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Design and High-Throughput Generation of Artificial Small RNA Constructs for Plants

  • Alberto CarbonellEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1932)

Abstract

Artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are two classes of 21-nucleotide artificial small RNAs (sRNAs) designed to selectively silence transcripts in plants with high efficacy and specificity. Despite their extensive use during the last decade, methods for designing and generating artificial sRNA constructs have not been optimized for time- and cost-effectiveness and high-throughput applicability since recently. In this chapter, I detail the protocols for both the rationale design and high-throughput generation of plant artificial sRNA constructs using the P-SAMS (“Plant Small RNA Maker Suite”) web tool and a new generation of BsaI/ccdB (B/c) vectors optimized for one-step cloning of artificial sRNA inserts. These protocols allow for the efficient generation of large number of amiRNA and syn-tasiRNA constructs for potent, selective, and specific gene silencing in plants.

Key words

Artificial small RNA amiRNA syn-tasiRNA RNA silencing P-SAMS 

Notes

Acknowledgments

This work was supported by grants BIO2014-54269-R and BIO2017-83184-R from the Ministerio de Economía, Industria y Competitividad (Spain). Alberto Carbonell was the recipient of an Individual Fellowship from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska Curie grant agreement No. 655841, and was selected in the Ramón y Cajal programme (RYC-2017-21648) from Ministerio de Ciencia, Innovación y Universidades (Spain).

References

  1. 1.
    Carbonell A (2017) Artificial small RNA-based strategies for effective and specific gene silencing in plants. In: Dalmay T (ed) Plant gene silencing: mechanisms and applications. CABI Publishing, Boston, MA, pp 110–127CrossRefGoogle Scholar
  2. 2.
    Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D (2006) Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell 18:1121–1133CrossRefGoogle Scholar
  3. 3.
    Montgomery TA, Howell MD, Cuperus JT, Li D, Hansen JE, Alexander AL, Chapman EJ, Fahlgren N, Allen E, Carrington JC (2008) Specificity of ARGONAUTE7-miR390 interaction and dual functionality in TAS3 trans-acting siRNA formation. Cell 133:128–141CrossRefGoogle Scholar
  4. 4.
    Montgomery TA, Yoo SJ, Fahlgren N, Gilbert SD, Howell MD, Sullivan CM, Alexander A, Nguyen G, Allen E, Ahn JH, Carrington JC (2008) AGO1-miR173 complex initiates phased siRNA formation in plants. Proc Natl Acad Sci U S A 105:20055–20062CrossRefGoogle Scholar
  5. 5.
    de la Luz Gutierrez-Nava M, Aukerman MJ, Sakai H, Tingey SV, Williams RW (2008) Artificial trans-acting siRNAs confer consistent and effective gene silencing. Plant Physiol 147:543–551CrossRefGoogle Scholar
  6. 6.
    Carbonell A (2017) Plant ARGONAUTEs: features, functions, and unknowns. Methods Mol Biol 1640:1–21CrossRefGoogle Scholar
  7. 7.
    Carbonell A, Takeda A, Fahlgren N, Johnson SC, Cuperus JT, Carrington JC (2014) New generation of artificial MicroRNA and synthetic trans-acting small interfering RNA vectors for efficient gene silencing in Arabidopsis. Plant Physiol 165:15–29CrossRefGoogle Scholar
  8. 8.
    Carbonell A, Fahlgren N, Mitchell S, Cox KL Jr, Reilly KC, Mockler TC, Carrington JC (2015) Highly specific gene silencing in a monocot species by artificial microRNAs derived from chimeric miRNA precursors. Plant J 82:1061–1075CrossRefGoogle Scholar
  9. 9.
    Fahlgren N, Hill ST, Carrington JC, Carbonell A (2016) P-SAMS: a web site for plant artificial microRNA and synthetic trans-acting small interfering RNA design. Bioinformatics 32:157–158PubMedGoogle Scholar
  10. 10.
    Carbonell A, Daros JA (2017) Artificial microRNAs and synthetic trans-acting small interfering RNAs interfere with viroid infection. Mol Plant Pathol 18:746–753CrossRefGoogle Scholar
  11. 11.
    Carbonell A, Carrington JC, Daros JA (2016) Fast-forward generation of effective artificial small RNAs for enhanced antiviral defense in plants. RNA Dis 3:e1130PubMedPubMedCentralGoogle Scholar
  12. 12.
    Carbonell A, Daros JA (2019) Design, synthesis and functional analysis of highly specific artificial small RNAs with antiviral activity in plants. Methods Mol Biol. (in press)Google Scholar
  13. 13.
    Carbonell A, López C, Daròs JA (2019) Fast-forward identification of highly effective artificial small RNAs against different Tomato spotted wilt virus isolates. Mol. Plant-Microbe Interact. (in press)Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Científicas-Universitat Politècnica de ValènciaValenciaSpain

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