RESUMEN
CRISPR-Cas systems are host-encoded pathways that protect microbes from viral infection using an adaptive RNA-guided mechanism. Using genome-resolved metagenomics, we find that CRISPR systems are also encoded in diverse bacteriophages, where they occur as divergent and hypercompact anti-viral systems. Bacteriophage-encoded CRISPR systems belong to all six known CRISPR-Cas types, though some lack crucial components, suggesting alternate functional roles or host complementation. We describe multiple new Cas9-like proteins and 44 families related to type V CRISPR-Cas systems, including the Casλ RNA-guided nuclease family. Among the most divergent of the new enzymes identified, Casλ recognizes double-stranded DNA using a uniquely structured CRISPR RNA (crRNA). The Casλ-RNA-DNA structure determined by cryoelectron microscopy reveals a compact bilobed architecture capable of inducing genome editing in mammalian, Arabidopsis, and hexaploid wheat cells. These findings reveal a new source of CRISPR-Cas enzymes in phages and highlight their value as genome editors in plant and human cells.
Asunto(s)
Bacteriófagos , Sistemas CRISPR-Cas , Animales , Humanos , Microscopía por Crioelectrón , Edición Génica , Genoma , Bacteriófagos/genética , ADN , ARN , Mamíferos/genéticaRESUMEN
The RNA-directed DNA methylation (RdDM) pathway in plants controls gene expression via cytosine DNA methylation. The ability to manipulate RdDM would shed light on the mechanisms and applications of DNA methylation to control gene expression. Here, we identified diverse RdDM proteins that are capable of targeting methylation and silencing in Arabidopsis when tethered to an artificial zinc finger (ZF-RdDM). We studied their order of action within the RdDM pathway by testing their ability to target methylation in different mutants. We also evaluated ectopic siRNA biogenesis, RNA polymerase V (Pol V) recruitment, targeted DNA methylation, and gene-expression changes at thousands of ZF-RdDM targets. We found that co-targeting both arms of the RdDM pathway, siRNA biogenesis and Pol V recruitment, dramatically enhanced targeted methylation. This work defines how RdDM components establish DNA methylation and enables new strategies for epigenetic gene regulation via targeted DNA methylation.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Metilación de ADN/fisiología , ARN Polimerasas Dirigidas por ADN/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Citosina/metabolismo , ADN/metabolismo , Metilación de ADN/genética , ARN Polimerasas Dirigidas por ADN/genética , Regulación de la Expresión Génica de las Plantas/genética , ARN Polimerasa II/metabolismo , ARN de Planta/genética , ARN Interferente Pequeño/metabolismoRESUMEN
RNA-directed DNA methylation in Arabidopsis thaliana is driven by the plant-specific RNA Polymerase IV (Pol IV). It has been assumed that a Pol IV transcript can give rise to multiple 24-nt small interfering RNAs (siRNAs) that target DNA methylation. Here, we demonstrate that Pol IV-dependent RNAs (P4RNAs) from wild-type Arabidopsis are surprisingly short in length (30 to 40 nt) and mirror 24-nt siRNAs in distribution, abundance, strand bias, and 5'-adenine preference. P4RNAs exhibit transcription start sites similar to Pol II products and are featured with 5'-monophosphates and 3'-misincorporated nucleotides. The 3'-misincorporation preferentially occurs at methylated cytosines on the template DNA strand, suggesting a co-transcriptional feedback to siRNA biogenesis by DNA methylation to reinforce silencing locally. These results highlight an unusual mechanism of Pol IV transcription and suggest a "one precursor, one siRNA" model for the biogenesis of 24-nt siRNAs in Arabidopsis.
Asunto(s)
Arabidopsis/metabolismo , ARN de Planta/genética , ARN Interferente Pequeño/genética , Adenina/metabolismo , Arabidopsis/enzimología , Arabidopsis/genética , Metilación de ADN , ARN Polimerasas Dirigidas por ADN/metabolismo , Modelos Biológicos , Sitio de Iniciación de la TranscripciónRESUMEN
DNA methylation is a conserved epigenetic gene-regulation mechanism. DOMAINS REARRANGED METHYLTRANSFERASE (DRM) is a key de novo methyltransferase in plants, but how DRM acts mechanistically is poorly understood. Here, we report the crystal structure of the methyltransferase domain of tobacco DRM (NtDRM) and reveal a molecular basis for its rearranged structure. NtDRM forms a functional homodimer critical for catalytic activity. We also show that Arabidopsis DRM2 exists in complex with the small interfering RNA (siRNA) effector ARGONAUTE4 (AGO4) and preferentially methylates one DNA strand, likely the strand acting as the template for RNA polymerase V-mediated noncoding RNA transcripts. This strand-biased DNA methylation is also positively correlated with strand-biased siRNA accumulation. These data suggest a model in which DRM2 is guided to target loci by AGO4-siRNA and involves base-pairing of associated siRNAs with nascent RNA transcripts.
Asunto(s)
Arabidopsis/enzimología , Metiltransferasas/metabolismo , Nicotiana/enzimología , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas/metabolismo , Dominio Catalítico , Metiltransferasas/química , Modelos Moleculares , Datos de Secuencia Molecular , Nicotiana/metabolismoRESUMEN
Histone variants play crucial roles in gene expression, genome integrity, and chromosome segregation. We report that the four H2A variants in Arabidopsis define different genomic features, contributing to overall genomic organization. The histone variant H2A.W marks heterochromatin specifically and acts in synergy with heterochromatic marks H3K9me2 and DNA methylation to maintain transposon silencing. In vitro, H2A.W enhances chromatin condensation by promoting fiber-to-fiber interactions via its conserved C-terminal motif. In vivo, H2A.W is required for heterochromatin condensation, demonstrating that H2A.W plays critical roles in heterochromatin organization. Similarities in conserved motifs between H2A.W and another H2A variant in metazoans suggest that plants and animals share common mechanisms for heterochromatin condensation.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ensamble y Desensamble de Cromatina , Heterocromatina/metabolismo , Histonas/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Metilación de ADN , Elementos Transponibles de ADN , Estudio de Asociación del Genoma Completo , Histonas/química , Histonas/genética , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
Cytosine methylation is involved in various biological processes such as silencing of transposable elements (TEs) and imprinting. Multiple pathways regulate DNA methylation in different sequence contexts, but the factors that regulate DNA methylation at a given site in the genome largely remain unknown. Here we have surveyed the methylomes of a comprehensive list of 86 Arabidopsis gene silencing mutants by generating single-nucleotide resolution maps of DNA methylation. We find that DNA methylation is site specifically regulated by different factors. Furthermore, we have identified additional regulators of DNA methylation. These data and analyses will serve as a comprehensive community resource for further understanding the control of DNA methylation patterning.
Asunto(s)
Arabidopsis/genética , Metilación de ADN , Genoma de Planta , Proteínas de Arabidopsis/metabolismo , Islas de CpG , Silenciador del Gen , Estudio de Asociación del Genoma Completo , Histonas/metabolismo , Interferencia de ARN , ARN Polimerasa II/metabolismo , Factores de Empalme de ARNRESUMEN
Methylation of DNA and of histone 3 at Lys 9 (H3K9) are highly correlated with gene silencing in eukaryotes from fungi to humans. Both of these epigenetic marks need to be established at specific regions of the genome and then maintained at these sites through cell division. Protein structural domains that specifically recognize methylated DNA and methylated histones are key for targeting enzymes that catalyse these marks to appropriate genome sites. Genetic, genomic, structural and biochemical data reveal connections between these two epigenetic marks, and these domains mediate much of the crosstalk.
Asunto(s)
Metilación de ADN , Epigénesis Genética , Histonas/metabolismo , Animales , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/fisiología , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Procesamiento Proteico-Postraduccional , Ubiquitina-Proteína LigasasRESUMEN
DNA methylation and histone modification exert epigenetic control over gene expression. CHG methylation by CHROMOMETHYLASE3 (CMT3) depends on histone H3K9 dimethylation (H3K9me2), but the mechanism underlying this relationship is poorly understood. Here, we report multiple lines of evidence that CMT3 interacts with H3K9me2-containing nucleosomes. CMT3 genome locations nearly perfectly correlated with H3K9me2, and CMT3 stably associated with H3K9me2-containing nucleosomes. Crystal structures of maize CMT3 homolog ZMET2, in complex with H3K9me2 peptides, showed that ZMET2 binds H3K9me2 via both bromo adjacent homology (BAH) and chromo domains. The structures reveal an aromatic cage within both BAH and chromo domains as interaction interfaces that capture H3K9me2. Mutations that abolish either interaction disrupt CMT3 binding to nucleosomes and show a complete loss of CMT3 activity in vivo. Our study establishes dual recognition of H3K9me2 marks by BAH and chromo domains and reveals a distinct mechanism of interplay between DNA methylation and histone modification.
Asunto(s)
Arabidopsis/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , ADN de Plantas/metabolismo , Nucleosomas/metabolismo , Zea mays/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Cristalografía por Rayos X , ADN (Citosina-5-)-Metiltransferasas/química , Heterocromatina/metabolismo , Histonas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de Secuencia , Zea mays/genéticaRESUMEN
DNA methylation and H3K9me are hallmarks of heterochromatin in plants and mammals, and are successfully maintained across generations. The biochemical and structural basis for this maintenance is poorly understood. The maintenance DNA methyltransferase from Zea mays, ZMET2, recognizes dimethylation of H3K9 via a chromodomain (CD) and a bromo adjacent homology (BAH) domain, which flank the catalytic domain. Here, we show that dinucleosomes are the preferred ZMET2 substrate, with DNA methylation preferentially targeted to linker DNA. Electron microscopy shows one ZMET2 molecule bridging two nucleosomes within a dinucleosome. We find that the CD stabilizes binding, whereas the BAH domain enables allosteric activation by the H3K9me mark. ZMET2 further couples recognition of H3K9me to an increase in the specificity for hemimethylated versus unmethylated DNA. We propose a model in which synergistic coupling between recognition of nucleosome spacing, H3K9 methylation, and DNA modification allows ZMET2 to maintain DNA methylation in heterochromatin with high fidelity.
Asunto(s)
Metilación de ADN , Metilasas de Modificación del ADN/metabolismo , Nucleosomas/enzimología , Proteínas de Plantas/metabolismo , Animales , Metilasas de Modificación del ADN/genética , Metilasas de Modificación del ADN/ultraestructura , Activación Enzimática , Escherichia coli/enzimología , Escherichia coli/genética , Microscopía Electrónica , Modelos Moleculares , Conformación de Ácido Nucleico , Nucleosomas/química , Nucleosomas/genética , Nucleosomas/ultraestructura , Proteínas de Plantas/genética , Proteínas de Plantas/ultraestructura , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Relación Estructura-Actividad , Especificidad por Sustrato , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) systems have been developed as important tools for plant genome engineering. Here, we demonstrate that the hypercompact CasΦ nuclease is able to generate stably inherited gene edits in Arabidopsis, and that CasΦ guide RNAs can be expressed with either the Pol-III U6 promoter or a Pol-II promoter together with ribozyme mediated RNA processing. Using the Arabidopsis fwa epiallele, we show that CasΦ displays higher editing efficiency when the target locus is not DNA methylated, suggesting that CasΦ is sensitive to chromatin environment. Importantly, two CasΦ protein variants, vCasΦ and nCasΦ, both showed much higher editing efficiency relative to the wild-type CasΦ enzyme. Consistently, vCasΦ and nCasΦ yielded offspring plants with inherited edits at much higher rates compared to WTCasΦ. Extensive genomic analysis of gene edited plants showed no off-target editing, suggesting that CasΦ is highly specific. The hypercompact size, T-rich minimal protospacer adjacent motif (PAM), and wide range of working temperatures make CasΦ an excellent supplement to existing plant genome editing systems.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Edición Génica , Arabidopsis/genética , Sistemas CRISPR-Cas , Plantas/genética , Genoma de Planta/genética , Factores de Transcripción/genética , Proteínas de Homeodominio/genética , Proteínas de Arabidopsis/genéticaRESUMEN
ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) AND ATXR6 are required for the deposition of H3K27me1 and for maintaining genomic stability in Arabidopsis Reduction of ATXR5/6 activity results in activation of DNA damage response genes, along with tissue-specific derepression of transposable elements (TEs), chromocenter decompaction, and genomic instability characterized by accumulation of excess DNA from heterochromatin. How loss of ATXR5/6 and H3K27me1 leads to these phenotypes remains unclear. Here we provide extensive characterization of the atxr5/6 hypomorphic mutant by comprehensively examining gene expression and epigenetic changes in the mutant. We found that the tissue-specific phenotypes of TE derepression and excessive DNA in this atxr5/6 mutant correlated with residual ATXR6 expression from the hypomorphic ATXR6 allele. However, up-regulation of DNA damage genes occurred regardless of ATXR6 levels and thus appears to be a separable process. We also isolated an atxr6-null allele which showed that ATXR5 and ATXR6 are required for female germline development. Finally, we characterize three previously reported suppressors of the hypomorphic atxr5/6 mutant and show that these rescue atxr5/6 via distinct mechanisms, two of which involve increasing H3K27me1 levels.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Elementos Transponibles de ADN , Regulación de la Expresión Génica de las Plantas , Inestabilidad Genómica , Metiltransferasas/genética , Alelos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Epigénesis Genética , Heterocromatina/metabolismo , Histonas/metabolismo , Metiltransferasas/metabolismo , Mutación , Fenotipo , TranscriptomaRESUMEN
Bioactive natural products have evolved to inhibit specific cellular targets and have served as lead molecules for health and agricultural applications for the past century1-3. The post-genomics era has brought a renaissance in the discovery of natural products using synthetic-biology tools4-6. However, compared to traditional bioactivity-guided approaches, genome mining of natural products with specific and potent biological activities remains challenging4. Here we present the discovery and validation of a potent herbicide that targets a critical metabolic enzyme that is required for plant survival. Our approach is based on the co-clustering of a self-resistance gene in the natural-product biosynthesis gene cluster7-9, which provides insight into the potential biological activity of the encoded compound. We targeted dihydroxy-acid dehydratase in the branched-chain amino acid biosynthetic pathway in plants; the last step in this pathway is often targeted for herbicide development10. We show that the fungal sesquiterpenoid aspterric acid, which was discovered using the method described above, is a sub-micromolar inhibitor of dihydroxy-acid dehydratase that is effective as a herbicide in spray applications. The self-resistance gene astD was validated to be insensitive to aspterric acid and was deployed as a transgene in the establishment of plants that are resistant to aspterric acid. This herbicide-resistance gene combination complements the urgent ongoing efforts to overcome weed resistance11. Our discovery demonstrates the potential of using a resistance-gene-directed approach in the discovery of bioactive natural products.
Asunto(s)
Productos Biológicos/metabolismo , Productos Biológicos/farmacología , Herbicidas/metabolismo , Herbicidas/farmacología , Compuestos Heterocíclicos con 3 Anillos/metabolismo , Compuestos Heterocíclicos con 3 Anillos/farmacología , Arabidopsis/efectos de los fármacos , Arabidopsis/enzimología , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Productos Biológicos/análisis , Inhibidores Enzimáticos/análisis , Inhibidores Enzimáticos/farmacología , Resistencia a los Herbicidas/genética , Herbicidas/análisis , Compuestos Heterocíclicos con 3 Anillos/análisis , Hidroliasas/antagonistas & inhibidores , Hidroliasas/química , Hidroliasas/metabolismo , Modelos Moleculares , Familia de Multigenes/genética , Reguladores del Crecimiento de las Plantas/análisis , Reguladores del Crecimiento de las Plantas/farmacología , Plantas Modificadas Genéticamente/genética , Transgenes/genéticaRESUMEN
CRISPR-based targeted modification of epigenetic marks such as DNA cytosine methylation is an important strategy to regulate the expression of genes and their associated phenotypes. Although plants have DNA methylation in all sequence contexts (CG, CHG, CHH, where H = A, T, C), methylation in the symmetric CG context is particularly important for gene silencing and is very efficiently maintained through mitotic and meiotic cell divisions. Tools that can directly add CG methylation to specific loci are therefore highly desirable but are currently lacking in plants. Here we have developed two CRISPR-based CG-specific targeted DNA methylation systems for plants using a variant of the bacterial CG-specific DNA methyltransferase MQ1 with reduced activity but high specificity. We demonstrate that the methylation added by MQ1 is highly target specific and can be heritably maintained in the absence of the effector. These tools should be valuable both in crop engineering and in plant genetic research.
Asunto(s)
Arabidopsis , Proteínas Bacterianas , Sistemas CRISPR-Cas , Metilación de ADN , ADN de Plantas/metabolismo , ADN-Citosina Metilasas , Plantas Modificadas Genéticamente , Tenericutes/genética , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN de Plantas/genética , ADN-Citosina Metilasas/genética , ADN-Citosina Metilasas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Tenericutes/enzimologíaRESUMEN
DNA methylation is a major epigenetic modification found across species and has a profound impact on many biological processes. However, its influence on chromatin accessibility and higher-order genome organization remains unclear, particularly in plants. Here, we present genome-wide chromatin accessibility profiles of 18 Arabidopsis mutants that are deficient in CG, CHG, or CHH DNA methylation. We find that DNA methylation in all three sequence contexts impacts chromatin accessibility in heterochromatin. Many chromatin regions maintain inaccessibility when DNA methylation is lost in only one or two sequence contexts, and signatures of accessibility are particularly affected when DNA methylation is reduced in all contexts, suggesting an interplay between different types of DNA methylation. In addition, we found that increased chromatin accessibility was not always accompanied by increased transcription, suggesting that DNA methylation can directly impact chromatin structure by other mechanisms. We also observed that an increase in chromatin accessibility was accompanied by enhanced long-range chromatin interactions. Together, these results provide a valuable resource for chromatin architecture and DNA methylation analyses and uncover a pivotal role for methylation in the maintenance of heterochromatin inaccessibility.
Asunto(s)
Arabidopsis/genética , Cromatina/genética , Metilación de ADN/genética , Genoma de Planta , Mutación/genética , Transcripción GenéticaRESUMEN
RNA polymerase V (Pol V) long noncoding RNAs (lncRNAs) have been proposed to guide ARGONAUTE4 (AGO4) to chromatin in RNA-directed DNA methylation (RdDM) in plants. Here, we provide evidence, based on laser UV-assisted zero-length cross-linking, for functionally relevant AGO4-DNA interaction at RdDM targets. We further demonstrate that Pol V lncRNAs or the act of their transcription are required to lock Pol V holoenzyme into a stable DNA-bound state that allows AGO4 recruitment via redundant glycine-tryptophan/tryptophan-glycine AGO hook motifs present on both Pol V and its associated factor, SPT5L. We propose a model in which AGO4-DNA interaction could be responsible for the unique specificities of RdDM.
Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Metilación de ADN , ADN de Plantas/metabolismo , ARN de Planta/metabolismo , Secuencias de Aminoácidos/genética , Proteínas de Arabidopsis/química , Proteínas Argonautas/química , Cromatina/metabolismo , Metilación de ADN/genética , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Silenciador del Gen , Modelos Biológicos , Interferencia de ARN , Transcripción Genética/genéticaRESUMEN
Comparison of biosynthetic gene clusters (BGCs) found in devastating plant pathogens and biocontrol fungi revealed an uncharacterized and conserved polyketide BGC. Genome mining identified the associated metabolite to be treconorin, which has a terpene-like, trans-fused 5,7-bicyclic core that is proposed to derive from a (4 + 3) cycloaddition. The core is esterified with d-glucose, which derives from the glycosidic cleavage of a trehalose ester precursor. This glycomodification strategy is different from the commonly observed glycosylation of natural products.
Asunto(s)
Policétidos , Terpenos , Familia de Multigenes , Hongos/genéticaRESUMEN
Tools for sequence-specific DNA binding have opened the door to new approaches in investigating fundamental questions in biology and crop development. While there are several platforms to choose from, many of the recent advances in sequence-specific targeting tools are focused on developing Clustered Regularly Interspaced Short Palindromic Repeats- CRISPR Associated (CRISPR-Cas)-based systems. Using a catalytically inactive Cas protein (dCas), this system can act as a vector for different modular catalytic domains (effector domains) to control a gene's expression or alter epigenetic marks such as DNA methylation. Recent trends in developing CRISPR-dCas systems include creating versions that can target multiple copies of effector domains to a single site, targeting epigenetic changes that, in some cases, can be inherited to the next generation in the absence of the targeting construct, and combining effector domains and targeting strategies to create synergies that increase the functionality or efficiency of the system. This review summarizes and compares DNA targeting technologies, the effector domains used to target transcriptional control and epi-mutagenesis, and the different CRISPR-dCas systems used in plants.
Asunto(s)
Sistemas CRISPR-Cas , Sistemas CRISPR-Cas/genética , MutagénesisRESUMEN
Studies of the model plant Arabidopsis thaliana may seem to have little impact on advances in medical research, yet a survey of the scientific literature shows that this is a misconception. Many discoveries with direct relevance to human health and disease have been elaborated using Arabidopsis, and several processes important to human biology are more easily studied in this versatile model plant.
Asunto(s)
Arabidopsis/metabolismo , Enfermedad de Alzheimer/metabolismo , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ritmo Circadiano , Humanos , Inmunidad , Modelos Biológicos , Neoplasias/metabolismoRESUMEN
Plant RNA viruses are used as delivery vectors for their high level of accumulation and efficient spread during virus multiplication and movement. Utilizing this concept, several viral-based guide RNA delivery platforms for CRISPR-Cas9 genome editing have been developed. The CRISPR-Cas9 system has also been adapted for epigenome editing. While systems have been developed for CRISPR-Cas9 based gene activation or site-specific DNA demethylation, viral delivery of guide RNAs remains to be developed for these purposes. To address this gap we have developed a tobacco rattle virus (TRV)-based single guide RNA delivery system for epigenome editing in Arabidopsis thaliana. Because tRNA-like sequences have been shown to facilitate the cell-to-cell movement of RNAs in plants, we used the tRNA-guide RNA expression system to express guide RNAs from the viral genome to promote heritable epigenome editing. We demonstrate that the tRNA-gRNA system with TRV can be used for both transcriptional activation and targeted DNA demethylation of the FLOWERING WAGENINGEN gene in Arabidopsis. We achieved up to ~8% heritability of the induced demethylation phenotype in the progeny of virus inoculated plants. We did not detect the virus in the next generation, indicating effective clearance of the virus from plant tissues. Thus, TRV delivery, combined with a specific tRNA-gRNA architecture, provides for fast and effective epigenome editing.
Asunto(s)
Proteínas de Arabidopsis/genética , Sistemas CRISPR-Cas , Metilación de ADN , Edición Génica/métodos , Marcación de Gen/métodos , Virus de Plantas/genética , ARN Guía de Kinetoplastida/genética , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Epigenoma , ARN de Transferencia/genética , Activación TranscripcionalRESUMEN
Transposable elements (TEs) are DNA repeats that must remain silenced to ensure cell integrity. Several epigenetic pathways including DNA methylation and histone modifications are involved in the silencing of TEs, and in the regulation of gene expression. In Arabidopsis thaliana, the TE-derived plant mobile domain (PMD) proteins have been involved in TE silencing, genome stability, and control of developmental processes. Using a forward genetic screen, we found that the PMD protein MAINTENANCE OF MERISTEMS (MAIN) acts synergistically and redundantly with DNA methylation to silence TEs. We found that MAIN and its close homolog MAIN-LIKE 1 (MAIL1) interact together, as well as with the phosphoprotein phosphatase (PPP) PP7-like (PP7L). Remarkably, main, mail1, pp7l single and mail1 pp7l double mutants display similar developmental phenotypes, and share common subsets of upregulated TEs and misregulated genes. Finally, phylogenetic analyses of PMD and PP7-type PPP domains among the Eudicot lineage suggest neo-association processes between the two protein domains to potentially generate new protein function. We propose that, through this interaction, the PMD and PPP domains may constitute a functional protein module required for the proper expression of a common set of genes, and for silencing of TEs.