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:To investigate how high temperature affects sRNA production during virus induced gene silencing (VIGS), we sequenced sRNAs from Tobacco Rattle Virus (TRV)-infected Nicotiana benthamiana kept at 29°C at an early (1 week post infection) and a late time point (3 weeks post infection). To compare sRNA production between virus induced transcriptional gene silencing (ViTGS) and virus induced post-translational gene silencing (ViPTGS) at 29°C, the N. benthamiana 16c plants were infected with TGS-inducing viruses (TRV-35S and TRV-35-2M) and PTGS-inducing viruses (TRV-GFP and TRV-GFP-2M). TRV-35S is a recombinant TRV containing a 120 nt segment of the 35S promoter. Its derivative, TRV-35S-2M, carrying single nucleotide substitutions (SNS) at every 10 nt within the 120 nt 35S target segment. Same strategy was used to create recombinant TRV-GFP and TRV-GFP-2M targeting GFP coding sequence. According to SNS content, sRNAs from TRV-35S-2M/TRV-GFP-2M infected plants can be separated to yield primary (containing SNSs) and secondary sRNAs (lacking SNSs). Wild Type TRV was used along as viral infection control. Libraries were indexed during PCR amplification (16 cycles) according to the Illumina protocol. See individual sample information for specific index primers used.
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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.
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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
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