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Characterization of small interfering RNAs derived from the geminivirus/betasatellite complex using deep sequencing

ABSTRACT: 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.

ORGANISM(S): Tomato yellow leaf curl China virus

PROVIDER: GSE26368 | GEO | 2011/08/21

SECONDARY ACCESSION(S): PRJNA136891

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.

Project description:Next-generation Illumina sequencing technology was used to analyze small RNA associated with post-transcriptional gene silencing induced by intron-spliced hairpin RNA (ihpRNA) in Arabidopsis. The experimental induction of RNA silencing in plants often involves expression of transgenes encoding inverted repeat (IR) sequences to produce abundant dsRNAs that are processed into small RNAs (sRNAs). These sRNAs are key mediators of post-transcriptional gene silencing (PTGS) and determine the specificity of the inhibition of gene expression. Despite its broad utility as a research tool, IR-PTGS is only a partially understood mechanism of RNA silencing in plants. We generated four sets of 60 Arabidopsis plants, each containing IR transgenes expressing different configurations of uidA and CHALCONE SYNTHASE (CHS) gene fragments. The levels of PTGS were dependent on the orientation and position of the fragment in the IR construct. To investigate these differences, we characterized the sRNA profiles by Illumina sequencing of seven libraries generated from transgenic families showing different levels of IR-PTGS. Mapping of sRNA sequences to their corresponding transgene-derived and endogenous transcripts identified distinctive patterns of differential sRNA accumulation. Analyses of these patterns and peaks revealed similarities among sRNAs associated with IR-PTGS and endogenous sRNAs linked to uncapped mRNA decay. We also found unexpected associations between sRNA accumulation and the presence of predicted open reading frames in the trigger sequence. Our observations provide new guidelines for designing constructs to increase the efficiency of IR-PTGS. In addition, strong IR-PTGS affected the prevalence of endogenous sRNAs, which has implications for the use of PTGS for experimental or applied purposes. Sequencing of small RNA from Arabidopsis plants transformed with ihpRNA constructs. Seven small RNA libraries were sequenced: Lib 0, made from non-transgenic Arabidopsis plants, and Libs 1-6, made from plants transformed with different configurations of ihpRNA.

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Characterization of small interfering RNAs derived from the geminivirus/betasatellite complex using deep sequencing

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