RESUMEN
N 6-methyladenosine (m6A) is a common epitranscriptional mRNA modification in eukaryotes. Thirteen putative m6A readers, mostly annotated as EVOLUTIONARILY CONSERVED C-TERMINAL REGION (ECT) proteins, have been identified in Arabidopsis (Arabidopsis thaliana), but few have been characterized. Here, we show that the Arabidopsis m6A reader ECT1 modulates salicylic acid (SA)-mediated plant stress responses. ECT1 undergoes liquid-liquid phase separation in vitro, and its N-terminal prion-like domain is critical for forming in vivo cytosolic biomolecular condensates in response to SA or bacterial pathogens. Fluorescence-activated particle sorting coupled with quantitative PCR analyses unveiled that ECT1 sequesters SA-induced m6A modification-prone mRNAs through its conserved aromatic cage to facilitate their decay in cytosolic condensates, thereby dampening SA-mediated stress responses. Consistent with this finding, ECT1 overexpression promotes bacterial multiplication in plants. Collectively, our findings unequivocally link ECT1-associated cytosolic condensates to SA-dependent plant stress responses, advancing the current understanding of m6A readers and the SA signaling network.
Asunto(s)
Adenina/análogos & derivados , Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Ácido Salicílico/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
Transposable elements (TEs) are mobile DNA elements that are particularly abundant in the plant genomes. They have long been considered as junk DNA; however, a growing body of evidence suggests that TE insertions promote genetic diversity that is essential for the adaptive evolution of a species. Thus far, studies have mainly investigated the cis-acting regulatory roles of TEs generated by their insertions nearby or within the host genes. However, the trans-acting effects of TE-derived RNA and DNA remained obscure to date. TEs contain various regulatory elements within their sequences that can accommodate the binding of specific RNAs and proteins. Recently, it was suggested that some of these cellular regulators are shared between TEs and the host genes, and the competition for the common host factors underlies the fine-tuned developmental reprogramming. In this review, we will highlight and discuss the latest discoveries on the biological functions of plant TEs, with a particular focus on their competitive binding with specific developmental regulators.
RESUMEN
Transposable elements (TEs) are mobile genetic elements that can impair the host genome stability and integrity. It has been well documented that activated transposons in plants are suppressed by small interfering (si) RNAs. However, transposon repression by the cytoplasmic RNA surveillance system is unknown. Here, we show that mRNA deadenylation is critical for controlling transposons in Arabidopsis. Trimming of poly(A) tail is a rate-limiting step that precedes the RNA decay and is primarily mediated by the CARBON CATABOLITE REPRESSION 4 (CCR4)-NEGATIVE ON TATA-LESS (NOT) complex. We found that the loss of CCR4a leads to strong derepression and mobilization of TEs in Arabidopsis. Intriguingly, CCR4a regulates a largely distinct set of TEs from those controlled by RNA-dependent RNA Polymerase 6 (RDR6), a key enzyme that produces cytoplasmic siRNAs. This indicates that the cytoplasmic RNA quality control mechanism targets the TEs that are poorly recognized by the previously well-characterized RDR6-mediated pathway, and thereby augments the host genome stability. Our study suggests a hitherto unknown mechanism for transposon repression mediated by RNA deadenylation and unveils a complex nature of the host's strategy to maintain the genome integrity.
Asunto(s)
Arabidopsis , Represión Catabólica , Arabidopsis/genética , Arabidopsis/metabolismo , ARN Interferente Pequeño/metabolismo , Elementos Transponibles de ADN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Inestabilidad Genómica , Estabilidad del ARN/genéticaRESUMEN
The tissue culture passage necessary for the generation of transgenic plants induces genome instability. This instability predominantly involves the uncontrolled mobilization of LTR retrotransposons (LTR-TEs), which are the most abundant class of mobile genetic elements in plant genomes. Here, we demonstrate that in conditions inductive for high LTR-TE mobilization, like abiotic stress in Arabidopsis (Arabidopsis thaliana) and callus culture in rice (Oryza sativa), application of the reverse transcriptase (RT) inhibitor known as Tenofovir substantially affects LTR-TE RT activity without interfering with plant development. We observed that Tenofovir reduces extrachromosomal DNA accumulation and prevents new genomic integrations of the active LTR-TE ONSEN in heat-stressed Arabidopsis seedlings, and transposons of O. sativa 17 and 19 (Tos17 and Tos19) in rice calli. In addition, Tenofovir allows the recovery of plants free from new LTR-TE insertions. We propose the use of Tenofovir as a tool for studies of LTR-TE transposition and for limiting genetic instabilities of plants derived from tissue culture.
Asunto(s)
Arabidopsis , Oryza , Retroelementos/genética , Arabidopsis/genética , Genoma de Planta/genética , Secuencias Repetidas Terminales/genética , Tenofovir , Oryza/genéticaRESUMEN
Gene expression can be modulated by epigenetic mechanisms, including chromatin modifications and small regulatory RNAs. These pathways are unevenly distributed within a cell and usually take place in specific intracellular regions. Unfortunately, the fundamental driving force and biological relevance of such spatial differentiation is largely unknown. Liquid-liquid phase separation (LLPS) is a natural propensity of demixing liquid phases and has been recently suggested to mediate the formation of biomolecular condensates that are relevant to diverse cellular processes. LLPS provides a mechanistic explanation for the self-assembly of subcellular structures by which the efficiency and specificity of certain cellular reactions are achieved. In plants, LLPS has been observed for several key factors in the chromatin and small RNA pathways. For example, the formation of facultative and obligate heterochromatin involves the LLPS of multiple relevant factors. In addition, phase separation is observed in a set of proteins acting in microRNA biogenesis and the small interfering RNA pathway. In this Focused Review, we highlight and discuss the recent findings regarding phase separation in the epigenetic mechanisms of plants.
Asunto(s)
Condensados Biomoleculares/metabolismo , Epigénesis Genética , Proteínas de Plantas/metabolismo , Plantas/metabolismo , ARN de Planta/metabolismo , Condensados Biomoleculares/genética , Cromatina/genética , Cromatina/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Proteínas de Plantas/genética , Plantas/genética , ARN de Planta/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
Plant small RNAs (sRNAs) are short non-coding RNAs that are implicated in various regulatory processes involving post-transcriptional gene silencing and epigenetic gene regulation. In epigenetic regulation, sRNAs are primarily involved in RNA-directed DNA methylation (RdDM) pathways. sRNAs in the RdDM pathways play a role not only in the suppression of transposable element (TE) activity but also in gene expression regulation. Although the major components of the RdDM pathways have been well studied in Arabidopsis, recent studies have revealed that the RdDM pathways in rice have important biological functions in stress response and developmental processes. In this review, we summarize and discuss recent literature on sRNA-mediated epigenetic regulation in rice. First, we describe the RdDM mechanisms in plants. We then introduce recent discoveries on the biological roles of rice genes involved in the RdDM pathway and TE-derived sRNAs working at specific genomic loci for epigenetic control in rice.
Asunto(s)
Arabidopsis , Oryza , Arabidopsis/genética , Metilación de ADN , Elementos Transponibles de ADN/genética , Epigénesis Genética , Regulación de la Expresión Génica de las Plantas , Oryza/genética , ARN/metabolismo , ARN Interferente Pequeño/metabolismoRESUMEN
Transposons are mobile and ubiquitous DNA molecules that can cause vast genomic alterations. In plants, it is well documented that transposon mobilization is strongly repressed by DNA methylation; however, its regulation at the posttranscriptional level remains relatively uninvestigated. Here, we suggest that transposon RNA is marked by m6A RNA methylation and can be localized in stress granules (SGs). Intriguingly, SG-localized AtALKBH9B selectively demethylates a heat-activated retroelement, Onsen, and thereby releases it from spatial confinement, allowing for its mobilization. In addition, we show evidence that m6A RNA methylation contributes to transpositional suppression by inhibiting virus-like particle assembly and extrachromosomal DNA production. In summary, this study unveils a previously unknown role for m6A in the suppression of transposon mobility and provides insight into how transposons counteract the m6A-mediated repression mechanism by hitchhiking the RNA demethylase of the host.
Asunto(s)
Arabidopsis , Arabidopsis/genética , Retroelementos/genética , ARN , Calor , Metilación de ADN , Secuencias Repetidas Terminales/genética , Regulación de la Expresión Génica de las PlantasRESUMEN
Transposable elements (TEs) comprise ~85% of the common wheat genome, which are highly diverse among subgenomes, possibly contribute to polyploid plasticity, but the causality is only assumed. Here, by integrating data from gene expression cap analysis and epigenome profiling via hidden Markov model in common wheat, we detect a large proportion of enhancer-like elements (ELEs) derived from TEs producing nascent noncoding transcripts, namely ELE-RNAs, which are well indicative of the regulatory activity of ELEs. Quantifying ELE-RNA transcriptome across typical developmental stages reveals that TE-initiated ELE-RNAs are mainly from RLG_famc7.3 specifically expanded in subgenome A. Acquisition of spike-specific transcription factor binding likely confers spike-specific expression of RLG_famc7.3-initiated ELE-RNAs. Knockdown of RLG_famc7.3-initiated ELE-RNAs resulted in global downregulation of spike-specific genes and abnormal spike development. These findings link TE expansion to regulatory specificity and polyploid developmental plasticity, highlighting the functional impact of TE-driven regulatory innovation on polyploid evolution.
Asunto(s)
Elementos Transponibles de ADN , Triticum , Elementos Transponibles de ADN/genética , Triticum/genética , Regulación de la Expresión Génica , Poliploidía , Transcriptoma , ARNRESUMEN
BACKGROUND: The epigenetic mechanisms play critical roles in a vast diversity of biological processes of plants, including development and response to environmental challenges. Particularly, DNA methylation is a stable epigenetic signature that supplements the genetics-based view of complex life phenomena. In crop breeding, the decrease in genetic diversity due to artificial selection of conventional breeding methods has been a long-standing concern. Therefore, the epigenetic diversity has been proposed as a new resource for future crop breeding, which will be hereinafter referred to as epibreeding. DISCUSSION: The induction of methylome changes has been performed in plants by several methods including chemical drugs treatment and tissue culture. Target-specific epigenetic engineering has been also attempted by exogenous RNAi mediated by virus-induced gene silencing and grafting. Importantly, the new and innovative techniques including the CRISPR-Cas9 system have recently been adopted in epigenetic engineering of plant genomes, facilitating the efforts for epibreeding. CONCLUSION: In this review, we introduce several examples of natural and induced epigenetic changes impacting on agronomic traits and discuss the methods for generating epigenomic diversity and site-specific epigenetic engineering.
Asunto(s)
Epigenómica , Fitomejoramiento , Productos Agrícolas/genética , Epigénesis Genética , Genoma de Planta , Fitomejoramiento/métodosRESUMEN
Heat-imposed crop failure is often attributed to reduced thermotolerance of floral tissues; however, the underlying mechanism remains unknown. Here, we demonstrate that m6A RNA methylation increases in Arabidopsis flowers and negatively regulates gene expression variability. Stochastic gene expression provides flexibility to cope with environmental stresses. We find that reduced transcriptional fluctuation is associated with compromised activation of heat-responsive genes. Moreover, disruption of an RNA demethylase AtALKBH10B leads to lower gene expression variability, suppression of heat-activated genes, and strong reduction of plant fertility. Our work proposes a novel role for RNA methylation in the bet-hedging strategy of heat stress response.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Termotolerancia , Arabidopsis/metabolismo , Termotolerancia/genética , Proteínas de Arabidopsis/metabolismo , Metilación , Regulación de la Expresión Génica de las Plantas , ARN/metabolismo , Expresión GénicaRESUMEN
Transposable elements (TEs, transposons) are mobile DNAs that are prevalent in most eukaryotic genomes. In plants, their mobility has vastly contributed to genetic diversity which is essential for adaptive changes and evolution of a species. Such mobile nature of transposon has been also actively exploited in plant science research by generating genetic mutants in non-model plant systems. On the other hand, transposon mobilization can bring about detrimental effects to host genomes and they are therefore mostly silenced by the epigenetic mechanisms. TEs have been studied as major silencing targets and acted a main feature in the remarkable growth of the plant epigenetics field. Despite the importance of transposon in plant biology and biotechnology, their mobilization and the underlying mechanisms are largely left unanswered. This is mainly because of the sequence repetitiveness of transposons, which makes their detection and analyses difficult and complicated. Recently, some attempts have been made to develop new experimental methods detecting active transposons and their mobilization behavior. These techniques reveal TE mobility in various levels, including the molecular, cellular, organismal and population scales. In this review, we will highlight the novel technical approaches in the study of mobile genetic elements and discuss how these techniques impacted on the advancement of transposon research and broadened our understanding of plant genome plasticity.
RESUMEN
Long terminal repeat retrotransposons (LTR-RTs) make up a considerable portion of plant genomes. New insertions of these active LTR-RTs modify gene structures and functions and play an important role in genome evolution. Therefore, identifying active forms of LTR-RTs could uncover the effects of these elements in plants. Extrachromosomal linear DNA (eclDNA) forms during LTR-RT replication; therefore, amplification LTRs of eclDNAs followed by sequencing (ALE-seq) uncover the current transpositional potential of the LTR-RTs. The ALE-seq protocol was validated by identification of Tos17 in callus of Nipponbare cultivar. Here, we identified two active LTR-RTs belonging to the Oryco family on chromosomes 6 and 9 in rice cultivar Dongjin callus based on the ALE-seq technology. Each Oryco family member has paired LTRs with identical sequences and internal domain regions. Comparison of the two LTR-RTs revealed 97% sequence identity in their internal domains and 65% sequence identity in their LTRs. These two putatively active Oryco LTR-RT family members could be used to expand our knowledge of retrotransposition mechanisms and the effects of LTR-RTs on the rice genome.
RESUMEN
Spontaneous proliferation of transposable elements contributes to genetic diversity at varying levels such as somatic mosaicism, genetic divergence in population, and genome evolution. Such genetic diversity is essential for plants' adaptation to changing environment and serves as a valuable resource for crop improvement. Therefore, measuring the copy number variation of transposable elements with precision and efficiency is important to understand the extent of their proliferation. Droplet Digital PCR (ddPCR) is an accurate and sensitive technique that allows measurement of copy number variation of a transposon. Briefly, genomic DNA is extracted, digested, and partitioned into thousands of nanoliter-scale droplets. The TaqMan real-time PCR followed by the end-point fluorescence detection enables the quantitative measurement of copy number of template DNAs. Here in this chapter, we describe the step-by-step procedure of ddPCR using EVADE retrotransposon of Arabidopsis as an example.
Asunto(s)
Arabidopsis/genética , Variaciones en el Número de Copia de ADN , Elementos Transponibles de ADN , ADN de Plantas/genética , Fluorescencia , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
ALE-seq is a method devised to identify pre-integration intermediates of LTR retrotransposons called extrachromosomal linear DNA, which can be used to predict retrotransposition activity. We describe here a bioinformatic methodology to process reads obtained from the ALE-seq protocol for the effective annotation of novel and active retroelements.
Asunto(s)
Biología Computacional/métodos , Retroelementos , Secuencias Repetidas Terminales , Análisis de Secuencia de ADN/métodos , Programas InformáticosRESUMEN
Extrachromosomal linear DNA (eclDNA) is the reverse-transcribed cDNA intermediate derived from long terminal repeat (LTR) transposable elements (TEs) (Cho et al., Nat Plants 5:26-33, 2018). Given that the eclDNAs are the final intermediate of LTR-TE life cycle prior to integration to the host chromosomes, their presence is considered a strong indication of active LTR retrotransposons (Cho et al., Nat Plants 5:26-33, 2018; Lanciano et al., PLoS Genet 13:e1006630, 2017). Here, we describe a method of amplification of LTR extrachromosomal DNA followed by sequencing (ALE-seq) which determines the 5' LTR sequences of eclDNAs. Briefly, ALE-seq consists of two steps of amplification, in vitro transcription of adaptor-ligated eclDNAs and subsequent reverse transcription to cDNAs primed at the conserved primer binding site (PBS) (Cho et al., Nat Plants 5:26-33, 2018). ALE-seq allows the high-throughput identification of novel LTR-TEs which are active in plants that could be potentially useful for crop biotechnology.
Asunto(s)
Plantas/genética , Retroelementos , Análisis de Secuencia de ADN/métodos , Secuencias Repetidas Terminales , Evolución Molecular , Genoma de Planta , Secuenciación de Nucleótidos de Alto Rendimiento , Transcripción ReversaRESUMEN
Mapping the genomic location to which transposons jumped is of greatest interest to transposon biologists. Transposon display (TD) is the technique of choice that is easy and fast in determining the neo-insertion positions of a target transposon. Essentially, tagging of transposon is performed by digesting genomic DNA, ligating adaptors to digested DNA ends and PCR amplifying genomic regions flanking the transposon of interest. In this chapter, the experimental procedure of TD is described using Onsen retrotransposon of Arabidopsis as an example.
Asunto(s)
Arabidopsis/genética , Mutagénesis Insercional/métodos , Retroelementos , Mapeo Cromosómico , ADN de Plantas/genéticaRESUMEN
RNA granules (RGs) are membraneless intracellular compartments that play important roles in the post-transcriptional control of gene expression. Stress granules (SGs) are a type of RGs that form under environmental challenges and/or internal cellular stresses. Stress treatments lead to strong mRNAs translational inhibition and storage in SGs until the normal growth conditions are restored. Intriguingly, we recently showed that plant stress granules are associated with siRNA bodies, where the RDR6-mediated and transposon-derived siRNA biogenesis occurs ( Kim et al., 2021 ). This protocol provides a technical workflow for the enrichment of cytoplasmic RGs from Arabidopsis seedlings. We used the DNA methylation-deficient ddm1 mutant in our study, but the method can be applied to any other plant samples with strong RG formation. The resulting RG fractions can be further tested for either RNAs or proteins using RNA-seq and mass spectrometry-based proteomics.
RESUMEN
BACKGROUND: Unlike peoples' belief that transposable elements (TEs) are "junk DNAs" or "genomic parasites", TEs are essential genomic elements that bring about genetic diversity and enable evolution of a species. In fact, transposons are major constituent of chromosome in crop genomes, particularly in major cereal crops, the primary type of which is long terminal repeat (LTR) retrotransposon. Since TE mobilization can be controlled by specific environmental stimulation and as the result can generate novel genetic variations, it has been suggested that controlled mobilization of TEs can be a plausible method for crop breeding. To achieve this goal, series of sequencing techniques have been recently established to identify TEs that are active in mobility. These methods target and detect extrachromosomal DNAs (ecDNAs), which are final products of integration. The newly identified TEs by these methods exhibit strong transpositional activity which can generate novel genetic diversity and provide useful breeding resources. CONCLUSIONS: In this mini review, we summarize and introduce ALE-seq, mobilome-seq, and VLP DNA-seq techniques employed to detect active TEs in plants.