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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]

ABSTRACT: 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.

ORGANISM(S): Nicotiana tabacum

PROVIDER: GSE85818 | GEO | 2019/11/08

REPOSITORIES: GEO

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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 [sRNA-seq]

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.

2019-11-08 | GSE85816 | GEO

Endogenous siRNA and miRNA targets identified by sequencing of the Arabidopsis degradome

Project description:MicroRNAs (miRNAs) regulate the expression of target mRNAs in plants and animals. Plant miRNA targets have been predicted based on their extensive and often conserved complementarity to the miRNAs, as well as from miRNA over-expression experiments; many of these target predictions have been confirmed by isolating the products of miRNA-directed cleavage. Here, we present a transcriptome-wide experimental method, called “degradome sequencing,” to directly detect cleaved miRNA targets without relying on predictions or over-expression. The 5' ends of poly-adenylated, uncapped mRNAs from Arabidopsis were directly sampled resulting in an empirical snapshot of the degradome. miRNA-mediated cleavage products were easily discerned from an extensive background of degraded mRNAs, which collectively covered the majority of the annotated transcriptome. Many previously known Arabidopsis miRNA targets were confirmed and several novel targets were also discovered. Quantification of cleavage fragments revealed that those derived from TAS transcripts, which are unusual in their production of abundant secondary siRNAs, accumulated to very high levels. A subset of secondary siRNAs are also known to direct cleavage of targets in trans; degradome sequencing revealed many cleaved targets of these trans-acting siRNAs (tasiRNAs). This empirical method is broadly applicable to discover and quantify cleaved targets of small RNAs without a priori predictions. Keywords: Degradome sequencing

2008-05-08 | GSE11007 | GEO

Endogenous siRNA and miRNA targets identified by sequencing of the Arabidopsis degradome

2008-05-08 | GSE11007 | GEO

High-pressure sprayed siRNAs influence the efficiency but not the profile of transitive silencing

Project description:In plants, small interfering RNAs (siRNAs) are a quintessential class of RNA interference (RNAi)-inducing molecules produced by the endonucleolytic cleavage of double stranded RNAs (dsRNAs). In order to ensure robust RNAi, siRNAs are amplified through a positive feedback mechanism called transitivity. Transitivity relies on RNA-DIRECTED-RNA POLYMERASE 6 (RDR6)-mediated dsRNA synthesis using siRNA-targeted RNA. The newly synthesized dsRNA is subsequently cleaved into secondary siRNAs by DICER-LIKE (DCL) endonucleases. Just like primary siRNAs, secondary siRNAs are also loaded into ARGONAUTE proteins (AGOs) to form an RNA-induced silencing complex (RISC) reinforcing the cleavage of the target RNA. Although the molecular players underlying transitivity are well established, the mode of action of transitivity remains elusive. In this study, we investigated the influence of primary target sites on transgene silencing and transitivity using the GFP-expressing Nicotiana benthamiana 16C line, high pressure spraying protocol (HPSP), and synthetic 22-nucleotide (nt) long siRNAs. We found that the 22-nt siRNA targeting the 3’ of the GFP transgene was less efficient in inducing silencing when compared to the siRNAs targeting the 5’ and middle region of the GFP. Moreover, sRNA sequencing of locally silenced leaves showed that the amount but not the profile of secondary RNAs is shaped by the occupancy of the primary siRNA triggers on the target RNA. Our findings suggest that RDR6-mediated dsRNA synthesis is not primed by primary siRNAs and that dsRNA synthesis appears to be generally initiated at the 3’ end of the target RNA.

2021-12-01 | GSE189777 | GEO

Tobacco RNA Dependent RNA Polymerase 1 affects the expression of defence related genes in Nicotiana benthamiana during geminivirus infection

Project description:RNA dependent RNA Polymerase 1 of Nicotiana tabacum modulates ToLCV pathogenesis by influencing a number of defence related genes in N. benthamiana plants.

2020-06-03 | GSE144074 | GEO

Degradome sequencing reveals endogenous small RNA targets in rice (Oryza sativa L. ssp. indica)

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

2010-01-01 | E-GEOD-19050 | biostudies-arrayexpress

Degradome sequencing reveals endogenous small RNA targets in rice (Oryza sativa L. ssp. indica)

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.

2010-01-01 | GSE19050 | GEO

Cold stress in 7 Solanaceae species, replicate 1 and 2

Project description:Seven different Solanaceae species, Potato (Solanum tubersosum), Tomato (Lycopersicum esculentum), Eggplant (Solanum melangena), Pepper (Capsicum annuum), Tobacco (Nicotiana tabacum), Petunia and Nicotiana benthamiana were subjected to cold stress. Plants were grown at 25 C for 4-6 weeks after wich cold stress was initiated by exposing the plants to 4 C for 4, 8, 12, 24 and 48 hours. Control samples were isolated from plants just before the cold stress was initated. RNA was isolated using Qiagen RNeasy. Keywords: Direct comparison

2007-09-01 | GSE8203 | GEO

Heat stress in 7 Solanaceae species, replicate 1 and 2

Project description:Seven different Solanaceae species, Potato (Solanum tuberosum), Tomato (Solanum lycopersicum), Eggplant (Solanum melongena), Pepper (Capsicum annuum), Tobacco (Nicotiana tabacum), Petunia and Nicotiana benthamiana were subjected to heat stress. Plants were grown for 4-6 weeks at 25 C after which heat stress was initiated by exposing the plants to 35 C for 6, 12, 24, 48 and 96 hours. Control samples were isolated from plants just before initiating the heat stress. RNA was isolated using Qiagen RNeasy. Keywords: Direct comparison

2007-09-01 | GSE8202 | GEO

The RIG-I like receptor LGP2 inhibits Dicer-dependent processing of long double-stranded RNA and blocks RNA interference in mammalian cells.

Project description:In vertebrates, the presence of viral RNA in the cytosol is sensed by members of the RIG-I like receptor (RLR) family , which signal to induce production of type I interferons (IFN). These key anti-viral cytokines act in a paracrine and autocrine manner to induce hundreds of interferon-stimulated genes (ISGs), whose protein products restrict viral entry, replication and budding. ISGs include the RLRs themselves: RIG-I, MDA5 and the least-studied family member, LGP2. In contrast, the IFN system is absent in plants and invertebrates, which defend themselves from viral intruders using RNA interference (RNAi). In RNAi, the endoribonuclease Dicer cleaves virus-derived double stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that target complementary viral RNA for cleavage. Interestingly, the RNAi machinery is conserved in mammals and we have recently demonstrated that it is able to participate in mammalian antiviral defence in conditions in which the IFN system is suppressed. In contrast, when the IFN system is active, one or more ISGs act to mask or suppress antiviral RNAi. Here, we demonstrate that LGP2 constitutes one of the ISGs that can inhibit antiviral RNAi in mammals. We identify Dicer as an LGP2-associated protein and show that LGP2 inhibits Dicer cleavage of dsRNA into siRNAs both in vitro and in vivo. Further, we show that in cells lacking an IFN response, ectopic expression of LGP2 interferes with RNAi-dependent suppression of gene expression. Thus, the inefficiency of RNAi as a mechanism of antiviral defence in mammalian somatic cells can be in part attributed to Dicer inhibition by LGP2 induced by type I IFNs.

2018-01-17 | PXD008364 | Pride

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