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The current technologies to place new DNA into specific locations in plant genomes are low frequency and error-prone, and this inefficiency hampers genome-editing approaches to develop improved crops 1,2 . Often considered to be genome 'parasites', transposable elements (TEs) evolved to insert their DNA seamlessly into genomes 3-5 . Eukaryotic TEs select their site of insertion based on preferences for chromatin contexts, which differ for each TE type 6-9 . Here we developed a genome engineering tool that controls the TE insertion site and cargo delivered, taking advantage of the natural ability of the TE to precisely excise and insert into the genome. Inspired by CRISPR-associated transposases that target transposition in a programmable manner in bacteria 10-12 , we fused the rice Pong transposase protein to the Cas9 or Cas12a programmable nucleases. We demonstrated sequence-specific targeted insertion (guided by the CRISPR gRNA) of enhancer elements, an open reading frame and a gene expression cassette into the genome of the model plant Arabidopsis. We then translated this system into soybean-a major global crop in need of targeted insertion technology. We have engineered a TE 'parasite' into a usable and accessible toolkit that enables the sequence-specific targeting of custom DNA into plant genomes., (© 2024. The Author(s).)

Plastid transformation technology has been widely used to express traits of potential commercial importance, though the technology has been limited to traits that function while sequestered in the organelle. Prior research indicates that plastid contents can escape from the organelle, suggesting a possible mechanism for engineering plastid transgenes to function in other cellular locations. To test this hypothesis, we created tobacco (Nicotiana tabacum cv. Petit Havana) plastid transformants that express a fragment of the nuclear-encoded Phytoene desaturase (PDS) gene capable of catalyzing post-transcriptional gene silencing if RNA escapes into the cytoplasm. We found multiple lines of direct evidence that plastid-encoded PDS transgenes affect nuclear PDS gene silencing: knockdown of the nuclear-encoded PDS mRNA and/or its apparent translational inhibition, biogenesis of 21-nucleotide (nt) phased small interfering RNAs (phasiRNAs), and pigment-deficient plants. Furthermore, plastid-expressed dsRNA with no cognate nuclear-encoded pairing partner also produced abundant 21-nt phasiRNAs in the cytoplasm, demonstrating that a nuclear-encoded template is not required for siRNA biogenesis. Our results indicate that RNA escape from plastids to the cytoplasm occurs generally, with functional consequences that include entry into the gene silencing pathway. Furthermore, we uncover a method to produce plastid-encoded traits with functions outside of the organelle and open additional fields of study in plastid development, compartmentalization, and small RNA biogenesis., Competing Interests: Conflict of interest statement: J.M.S. is Chief Scientific Officer of Plastomics Inc. J.M.S. and B.C.M. have filed a patent application based on results reported in this paper. The remaining authors declare no competing interests., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Plant responses to abiotic environmental challenges are known to have lasting effects on the plant beyond the initial stress exposure. Some of these lasting effects are transgenerational, affecting the next generation. The plant response to elevated carbon dioxide (CO 2 ) levels has been well studied. However, these investigations are typically limited to plants grown for a single generation in a high CO 2 environment while transgenerational studies are rare. We aimed to determine transgenerational growth responses in plants after exposure to high CO 2 by investigating the direct progeny when returned to baseline CO 2 levels. We found that both the flowering plant Arabidopsis thaliana and seedless nonvascular plant Physcomitrium patens continue to display accelerated growth rates in the progeny of plants exposed to high CO 2 . We used the model species Arabidopsis to dissect the molecular mechanism and found that DNA methylation pathways are necessary for heritability of this growth response. More specifically, the pathway of RNA-directed DNA methylation is required to initiate methylation and the proteins CMT2 and CMT3 are needed for the transgenerational propagation of this DNA methylation to the progeny plants. Together, these two DNA methylation pathways establish and then maintain a cellular memory to high CO 2 exposure., (© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

The plant-specific RNA Polymerase V (Pol V) plays a key role in gene silencing, but its role in repair of double stranded DNA breaks is unclear. Excision of the transposable element mPing creates double stranded breaks that are repaired by NHEJ. We measured mPing excision site repair in multiple DNA methylation mutants including pol V using an mPing : GFP reporter. Two independent mutant alleles of pol V showed less GFP expression, indicating that the Pol V protein plays a role in excision site repair. Sequence analysis of the pol V excision sites indicated an elevated rate of large deletions consistent with less efficient repair. These results clarify the role of Pol V, but not other RNA-directed DNA methylation proteins (Pol IV) or maintenance DNA methylation pathways ( MET1 ), in the repair of double-strand DNA breaks., Competing Interests: The authors declare that there are no conflicts of interest present., (Copyright: © 2023 by the authors.)

Throughout the eukaryotic kingdoms, small RNAs direct chromatin modification. ARGONAUTE proteins sit at the nexus of this process, linking the small RNA information to the programming of chromatin. ARGONAUTE proteins physically incorporate the small RNAs as guides to target specific regions of the genome. In this issue of Genes & Development , Wang and colleagues (pp. 103-118) add substantial new detail to the processes of ARGONAUTE RNA loading, preference, cleavage, and retention, which together accomplish RNA-directed chromatin modification. They show that after catalytic cleavage by the plant ARGONAUTE protein AGO4, the cleaved fragment remains bound. This happens during two distinct RNA cleavage reactions performed by AGO4: first for a passenger RNA strand of the siRNA duplex, and second for a nascent transcript at the target DNA locus. Cleaved fragment retention of the nascent transcript explains how the protein complex accumulates to high levels at the target locus, amplifying chromatin modification., (© 2023 Edwards and Slotkin; Published by Cold Spring Harbor Laboratory Press.)

Within the life cycle of a living organism, another life cycle exists for the selfish genome inhabitants, which are called transposable elements (TEs). These mobile sequences invade, duplicate, amplify, and diversify within a genome, increasing the genome's size and generating new mutations. Cells act to defend their genome, but rather than permanently destroying TEs, they use chromatin-level repression and epigenetic inheritance to silence TE activity. This level of silencing is ephemeral and reversible, leading to a dynamic equilibrium between TE suppression and reactivation within a host genome. The coexistence of the TE and host genome can also lead to the domestication of the TE to serve in host genome evolution and function. In this review, we describe the life cycle of a TE, with emphasis on how epigenetic regulation is harnessed to control TEs for host genome stability and innovation.

The sorting of RNA transcripts dictates their ultimate post-transcriptional fates, such as translation, decay or degradation by RNA interference (RNAi). This sorting of RNAs into distinct fates is mediated by their interaction with RNA-binding proteins. While hundreds of RNA binding proteins have been identified, which act to sort RNAs into different pathways is largely unknown. Particularly in plants, this is due to the lack of reliable protein-RNA artificial tethering tools necessary to determine the mechanism of protein action on an RNA in vivo. Here we generated a protein-RNA tethering system which functions on an endogenous Arabidopsis RNA that is tracked by the quantitative flowering time phenotype. Unlike other protein-RNA tethering systems that have been attempted in plants, our system circumvents the inadvertent triggering of RNAi. We successfully in vivo tethered a protein epitope, deadenylase protein and translation factor to the target RNA, which function to tag, decay and boost protein production, respectively. We demonstrated that our tethering system (1) is sufficient to engineer the downstream fate of an RNA, (2) enables the determination of any protein's function upon recruitment to an RNA, and (3) can be used to discover new interactions with RNA-binding proteins., (© 2022. The Author(s).)

Animal and plant microRNAs (miRNAs) are essential for the spatio-temporal regulation of development. Together with this role, plant miRNAs have been proposed to target transposable elements (TEs) and stimulate the production of epigenetically active small interfering RNAs. This activity is evident in the plant male gamete containing structure, the male gametophyte or pollen grain. How the dual role of plant miRNAs, regulating both genes and TEs, is integrated during pollen development and which mRNAs are regulated by miRNAs in this cell type at a genome-wide scale are unknown. Here, we provide a detailed analysis of miRNA dynamics and activity during pollen development in Arabidopsis thaliana using small RNA and degradome parallel analysis of RNA end high-throughput sequencing. Furthermore, we uncover miRNAs loaded into the two main active Argonaute (AGO) proteins in the uninuclear and mature pollen grain, AGO1 and AGO5. Our results indicate that the developmental progression from microspore to mature pollen grain is characterized by a transition from miRNAs targeting developmental genes to miRNAs regulating TE activity., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

In mammals and plants, cytosine DNA methylation is essential for the epigenetic repression of transposable elements and foreign DNA. In plants, DNA methylation is guided by small interfering RNAs (siRNAs) in a self-reinforcing cycle termed RNA-directed DNA methylation (RdDM). RdDM requires the specialized RNA polymerase V (Pol V), and the key unanswered question is how Pol V is first recruited to new target sites without pre-existing DNA methylation. We find that Pol V follows and is dependent on the recruitment of an AGO4-clade ARGONAUTE protein, and any siRNA can guide the ARGONAUTE protein to the new target locus independent of pre-existing DNA methylation. These findings reject long-standing models of RdDM initiation and instead demonstrate that siRNA-guided ARGONAUTE targeting is necessary, sufficient and first to target Pol V recruitment and trigger the cycle of RdDM at a transcribed target locus, thereby establishing epigenetic silencing., (© 2021. The Author(s).)

When an mRNA enters into the RNA degradation pathway called RNA interference (RNAi), it is cleaved into small interfering RNAs (siRNAs) that then target complementary mRNAs for destruction. The consequence of entry into RNAi is mRNA degradation, post-transcriptional silencing and in some cases transcriptional silencing. RNAi functions as a defense against transposable element and virus activity, and in plants, RNAi additionally plays a role in development by regulating some genes. However, it is unknown how specific transcripts are selected for RNAi, and how most genic mRNAs steer clear. This Current Opinion article explores the key question of how RNAs are selected for entry into RNAi, and proposes models that enable the cell to distinguish between transcripts to translate versus destroy., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Environmentally induced changes in the epigenome help individuals to quickly adapt to fluctuations in the conditions of their habitats. We explored those changes in Arabidopsis thaliana plants subjected to multiple biotic and abiotic stresses, and identified transposable element (TE) activation in plants infested with the green peach aphid, Myzus persicae. We performed a genome-wide analysis mRNA expression, small RNA accumulation and DNA methylation Our results demonstrate that aphid feeding induces loss of methylation of hundreds of loci, mainly TEs. This loss of methylation has the potential to regulate gene expression and we found evidence that it is involved in the control of plant immunity genes. Accordingly, mutant plants deficient in DNA and H3K9 methylation (kyp) showed increased resistance to M. persicae infestation. Collectively, our results show that changes in DNA methylation play a significant role in the regulation of the plant transcriptional response and induction of defense response against aphid feeding., (© 2021 The Authors New Phytologist ©2021 New Phytologist Foundation.)

Histone modifications deposited by the Polycomb repressive complex 2 (PRC2) play a critical role in the control of growth, development, and adaptation to environmental fluctuations of most multicellular eukaryotes. The catalytic activity of PRC2 is counteracted by Jumonji-type (JMJ) histone demethylases, which shapes the genomic distribution of H3K27me3. Here, we show that two JMJ histone demethylases in Arabidopsis , EARLY FLOWERING 6 (ELF6) and RELATIVE OF EARLY FLOWERING 6 (REF6), play distinct roles in H3K27me3 and H3K27me1 homeostasis. We show that failure to reset these chromatin marks during sexual reproduction results in the transgenerational inheritance of histone marks, which cause a loss of DNA methylation at heterochromatic loci and transposon activation. Thus, Jumonji-type histone demethylases play a dual role in plants by helping to maintain transcriptional states through development and safeguard genome integrity during sexual reproduction., Competing Interests: JA, MN, JR, SO, YH, AD, KO, DM, FA, CR, AW, JD, MU, DL, RS, MB, JG No competing interests declared, DW Senior editor, eLife, (© 2020, Antunez-Sanchez et al.)

Competing Interests: The authors declare no competing interest.

Plant NLR-type receptors serve as sensitive triggers of host immunity. Their expression has to be well-balanced, due to their interference with various cellular processes and dose-dependency of their defense-inducing activity. A genetic "arms race" with fast-evolving pathogenic microbes requires plants to constantly innovate their NLR repertoires. We previously showed that insertion of the COPIA-R7 retrotransposon into RPP7 co-opted the epigenetic transposon silencing signal H3K9me2 to a new function promoting expression of this Arabidopsis thaliana NLR gene. Recruitment of the histone binding protein EDM2 to COPIA-R7-associated H3K9me2 is required for optimal expression of RPP7. By profiling of genome-wide effects of EDM2, we now uncovered additional examples illustrating effects of transposons on NLR gene expression, strongly suggesting that these mobile elements can play critical roles in the rapid evolution of plant NLR genes by providing the "raw material" for gene expression mechanisms. We further found EDM2 to have a global role in NLR expression control. Besides serving as a positive regulator of RPP7 and a small number of other NLR genes, EDM2 acts as a suppressor of a multitude of additional NLR genes. We speculate that the dual functionality of EDM2 in NLR expression control arose from the need to compensate for fitness penalties caused by high expression of some NLR genes by suppression of others. Moreover, we are providing new insights into functional relationships of EDM2 with its interaction partner, the RNA binding protein EDM3/AIPP1, and its target gene IBM1, encoding an H3K9-demethylase., Competing Interests: The authors have declared that no competing interests exist.

Transcript-based annotations of genes facilitate both genome-wide analyses and detailed single-locus research. In contrast, transposable element (TE) annotations are rudimentary, consisting of information only on TE location and type. The repetitiveness and limited annotation of TEs prevent the ability to distinguish between potentially functional expressed elements and degraded copies. To improve genome-wide TE bioinformatics, we performed long-read sequencing of cDNAs from Arabidopsis ( Arabidopsis thaliana ) lines deficient in multiple layers of TE repression. These uniquely mapping transcripts were used to identify the set of TEs able to generate polyadenylated RNAs and create a new transcript-based annotation of TEs that we have layered upon the existing high-quality community standard annotation. We used this annotation to reduce the bioinformatic complexity associated with multimapping reads from short-read RNA sequencing experiments, and we show that this improvement is expanded in a TE-rich genome such as maize ( Zea mays ). Our TE annotation also enables the testing of specific standing hypotheses in the TE field. We demonstrate that inaccurate TE splicing does not trigger small RNA production, and the cell more strongly targets DNA methylation to TEs that have the potential to make mRNAs. This work provides a transcript-based TE annotation for Arabidopsis and maize, which serves as a blueprint to reduce the bioinformatic complexity associated with repetitive TEs in any organism., (© 2020 American Society of Plant Biologists. All rights reserved.)

In agricultural systems, crops equipped with host-plant resistance (HPR) have enhanced protection against pests, and are used as a safe and sustainable tool in pest management. In soybean, HPR can control the soybean aphid (Aphis glycines), but certain aphid populations have overcome this resistance (i.e., virulence). The molecular mechanisms underlying aphid virulence to HPR are unknown, but likely involve effector proteins that are secreted by aphids to modulate plant defenses. Another mechanism to facilitate adaptation is through the activity of transposable elements, which can become activated by stress. In this study, we performed RNA sequencing of virulent and avirulent soybean aphids fed susceptible or resistant (Rag1 + Rag2) soybean. Our goal was to better understand the molecular mechanisms underlying soybean aphid virulence. Our data showed that virulent aphids mostly down regulate putative effector genes relative to avirulent aphids, especially when aphids were fed susceptible soybean. Decreased expression of effectors may help evade HPR plant defenses. Virulent aphids also transcriptionally up regulate a diverse set of transposable elements and nearby genes, which is consistent with stress adaptation. Our work demonstrates two mechanisms of pest adaptation to resistance, and identifies effector gene targets for future functional testing., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In flowering plants, gene expression in the haploid male gametophyte (pollen) is essential for sperm delivery and double fertilization. Pollen also undergoes dynamic epigenetic regulation of expression from transposable elements (TEs), but how this process interacts with gene expression is not clearly understood. To explore relationships among these processes, we quantified transcript levels in four male reproductive stages of maize (tassel primordia, microspores, mature pollen, and sperm cells) via RNA-seq. We found that, in contrast with vegetative cell-limited TE expression in Arabidopsis pollen, TE transcripts in maize accumulate as early as the microspore stage and are also present in sperm cells. Intriguingly, coordinate expression was observed between highly expressed protein-coding genes and their neighboring TEs, specifically in mature pollen and sperm cells. To investigate a potential relationship between elevated gene transcript level and pollen function, we measured the fitness cost (male-specific transmission defect) of GFP-tagged coding sequence insertion mutations in over 50 genes identified as highly expressed in the pollen vegetative cell, sperm cell, or seedling (as a sporophytic control). Insertions in seedling genes or sperm cell genes (with one exception) exhibited no difference from the expected 1:1 transmission ratio. In contrast, insertions in over 20% of vegetative cell genes were associated with significant reductions in fitness, showing a positive correlation of transcript level with non-Mendelian segregation when mutant. Insertions in maize gamete expressed2 (Zm gex2), the sole sperm cell gene with measured contributions to fitness, also triggered seed defects when crossed as a male, indicating a conserved role in double fertilization, given the similar phenotype previously demonstrated for the Arabidopsis ortholog GEX2. Overall, our study demonstrates a developmentally programmed and coordinated transcriptional activation of TEs and genes in pollen, and further identifies maize pollen as a model in which transcriptomic data have predictive value for quantitative phenotypes., Competing Interests: The authors have declared that no competing interests exist.

Technological advancement in DNA sequencing read-length has drastically changed the quality and completeness of decoded genomes. The aim of this article is not to describe the different technologies of long-read sequencing, or the widely appreciated power of this technology in genome sequencing, assembly, and gene annotation. Instead, in this article, we provide our opinion that with the exception of genome production, transposable element biology is the most radically altered field as a consequence of the advent of long-read sequencing technology. We review how long-reads have been used to answer key questions in transposable element biology, and how in the future long-reads will help elucidate the function of the repetitive fraction of genomes., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The plant-specific RNA Polymerase IV (Pol IV) transcribes heterochromatic regions, including many transposable elements (TEs), with the well-described role of generating 24 nucleotide (nt) small interfering RNAs (siRNAs). These siRNAs target DNA methylation back to TEs to reinforce the boundary between heterochromatin and euchromatin. In the male gametophytic phase of the plant life cycle, pollen, Pol IV switches to generating primarily 21-22 nt siRNAs, but the biogenesis and function of these siRNAs have been enigmatic. In contrast to being pollen-specific, we identified that Pol IV generates these 21-22 nt siRNAs in sporophytic tissues, likely from the same transcripts that are processed into the more abundant 24 nt siRNAs. The 21-22 nt forms are specifically generated by the combined activities of DICER proteins DCL2/DCL4 and can participate in RNA-directed DNA methylation. These 21-22 nt siRNAs are also loaded into ARGONAUTE1 (AGO1), which is known to function in post-transcriptional gene regulation. Like other plant siRNAs and microRNAs incorporated into AGO1, we find a signature of genic mRNA cleavage at the predicted target site of these siRNAs, suggesting that Pol IV-generated 21-22 nt siRNAs may function to regulate gene transcript abundance. Our data provide support for the existing model that in pollen Pol IV functions in gene regulation. This article is part of a discussion meeting issue 'Crossroads between transposons and gene regulation'.

Somatic embryogenesis is an important tissue culture technique that sometimes leads to phenotypic variation via genetic and/or epigenetic changes. To understand the genomic and epigenomic impacts of somatic embryogenesis, we characterized soybean ( Glycine max ) epigenomes sampled from embryos at 10 different stages ranging from 6 weeks to 13 years of continuous culture. We identified genome-wide increases in DNA methylation from cultured samples, especially at CHH sites. The hypermethylation almost exclusively occurred in regions previously possessing non-CG methylation and was accompanied by increases in the expression of genes encoding the RNA-directed DNA methylation (RdDM) machinery. The epigenomic changes were similar between somatic and zygotic embryogenesis. Following the initial global wave of hypermethylation, rare decay events of maintenance methylation were observed, and the extent of the decay increased with time in culture. These losses in DNA methylation were accompanied by downregulation of genes encoding the RdDM machinery and transcriptome reprogramming reminiscent of transcriptomes during late-stage seed development. These results reveal a process for reinforcing already silenced regions to maintain genome integrity during somatic embryogenesis over the short term, which eventually decays at certain loci over longer time scales., (© 2019 American Society of Plant Biologists. All rights reserved.)

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., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

RNA-directed DNA methylation (RdDM) is a set of mechanisms by which transcriptionally repressive DNA and histone methylation are targeted to viruses, transposable elements, and some transgenes. We identified an Arabidopsis ( Arabidopsis thaliana ) mutant in which all forms of RdDM are deficient, leading to transcriptional activation of some transposable elements and the inability to initiate transgene silencing. The corresponding gene, ALY1 , encodes an RNA binding nuclear export protein. Arabidopsis ALY proteins function together to export many messenger RNAs (mRNAs), but we found that ALY1 is unique among this family for its ability to enable RdDM. Through the identification of ALY1 direct targets via RNA immunoprecipitation sequencing, coupled with mRNA sequencing of nuclear and cytoplasmic fractions, we identified mRNAs of known RdDM factors that fail to efficiently export from the nucleus in aly1 mutants. We found that loss of RdDM in aly1 is a result of deficient nuclear export of the ARGONAUTE6 mRNA and subsequent decreases in ARGONAUTE6 protein, a key effector of RdDM. One aly1 allele was more severe due to an additional loss of RNA Polymerase V function, which is also necessary for RdDM. Together, our data reconcile the broad role of ALY1 in mRNA export with the specific loss of RdDM through the activities of ARGONAUTE6 and RNA Polymerase V., (© 2019 American Society of Plant Biologists. All rights reserved.)

The bacterium Agrobacterium tumefaciens has been the workhorse in plant genome engineering. Customized replacement of native tumor-inducing (Ti) plasmid elements enabled insertion of a sequence of interest called Transfer-DNA (T-DNA) into any plant genome. Although these transfer mechanisms are well understood, detailed understanding of structure and epigenomic status of insertion events was limited by current technologies. Here we applied two single-molecule technologies and analyzed Arabidopsis thaliana lines from three widely used T-DNA insertion collections (SALK, SAIL and WISC). Optical maps for four randomly selected T-DNA lines revealed between one and seven insertions/rearrangements, and the length of individual insertions from 27 to 236 kilobases. De novo nanopore sequencing-based assemblies for two segregating lines partially resolved T-DNA structures and revealed multiple translocations and exchange of chromosome arm ends. For the current TAIR10 reference genome, nanopore contigs corrected 83% of non-centromeric misassemblies. The unprecedented contiguous nucleotide-level resolution enabled an in-depth study of the epigenome at T-DNA insertion sites. SALK_059379 line T-DNA insertions were enriched for 24nt small interfering RNAs (siRNA) and dense cytosine DNA methylation, resulting in transgene silencing via the RNA-directed DNA methylation pathway. In contrast, SAIL_232 line T-DNA insertions are predominantly targeted by 21/22nt siRNAs, with DNA methylation and silencing limited to a reporter, but not the resistance gene. Additionally, we profiled the H3K4me3, H3K27me3 and H2A.Z chromatin environments around T-DNA insertions using ChIP-seq in SALK_059379, SAIL_232 and five additional T-DNA lines. We discovered various effect s ranging from complete loss of chromatin marks to the de novo incorporation of H2A.Z and trimethylation of H3K4 and H3K27 around the T-DNA integration sites. This study provides new insights into the structural impact of inserting foreign fragments into plant genomes and demonstrates the utility of state-of-the-art long-range sequencing technologies to rapidly identify unanticipated genomic changes., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: FJ is an employee of Bayer Crop Science. JRE serves on the scientific advisory boards of Cibus, Zymo Research and Pathway Genomics Inc.

The regulation of parental genome dosage is of fundamental importance in animals and plants, as exemplified by X-chromosome inactivation and dosage compensation. The 'triploid block' is a classic example of dosage regulation in plants that establishes a reproductive barrier between species differing in chromosome number 1,2 . This barrier acts in the embryo-nourishing endosperm tissue and induces the abortion of hybrid seeds through a yet unknown mechanism 3 . Here we show that depletion of paternal epigenetically activated small interfering RNAs (easiRNAs) bypasses the triploid block in response to increased paternal ploidy in Arabidopsis thaliana. Paternal loss of the plant-specific RNA polymerase IV suppressed easiRNA formation and rescued triploid seeds by restoring small-RNA-directed DNA methylation at transposable elements (TEs), correlating with reduced expression of paternally expressed imprinted genes (PEGs). Our data suggest that easiRNAs form a quantitative signal for paternal chromosome number and that their balanced dosage is required for post-fertilization genome stability and seed viability.

tRNA-derived RNA fragments (tRFs) are 18-26 nucleotide small RNAs that are not random degradation products, but are rather specifically cleaved from mature tRNA transcripts. Abundant in stressed or viral-infected cells, the function and potential targets of tRFs are not known. We identified that in the unstressed wild-type male gamete containing pollen of flowering plants, and analogous reproductive structure in non-flowering plant species, tRFs accumulate to high levels. In the reference plant Arabidopsis thaliana, tRFs are processed by Dicer-like 1 and incorporated into Argonaute1 (AGO1), akin to a microRNA. We utilized the fact that many plant small RNAs direct cleavage of their target transcripts to demonstrate that the tRF-AGO1 complex acts to specifically target and cleave endogenous transposable element (TE) mRNAs produced from transcriptionally active TEs. The data presented here demonstrate that tRFs are bona-fide regulatory microRNA-like small RNAs involved in the regulation of genome stability through the targeting of TE transcripts., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The genome-wide investigation of DNA methylation levels has been limited to reference transposable element positions. The methylation analysis of non-reference and mobile transposable elements has only recently been performed, but required both genome resequencing and MethylC-seq datasets. We have created epiTEome, a program that detects both new transposable element insertion sites and their methylation states from a single MethylC-seq dataset. EpiTEome outperforms other split-read insertion site detection programs, even while functioning on bisulfite-converted reads. EpiTEome characterizes the previously discarded fraction of DNA methylation at sites of new insertions, enabling future investigation into the epigenetic regulation of non-reference and transposed elements.

The propagation of epigenetic marks has received a great deal of attention, yet the initiation of epigenetic silencing of a new transgene, virus, or transposable element (TE) remains enigmatic. The overlapping and simultaneous function of multiple silencing mechanisms has obscured this area of investigation. Here, we revealed two broad mechanisms that can initiate silencing independently: identity-based and expression-dependent silencing. We found that identity-based silencing is targeted by 21- to 22-nucleotide or 24-nucleotide small interfering RNAs (siRNAs) generated from previously silenced regions of the genome. By transforming exogenous TEs into Arabidopsis thaliana , we circumvented identity-based silencing, allowing us to isolate and investigate the molecular mechanism of expression-dependent silencing. We found that several siRNA-generating mechanisms all trigger de novo expression-dependent RNA-directed DNA methylation (RdDM) through RNA Polymerase V. In addition, while full-length TEs quickly progress beyond RdDM to heterochromatin formation and the final maintenance methylation state, TE fragments stall at the RdDM phase. Lastly, we found that transformation into a mutant genotype followed by introgression into the wild type does not result in the same level of silencing as direct transformation into the wild type. This demonstrates that the plant genotype during a narrow window of time at TE insertion (or transgene transformation) is key for establishing the transgenerational extent of epigenetic silencing., (© 2017 American Society of Plant Biologists. All rights reserved.)

Small RNA-directed DNA methylation (RdDM) has been extensively studied in plants, resulting in a deep understanding of a major 'canonical RdDM' mechanism. However, current models of canonical RdDM cannot explain how this self-perpetuating mechanism is initiated. Recent investigations into the initiation of epigenetic silencing have determined that there are several alternative 'non-canonical RdDM' pathways that function through distinct mechanisms to modify chromatin. This Review aims to illustrate the diversity of non-canonical RdDM mechanisms described to date, recognize common themes within this dizzying array of interconnected pathways, and identify the key unanswered questions remaining in this field.

Please note that in the published editorial [1], affiliations 1, and 8 contained errors.[...].

Background: Chromatin modifications such as DNA methylation are targeted to transposable elements by small RNAs in a process termed RNA-directed DNA methylation (RdDM). In plants, canonical RdDM functions through RNA polymerase IV to reinforce pre-existing transposable element silencing. Recent investigations have identified a "non-canonical" form of RdDM dependent on RNA polymerase II expression to initiate and re-establish silencing of active transposable elements. This expression-dependent RdDM mechanism functions through RNAi degradation of transposable element mRNAs into small RNAs guided by the RNA-dependent RNA polymerase 6 (RDR6) protein and is therefore referred to as RDR6-RdDM., Results: We performed whole-genome MethylC-seq in 20 mutants that distinguish RdDM mechanisms when transposable elements are either transcriptionally silent or active. We identified a new mechanism of expression-dependent RdDM, which functions through DICER-LIKE3 (DCL3) but bypasses the requirement of both RNA polymerase IV and RDR6 (termed DCL3-RdDM). We found that RNA polymerase II expression-dependent forms of RdDM function on over 20 % of transcribed transposable elements, including the majority of full-length elements with all of the domains required for autonomous transposition. Lastly, we find that RDR6-RdDM preferentially targets long transposable elements due to the specificity of primary small RNAs to cleave full-length mRNAs., Conclusions: Expression-dependent forms of RdDM function to critically target DNA methylation to full-length and transcriptionally active transposable elements, suggesting that these pathways are key to suppressing mobilization. This targeting specificity is initiated on the mRNA cleavage-level, yet manifested as chromatin-level silencing that in plants is epigenetically inherited from generation to generation.

Plant small interfering RNAs (siRNAs) communicate from cell to cell and travel long distances through the vasculature. However, siRNA movement into germ cells has remained controversial, and has gained interest because the terminally differentiated pollen vegetative nurse cell surrounding the sperm cells undergoes a programmed heterochromatin decondensation and transcriptional reactivation of transposable elements (TEs). Transcription of TEs leads to their post-transcriptional degradation into siRNAs, and it has been proposed that the purpose of this TE reactivation is to generate and load TE siRNAs into the sperm cells. Here, we identify the molecular pathway of TE siRNA production in the pollen grain and demonstrate that siRNAs produced from pollen vegetative cell transcripts can silence TE reporters in the sperm cells. Our data demonstrates that TE siRNAs act non-cell-autonomously, inhibiting TE activity in the germ cells and potentially the next generation.

Transposable elements (TEs) are mobile units of DNA that comprise large portions of plant genomes. Besides creating mutations via transposition and contributing to genome size, TEs play key roles in chromosome architecture and gene regulation. TE activity is repressed by overlapping mechanisms of chromatin condensation, epigenetic transcriptional silencing, and targeting by small interfering RNAs. The specific regulation of different TEs, as well as their different roles in chromosome architecture and gene regulation, is specified by where on the chromosome the TE is located: near a gene, within a gene, in a pericentromere/TE island, or at the centromere core. In this Review, we investigate the silencing mechanisms responsible for inhibiting TE activity for each of these chromosomal contexts, emphasizing that chromosomal location is the first rule dictating the specific regulation of each TE., (© 2016 American Society of Plant Biologists. All rights reserved.)

In plant genomes the vast majority of transposable elements (TEs) are found in a transcriptionally silenced state that is epigenetically propagated from generation to generation. Although the mechanism of this maintenance of silencing has been well studied, it is now clear that the pathways responsible for maintaining TEs in a silenced state differ from the pathways responsible for initially targeting the TE for silencing. Recently, attention in this field has focused on investigating the molecular mechanisms that initiate and establish TE silencing. Here we review the current models of how TEs are triggered for silencing, the data supporting each model, and the key future questions in this fast moving field., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Transposable elements (TEs) generate mutations and chromosomal instability when active. To repress TE activity, eukaryotic cells evolved mechanisms to both degrade TE mRNAs into small interfering RNAs (siRNAs) and modify TE chromatin to epigenetically inhibit transcription. Since the populations of small RNAs that participate in TE post-transcriptional regulation differ from those that establish RNA-directed DNA methylation (RdDM), the mechanism through which transcriptionally active TEs transition from post-transcriptional RNAi regulation to chromatin level control has remained unclear. We have identified the molecular mechanism of a plant pathway that functions to direct DNA methylation to transcriptionally active TEs. We demonstrated that 21-22 nucleotide (nt) siRNA degradation products from the RNAi of TE mRNAs are directly incorporated into the ARGONAUTE 6 (AGO6) protein and direct AGO6 to TE chromatin to guide its function in RdDM. We find that this pathway functions in reproductive precursor cells to primarily target long centromeric high-copy transcriptionally active TEs for RdDM prior to gametogenesis. This study provides a direct mechanism that bridges the gap between the post-transcriptional regulation of TEs and the establishment of TE epigenetic silencing., (© 2014 The Authors.)

The Mobile Genetic Elements and Genome Evolution conference was hosted by Keystone Symposia in Santa Fe, NM USA, 9 March through 14 March 2014. The goal of this conference was to bring together scientists from around the world who study transposable elements in diverse organisms and researchers who study the impact these elements have on genome evolution. The meeting included over 200 scientists who participated through poster presentations, short talks selected from abstracts, and invited speakers. The talks were organized into eight sessions and two workshops. The topics varied from diverse mechanisms of mobilization to the evolution of genomes and their defense strategies against transposable elements.

Retrotransposons are transposable elements that duplicate themselves by converting their transcribed RNA genome into cDNA, which is then integrated back into the genome. Retrotransposons can be divided into two major classes based on their mechanism of transposition and the presence or absence of long terminal repeats (LTRs). In contrast to mammalian genomes, in which non-LTR retrotransposons have proliferated, plant genomes show evolutionary evidence of an explosion in LTR retrotransposon copy number. These retrotransposons can comprise a large fraction of the genome (75 % in maize). Although often viewed as molecular parasites, retrotransposons have been shown to influence neighboring gene expression and play a structural and potential regulatory role in the centromere. To prevent retrotransposon activity, eukaryotic cells have evolved overlapping mechanisms to repress transposition. Plants are an excellent system for studying the mechanisms of LTR retrotransposon inhibition such as DNA methylation and small RNA-mediated degradation of retrotransposon transcripts. However, analysis of these multi-copy, mobile elements is considerably more difficult than analysis of single-copy genes located in stable regions of the genome. In this chapter we outline methods for analyzing the progress of LTR retrotransposons through their replication cycle in plants. We describe a mixture of traditional molecular biology experiments, such as Southern, Northern, and Western blotting, in addition to nontraditional techniques designed to take advantage of the specific mechanism of LTR retrotransposition.

Transposable elements (TEs) are known to influence the regulation of neighboring genes through a variety of mechanisms. Additionally, it was recently discovered that TEs can regulate non-neighboring genes through the trans-acting nature of small interfering RNAs (siRNAs). When the epigenetic repression of TEs is lost, TEs become transcriptionally active, and the host cell acts to repress mutagenic transposition by degrading TE mRNAs into siRNAs. In this study, we have performed a genome-wide analysis in the model plant Arabidopsis thaliana and found that TE siRNA-based regulation of genic mRNAs is more pervasive than the two formerly characterized proof-of-principle examples. We identified 27 candidate genic mRNAs that do not contain a TE fragment but are regulated through partial complementarity by the accumulation of TE siRNAs and are therefore influenced by TE epigenetic activation. We have experimentally confirmed several gene targets and demonstrated that they respond to the accumulation of specific 21 nucleotide TE siRNAs that are incorporated into the Arabidopsis Argonaute1 protein. Additionally, we found that one TE siRNA specifically targets and inhibits the formation of a host protein that acts to repress TE activity, suggesting that TEs harbor and potentially evolutionarily select short sequences to act as suppressors of host TE repression.

In plants, RNA silencing is a fundamental regulator of gene expression, heterochromatin formation, suppression of transposable elements, and defense against viruses. The sequence specificity of these processes relies on small noncoding RNA (sRNA) molecules. Although the spreading of RNA silencing across the plant has been recognized for nearly two decades, only recently have sRNAs been formally demonstrated as the mobile silencing signals. Here, we discuss the various types of mobile sRNA molecules, their short- and long-range movement, and their function in recipient cells.

Transposable elements (TEs) are mobile fragments of DNA that are repressed in both plant and animal genomes through the epigenetic inheritance of repressed chromatin and expression states. The epigenetic silencing of TEs in plants is mediated by a process of RNA-directed DNA methylation (RdDM). Two pathways of RdDM have been identified: RNA Polymerase IV (Pol IV)-RdDM, which has been shown to be responsible for the de novo initiation, corrective reestablishment, and epigenetic maintenance of TE and/or transgene silencing; and RNA-dependent RNA Polymerase6 (RDR6)-RdDM, which was recently identified as necessary for maintaining repression for a few TEs. We have further characterized RDR6-RdDM using a genome-wide search to identify TEs that generate RDR6-dependent small interfering RNAs. We have determined that TEs only produce RDR6-dependent small interfering RNAs when transcriptionally active, and we have experimentally identified two TE subfamilies as direct targets of RDR6-RdDM. We used these TEs to test the function of RDR6-RdDM in assays for the de novo initiation, corrective reestablishment, and maintenance of TE silencing. We found that RDR6-RdDM plays no role in maintaining TE silencing. Rather, we found that RDR6 and Pol IV are two independent entry points into RdDM and epigenetic silencing that perform distinct functions in the silencing of TEs: Pol IV-RdDM functions to maintain TE silencing and to initiate silencing in an RNA Polymerase II expression-independent manner, while RDR6-RdDM functions to recognize active Polymerase II-derived TE mRNA transcripts to both trigger and correctively reestablish TE methylation and epigenetic silencing.

Transposable elements (TEs) are a source of endogenous small RNAs in animals and plants. These TE-derived small RNAs have been traditionally treated as functionally distinct from gene-regulating small RNAs, such as miRNAs. Two recent reports in Drosophila and Arabidopsis have blurred the lines of this distinction. In both examples, epigenetically and developmentally regulated bursts in TE expression produce gene-regulating small RNAs. In the Drosophila early embryo, maternally deposited TE-derived PIWI-interacting small RNAs (piRNAs) play a role in regulating the nanos mRNA through small RNA binding sites in the nanos 3' untranslated region (UTR). In Arabidopsis, when Athila retrotransposons are epigenetically activated, their transcripts are processed into small RNAs, which directly target the 3'UTR of the genic oligouridylate binding protein 1B (UBP1b) mRNA. Based on these two examples, we suggest that other TE-derived small RNAs regulate additional genes and propose that, through small RNAs, the epigenetic status of TEs could widely influence the genic transcriptome., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In plants, the developmental relaxation of transposable element silencing (DRTS) occurs at distinct spatial and temporal points in the normal development of a wild-type individual. Several examples of DRTS have now been described, including in maize shoot apical meristems, and in Arabidopsis meiocytes, endosperm and nurse cells of gametophytes. In this opinion article, we review the known DRTS events and speculate on the function, if any, of DRTS in plants., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: The male germline in flowering plants differentiates by asymmetric division of haploid uninucleated microspores, giving rise to a vegetative cell enclosing a smaller generative cell, which eventually undergoes a second mitosis to originate two sperm cells. The vegetative cell and the sperm cells activate distinct genetic and epigenetic mechanisms to control pollen tube growth and germ cell specification, respectively. Therefore, a comprehensive characterization of these processes relies on efficient methods to isolate each of the different cell types throughout male gametogenesis., Results: We developed stable transgenic Arabidopsis lines and reliable purification tools based on Fluorescence-Activated Cell Sorting (FACS) in order to isolate highly pure and viable fractions of each cell/nuclei type before and after pollen mitosis. In the case of mature pollen, this was accomplished by expressing GFP and RFP in the sperm and vegetative nuclei, respectively, resulting in 99% pure sorted populations. Microspores were also purified by FACS taking advantage of their characteristic small size and autofluorescent properties, and were confirmed to be 98% pure., Conclusions: We provide simple and efficient FACS-based purification protocols for Arabidopsis microspores, vegetative nuclei and sperm cells. This paves the way for subsequent molecular analysis such as transcriptomics, DNA methylation analysis and chromatin immunoprecipitation, in the developmental context of microgametogenesis in Arabidopsis.

The epigenetic activity of transposable elements (TEs) can influence the regulation of genes; though, this regulation is confined to the genes, promoters, and enhancers that neighbor the TE. This local cis regulation of genes therefore limits the influence of the TE's epigenetic regulation on the genome. TE activity is suppressed by small RNAs, which also inhibit viruses and regulate the expression of genes. The production of TE heterochromatin-associated endogenous small interfering RNAs (siRNAs) in the reference plant Arabidopsis thaliana is mechanistically distinct from gene-regulating small RNAs, such as microRNAs or trans-acting siRNAs (tasiRNAs). Previous research identified a TE small RNA that potentially regulates the UBP1b mRNA, which encodes an RNA-binding protein involved in stress granule formation. We demonstrate that this siRNA, siRNA854, is under the same trans-generational epigenetic control as the Athila family LTR retrotransposons from which it is produced. The epigenetic activation of Athila elements results in a shift in small RNA processing pathways, and new 21-22 nucleotide versions of Athila siRNAs are produced by protein components normally not responsible for processing TE siRNAs. This processing results in siRNA854's incorporation into ARGONAUTE1 protein complexes in a similar fashion to gene-regulating tasiRNAs. We have used reporter transgenes to demonstrate that the UPB1b 3' untranslated region directly responds to the epigenetic status of Athila TEs and the accumulation of siRNA854. The regulation of the UPB1b 3' untranslated region occurs both on the post-transcriptional and translational levels when Athila TEs are epigenetically activated, and this regulation results in the phenocopy of the ubp1b mutant stress-sensitive phenotype. This demonstrates that a TE's epigenetic activity can modulate the host organism's stress response. In addition, the ability of this TE siRNA to regulate a gene's expression in trans blurs the lines between TE and gene-regulating small RNAs., Competing Interests: The authors have declared that no competing interests exist.