RESUMEN
Precise genetic modification can be achieved via a sequence homology-mediated process known as gene targeting (GT). Whilst established for genome engineering purposes, the application of GT in plants still suffers from a low efficiency for which an explanation is currently lacking. Recently reported reduced rates of GT in A. thaliana deficient in polymerase theta (Polθ), a core component of theta-mediated end joining (TMEJ) of DNA breaks, have led to the suggestion of a direct involvement of this enzyme in the homology-directed process. Here, by monitoring homology-driven gene conversion in plants with CRISPR reagent and donor sequences pre-integrated at random sites in the genome (in planta GT), we demonstrate that Polθ action is not required for GT, but instead suppresses the process, likely by promoting the repair of the DNA break by end-joining. This finding indicates that lack of donor integration explains the previously established reduced GT rates seen upon transformation of Polθ-deficient plants. Our study additionally provides insight into ectopic gene targeting (EGT), recombination events between donor and target that do not map to the target locus. EGT, which occurs at similar frequencies as "true" GT during transformation, was rare in our in planta GT experiments arguing that EGT predominantly results from target locus recombination with nonintegrated T-DNA molecules. By describing mechanistic features of GT our study provides directions for the improvement of precise genetic modification of plants.
Asunto(s)
Arabidopsis , Arabidopsis/genética , Marcación de Gen/métodos , Edición Génica , Plantas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Reparación del ADN por Unión de Extremidades/genéticaRESUMEN
MSI1 belongs to a family of histone binding WD40-repeat proteins. Arabidopsis thaliana contains five genes encoding MSI1-like proteins, but their functions in diverse chromatin-associated complexes are poorly understood. Here, we show that MSI1 is part of a histone deacetylase complex. We copurified HISTONE DEACETYLASE19 (HDA19) with MSI1 and transcriptional regulatory SIN3-like proteins and provide evidence that MSI1 and HDA19 associate into the same complex in vivo. These data suggest that MSI1, HDA19, and HISTONE DEACETYLATION COMPLEX1 protein form a core complex that can integrate various SIN3-like proteins. We found that reduction of MSI1 or HDA19 causes upregulation of abscisic acid (ABA) receptor genes and hypersensitivity of ABA-responsive genes. The MSI1-HDA19 complex fine-tunes ABA signaling by binding to the chromatin of ABA receptor genes and by maintaining low levels of acetylation of histone H3 at lysine 9, thereby affecting the expression levels of ABA receptor genes. Reduced MSI1 or HDA19 levels led to increased tolerance to salt stress corresponding to the increased ABA sensitivity of gene expression. Together, our results reveal the presence of an MSI1-HDA19 complex that fine-tunes ABA signaling in Arabidopsis.
Asunto(s)
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Histona Desacetilasas/metabolismo , Transducción de Señal , Ácido Abscísico/farmacología , Acetilación/efectos de los fármacos , Adaptación Fisiológica/efectos de los fármacos , Adaptación Fisiológica/genética , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cromatina/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas , Histonas/metabolismo , Lisina/metabolismo , Modelos Biológicos , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Subunidades de Proteína/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Cloruro de Sodio/farmacología , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
During the last two centuries, the North American common ragweed (Ambrosia artemisiifolia L.) invaded a large part of the globe. Local adaptation of this species was revealed by a common garden experiment, demonstrating that the distribution of the species in Europe could extend considerably to the North. Our study compares two populations of common ragweed (one from the native range and one from the invaded range) that differ in flowering time in the wild: the invasive population flowers earlier than the native population under non-inductive long-day photoperiods. Experiments conducted in controlled environments established that the two populations differ in their flowering time even under inductive short-day photoperiods, suggesting a change in autonomous flowering control. Genetic analysis revealed that early flowering is dominantly inherited and accompanied by the increased expression of the floral activator AaFTL1 and decreased expression of the floral repressor AaFTL2. Early flowering is also accompanied by reduced reproductive output, which is evolutionarily disadvantageous under long vegetation periods. In contrast, under short vegetation periods, only early-flowering plants can produce any viable seeds, making the higher seed set of late-flowering plants irrelevant. Thus, earlier flowering appears to be a specific adaptation to the higher latitudes of northern Europe.
Asunto(s)
Ambrosia/fisiología , Flores/crecimiento & desarrollo , Fotoperiodo , Ambrosia/genética , Europa (Continente) , Flores/genética , Expresión Génica , Aptitud Genética , Especies Introducidas , Reproducción , Estaciones del AñoRESUMEN
A practical and powerful approach for genome editing in plants is delivery of CRISPR reagents via Agrobacterium tumefaciens transformation. The double-strand break (DSB)-inducing enzyme is expressed from a transferred segment of bacterial DNA, the T-DNA, which upon transformation integrates at random locations into the host genome or is captured at the self-inflicted DSB site. To develop efficient strategies for precise genome editing, it is thus important to define the mechanisms that repair CRISPR-induced DSBs, as well as those that govern random and targeted integration of T-DNA. In this study, we present a detailed and comprehensive genetic analysis of Cas9-induced DSB repair and T-DNA capture in the model plant Arabidopsis thaliana. We found that classical nonhomologous end joining (cNHEJ) and polymerase theta-mediated end joining (TMEJ) are both, and in part redundantly, acting on CRISPR-induced DSBs to produce very different mutational outcomes. We used newly developed CISGUIDE technology to establish that 8% of mutant alleles have captured T-DNA at the induced break site. In addition, we find T-DNA shards within genomic DSB repair sites indicative of frequent temporary interactions during TMEJ. Analysis of thousands of plant genome-T-DNA junctions, followed up by genetic dissection, further reveals that TMEJ is responsible for attaching the 3' end of T-DNA to a CRISPR-induced DSB, while the 5' end can be attached via TMEJ as well as cNHEJ. By identifying the mechanisms that act to connect recombinogenic ends of DNA molecules at chromosomal breaks, and quantifying their contributions, our study supports the development of tailor-made strategies toward predictable engineering of crop plants.
RESUMEN
Agrobacterium tumefaciens, a pathogenic bacterium capable of transforming plants through horizontal gene transfer, is nowadays the preferred vector for plant genetic engineering. The vehicle for transfer is the T-strand, a single-stranded DNA molecule bound by the bacterial protein VirD2, which guides the T-DNA into the plant's nucleus where it integrates. How VirD2 is removed from T-DNA, and which mechanism acts to attach the liberated end to the plant genome is currently unknown. Here, using newly developed technology that yields hundreds of T-DNA integrations in somatic tissue of Arabidopsis thaliana, we uncover two redundant mechanisms for the genomic capture of the T-DNA 5' end. Different from capture of the 3' end of the T-DNA, which is the exclusive action of polymerase theta-mediated end joining (TMEJ), 5' attachment is accomplished either by TMEJ or by canonical non-homologous end joining (cNHEJ). We further find that TMEJ needs MRE11, whereas cNHEJ requires TDP2 to remove the 5' end-blocking protein VirD2. As a consequence, T-DNA integration is severely impaired in plants deficient for both MRE11 and TDP2 (or other cNHEJ factors). In support of MRE11 and cNHEJ specifically acting on the 5' end, we demonstrate rescue of the integration defect of double-deficient plants by using T-DNAs that are capable of forming telomeres upon 3' capture. Our study provides a mechanistic model for how Agrobacterium exploits the plant's own DNA repair machineries to transform it.
Asunto(s)
Agrobacterium tumefaciens , Arabidopsis , Agrobacterium tumefaciens/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas Bacterianas/genética , Reparación del ADN por Unión de Extremidades , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Genómica , Plantas/genéticaRESUMEN
BACKGROUND: Stable gene repression is essential for normal growth and development. Polycomb repressive complexes 1 and 2 (PRC1&2) are involved in this process by establishing monoubiquitination of histone 2A (H2Aub1) and subsequent trimethylation of lysine 27 of histone 3 (H3K27me3). Previous work proposed that H2Aub1 removal by the ubiquitin-specific proteases 12 and 13 (UBP12 and UBP13) is part of the repressive PRC1&2 system, but its functional role remains elusive. RESULTS: We show that UBP12 and UBP13 work together with PRC1, PRC2, and EMF1 to repress genes involved in stimulus response. We find that PRC1-mediated H2Aub1 is associated with gene responsiveness, and its repressive function requires PRC2 recruitment. We further show that the requirement of PRC1 for PRC2 recruitment depends on the initial expression status of genes. Lastly, we demonstrate that removal of H2Aub1 by UBP12/13 prevents loss of H3K27me3, consistent with our finding that the H3K27me3 demethylase REF6 is positively associated with H2Aub1. CONCLUSIONS: Our data allow us to propose a model in which deposition of H2Aub1 permits genes to switch between repression and activation by H3K27me3 deposition and removal. Removal of H2Aub1 by UBP12/13 is required to achieve stable PRC2-mediated repression.