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RNA sequencing of plant microRNAs precipitated by the tombusviral suppressor of RNA silencing p19.

ABSTRACT: Viral suppressors of RNA silencing, VSRs, counteract the antiviral RNA silencing of host plants by sequestration of virus-derived siRNAs. A central question concerns whether and how VSRs associate cellular miRNAs and thus modulate plant gene expression during a viral infection. In this study we characterize the binding behaviour of the tombusviral p19 protein to miRNAs by performing an RNA-pull down experiment with bead-associated p19 protein from carnation italian ringspot virus. For this, we used cytoplasmatic extracs of Nicotiana tabacum protoplasts as an RNA source. By applying Next Generation Sequencing (NGS) to the precipitated small RNAs, we were able to identify miRNAs specifically associating with the protein and other that were not efficiently bound by p19.

ORGANISM(S): Nicotiana tabacum

PROVIDER: GSE98956 | GEO | 2018/05/09

REPOSITORIES: GEO

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Project description:RNA silencing is one of the main defense mechanisms employed by plants to fight viruses. In change, viruses have evolved silencing suppressor proteins to neutralize antiviral silencing. Since the endogenous and antiviral functions of RNA silencing pathway rely on common components, it was suggested that viral suppressors interfere with endogenous silencing pathway contributing to viral symptom development. In this work, we aimed to understand the effects of the tombusviral p19 suppressor on endogenous and antiviral silencing during genuine virus infection. We showed that ectopically expressed p19 sequesters endogenous small RNAs (sRNAs) in the absence, but not in the presence of virus infection. Our presented data question the generalized model in which the sequestration of endogenous sRNAs by the viral suppressor contributes to the viral symptom development. We further showed that p19 preferentially binds the perfectly-paired ds-viral small interfering RNAs (vsiRNAs) but does not select based on their sequence or the type of the 5’ nucleotide. Finally, co-immunoprecipitation of sRNAs with AGO1 or AGO2 from virus-infected plants revealed that p19 specifically impairs vsiRNA loading into AGO1 but not AGO2. Our findings, coupled with the fact that p19-expressing wild type Cymbidium ringspot virus (CymRSV) overcomes the Nicotiana benthamiana silencing based defense killing the host, suggest that AGO1 is the main effector of antiviral silencing in this host-virus combination. To further support our hypothesis we investigate whether the ability of p19 to bind endogenous sRNA without virus infection has biological important impact on endogenous pathways and is this reduced if the virus is present. To asses this we made mRNA sequencing from mock inoculated and Cym19stop infected p19syn plants. Cym19stop infected wild type plant was sequenced as a control. The sequencing data results supports our claims. An increase in transcriptional levels were found in case of genes known to be under small RNA regulation in uninfected p19syn plants and expressional levels return to normal Cym19stop p19syn plants.

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RNA sequencing of plant microRNAs precipitated by the tombusviral suppressor of RNA silencing p19.

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