RESUMEN
Histone deacetylation regulates gene expression during plant stress responses and is therefore an interesting target for epigenetic manipulation of stress sensitivity in plants. Unfortunately, overexpression of the core enzymes (histone deacetylases [HDACs]) has either been ineffective or has caused pleiotropic morphological abnormalities. In yeast and mammals, HDACs operate within multiprotein complexes. Searching for putative components of plant HDAC complexes, we identified a gene with partial homology to a functionally uncharacterized member of the yeast complex, which we called Histone Deacetylation Complex1 (HDC1). HDC1 is encoded by a single-copy gene in the genomes of model plants and crops and therefore presents an attractive target for biotechnology. Here, we present a functional characterization of HDC1 in Arabidopsis thaliana. We show that HDC1 is a ubiquitously expressed nuclear protein that interacts with at least two deacetylases (HDA6 and HDA19), promotes histone deacetylation, and attenuates derepression of genes under water stress. The fast-growing HDC1-overexpressing plants outperformed wild-type plants not only on well-watered soil but also when water supply was reduced. Our findings identify HDC1 as a rate-limiting component of the histone deacetylation machinery and as an attractive tool for increasing germination rate and biomass production of plants.
Asunto(s)
Ácido Abscísico/farmacología , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Regulación de la Expresión Génica de las Plantas , Arabidopsis/efectos de los fármacos , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Biomasa , Sequías , Flores/efectos de los fármacos , Flores/enzimología , Flores/genética , Flores/fisiología , Expresión Génica , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Modelos Biológicos , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/enzimología , Raíces de Plantas/genética , Raíces de Plantas/fisiología , Brotes de la Planta/efectos de los fármacos , Brotes de la Planta/enzimología , Brotes de la Planta/genética , Brotes de la Planta/fisiología , Plantas Modificadas Genéticamente , Proteínas Recombinantes de Fusión , Plantones/efectos de los fármacos , Plantones/enzimología , Plantones/genética , Plantones/fisiología , Semillas/efectos de los fármacos , Semillas/enzimología , Semillas/genética , Semillas/fisiología , Estrés FisiológicoRESUMEN
This unit describes the use of satellite tobacco mosaic virus (STMV) vectors in combination with native TMV particles for inducing transient gene silencing in tobacco plants. Target gene fragment selection and insertion, virus delivery procedures, and phenotype screening of silenced plants are described in detail. All critical parameters for tobacco plant cultivation, virus infection, and RNA silencing efficiency are discussed.
Asunto(s)
Silenciador del Gen , Técnicas Genéticas , Vectores Genéticos/genética , Nicotiana/virología , Virus Satélite del Mosaico del Tabaco/genética , Marcación de Gen , Virus Helper/genética , Virus Helper/aislamiento & purificación , Fenotipo , Enfermedades de las Plantas/virología , Hojas de la Planta/genética , Hojas de la Planta/virología , Plantas Modificadas Genéticamente/genética , ARN Viral/genética , ARN Viral/aislamiento & purificación , Nicotiana/genética , Nicotiana/crecimiento & desarrollo , Virus del Mosaico del Tabaco/genética , Virus del Mosaico del Tabaco/aislamiento & purificaciónRESUMEN
We developed a novel, two-component transient gene silencing system in which the satellite tobacco mosaic virus (STMV) is used as vector for the delivery of inhibitory RNA into tobacco plants and the tobacco mosaic virus strain U2 (TMV-U2) is used as helper virus for supplying replication and movement proteins in trans. The main advantage of the system is that by uncoupling virus replication components from silencing induction components, the intensity of silencing becomes more pronounced. We call this system satellite virus-induced silencing system (SVISS) and will demonstrate here its robustness, speed and effectiveness. We were able to obtain pronounced and severe knockout phenotypes for a range of targeted endogenous genes belonging to various biochemical pathways and expressed in different plant tissues, such as genes involved in leaf and flower pigmentation, genes for cell wall synthesis in leaf, stem and root tissues or a ubiquitous RNA polymerase gene. By tandem insertion of more than one target gene sequence into the vector, we were able to induce simultaneous knockouts of an endogenous gene and a transgene. SVISS is the first transient gene silencing system for Nicotiana tabacum, which is a genetically well-characterized bridging species for the Solanaceae plant family.
