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Assorted Processing of Synthetic Trans-Acting siRNAs and Its Activity in Antiviral Resistance.

Zhao M, San León D, Mesel F, García JA, Simón-Mateo C - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética Molecular de Plantas, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, 28049, Madrid, Spain.

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.

No MeSH data available.


Related in: MedlinePlus

Syn-tasiRNA constructs and miR173 accumulation in N. benthamiana transient assays.A) Diagram of TAS1-derived syn-tasi constructs containing PPV sequences. The tasiRNA-spawning region is indicated by brackets and the length of the PPV regions included in the constructs is shown. The miR173 target site is indicated by a line. B) Agroinfiltration transient assay in N. benthamiana. Syn-tasiRNA constructs were expressed individually or in combination with miR173 precursor. C) Blot assay to assess the accumulation of miR173 in the plant tissue three days after agroinfiltration. Two biological replicates are shown. EtBr-stained 5S rRNA/tRNA is shown.
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pone.0132281.g001: Syn-tasiRNA constructs and miR173 accumulation in N. benthamiana transient assays.A) Diagram of TAS1-derived syn-tasi constructs containing PPV sequences. The tasiRNA-spawning region is indicated by brackets and the length of the PPV regions included in the constructs is shown. The miR173 target site is indicated by a line. B) Agroinfiltration transient assay in N. benthamiana. Syn-tasiRNA constructs were expressed individually or in combination with miR173 precursor. C) Blot assay to assess the accumulation of miR173 in the plant tissue three days after agroinfiltration. Two biological replicates are shown. EtBr-stained 5S rRNA/tRNA is shown.

Mentions: TAS1-like constructs designed to produce siRNAs targeting the different regions of the PPV genome were engineered. We generated two constructs that included the miR173 target site (22 nt) and two upstream nucleotides of TAS1a and c, immediately followed by 126 nt from either the 3’NCR (nt 9529–9654) or the CP coding region (nt 9242–9367) of PPV. These constructs were cloned downstream a 35S promotor into pMDC32 yielding syn-tasiR-3NCR and syn-tasiR-CP (Fig 1A). In order to produce in N. benthamiana the miR173 required to trigger syn-tasiRNA formation from syn-tasiR-3NCR and syn-tasiR-CP, we generated a construct, MIR173, containing the AtmiR173 precursor (509 nt) [29]. The combined expression of either syn-tasiR-3NCR or syn-tasiR-CP and MIR173 is predicted to yield up to six phased syn-tasiRNAs designed to target PPV RNA.


Assorted Processing of Synthetic Trans-Acting siRNAs and Its Activity in Antiviral Resistance.

Zhao M, San León D, Mesel F, García JA, Simón-Mateo C - PLoS ONE (2015)

Syn-tasiRNA constructs and miR173 accumulation in N. benthamiana transient assays.A) Diagram of TAS1-derived syn-tasi constructs containing PPV sequences. The tasiRNA-spawning region is indicated by brackets and the length of the PPV regions included in the constructs is shown. The miR173 target site is indicated by a line. B) Agroinfiltration transient assay in N. benthamiana. Syn-tasiRNA constructs were expressed individually or in combination with miR173 precursor. C) Blot assay to assess the accumulation of miR173 in the plant tissue three days after agroinfiltration. Two biological replicates are shown. EtBr-stained 5S rRNA/tRNA is shown.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4492489&req=5

pone.0132281.g001: Syn-tasiRNA constructs and miR173 accumulation in N. benthamiana transient assays.A) Diagram of TAS1-derived syn-tasi constructs containing PPV sequences. The tasiRNA-spawning region is indicated by brackets and the length of the PPV regions included in the constructs is shown. The miR173 target site is indicated by a line. B) Agroinfiltration transient assay in N. benthamiana. Syn-tasiRNA constructs were expressed individually or in combination with miR173 precursor. C) Blot assay to assess the accumulation of miR173 in the plant tissue three days after agroinfiltration. Two biological replicates are shown. EtBr-stained 5S rRNA/tRNA is shown.
Mentions: TAS1-like constructs designed to produce siRNAs targeting the different regions of the PPV genome were engineered. We generated two constructs that included the miR173 target site (22 nt) and two upstream nucleotides of TAS1a and c, immediately followed by 126 nt from either the 3’NCR (nt 9529–9654) or the CP coding region (nt 9242–9367) of PPV. These constructs were cloned downstream a 35S promotor into pMDC32 yielding syn-tasiR-3NCR and syn-tasiR-CP (Fig 1A). In order to produce in N. benthamiana the miR173 required to trigger syn-tasiRNA formation from syn-tasiR-3NCR and syn-tasiR-CP, we generated a construct, MIR173, containing the AtmiR173 precursor (509 nt) [29]. The combined expression of either syn-tasiR-3NCR or syn-tasiR-CP and MIR173 is predicted to yield up to six phased syn-tasiRNAs designed to target PPV RNA.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética Molecular de Plantas, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, 28049, Madrid, Spain.

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.

No MeSH data available.


Related in: MedlinePlus