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| Status |
Public on Oct 01, 2019 |
| Title |
Highly efficacious antiviral protection of plants by siRNAs identified in vitro |
| Organism |
Nicotiana tabacum |
| Experiment type |
Non-coding RNA profiling by high throughput sequencing
|
| Summary |
In response to a viral infection, the plant’s RNA silencing machinery processes viral RNAs into a huge number of small interfering RNAs (siRNAs). However, very few of these siRNAs actually interfere with viral replication. A reliable approach to define the characteristics underlying the activity of these immunologically effective siRNAs (esiRNAs) has not been available so far.
We developed a novel screening approach that enables a rapid functional identification of antiviral esiRNAs. The approach is essentially based on the use of a cytoplasmic extract from Nicotiana tabacum BY-2 protoplasts (BY-2 lysate, BYL), that shows Dicer-like (DCL) activity and facilitates the assembly of active RNA-induced silencing complexes (RISC) with an in vitro-translated Argonaute (AGO) protein of choice.
We exposed double-stranded (ds) RNA of Tomato bushy stunt virus (TBSV) to the BYL to generate viral siRNAs (DCL assay). Total RNA was isolated from the reactions and DCL-generated TBSV siRNAs were identified by NGS. In another approach, AGO1/RISC- or AGO2/RISC-associated siRNAs were isolated using FLAG-AGO immunoprecipitation (AGO-IP) and analyzed by NGS. Subsequently, the antiviral activity of siRNAs with high affinity to AGO proteins was characterized in vitro and in vivo.
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| Overall design |
siRNA sequence data was obtained from three different DCL assays, two AGO1-IPs and three AGO2-IPs
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| Contributor(s) |
Gago-Zachert S, Schuck J, Weinholdt C, Knoblich M, Pantaleo V, Grosse I, Gursinsky T, Behrens S |
| Citation(s) |
31433052 |
| |
| Submission date |
Oct 23, 2018 |
| Last update date |
Oct 01, 2019 |
| Contact name |
Claus Weinholdt |
| E-mail(s) |
claus.weinholdt@informatik.uni-halle.de
|
| Organization name |
Martin Luther University Halle-Wittenberg
|
| Department |
Institute of Computer Science
|
| Street address |
Von-Seckendorff-Platz 1
|
| City |
Halle |
| ZIP/Postal code |
06120 |
| Country |
Germany |
| |
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| Platforms (1) |
| GPL23484 |
Illumina HiScanSQ (Nicotiana tabacum) |
|
| Samples (8)
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| Relations |
| BioProject |
PRJNA497995 |
| SRA |
SRP166418 |
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