Abstract
The use of syn-tasiRNAs has been proposed as an RNA interference technique alternative to those previously described: hairpin based, virus induced gene silencing or artificial miRNAs. In this study we engineered the TAS1c locus to impair Plum pox virus (PPV) infection by replacing the five native siRNAs with two 210-bp fragments from the CP and the 3´NCR regions of the PPV genome. Deep sequencing analysis of the small RNA species produced by both constructs in planta has shown that phased processing of the syn-tasiRNAs is construct-specific. While in syn-tasiR-CP construct the processing was as predicted 21-nt phased in register with miR173-guided cleavage, the processing of syn-tasiR-3NCR is far from what was expected. A 22-nt species from the miR173-guided cleavage was a guide of two series of phased small RNAs, one of them in an exact 21-nt register, and the other one in a mixed of 21-/22-nt frame. In addition, both constructs produced abundant PPV-derived small RNAs in the absence of miR173 as a consequence of a strong sense post-transcriptional gene silencing induction. The antiviral effect of both constructs was also evaluated in the presence or absence of miR173 and showed that the impairment of PPV infection was not significantly higher when miR173 was present. The results show that syn-tasiRNAs processing depends on construct-specific factors that should be further studied before the so-called MIGS (miRNA-induced gene silencing) technology can be used reliably.
Highlights
The term RNA silencing describes an ensemble of regulatory pathways that share the key role played by diverse sets of endogenous small RNAs of 21–24 nt in length [1]
In order to gain deeper information on how a miR173 target site contributes to downstream production of phased small interfering RNAs (siRNAs), we checked by high throughput sequencing the production of small RNAs from syn-tasiR-3NCR or syn-tasiR-capsid protein (CP) transiently expressed by agroinfiltration in N. benthamiana leaves
In this paper we present evidence showing that phased processing of pre-syntasi RNAs can be more complex and construct-specific that previously speculated, and that syn-tasi-RNA technology might be not very advantageous when compared with other RNA silencing-based approaches
Summary
The term RNA silencing describes an ensemble of regulatory pathways that share the key role played by diverse sets of endogenous small RNAs (sRNAs) of 21–24 nt in length [1] These sRNAs are initially produced by Dicer-like (DCL) endonucleases that process double-stranded RNA precursors [2]. Primary products of some miRNA- and siRNA-directed cleavages serve as templates for RNA-dependent RNA polymerases (RDR), to generate double stranded RNA, which is processed into a second wave of siRNAs [7,8,9] These so-called secondary siRNAs amplify and reinforce RNA silencing, and virus-derived secondary siRNAs have been shown to play a main role in antiviral immunity [10]. We used a transient agro-infiltration assay in Nicotiana benthamiana and Illumina deep-sequencing analysis, to assess the small RNA accumulation and the susceptibility to PPV infection of plants expressing the PPV-targeted syn-tasiRNA constructs alone or co-expressed with A. thaliana miR173 precursor
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