Nicotiana tabacum
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA339477 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR4044040.fastq.gz | Fastqsanger.gz | |||
| SRR4044039.fastq.gz | Fastqsanger.gz |
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Project description:RNA interference (RNAi) is a conserved, RNA-mediated, regulatory mechanism in eukaryotes. In plants, it plays an important role in growth, development and resistance against viral infections. As a counter-defence, plant viruses, e.g. geminiviruses, encode RNAi suppressors, such as AC2, AC4 and AV2. To obtain Nicotiana tabacum virus resistant plants against Tomato leaf curl New Delhi virus (ToLCNDV), we employ the biogenesis pathway of a class of endogenous siRNAs, the trans-acting siRNAs (ta-siRNAs), by engineering artificial ta-siRNAs (ata-siRNAs) targeting the AC2 (TRiV-AC2) and AC4 (TRiV-AC4) RNAi suppressors using miRNA390 dual target sites. The mode of action of ta-siRNAs comprises of the cleavage of the target (similar to the miRNA targeting). Using degradome approaches, the abundance of the resulting 3' fragment of the cleaved transcript can be quantified and the precise localization of the cleavage on the target mRNA can be identified. We sequenced degradome libraries of Nicotiana tabacum plants infected with ToLCNDV which were treated with the ata-siRNA-AC2 construct; mock-treated plants were used as controls. Following quality checks, the abundance distributions of the degradation fragments were normalized. The transcripts with different cleavage patterns was the AC2, supporting the conclusion that an efficient cleavage of the target occurred, without significant off-target effects.
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Project description:RNA interference (RNAi) is a conserved, RNA-mediated, regulatory mechanism in eukaryotes. In plants, it plays an important role in growth, development and resistance against viral infections. As a counter-defence, plant viruses, e.g. geminiviruses, encode RNAi suppressors, such as AC2, AC4 and AV2. To obtain Nicotiana tabacum virus resistant plants against Tomato leaf curl New Delhi virus (ToLCNDV), we employ the biogenesis pathway of a class of endogenous siRNAs, the trans-acting siRNAs (ta-siRNAs), by engineering artificial ta-siRNAs (ata-siRNAs) targeting the AC2 (TRiV-AC2) and AC4 (TRiV-AC4) RNAi suppressors using miRNA390 dual target sites. The mode of action of ta-siRNAs comprises of the cleavage of the target (similar to the miRNA targeting). Using degradome approaches, the abundance of the resulting 3' fragment of the cleaved transcript can be quantified and the precise localization of the cleavage on the target mRNA can be identified. We sequenced degradome libraries of Nicotiana tabacum plants infected with ToLCNDV which were treated with the ata-siRNA-AC2 construct; mock-treated plants were used as controls. Following quality checks, the abundance distributions of the degradation fragments were normalized. The transcripts with different cleavage patterns was the AC2, supporting the conclusion that an efficient cleavage of the target occurred, without significant off-target effects.
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Project description:Nicotiana tabacum degradome analysis using high definition adapters proves an efficient targeting method against the Tomato leaf curl New Delhi virus using artificial ta-siRNAs [PARE]
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Project description:Nicotiana tabacum degradome analysis using high definition adapters proves an efficient targeting method against the Tomato leaf curl New Delhi virus using artificial ta-siRNAs
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Project description:Transcriptome analysis of Nicotiana tabacum infected by Tomato leaf curl Gujarat virus
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Project description:Small RNAs (21-24 nt) are pivotal regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in diverse eukaryotes, including most if not all plants. MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are the two major types, both of which have a demonstrated and important role in plant development, stress responses and pathogen resistance. In this work, we used a deep sequencing approach (Sequencing-By-Synthesis, or SBS) to develop sequence resources of small RNAs from Nicotiana tabacum tissues (including leaves, flowers and pods). The high depth of the resulting datasets enabled us to examine in detail critical small RNA features as size distribution, tissue-specific regulation and sequence conservation between different organs in this species. We also developed database resources and a dedicated website (http://smallrna.udel.edu/) with computational tools for allowing other users to identify new miRNAs or siRNAs involved in specific regulatory pathways, verify the degree of conservation of these sequences in other plant species and map small RNAs on genes or larger regions of the maize genome under study. Small RNA libraries were derived from leaves, flowers and pods of Nicotiana tabacum. Total RNA was isolated using the TriReagent (Molecular Research Center) for leaves and flowers, and the Plant RNA Purification Reagent (Invitrogen) for pods, and submitted to Illumina (Hayward, CA, http://www.illumina.com) for small RNA library construction using approaches described in (Lu et al., 2007) with minor modifications. The small RNA libraries were sequenced with the Sequencing-By-Synthesis (SBS) technology by Illumina. PERL scripts were designed to remove the adapter sequences and determine the abundance of each distinct small RNA. We thank Barbara Baker for providing the plant material as well as Kan Nobuta and Gayathri Mahalingam for assistance with the computational methods.
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Project description:MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) regulate gene expression in eukaryotes. Plant miRNAs modulate their targets mainly via messenger RNA (mRNA) cleavage. Small RNA targets have been extensively investigated in Arabidopsis using computational prediction, experimental validation, and degradome sequencing. However, small RNA targets are largely unknown in rice (Oryza sativa). Here, we report global identification of small RNA targets using high throughput degradome sequencing in the rice indica cultivar 93-11 (Oryza sativa L. ssp. indica). 177 transcripts targeted by total of 87 unique miRNAs were identified. Of targets for the conserved miRNAs between Arabidopsis and rice, transcription factors comprise around 70% (58 in 82), indicating that these miRNAs act as masters of gene regulatory nodes in rice. In contrast, non-conserved miRNAs targeted diverse genes which provide more complex regulatory networks. In addition, 5 AUXIN RESPONSE FACTORS (ARF) cleaved by the TAS3 derived ta-siRNAs were also detected. A total of 40 sRNA targets were further validated via RNA ligase-mediated 5M-bM-^@M-^Y rapid amplification of cDNA ends (RLM 5M-bM-^@M-^Y-RACE). Our degradome results present a detailed sRNA-target interaction atlas, which provides a guide for the study of the roles of sRNAs and their targets in rice. The degradome sequence of Young inflorescences from Oryza sativa L. ssp. indica (93-11) was sequenced
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Project description:MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) regulate gene expression in eukaryotes. Plant miRNAs modulate their targets mainly via messenger RNA (mRNA) cleavage. Small RNA targets have been extensively investigated in Arabidopsis using computational prediction, experimental validation, and degradome sequencing. However, small RNA targets are largely unknown in rice (Oryza sativa). Here, we report global identification of small RNA targets using high throughput degradome sequencing in the rice indica cultivar 93-11 (Oryza sativa L. ssp. indica). 177 transcripts targeted by total of 87 unique miRNAs were identified. Of targets for the conserved miRNAs between Arabidopsis and rice, transcription factors comprise around 70% (58 in 82), indicating that these miRNAs act as masters of gene regulatory nodes in rice. In contrast, non-conserved miRNAs targeted diverse genes which provide more complex regulatory networks. In addition, 5 AUXIN RESPONSE FACTORS (ARF) cleaved by the TAS3 derived ta-siRNAs were also detected. A total of 40 sRNA targets were further validated via RNA ligase-mediated 5’ rapid amplification of cDNA ends (RLM 5’-RACE). Our degradome results present a detailed sRNA-target interaction atlas, which provides a guide for the study of the roles of sRNAs and their targets in rice.
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