retrotransposon to those observed in wild-type Arabidopsis plants. Our study provides insight into the distinct binding specificities and involvement in TE regulation of barley AGO4 proteins in Arabidopsis by heterologous complementation. [ABSTRACT FROM AUTHOR]

RNA-directed DNA methylation (RdDM) is a plant-specific de novo DNA methylation pathway that relies on the atypical RNA polymerases Pol IV-RDR2 complex and Pol V to produce the siRNA precursor and lncRNA, respectively. Both Pol IV and Pol V have evolved from Pol II, but adopt unique biochemical features relevant for their functions in siRNA precursor (Pol IV) and scaffold lncRNA (Pol V) production. Recent structural studies suggest that the Pol IV and Pol V NRPD(E)2 subunit-induced enhanced backtracking plays an important role in their ncRNA production function. A dual measurement mechanism was proposed for the siRNA precursor production by Pol IV-RDR2 holoenzyme in RdDM. In plants, two atypical DNA-dependent RNA polymerases, RNA polymerase IV (Pol IV) and Pol V, and an RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) together produce noncoding RNAs (ncRNAs) to guide the plant-specific RNA-directed DNA methylation (RdDM). Although both Pol IV and Pol V have evolved from the canonical Pol II, they have adapted to different roles in RdDM. The mechanisms of their adaptation are key to understanding plant DNA methylation and the divergent evolution of polymerases. In this review, we summarize insights that have emerged from recent structural studies of Pol IV, Pol V, and RDR2 and discuss their structural features critical for efficient ncRNA production in RdDM. [ABSTRACT FROM AUTHOR]

SUMMARY: Chromatin in eukaryotes folds into a complex three‐dimensional (3D) structure that is essential for controlling gene expression and cellular function and is dynamically regulated in biological processes. Studies on plant phosphorus signaling have concentrated on single genes and gene interactions. It is critical to expand the existing signaling pathway in terms of its 3D structure. In this study, low‐Pi treatment led to greater chromatin volume. Furthermore, low‐Pi stress increased the insulation score and the number of TAD‐like domains, but the effects on the A/B compartment were not obvious. The methylation levels of target sites (hereafter as RdDM levels) peaked at specific TAD‐like boundaries, whereas RdDM peak levels at conserved TAD‐like boundaries shifted and decreased sharply. The distribution pattern of RdDM sites originating from the Helitron transposons matched that of genome‐wide RdDM sites near TAD‐like boundaries. RdDM pathway genes were upregulated in the middle or early stages and downregulated in the later stages under low‐Pi conditions. The RdDM pathway mutant ddm1a showed increased tolerance to low‐Pi stress, with shortened and thickened roots contributing to higher Pi uptake from the shallow soil layer. ChIP‐seq results revealed that ZmDDM1A could bind to Pi‐ and root development‐related genes. Strong associations were found between interacting genes in significantly different chromatin‐interaction regions and root traits. These findings not only expand the mechanisms by which plants respond to low‐Pi stress through the RdDM pathway but also offer a crucial framework for the analysis of biological issues using 3D genomics. Significance Statement: This study is a pioneering study in the field of plant nutrient utilization. For the first time, this study discovered that the plant's low‐Pi response was closely linked to the RdDM pathway and chromatin spatial structure, and the low‐Pi response was validated by a mutant of chromatin remodeling gene ZmDDM1a (RdDM pathway gene). In addition, the results of ChIP‐seq and multi‐point GWAS for 2 years revealed the interaction of a large number of candidate genes related to Pi and roots with ZmDDM1A. We believe that the results presented in this article will have a great influence in this field and will be concerned and cited by many researchers. [ABSTRACT FROM AUTHOR]

Gamete development is a complex process during the plant reproductive life cycle. A global vision of the molecular mechanisms regulating gametic development would enhance our understanding of developmental similarities and differences between male and female reproductive pathways in plants. In this study, we applied comparative transcriptomics to characterize gene expression during sporogenesis and gametogenesis in Arabidopsis. Compared with genes expressed in leaves, 11,112 and 10,133 differentially expressed genes (DEGs) were identified in the anthers and ovules, respectively. The shared DEGs exhibited a similar expression pattern regarding cell cycle and chromatin dynamics, but DEGs associated with cell wall formation and pectin modifications were prevalent during male development, reflecting dynamic changes during pollen wall development in anthers. In contrast, DEGs related to DNA repair, homologous recombination, and RNA-directed DNA methylation (RdDM) were comparatively over-represented in ovules, probably indicating a relatively intensified mechanism for supervising genome fidelity during female gametogenesis. In addition, DEGs related to brassinolide (BR) synthesis and signaling pathway were also highly enriched in ovules, suggesting a pivotal role in female gamete development. Collectively, our results provide a contrasting transcriptional atlas in developing anthers and ovules, and shed new light on our global understanding of gamete development in plants. [ABSTRACT FROM AUTHOR]

The outbreak of Fusarium head blight (FHB) poses a serious threat to wheat production as it leads to both significant yield losses and accumulation of several mycotoxins including deoxynivalenol (DON) in the grains, which are harmful to human and livestock. To date, hundreds of FHB-resistance-related quantitative trait loci (QTLs) have been reported, but only a few of them have been cloned and used for breeding. Small interfering RNAs (siRNA) have been reported in plants to mediate host defense against pathogens, but they have rarely been reported in wheat-FHB interaction. In order to identify the key siRNAs that can potentially be used in the improvement of resistance to FHB, siRNAs from the spikes of an FHB-resistant variety Sumai 3 and an FHB-susceptible variety of Chinese Spring (CS) were sequenced after F. graminearum infection and mock inoculation, respectively. The expression patterns of the siRNAs of interest were analyzed. A total of 4019 siRNAs of high-confidence were identified, with 131 being CS-specific, 309 Sumai 3-specific and 3071 being common in both varieties. More than 87% of these siRNAs were 24 nt in length. An overall down-regulation trend was found for siRNAs in the spikes of both varieties after being infected with F. graminearum. The expression patterns for Triticum aestivum Dicer-like 3 (TaDCL3) that synthesizes 24 nt siRNAs were validated by qRT-PCR, which were positively correlated with those of the siRNAs. A total of 85% of the differentially expressed genes putatively targeted by the siRNAs were significantly up-regulated after infection, showing a negative correlation with the overall down-regulated expression of siRNAs. Interestingly, the majority of the up-regulated genes are annotated as disease resistance. These results suggested that the inhibition of siRNA by F. graminearum up-regulated the disease resistance genes, which were putatively suppressed by siRNAs through RNA-directed DNA methylation (RdDM). Consequently, the resistant capability to F. graminearum infection was enhanced. This study provides novel clues for investigating the function of siRNA in wheat-F. graminearum interaction. [ABSTRACT FROM AUTHOR]

Small RNAs are abundant in plant reproductive tissues, especially 24-nucleotide (nt) small interfering RNAs (siRNAs). Most 24-nt siRNAs are dependent on RNA Pol IV and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and establish DNA methylation at thousands of genomic loci in a process called RNA-directed DNA methylation (RdDM). In Brassica rapa, RdDM is required in the maternal sporophyte for successful seed development. Here, we demonstrate that a small number of siRNA loci account for over 90% of siRNA expression during B. rapa seed development. These loci exhibit unique characteristics with regard to their copy number and association with genomic features, but they resemble canonical 24-nt siRNA loci in their dependence on RNA Pol IV/RDR2 and role in RdDM. These loci are expressed in ovules before fertilization and in the seed coat, embryo, and endosperm following fertilization. We observed a similar pattern of 24-nt siRNA expression in diverse angiosperms despite rapid sequence evolution at siren loci. In the endosperm, siren siRNAs show a marked maternal bias, and siren expression in maternal sporophytic tissues is required for siren siRNA accumulation. Together, these results demonstrate that seed development occurs under the influence of abundant maternal siRNAs that might be transported to, and function in, filial tissues.

DNA methylation is an important epigenetic mark involved in gene regulation and silencing of transposable elements. The presence or absence of DNA methylation at specific sites can influence nearby gene expression and cause phenotypic changes that remain stable over generations. Recently, development of new technologies has enabled the targeted addition or removal of DNA methylation at specific sites of the genome. Of these new technologies, the targeting of the catalytic domain of Nicotiana tabacum DOMAINS REARRANGED METHYLTRANSFERASE 2 (ntDRM2cd) offers a promising tool for the addition of DNA methylation as it can directly methylate DNA. However, the methylation targeting efficiency of constructs using ntDRM2cd thus far has been relatively low. Previous studies have shown that the use of different promoters or terminators can greatly improve genome-editing efficiencies. In this study, we systematically survey a variety of promoter and terminator combinations to identify optimal combinations to use when targeting the addition of DNA methylation in Arabidopsis thaliana.

Abstract 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.

RNA-directed DNA methylation (RdDM) is a plant-specific de novo methylation pathway that is responsible for maintenance of asymmetric methylation (CHH, H = A, T or G) in euchromatin. Loci with CHH methylation produce 24 nucleotide (nt) short interfering (si) RNAs. These siRNAs direct additional CHH methylation to the locus, maintaining methylation states through DNA replication. To understand the necessary conditions to produce stable methylation, we developed a stochastic mathematical model of RdDM. The model describes DNA target search by siRNAs derived from CHH methylated loci bound by an Argonaute. Methylation reinforcement occurs either throughout the cell cycle (steady) or immediately following replication (bursty). We compare initial and final methylation distributions to determine simulation conditions that produce stable methylation. We apply this method to the low CHH methylation case. The resulting model predicts that siRNA production must be linearly proportional to methylation levels, that bursty reinforcement is more stable and that slightly higher levels of siRNA production are required for searching DNA, compared to RNA. Unlike CG methylation, which typically exhibits bi-modality with loci having either 100% or 0% methylation, CHH methylation exists across a range. Our model predicts that careful tuning of the negative feedback in the system is required to enable stable maintenance.

Abiotic and biotic stresses can lead to changes in host DNA methylation, which in plants is also mediated by an RNA-directed DNA methylation mechanism. Infections with viroids have been shown to affect DNA methylation dynamics in different plant hosts. The aim of our research was to determine the content of 5-methylcytosine (5-mC) in genomic DNA at the whole genome level of hop plants (Humulus lupulus Var. 'Celeia') infected with different viroids and their combinations and to analyse the expression of the selected genes to improve our understanding of DNA methylation dynamics in plant-viroid systems. The adapted HPLC-UV method used proved to be suitable for this purpose, and thus we were able to estimate for the first time that the cytosine methylation level in viroid-free hop plants was 26.7%. Interestingly, the observed 5-mC level was the lowest in hop plants infected simultaneously with CBCVd, HLVd and HSVd (23.7%), whereas the highest level was observed in plants infected with HLVd (31.4%). In addition, we identified three DNA methylases and one DNA demethylase gene in the hop's draft genome. The RT-qPCR revealed upregulation of all newly identified genes in hop plants infected with all three viroids, while no altered expression was observed in any of the other hop plants tested, except for CBCVd-infected hop plants, in which one DNA methylase was also upregulated. [ABSTRACT FROM AUTHOR]

SUMMARY: All eukaryotes possess three DNA‐dependent RNA polymerases, Pols I–III, while land plants possess two additional polymerases, Pol IV and Pol V. Derived through duplication of Pol II subunits, Pol IV produces 24‐nt short interfering RNAs that interact with Pol V transcripts to target de novo DNA methylation and silence transcription of transposons. Members of the grass family encode additional duplicated subunits of Pol IV and V, raising questions regarding the function of each paralog. In this study, we identify a null allele of the putative Pol IV second subunit, NRPD2, and demonstrate that NRPD2 is the sole subunit functioning with NRPD1 in small RNA production and CHH methylation in leaves. Homozygous nrpd2 mutants have neither gametophytic defects nor embryo lethality, although adult plants are dwarf and sterile. Significance Statement: Loss of NRPD2 function in rice causes loss of 24‐nt short interfering RNAs and CHH methylation from thousands of genomic regions, including small intergenic transposons. This results in dwarfism and defects in reproductive development, but does not cause embryo lethality. [ABSTRACT FROM AUTHOR]

DNA methylation is associated with gene silencing in eukaryotic organisms. Although pathways controlling the establishment, maintenance and removal of DNA methylation are known, relatively little is understood about how DNA methylation influences gene expression. Here we identified a METHYL-CpG-BINDING DOMAIN 7 (MBD7) complex in Arabidopsis thaliana that suppresses the transcriptional silencing of two LUCIFERASE (LUC) reporters via a mechanism that is largely downstream of DNA methylation. Although mutations in components of the MBD7 complex resulted in modest increases in DNA methylation concomitant with decreased LUC expression, we found that these hyper-methylation and gene expression phenotypes can be genetically uncoupled. This finding, along with genome-wide profiling experiments showing minimal changes in DNA methylation upon disruption of the MBD7 complex, places the MBD7 complex amongst a small number of factors acting downstream of DNA methylation. This complex, however, is unique as it functions to suppress, rather than enforce, DNA methylation-mediated gene silencing.

Abstract Background RNA-directed DNA methylation (RdDM) initiates cytosine methylation in all contexts and maintains asymmetric CHH methylation. Mature plant embryos show one of the highest levels of CHH methylation, and it has been suggested that RdDM is responsible for this hypermethylation. Because loss of RdDM in Brassica rapa causes seed abortion, embryo methylation might play a role in seed development. RdDM is required in the maternal sporophyte, suggesting that small RNAs from the maternal sporophyte might translocate to the developing embryo, triggering DNA methylation that prevents seed abortion. This raises the question of whether embryo hypermethylation is autonomously regulated by the embryo itself or influenced by the maternal sporophyte. Results Here, we demonstrate that B. rapa embryos are hypermethylated in both euchromatin and heterochromatin and that this process requires RdDM. Contrary to the current models, B. rapa embryo hypermethylation is not correlated with demethylation of the endosperm. We also show that maternal somatic RdDM is not sufficient for global embryo hypermethylation, and we find no compelling evidence for maternal somatic influence over embryo methylation at any locus. Decoupling of maternal and zygotic RdDM leads to successful seed development despite the loss of embryo CHH hypermethylation. Conclusions We conclude that embryo CHH hypermethylation is conserved, autonomously controlled, and not required for embryo development. Furthermore, maternal somatic RdDM, while required for seed development, does not directly influence embryo methylation patterns.

Abstract Background Plants are naturally associated with root microbiota, which are microbial communities influential to host fitness. Thus, it is important to understand how plants control root microbiota. Epigenetic factors regulate the readouts of genetic information and consequently many essential biological processes. However, it has been elusive whether RNA-directed DNA methylation (RdDM) affects root microbiota assembly. Results By applying 16S rRNA gene sequencing, we investigated root microbiota of Arabidopsis mutants defective in the canonical RdDM pathway, including dcl234 that harbors triple mutation in the Dicer-like proteins DCL3, DCL2, and DCL4, which produce small RNAs for RdDM. Alpha diversity analysis showed reductions in microbe richness from the soil to roots, reflecting the selectivity of plants on root-associated bacteria. The dcl234 triple mutation significantly decreases the levels of Aeromonadaceae and Pseudomonadaceae, while it increases the abundance of many other bacteria families in the root microbiota. However, mutants of the other examined key players in the canonical RdDM pathway showed similar microbiota as Col-0, indicating that the DCL proteins affect root microbiota in an RdDM-independent manner. Subsequently gene analysis by shotgun sequencing of root microbiome indicated a selective pressure on microbial resistance to plant defense in the dcl234 mutant. Consistent with the altered plant-microbe interactions, dcl234 displayed altered characters, including the mRNA and sRNA transcriptomes that jointly highlighted altered cell wall organization and up-regulated defense, the decreased cellulose and callose deposition in root xylem, and the restructured profile of root exudates that supported the alterations in gene expression and cell wall modifications. Conclusion Our findings demonstrate an important role of the DCL proteins in influencing root microbiota through integrated regulation of plant defense, cell wall compositions, and root exudates. Our results also demonstrate that the canonical RdDM is dispensable for Arabidopsis root microbiota. These findings not only establish a connection between root microbiota and plant epigenetic factors but also highlight the complexity of plant regulation of root microbiota. Video abstract

SUMMARY: Trichoderma atroviride is a root‐colonizing fungus that confers multiple benefits to plants. In plants, small RNA (sRNA)‐mediated gene silencing (sRNA‐MGS) plays pivotal roles in growth, development, and pathogen attack. Here, we explored the role of core components of Arabidopsis thaliana sRNA‐MGS pathways during its interaction with Trichoderma. Upon interaction with Trichoderma, sRNA‐MGS‐related genes paralleled the expression of Arabidopsis defense‐related genes, linked to salicylic acid (SA) and jasmonic acid (JA) pathways. SA‐ and JA‐related genes were primed by Trichoderma in leaves after the application of the well‐known pathogen‐associated molecular patterns flg22 and chitin, respectively. Defense‐related genes were primed in roots as well, but to different extents and behaviors. Phenotypical characterization of mutants in AGO genes and components of the RNA‐dependent DNA methylation (RdDM) pathway revealed that different sets of sRNA‐MGS‐related genes are essential for (i) the induction of systemic acquired resistance against Botrytis cinerea, (ii) the activation of the expression of plant defense‐related genes, and (iii) root colonization by Trichoderma. Additionally, plant growth induced by Trichoderma depends on functional RdDM. Profiling of DNA methylation and histone N‐tail modification patterns at the Arabidopsis Nitrile‐Specifier Protein‐4 (NSP4) locus, which is responsive to Trichoderma, showed altered epigenetic modifications in RdDM mutants. Furthermore, NSP4 is required for the induction of systemic acquired resistance against Botrytis and avoidance of enhanced root colonization by Trichoderma. Together, our results indicate that RdDM is essential in Arabidopsis to establish a beneficial relationship with Trichoderma. We propose that DNA methylation and histone modifications are required for plant priming by the beneficial fungus against B. cinerea. Significance Statement: The root‐colonizing fungi Trichoderma spp. exert beneficial effects on plants. Great efforts have been made to understand how such relationship is established from the fungal side; however, little is known about the molecular mechanisms used by plants. Here, we provide evidence of the requirement of core components of the plant gene silencing machinery for growth and the induction of systemic resistance against phytopathogens promoted by Trichoderma, mainly those of the RNA‐directed DNA methylation pathway. [ABSTRACT FROM AUTHOR]

Alu (B1 in rodents) hypomethylation, commonly found in diabetes mellitus patients, increases DNA damage and, consequently, delays the healing process. Alu siRNA increases Alu methylation, reduces DNA damage, and promotes cell proliferation.Aim: To explore whether B1 siRNA treatment restores B1 hypomethylation, resulting in a reduction in DNA damage and acceleration of the healing process in diabetic rat wounds.Methods: We generated splinted-excisional wounds in a streptozotocin (STZ)-induced type I diabetic rat model and treated the wounds with B1 siRNA/Ca-P nanoparticles to generate de novo DNA methylation in B1 intersperse elements. After treatment, we investigated B1 methylation levels, wound closure rate, wound histopathological structure, and DNA damage markers in diabetic wounds compared to nondiabetic wounds.Results: We reported that STZ-induced diabetic rat wounds exhibited B1 hypomethylation, wound repair defects, anatomical feature defects, and greater DNA damage compared to normal rats. We also determined that B1 siRNA treatment by Ca-P nanoparticle delivery restored a decrease in B1 methylation levels, remedied delayed wound healing, and improved the histological appearance of the wounds by reducing DNA damage.Conclusion: B1 hypomethylation is inducible in an STZ-induced type I diabetes rat model. Restoration of B1 hypomethylation using B1 siRNA leads to increased genome stability and improved wound repair in diabetes. Thus, B1 siRNA intervention may be a promising strategy for reprogramming DNA methylation to treat or prevent DNA damage-related diseases.

In plants, selfish genetic elements, including retrotransposons and DNA viruses, are transcriptionally silenced by RNA-directed DNA methylation. Guiding the process are short interfering RNAs (siRNAs) cut by DICER-LIKE 3 (DCL3) from double-stranded precursors of ~30 bp that are synthesized by NUCLEAR RNA POLYMERASE IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). We show that Pol IV’s choice of initiating nucleotide, RDR2’s initiation 1–2 nt internal to Pol IV transcript ends and RDR2’s terminal transferase activity collectively yield a code that influences which precursor end is diced and whether 24 or 23 nt siRNAs are produced. By diversifying the size, sequence, and strand specificity of siRNAs derived from a given precursor, alternative patterns of DCL3 dicing allow for maximal siRNA coverage at methylated target loci.

SUMMARY: DNA methylation is an important epigenetic mark. In plants, de novo DNA methylation occurs mainly through the RNA‐directed DNA methylation (RdDM) pathway. Researchers have previously inferred that a flowering regulator, MULTICOPY SUPPRESSOR OF IRA1 4 (MSI4)/FVE, is involved in non‐CG methylation at several RdDM targets, suggesting a role of FVE in RdDM. However, whether and how FVE affects RdDM genome‐wide is not known. Here, we report that FVE is required for DNA methylation at thousands of RdDM target regions. In addition, dysfunction of FVE significantly reduces 24‐nucleotide siRNA accumulation that is dependent on factors downstream in the RdDM pathway. By using chromatin immunoprecipitation and sequencing (ChIP‐seq), we show that FVE directly binds to FVE‐dependent 24‐nucleotide siRNA cluster regions. Our results also indicate that FVE may function in RdDM by physically interacting with RDM15, a downstream factor in the RdDM pathway. Our study has therefore revealed that FVE, by associating with RDM15, directly regulates DNA methylation and siRNA accumulation at a subset of RdDM targets. Significance Statement: The RNA‐directed DNA methylation (RdDM) pathway is one of the most important DNA methylation pathways in plants. In this manuscript, we show that FVE is required for DNA methylation and for 24‐nucleotide siRNA accumulation at a subset of RdDM targets. Moreover, FVE physically interacts with RDM15 and shares DNA methylation targets with RDM15 in the genome. Our results support the notion that FVE functions in RdDM, particularly in RNA polymerase V‐mediated downstream step. [ABSTRACT FROM AUTHOR]

Plants respond to abiotic stress stimuli, such as water deprivation, through a hierarchical cascade that includes detection and signaling to mediate transcriptional and physiological changes. The phytohormone abscisic acid (ABA) is well-characterized for its regulatory role in these processes in response to specific environmental cues. ABA-mediated changes in gene expression have been demonstrated to be temporally-dependent, however, the genome-wide timing of these responses are not well-characterized in the agronomically important crop plant Zea mays (maize). ABA-mediated responses are synergistic with other regulatory mechanisms, including the plant-specific RNA-directed DNA methylation (RdDM) epigenetic pathway. Our prior work demonstrated that after relatively long-term ABA induction (8 h), maize plants homozygous for the mop1-1 mutation, defective in a component of the RdDM pathway, exhibit enhanced transcriptional sensitivity to the phytohormone. At this time-point, many hierarchically positioned transcription factors are differentially expressed resulting in primary (direct) and secondary (indirect) transcriptional outcomes. To identify more immediate and direct MOP1-dependent responses to ABA, we conducted a transcriptomic analysis using mop1-1 mutant and wild type plants treated with ABA for 1 h. One h of ABA treatment was sufficient to induce unique categories of differentially expressed genes (DEGs) in mop1-1. A comparative analysis between the two time-points revealed that distinct epigenetically-regulated changes in gene expression occur within the early stages of ABA induction, and that these changes are predicted to influence less immediate, indirect transcriptional responses. Homology with MOP1-dependent siRNAs and a gene regulatory network (GRN) were used to identify putative immediate and indirect targets, respectively. By manipulating two key regulatory networks in a temporal dependent manner, we identified genes and biological processes regulated by RdDM and ABA-mediated stress responses. Consistent with mis-regulation of gene expression, mop1-1 homozygous plants are compromised in their ability to recover from water deprivation. Collectively, these results indicate transcriptionally and physiologically relevant roles for MOP1-mediated regulation of gene expression of plant responses to environmental stress.

Plants respond to abiotic stress stimuli, such as water deprivation, through a hierarchical cascade that includes detection and signaling to mediate transcriptional and physiological changes. The phytohormone abscisic acid (ABA) is well-characterized for its regulatory role in these processes in response to specific environmental cues. ABA-mediated changes in gene expression have been demonstrated to be temporally-dependent, however, the genome-wide timing of these responses are not well-characterized in the agronomically important crop plant Zea mays (maize). ABA-mediated responses are synergistic with other regulatory mechanisms, including the plant-specific RNA-directed DNA methylation (RdDM) epigenetic pathway. Our prior work demonstrated that after relatively long-term ABA induction (8 h), maize plants homozygous for the mop1-1 mutation, defective in a component of the RdDM pathway, exhibit enhanced transcriptional sensitivity to the phytohormone. At this time-point, many hierarchically positioned transcription factors are differentially expressed resulting in primary (direct) and secondary (indirect) transcriptional outcomes. To identify more immediate and direct MOP1-dependent responses to ABA, we conducted a transcriptomic analysis using mop1-1 mutant and wild type plants treated with ABA for 1 h. One h of ABA treatment was sufficient to induce unique categories of differentially expressed genes (DEGs) in mop1-1. A comparative analysis between the two time-points revealed that distinct epigenetically-regulated changes in gene expression occur within the early stages of ABA induction, and that these changes are predicted to influence less immediate, indirect transcriptional responses. Homology with MOP1-dependent siRNAs and a gene regulatory network (GRN) were used to identify putative immediate and indirect targets, respectively. By manipulating two key regulatory networks in a temporal dependent manner, we identified genes and biological processes regulated by RdDM and ABA-mediated stress responses. Consistent with mis-regulation of gene expression, mop1-1 homozygous plants are compromised in their ability to recover from water deprivation. Collectively, these results indicate transcriptionally and physiologically relevant roles for MOP1-mediated regulation of gene expression of plant responses to environmental stress. [ABSTRACT FROM AUTHOR]

Abstract Background De novo DNA methylation triggered by short interfering RNAs is called RNA-directed DNA methylation (RdDM). Transcriptional gene silencing (TGS) through RdDM can be induced using a viral vector. We have previously induced RdDM on the 35S promoter in the green fluorescent protein (GFP)-expressing Nicotiana benthamiana line 16c using the cucumber mosaic virus vector. The GFP fluorescence phenotype segregated into two types, “red” and “orange” in the first self-fertilized (S1) progeny plants by the difference in degree of recovery from TGS on GFP expression. In the second self-fertilized generation (S2 plants), the phenotypes again segregated. Explaining what generates the red and orange types could answer a very important question in epigenetics: How is the robustness of TGS maintained after RdDM induction? Results In bisulfite sequencing analyses, we found a significant difference in the overall promoter hypermethylation pattern between the red and orange types in S1 plants but little difference in S2 plants. Therefore, we assumed that methylation at some specific cytosine residues might be important in determining the two phenotypes. To find the factor that discriminates stable, robust TGS from the unstable TGS with incomplete inheritance, we analyzed the direct effect of methylated cytosine residues on TGS. Because it has not yet been demonstrated that DNA methylation at a few specific cytosine residues on known sequence elements can indeed determine TGS robustness, we newly developed a method by which we can directly evaluate the effect of specific methylation on promoter activity. In this assay, we found that the effects of the specific cytosine methylation on TGS differed between the plus- and minus-strands. Conclusions We found two distinct phenotypes, the stable and unstable TGS in the progenies of virus-induced TGS plants. Our bisulfite sequencing analyses suggested that methylation at some specific cytosine residues in the 35S promoter played a role in determining whether stable or unstable TGSs are induced. Using the developed method, we inferred that DNA methylation heterogeneity in and between the plus- and minus-strands can differentially determine TGS.

RNA‐directed DNA methylation (RdDM) is a plant‐specific de novo DNA methylation pathway, which has extensive cross‐talk with histone modifications. Here, we report that the maize RdDM regulator SAWADEE HOMEODOMAIN HOMOLOG 2 (SHH2) is an H3K9me1 reader. Our structural studies reveal that H3K9me1 recognition is achieved by recognition of the methyl group via a classic aromatic cage and hydrogen‐bonding and salt‐bridge interactions with the free protons of the mono‐methyllysine. The di‐ and tri‐methylation states disrupt the polar interactions, decreasing the binding affinity. Our study reveals a mono‐methyllysine recognition mechanism which potentially links RdDM to H3K9me1 in maize. [ABSTRACT FROM AUTHOR]

ONSEN is a heat-activated LTR retrotransposon in Arabidopsis thaliana. Screens to identify transcriptional regulatory factors of ONSEN revealed a SWI/SNF-like chromatin remodeling protein, DRD1, which cooperates with plant-specific RNA polymerase and is involved in RNA-directed DNA methylation. ONSEN transcript level was increased in the drd1 mutant relative to wild-type under heat stress, indicating that DRD1 plays a significant role in the silencing of activated ONSEN under the stress condition. The transcript level of HsfA2, which is directly involved in transcriptional activation of ONSEN, was not higher in the drd1 mutant than in the wild-type. Interestingly, no transgenerational transposition of ONSEN was observed in the drd1 mutant, even though DNA methylation levels were significantly reduced and expression levels were increased compared to the wild-type. These results suggest that other factors are involved in the regulation of ONSEN transposition in addition to the transcript level of ONSEN. [ABSTRACT FROM AUTHOR]

In plants, transcription of selfish genetic elements such as transposons and DNA viruses is suppressed by RNA-directed DNA methylation. This process is guided by 24-nt short-interfering RNAs (siRNAs) whose double-stranded precursors are synthesized by DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). Pol IV and RDR2 coimmunoprecipitate, and their activities are tightly coupled, yet the basis for their association is unknown. Here, we show that an interval near the RDR2 active site contacts the Pol IV catalytic subunit, NRPD1, the largest of Pol IV's 12 subunits. Contacts between the catalytic regions of the two enzymes suggests that RDR2 is positioned to rapidly engage the free 3' ends of Pol IV transcripts and convert these single-stranded transcripts into double-stranded RNAs (dsRNAs). [ABSTRACT FROM AUTHOR]

Background: Plants are naturally associated with root microbiota, which are microbial communities influential to host fitness. Thus, it is important to understand how plants control root microbiota. Epigenetic factors regulate the readouts of genetic information and consequently many essential biological processes. However, it has been elusive whether RNA-directed DNA methylation (RdDM) affects root microbiota assembly. Results: By applying 16S rRNA gene sequencing, we investigated root microbiota of Arabidopsis mutants defective in the canonical RdDM pathway, including dcl234 that harbors triple mutation in the Dicer-like proteins DCL3, DCL2, and DCL4, which produce small RNAs for RdDM. Alpha diversity analysis showed reductions in microbe richness from the soil to roots, reflecting the selectivity of plants on root-associated bacteria. The dcl234 triple mutation significantly decreases the levels of Aeromonadaceae and Pseudomonadaceae, while it increases the abundance of many other bacteria families in the root microbiota. However, mutants of the other examined key players in the canonical RdDM pathway showed similar microbiota as Col-0, indicating that the DCL proteins affect root microbiota in an RdDM-independent manner. Subsequently gene analysis by shotgun sequencing of root microbiome indicated a selective pressure on microbial resistance to plant defense in the dcl234 mutant. Consistent with the altered plant-microbe interactions, dcl234 displayed altered characters, including the mRNA and sRNA transcriptomes that jointly highlighted altered cell wall organization and up-regulated defense, the decreased cellulose and callose deposition in root xylem, and the restructured profile of root exudates that supported the alterations in gene expression and cell wall modifications. Conclusion: Our findings demonstrate an important role of the DCL proteins in influencing root microbiota through integrated regulation of plant defense, cell wall compositions, and root exudates. Our results also demonstrate that the canonical RdDM is dispensable for Arabidopsis root microbiota. These findings not only establish a connection between root microbiota and plant epigenetic factors but also highlight the complexity of plant regulation of root microbiota. 9t4aP5wZAjZ4XcwBxRVNPd Video abstract [ABSTRACT FROM AUTHOR]

SUMMARY: Plants respond to adverse environmental cues by adjusting a wide variety of processes through highly regulated mechanisms to maintain plant homeostasis for survival. As a result of the sessile nature of plants, their response, adjustment and adaptation to the changing environment is intimately coordinated with their developmental programs through the crosstalk of regulatory networks. Germination is a critical process in the plant life cycle, and thus plants have evolved various strategies to control the timing of germination according to their local environment. The mechanisms involved in these adjustment responses are largely unknown, however. Here, we report that mutations in core elements of canonical RNA‐directed DNA methylation (RdDM) affect the germination and post‐germination growth of Arabidopsis seeds grown under salinity stress. Transcriptomic and whole‐genome bisulfite sequencing (WGBS) analyses support the involvement of this pathway in the control of germination timing and post‐germination growth under salinity stress by preventing the transcriptional activation of genes implicated in these processes. Subsequent transcriptional effects on genes that function in relation to these developmental events support this conclusion. Significance Statement: Germination is a key process in the plant life cycle, such that plants have evolved various strategies to control the timing of germination according to their local environment. We show that canonical RNA‐directed DNA methylation (RdDM), one of the transcriptional regulation mechanisms in plants, participates in the control of germination timing and post‐germination growth under salinity stress in Arabidopsis thaliana by preventing the transcriptional activation of genes implicated in these processes. [ABSTRACT FROM AUTHOR]

Plant tissue culture techniques have been extensively employed in commercial micropropagation to provide year-round production. Tissue culture regenerants are not always genotypically and phenotypically similar. Due to the changes in the tissue culture microenvironment, plant cells are exposed to additional stress which induces genetic and epigenetic instabilities in the regenerants. These changes lead to tissue culture-induced variations (TCIV) which are also known as somaclonal variations to categorically specify the inducing environment. TCIV includes molecular and phenotypic changes persuaded in the in vitro culture due to continuous sub-culturing and tissue culture-derived stress. Epigenetic variations such as altered DNA methylation pattern are induced due to the above-mentioned factors. Reportedly, alteration in DNA methylation pattern is much more frequent in the plant genome during the tissue culture process. DNA methylation plays an important role in gene expression and regulation of plant development. Variants originated in tissue culture process due to heritable methylation changes, can contribute to intra-species phenotypic variation. Several molecular techniques are available to detect DNA methylation at different stages of in vitro culture. Here, we review the aspects of TCIV with respect to DNA methylation and its effect on crop improvement programs. It is anticipated that a precise and comprehensive knowledge of molecular basis of in vitro-derived DNA methylation will help to design strategies to overcome the bottlenecks of micropropagation system and maintain the clonal fidelity of the regenerants. [ABSTRACT FROM AUTHOR]

In plants, heterochromatin is maintained by a small RNA-based gene silencing mechanism known as RNA-directed DNA methylation (RdDM). RdDM requires the non-redundant functions of two plant-specific DNA-dependent RNA polymerases (RNAP), RNAP IV and RNAP V. RNAP IV plays a major role in siRNA biogenesis, while RNAP V may recruit DNA methylation machinery to target endogenous loci for silencing. Although small RNA-generating regions that are dependent on both RNAP IV and RNAP V have been identified previously, the genomic loci targeted by RNAP V for siRNA accumulation and silencing have not been described extensively. To characterize the RNAP V-dependent, heterochromatic siRNA-generating regions in the Arabidopsis genome, we deeply sequenced the small RNA populations of wild-type and RNAP V null mutant (nrpe1) plants. Our results showed that RNAP V-dependent siRNA-generating loci are associated predominately with short repetitive sequences in intergenic regions. Suppression of small RNA production from short repetitive sequences was also prominent in RdDM mutants including dms4, drd1, dms3 and rdm1, reflecting the known association of these RdDM effectors with RNAP V. The genomic regions targeted by RNAP V were small, with an estimated average length of 238 bp. Our results suggest that RNAP V affects siRNA production from genomic loci with features dissimilar to known RNAP IV-dependent loci. RNAP V, along with RNAP IV and DRM1/2, may target and silence a set of small, intergenic transposable elements located in dispersed genomic regions for silencing. Silencing at these loci may be actively reinforced by RdDM.

Abstract Background Freakish and rare or the tip of the iceberg? Both phrases have been used to refer to paramutation, an epigenetic drive that contravenes Mendel’s first law of segregation. Although its underlying mechanisms are beginning to unravel, its understanding relies only on a few examples that may involve transgenes or artificially generated epialleles. Results By using DNA methylation of introgression lines as an indication of past paramutation, we reveal that the paramutation-like properties of the H06 locus in hybrids of Solanum lycopersicum and a range of tomato relatives and cultivars depend on the timing of sRNA production and conform to an RNA-directed mechanism. In addition, by scanning the methylomes of tomato introgression lines for shared regions of differential methylation that are absent in the S. lycopersicum parent, we identify thousands of candidate regions for paramutation-like behaviour. The methylation patterns for a subset of these regions segregate with non Mendelian ratios, consistent with secondary paramutation-like interactions to variable extents depending on the locus. Conclusion Together these results demonstrate that paramutation-like epigenetic interactions are common for natural epialleles in tomato, but vary in timing and penetrance.

Summary: DNA methylation is an epigenetic mark that regulates multiple processes, such as gene expression and genome stability. Mutants and pharmacological treatments have been instrumental in the study of this mark in plants, although their genome‐wide effect complicates the direct association between changes in methylation and a particular phenotype. A variety of tools that allow locus‐specific manipulation of DNA methylation can be used to assess its direct role in specific processes, as well as to create novel epialleles. Recently, new tools that recruit the methylation machinery directly to target loci through programmable DNA‐binding proteins have expanded the tool kit available to researchers. This review provides an overview of DNA methylation in plants and discusses the tools that have recently been developed for its manipulation. [ABSTRACT FROM AUTHOR]

Although epigenetic factors may influence the expression of defense genes in plants, their role in antiviral responses and the impact of viral adaptation and evolution in shaping these interactions are still poorly explored. We used two isolates of turnip mosaic potyvirus with varying degrees of adaptation to Arabidopsis thaliana to address these issues. One of the isolates was experimentally evolved in the plant and presented increased load and virulence relative to the ancestral isolate. The magnitude of the transcriptomic responses was larger for the evolved isolate and indicated a role of innate immunity systems triggered by molecular patterns and effectors in the infection process. Several transposable elements located in different chromatin contexts and epigenetic-related genes were also affected. Correspondingly, mutant plants having loss or gain of repressive marks were, respectively, more tolerant and susceptible to turnip mosaic potyvirus, with a more efficient response against the ancestral isolate. In wild-type plants, both isolates induced similar levels of cytosine methylation changes, including in and around transposable elements and stress-related genes. Results collectively suggested that apart from RNA silencing and basal immunity systems, DNA methylation and histone modification pathways may also be required for mounting proper antiviral defenses and that the effectiveness of this type of regulation strongly depends on the degree of viral adaptation to the host. [ABSTRACT FROM AUTHOR]

Multisubunit RNA polymerase (Pol) complexes are the core machinery for gene expression in eukaryotes. The enzymes Pol I, Pol II and Pol III transcribe distinct subsets of nuclear genes. This family of nuclear RNA polymerases expanded in terrestrial plants by the duplication of Pol II subunit genes. Two Pol II-related enzymes, Pol IV and Pol V, are highly specialized in the production of regulatory, non-coding RNAs. Pol IV and Pol V are the central players of RNA-directed DNA methylation (RdDM), an RNA interference pathway that represses transposable elements (TEs) and selected genes. Genetic and biochemical analyses of Pol IV/V subunits are now revealing how these enzymes evolved from ancestral Pol II to sustain non-coding RNA biogenesis in silent chromatin. Intriguingly, Pol IV-RdDM regulates genes that influence flowering time, reproductive development, stress responses and plant–pathogen interactions. Pol IV target genes vary among closely related taxa, indicating that these regulatory circuits are often species-specific. Data from crops like maize, rice, tomato and Brassicarapa suggest that dynamic repositioning of TEs, accompanied by Pol IV targeting to TE-proximal genes, leads to the reprogramming of plant gene expression over short evolutionary timescales. [ABSTRACT FROM AUTHOR]

Viroids are plant pathogenic, circular, non-coding, single-stranded RNAs (ssRNAs). Members of the Pospiviroidae family replicate in the nucleus of plant cells through double-stranded RNA (dsRNA) intermediates, thus triggering the host’s RNA interference (RNAi) machinery. In plants, the two RNAi pillars are Post-Transcriptional Gene Silencing (PTGS) and RNA-directed DNA Methylation (RdDM), and the latter has the potential to trigger Transcriptional Gene Silencing (TGS). Over the last three decades, the employment of viroid-based systems has immensely contributed to our understanding of both of these RNAi facets. In this review, we highlight the role of Pospiviroidae in the discovery of RdDM, expound the gradual elucidation through the years of the diverse array of RdDM’s mechanistic details and propose a revised RdDM model based on the cumulative amount of evidence from viroid and non-viroid systems.

Summary: BIR1 is a receptor‐like kinase that functions as a negative regulator of basal immunity and cell death in Arabidopsis.Using Arabidopsis thaliana and Tobacco rattle virus (TRV), we investigate the antiviral role of BIR1, the molecular mechanisms of BIR1 gene expression regulation during viral infections, and the effects of BIR1 overexpression on plant immunity and development.We found that SA acts as a signal molecule for BIR1 activation during infection. Inactivating mutations of BIR1 in the bir1‐1 mutant cause strong antiviral resistance independently of constitutive cell death or SA defense priming. BIR1 overexpression leads to severe developmental defects, cell death and premature death, which correlate with the constitutive activation of plant immune responses.Our findings suggest that BIR1 acts as a negative regulator of antiviral defense in plants, and indicate that RNA silencing contributes, alone or in conjunction with other regulatory mechanisms, to define a threshold expression for proper BIR1 function beyond which an autoimmune response may occur. This work provides novel mechanistic insights into the regulation of BIR1 homeostasis that may be common for other plant immune components. [ABSTRACT FROM AUTHOR]

Summary: RNA‐based processes play key roles in the regulation of eukaryotic gene expression. This includes both the processing of pre‐mRNAs into mature mRNAs ready for translation and RNA‐based silencing processes, such as RNA‐directed DNA methylation (RdDM). Polyadenylation of pre‐mRNAs is one important step in their processing and is carried out by three functionally specialized canonical nuclear poly(A) polymerases in Arabidopsis thaliana. Null mutations in one of these, termed PAPS1, result in a male gametophytic defect. Using a fluorescence‐labelling strategy, we have characterized this defect in more detail using RNA and small‐RNA sequencing. In addition to global defects in the expression of pollen‐differentiation genes, paps1 null‐mutant pollen shows a strong overaccumulation of transposable element (TE) transcripts, yet a depletion of 21‐ and particularly 24‐nucleotide‐long short interfering RNAs (siRNAs) and microRNAs (miRNAs) targeting the corresponding TEs. Double‐mutant analyses support a specific functional interaction between PAPS1 and components of the RdDM pathway, as evident from strong synergistic phenotypes in mutant combinations involving paps1, but not paps2 paps4, mutations. In particular, the double‐mutant of paps1 and rna‐dependent rna polymerase 6 (rdr6) shows a synergistic developmental phenotype disrupting the formation of the transmitting tract in the female gynoecium. Thus, our findings in A. thaliana uncover a potentially general link between canonical poly(A) polymerases as components of mRNA processing and RdDM, reflecting an analogous interaction in fission yeast. Significance Statement: This work identifies a functional connection between mRNA processing and RNA‐directed DNA methylation, involving a specific poly(A) polymerase in A. thaliana. [ABSTRACT FROM AUTHOR]

DNA methylation is an epigenetic mark that ensures silencing of transposable elements (TEs) and affects gene expression in many organisms. The function of different DNA methylation regulatory pathways has been largely characterized in the model plant Arabidopsis thaliana. However, far less is known about DNA methylation regulation and functions in basal land plants. Here we focus on the liverwort Marchantia polymorpha, an emerging model species that represents a basal lineage of land plants. We identified MpMET, the M. polymorpha ortholog of the METHYLTRANSFERASE 1 (MET1) gene required for maintenance of methylation at CG sites in angiosperms. We generated Mpmet mutants using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein9) system, which showed a significant loss of CG methylation and severe morphological changes and developmental defects. The mutants developed many adventitious shoot-like structures, suggesting that MpMET is required for maintaining differentiated cellular identities in the gametophyte. Even though numerous TEs were up-regulated, non-CG methylation was generally highly increased at TEs in the Mpmet mutants. Closer inspection of CHG methylation revealed features unique to M. polymorpha. Methylation of CCG sites in M. polymorpha does not depend on MET1, unlike in A. thaliana and Physcomitrella patens. Our results highlight the diversity of non-CG methylation regulatory mechanisms in plants. [ABSTRACT FROM AUTHOR]