High-pressure-sprayed double stranded RNA does not induce RNA interference of a reporter gene
Ontology highlight
ABSTRACT:
ORGANISM(S): Nicotiana benthamiana
PROVIDER: GSE160110 | GEO | 2020/12/01
REPOSITORIES: GEO
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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.
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Project description:Provided data came from a detailed study on Nicotiana benthamiana 16c plants where we use Tobacco Rattle Virus (TRV) as a molecular switch to change the chromatin state of a reporter gene (P35S::GFP) from an actively transcribed to a transcriptionally silenced state. Our approach enables us to interrogate different chromatin states of the same locus with the same set of CRISPR/Cas9 genome editing reagents and systematically describe the effect of chromatin state on the frequency and type of mutations induced at various Cas9 targets in a huge set of independently edited cells.
2022-05-04 | E-MTAB-10256 | biostudies-arrayexpress
Project description:Small RNA (sRNA)-guided RNA silencing is a critical antiviral defense mechanism employed by a variety of eukaryotic organisms. Although the induction of RNA silencing by bipartite and monopartite begomoviruses has been described in plants, the nature of begomovirus/betasatellite complexes remains undefined. We profiled Tomato yellow leaf curl China virus (TYLCCNV) and its associated betasatellite (TYLCCNB)-derived small RNAs (V-sRNAs and S-sRNAs) using Solexa-based deep sequencing to evaluate the role of betasatellites in V-sRNA modulation. Both sense and anti-sense V-sRNAs and S-sRNAs accumulated preferentially as 22 nucleotide species in infected Solanum lycopersicum and Nicotiana benthamiana plants, indicating that secondary siRNAs were triggered. High resolution mapping of V-sRNA and S-sRNA revealed heterogeneous distribution of V-sRNA and S-sRNA sequences across the TYLCCNV and TYLCCNB genomes. In TYLCCNV-infected S. lycopersicum or N. benthamiana and TYLCCNV and betaC1-mutant TYLCCNB co-infected N. benthamiana plants, the primary TYLCCNV targets were AV2 and the 5’ terminus of AV1. In TYLCCNV and betasatellite-infected plants, the number of V-sRNAs targeting this region decreased and the production of V-sRNAs increased corresponding to the overlapping regions of AC2 and AC3, as well as the 3’ terminal of AC1. betaC1 is the primary determinant mediating symptom induction and also the primary silencing target of the TYLCCNB genome even in its mutated form. In addition, the betasatellite affected the amount of V-sRNAs detected in S. lycopersicum and N. benthamiana plants. characterization of Tomato yellow leaf curl China virus and Tomato yellow leaf curl China betasatellite-derived small interfering RNAs from five cDNA libraries of two plant species
2011-08-21 | E-GEOD-26368 | biostudies-arrayexpress
Project description:In plants, RNA interference (RNAi) is an effective defense mechanism against pathogens and pests. RNAi mainly involves the micro RNA and the small interfering RNA (siRNA) pathways. The latter pathway is generally based on the processing of long double stranded RNAs (dsRNA) into siRNAs by DICER-LIKE endonucleases (DCLs). SiRNAs are loaded onto ARGONAUTE proteins to constitute the RNA-induced silencing complex (RISC). Natural dsRNAs derive from transcription of inverted repeats or of specific RNA molecules that are transcribed by RNA-directed RNA polymerase 6 (RDR6). Moreover, replication of infecting viruses/viroids results in the production of dsRNA intermediates that can serve as substrates for DCLs. The high effectiveness of RNAi both locally and systemically implicated that plants could become resistant to pathogens, including viruses, through artificial activation of RNAi by topical exogenous application of dsRNA. The most preferable procedure to exploit RNAi would be to simply spray naked dsRNAs onto mature plants that are specific for the attacking pathogens serving as a substitute for pesticides applications. However, the plant cell wall is a difficult barrier to overcome and only few reports claim that topical application of naked dsRNA triggers RNAi in plants. Using a transgenic Nicotiana benthamiana line, we found that high-pressure-sprayed naked dsRNA did not induce silencing of a green fluorescence protein (GFP) reporter gene. Small RNA sequencing (sRNA-seq) of the samples from dsRNA sprayed leaves revealed that the dsRNA was, if at all, not efficiently processed into siRNAs indicating that the dsRNA was insufficiently taken up by plant cells.
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