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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

Antiviral effect of PPV-specific syn-tasiRNA constructs.(A and B) The indicated combinations of MIR173 (miR173), MIR159 (miR159), empty pMDC32 (vector) syn-tasiR-3NCR and syn-tasiR-CP were transiently expressed by agroinfiltration in N. benthamiana plants. Three days post-agroinfiltration plants were inoculated with PPV-GFP and 5 days post-inoculation (dpi) infection foci were observed under a fluorescence microscope. Number of foci/leaves is indicated. (C) Agroinfiltrated leaves harvested at 5 dpi were subjected to immunoblot analysis with anti-CP serum. The membrane stained with Ponceau red is included as loading control.
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pone.0132281.g005: Antiviral effect of PPV-specific syn-tasiRNA constructs.(A and B) The indicated combinations of MIR173 (miR173), MIR159 (miR159), empty pMDC32 (vector) syn-tasiR-3NCR and syn-tasiR-CP were transiently expressed by agroinfiltration in N. benthamiana plants. Three days post-agroinfiltration plants were inoculated with PPV-GFP and 5 days post-inoculation (dpi) infection foci were observed under a fluorescence microscope. Number of foci/leaves is indicated. (C) Agroinfiltrated leaves harvested at 5 dpi were subjected to immunoblot analysis with anti-CP serum. The membrane stained with Ponceau red is included as loading control.

Mentions: The results presented above clearly show that the sRNAs accumulation patterns observed upon expression of syn-tasiR-CP and syn-tasiR-3NCR constructs are significantly different. The sRNAs species observed from the processing of syn-tasiR-CP construct fulfill with the expected ones, that is, seven 21-nt phased sRNAs in register with the predicted miR173-guided cleavage site (Fig 4A). However, this accumulation pattern is not the one favored in the processing of syn-tasiR-3NCR constructs (Fig 5B). Two different patterns can be envisaged in which a 22nt sRNA specie [D1a(+)] is the first observed in register with the predicted miR173-guided cleavage site (Fig 4C). In one pattern, 21-nt sRNAs (species a and aa) in phase with D1a accumulate while in a second pattern D1a species are followed by phased sRNAs (species a and ab) in which species of 21-nt and 22-nt alternate (Fig 4C). These results highlight that, although production of phased siRNAs in register with a miR173 cleavage happens, specific features of the phased series might depend on each particular downstream sequence.


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)

Antiviral effect of PPV-specific syn-tasiRNA constructs.(A and B) The indicated combinations of MIR173 (miR173), MIR159 (miR159), empty pMDC32 (vector) syn-tasiR-3NCR and syn-tasiR-CP were transiently expressed by agroinfiltration in N. benthamiana plants. Three days post-agroinfiltration plants were inoculated with PPV-GFP and 5 days post-inoculation (dpi) infection foci were observed under a fluorescence microscope. Number of foci/leaves is indicated. (C) Agroinfiltrated leaves harvested at 5 dpi were subjected to immunoblot analysis with anti-CP serum. The membrane stained with Ponceau red is included as loading control.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132281.g005: Antiviral effect of PPV-specific syn-tasiRNA constructs.(A and B) The indicated combinations of MIR173 (miR173), MIR159 (miR159), empty pMDC32 (vector) syn-tasiR-3NCR and syn-tasiR-CP were transiently expressed by agroinfiltration in N. benthamiana plants. Three days post-agroinfiltration plants were inoculated with PPV-GFP and 5 days post-inoculation (dpi) infection foci were observed under a fluorescence microscope. Number of foci/leaves is indicated. (C) Agroinfiltrated leaves harvested at 5 dpi were subjected to immunoblot analysis with anti-CP serum. The membrane stained with Ponceau red is included as loading control.
Mentions: The results presented above clearly show that the sRNAs accumulation patterns observed upon expression of syn-tasiR-CP and syn-tasiR-3NCR constructs are significantly different. The sRNAs species observed from the processing of syn-tasiR-CP construct fulfill with the expected ones, that is, seven 21-nt phased sRNAs in register with the predicted miR173-guided cleavage site (Fig 4A). However, this accumulation pattern is not the one favored in the processing of syn-tasiR-3NCR constructs (Fig 5B). Two different patterns can be envisaged in which a 22nt sRNA specie [D1a(+)] is the first observed in register with the predicted miR173-guided cleavage site (Fig 4C). In one pattern, 21-nt sRNAs (species a and aa) in phase with D1a accumulate while in a second pattern D1a species are followed by phased sRNAs (species a and ab) in which species of 21-nt and 22-nt alternate (Fig 4C). These results highlight that, although production of phased siRNAs in register with a miR173 cleavage happens, specific features of the phased series might depend on each particular downstream sequence.

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