RESUMEN
In plants, Potato spindle tuber viroid (PSTVd) replication triggers post-transcriptional gene silencing (PTGS) and RNA-directed DNA methylation (RdDM) of homologous RNA and DNA sequences, respectively. PTGS predominantly occurs in the cytoplasm, but nuclear PTGS has been also reported. In this study, we investigated whether the nuclear replicating PSTVd is able to trigger nuclear PTGS. Transgenic tobacco plants carrying cytoplasmic and nuclear PTGS sensor constructs were PSTVd-infected resulting in the generation of abundant PSTVd-derived small interfering RNAs (vd-siRNAs). Northern blot analysis revealed that, in contrast to the cytoplasmic sensor, the nuclear sensor transcript was not targeted for RNA degradation. Bisulfite sequencing analysis showed that the nuclear PTGS sensor transgene was efficiently targeted for RdDM. Our data suggest that PSTVd fails to trigger nuclear PTGS, and that RdDM and nuclear PTGS are not necessarily coupled.
Asunto(s)
Nicotiana/virología , Células Vegetales/virología , Edición de ARN , Precursores del ARN/metabolismo , ARN Interferente Pequeño/biosíntesis , ARN Viral/metabolismo , Secuencia de Bases , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/virología , Citoplasma/genética , Citoplasma/metabolismo , Citoplasma/virología , Metilación de ADN , Intrones , Datos de Secuencia Molecular , Tubérculos de la Planta/virología , Plantas Modificadas Genéticamente/virología , Precursores del ARN/genética , ARN Interferente Pequeño/genética , ARN Viral/genética , Solanum tuberosum/virología , Viroides/genética , Viroides/metabolismo , Replicación Viral/genéticaRESUMEN
In plants, endogenes are less prone to RNA silencing than transgenes. While both can be efficiently targeted by small RNAs for post-transcriptional gene silencing (PTGS), generally only transgene PTGS is accompanied by transitivity, RNA-directed DNA methylation (RdDM) and systemic silencing. In order to investigate whether a transgene could mimick an endogene and thus be less susceptible to RNA silencing, we generated an intron-containing, endogene-resembling GREEN FLUORESCENT PROTEIN (GFP) transgene (GFP(endo)). Upon agroinfiltration of a hairpin GFP (hpF) construct, transgenic Nicotiana benthamiana plants harboring GFP(endo) (Nb-GFP(endo)) were susceptible to local PTGS. Yet, in the local area, PTGS was not accompanied by RdDM of the GFP(endo) coding region. Importantly, hpF-agroinfiltrated Nb-GFP(endo) plants were resistant to systemic silencing. For reasons of comparison, transgenic N. benthamiana plants (Nb-GFP(cDNA)) carrying a GFP cDNA transgene (GFP(cDNA)) were included in the analysis. HpF-agroinfiltrated Nb-GFP(cDNA) plants exhibited local PTGS and RdDM. In addition, systemic silencing was established in Nb-GFP(cDNA) plants. In agreement with previous reports using grafted scions, in systemically silenced tissue, siRNAs mapping to the 3' of GFP were predominantly detectable by Northern blot analysis. Yet, in contrast to other reports, in systemically silenced leaves, PTGS was also accompanied by dense RdDM comprising the entire GFP(cDNA) coding region. Overall, our analysis indicated that cDNA transgenes are prone to systemic PTGS and RdDM, while endogene-resembling ones are resistant to RNA silencing.
Asunto(s)
Metilación de ADN , Nicotiana/genética , Hojas de la Planta/crecimiento & desarrollo , Transgenes , Silenciador del Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Intrones , Hojas de la Planta/genética , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Nicotiana/crecimiento & desarrolloRESUMEN
In plants, transgenes are generally more sensitive against RNA silencing than endogenes are. In this study, we generated a transgene that structurally mimicks an endogene. It is composed of endogenous promoter, 5'-UTR, introns, 3'-UTR and terminator elements. Our data revealed that, in contrast to a conventional transgene, an endogene-resembling transgene was more stably expressed and poorly processed into small RNAs. In addition, although both constructs triggered methylation of homologous DNA sequences at similar levels, the endogene-resembling transgene exhibited significantly delayed onset of local and systemic silencing.
Asunto(s)
Silenciador del Gen , Nicotiana/genética , ARN Interferente Pequeño/genética , Transgenes , Regiones no Traducidas 3' , Regiones no Traducidas 5' , Agrobacterium tumefaciens/genética , Metilación de ADN , Técnicas de Transferencia de Gen , Genes Reporteros , Ingeniería Genética , Proteínas Fluorescentes Verdes , Intrones , Imitación Molecular , Plásmidos , Regiones Promotoras GenéticasRESUMEN
In plants, RNA-directed DNA methylation (RdDM) can target both transgene promoters and coding regions/gene bodies. RdDM leads to methylation of cytosines in all sequence contexts: CG, CHG and CHH. Upon segregation of the RdDM trigger, at least CG methylation can be maintained at promoter regions in the progeny. So far, it is not clear whether coding region methylation can be also maintained. We showed that the body of Potato spindle tuber viroid (PSTVd) transgene constructs became densely de novo methylated at CG, CHG and CHH sites upon PSTVd infection. In this study, we demonstrate that in viroid-free progeny plants, asymmetric CHH and CHG methylation was completely lost. However, symmetric CG methylation was stably maintained for at least two generations. Importantly, the presence of transgene body methylation did not lead to an increase of dimethylation of histone H3 lysine 9 or a decrease of acetylation of H3. Our data supports the view that CG methylation can be maintained not only in promoters but also in the body of transgenes. They further suggest that maintenance of methylation may occur independently of tested chromatin modifications.
Asunto(s)
Citosina/metabolismo , Metilación de ADN , Plantas Modificadas Genéticamente/genética , Acetilación , Islas de CpG , Histonas/genética , Histonas/metabolismo , Lisina/metabolismo , Motivos de Nucleótidos , Sistemas de Lectura Abierta/genética , Virus de Plantas/genética , Plantas Modificadas Genéticamente/metabolismo , Regiones Promotoras Genéticas , Nicotiana/genética , Transgenes/genética , Viroides/genéticaRESUMEN
Ectopically expressed rice yellow mottle virus P1 fusion proteins were found to be cleaved in planta and in Escherichia coli. Cleavage takes place in the absence of bacterial protease activity, indicating that the P1 fusion is autocatalytically processed independently of host factors. N-terminal sequencing of the C-terminal cleavage product of transiently expressed P1/GFP (green fluorescence protein) in Nicotiana benthamiana showed that the cleavage site is located between the first two amino acids (aa) downstream of the P1 sequence. Mutagenesis experiments revealed that a phenylalanine to valine substitution at position 157 of the P1 aa sequence impairs proper cleavage, which is nearly unaffected by replacement of phenylalanine with tyrosine. Deletion of methionine(159) (first GFP aa residue) appeared to not affect P1/GFP cleavage. N-terminal P1-tagging with GFP turned out to impair autocleavage, whereas a small His-tag could not fully prevent cleavage. Additionally, a modified P1/GFP carrying an N-terminal deletion of 81 aa was not cleaved. These findings indicate that this region is involved in the proteolysis mechanism and that large N-terminal fusion partners might affect correct folding of the P1 necessary for self-catalysis.
Asunto(s)
Oryza/virología , Virus de Plantas/genética , Procesamiento Proteico-Postraduccional , Selección Genética , Proteínas Virales/genética , Secuencia de Aminoácidos , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación de la Expresión Génica de las Plantas , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Datos de Secuencia Molecular , Oryza/genética , Oryza/metabolismo , Virus de Plantas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Nicotiana/genética , Nicotiana/metabolismo , Proteínas Virales/metabolismoRESUMEN
So far, conventional hairpin RNA (hpRNA) constructs consisting of an inverted repeat (IR) of target promoters directly introduced into an expression cassette have been used to mediate de novo DNA methylation. Transcripts of such constructs resemble mRNA molecules, and are likely to be exported to the cytoplasm. The presence of hpRNAs in the cytoplasm and the nucleus may account for the simultaneous activation of post-transcriptional gene silencing (PTGS) and RNA-directed DNA methylation (RdDM). We hypothesized that by retaining hpRNAs in the nucleus, efficient induction of only RdDM may be achieved. Thus, we introduced into tobacco a transgene containing an intron into which an IR of a target promoter was inserted. The intronic hpRNA initiated highly specific cis- and trans-methylation, but did not induce PTGS. No spreading of methylation into sequences flanking the region of homology between the hpRNA and the target DNA was detectable. The efficient methylation-directing activity of the intronic hpRNA may indicate a previously unrecognized role of introns, potentially regulating gene expression at the transcriptional level.
Asunto(s)
Metilación de ADN , Secuencias Invertidas Repetidas/fisiología , Nicotiana/genética , ARN de Planta/fisiología , Secuencia de Bases , Proteínas Fluorescentes Verdes/análisis , Intrones/fisiología , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente/metabolismo , Interferencia de ARN , Nicotiana/metabolismo , TransgenesRESUMEN
Because of their highly ordered structure, mature viroid RNA molecules are assumed to be resistant to degradation by RNA interference (RNAi). In this article, we report that transgenic tomato plants expressing a hairpin RNA (hpRNA) construct derived from Potato spindle tuber viroid (PSTVd) sequences exhibit resistance to PSTVd infection. Resistance seems to be correlated with high-level accumulation of hpRNA-derived short interfering RNAs (siRNAs) in the plant. Thus, although small RNAs produced by infecting viroids [small RNAs of PSTVd (srPSTVds)] do not silence viroid RNAs efficiently to prevent their replication, hpRNA-derived siRNAs (hp-siRNAs) appear to effectively target the mature viroid RNA. Genomic mapping of the hp-siRNAs revealed an unequal distribution of 21- and 24-nucleotide siRNAs of both (+)- and (-)-strand polarities along the PSTVd genome. These data suggest that RNAi can be employed to engineer plants for viroid resistance, as has been well established for viruses.