RESUMEN
Small RNAs are key molecules in RNA silencing pathways that exert sequence-specific regulation of gene expression and chromatin modifications in many eukaryotes. In plants, endogenous small RNAs, including microRNAs (miRNAs) and trans-acting small interfering RNAs (tasiRNAs) play an important role in biological processes such as development and stress responses. In addition, viral genome-derived siRNAs are produced during viral infection, and they exhibit anti-viral defense by an RNA silencing pathway. These endogenous and exogenous small RNAs are mainly 21-24 nucleotides in length. Here, we describe a method to identify small RNA sequences from plant tissues. Small RNAs are purified by column fractionation and gel excision from total RNAs. These small RNAs are ligated at both termini to DNA/RNA chimeric adapters and reverse-transcribed to produce cDNAs. By the following PCR amplification, BanI restriction sites are added to cDNAs, which enables directional concatamerization. Concatamerized-fragments are cloned and sequenced. This method could be applied to identify small RNA sequences from many sources, e.g., mutant plants, plants in various stress environments, and virus-infected plants.
Asunto(s)
Clonación Molecular/métodos , MicroARNs/genética , ARN de Planta/química , ARN Interferente Pequeño/genética , Análisis de Secuencia de ARN/métodos , Secuencia de Bases , ADN Complementario , ADN de Plantas , Datos de Secuencia Molecular , PlantasRESUMEN
MicroRNAs (miRNAs) are sequence-specific negative regulators of gene expression generated by DICER-LIKE1 (DCL1) with the assistance of a double-stranded RNA-binding protein, HYPONASTIC LEAVES1 (HYL1), in Arabidopsis. To achieve a better understanding of miRNA biogenesis, we isolated hyl1 suppressors. Our genetic screening identified a novel semidominant mutation in DCL1 (dcl1-13), which causes an amino acid substitution of Glu-395 with Lys in the ATPase/DExH-box RNA helicase domain. This mutation confers significant restoration from the developmental abnormality and a reduction in the level of miRNA in the loss-of-function mutant of HYL1. However, the dcl1-13 single mutant, exhibiting a decreased number of leaves, showed a slight decrease in miRNA accumulation. Thus, the effect of the dcl1-13 mutation is HYL1 dependent: it promotes miRNA processing in the absence of HYL1, but conversely, impairs it in the presence of HYL1. Our results suggest significant roles of the helicase domain of DCL1, which remain unclear to date, possibly in relation with HYL1.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ARN/genética , Ribonucleasa III/genética , Sustitución de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , MicroARNs/metabolismo , Modelos Moleculares , Mutación , Fenotipo , ARN de Planta/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Ribonucleasa III/química , Ribonucleasa III/metabolismoRESUMEN
RNA silencing is a broadly conserved machinery and is involved in many biological events. Small RNAs are key molecules in RNA silencing pathway that guide sequence-specific gene regulations and chromatin modifications. The silencing machinery works as an anti-viral defense in virus-infected plants. It is generally accepted that virus-specific small interfering (si) RNAs bind to the viral genome and trigger its cleavage. Previously, we have cloned and obtained sequences of small RNAs from Arabidopsis thaliana infected or uninfected with crucifer Tobacco mosaic virus. MicroRNAs (miRNAs) accumulated to a higher percentage of total small RNAs in the virus-infected plants. This was partly because the viral replication protein binds to the miRNA/miRNA* duplexes. In the present study, we mapped the sequences of small RNAs other than virus-derived siRNAs to the Arabidopsis genome and assigned each small RNA. It was demonstrated that only miRNAs increased as a result of viral infection. Furthermore, some newly identified miRNAs and miRNA candidates were found from the virus-infected plants despite a limited number of examined sequences. We propose that it is advantageous to use virus-infected plants as a source for cloning and identifying new miRNAs.
Asunto(s)
Arabidopsis/genética , Arabidopsis/virología , MicroARNs/genética , MicroARNs/aislamiento & purificación , Virus del Mosaico del Tabaco/patogenicidad , Secuencia de Bases , Silenciador del Gen , MicroARNs/química , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/virología , ARN de Planta/química , ARN de Planta/genética , ARN de Planta/aislamiento & purificación , Virus del Mosaico del Tabaco/genéticaRESUMEN
The sequence profiles of small interfering RNAs (siRNAs) in Arabidopsis infected with the crucifer tobamovirus tobacco mosaic virus (TMV)-Cg were determined by using a small RNA cloning technique. The majority of TMV-derived siRNAs were 21 nt in length. The size of the most abundant endogenous small RNAs in TMV-infected plants was 21 nt, whilst in mock-inoculated plants, it was 24 nt. Northern blot analysis revealed that some microRNAs (miRNAs) accumulated more in TMV-infected plants than in mock-inoculated plants. The question of whether the TMV-Cg-encoded 126K replication protein, an RNA-silencing suppressor, caused small RNA enrichment was examined. Transient expression of the replication protein did not change the pattern of miRNA processing. However, miRNA, miRNA* (the opposite strand of the miRNA duplex) and hairpin-derived siRNA all co-immunoprecipitated with the replication protein. Gel mobility-shift assays indicated that the replication protein binds small RNA duplexes. These results suggest that the tobamovirus replication protein functions as a silencing suppressor by binding small RNA duplexes, changing the small RNA profile in infected plants.
Asunto(s)
Arabidopsis/metabolismo , Arabidopsis/virología , MicroARNs/metabolismo , ARN de Planta/metabolismo , ARN Interferente Pequeño/metabolismo , Virus del Mosaico del Tabaco/química , Proteínas Virales/metabolismo , Citoplasma/metabolismo , MicroARNs/química , Peso Molecular , Hojas de la Planta/metabolismo , Hojas de la Planta/virología , Unión Proteica , Complejo Silenciador Inducido por ARN/metabolismoRESUMEN
It has been demonstrated that the subcellular location of Tobamovirus movement protein (MP) which was fused with green fluorescent protein (MP:GFP) changed during the infection process. However, the intracellular route through which MP is transported and its biological meaning are still obscure. Treatment with brefeldin A (BFA), which disrupts ER-to-Golgi transport, inhibited the formation of irregularly shaped and filamentous structures of MP. In this condition, MP was still targeted to plasmodesmata in leaf cells. Furthermore, the viral cell-to-cell movement was not inhibited by BFA treatment. These data indicated that the targeting of viral replication complexes (VRCs) to plasmodesmata is mediated by a BFA-insensitive pathway and that the ER-to-Golgi transport pathway is not involved in viral intercellular movement.
Asunto(s)
Antivirales/farmacología , Brefeldino A/farmacología , Nicotiana/metabolismo , Proteínas de Movimiento Viral en Plantas/metabolismo , Tobamovirus/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Locomoción/efectos de los fármacos , Hojas de la Planta/metabolismo , Nicotiana/virología , Tobamovirus/químicaRESUMEN
A method for the numerical assessment of the foliar injury caused by the photochemical oxidant, peroxyacetyl nitrate (PAN), was devised, using three injury indices: fresh weight (FW) loss, decreased photosynthetic pigment content, and increased ion leakage, which can be measured using the same leaves. The injury indices clearly indicated a larger number of PAN-sensitive leaves and a more severe level of injury in the PAN-sensitive variety of Petunia hybrida, White Champion (WHITE), compared to the PAN-tolerant variety, Blue Champion (BLUE). FW and photosynthetic pigment content decreased correlatively in both varieties, but ion leakage increased only in WHITE. Morphological observations revealed that ion leakage started concurrently with the start of plasmolysis-like symptoms at the mesophyll cells of injured WHITE leaves, whereas FW loss corresponded to the shrinkage of cells without loss of their round shape in BLUE leaves. PAN injuries measured by the injury indices were markedly increased in the presence of light, and the morphological changes following PAN exposure were similar to those caused by the superoxide-generating chemical, paraquat. The results suggested that PAN injuries indicated by the three injury indices are all light-dependent, but are caused through several independent mechanisms.