Skip to main content
SpringerLink
Log in

In planta silencing of NSs and Hc-Pro through RNAi constructs: to develop durable resistance

  • Original Article
  • Published:
Indian Journal of Plant Physiology Aims and scope Submit manuscript

Abstract

Groundnut bud necrosis disease and Papaya ring spot disease are devastating disease of tomato and papaya/cucumber. In order to develop transgenic resistance against these viruses, gene silencing approach was followed to target the conserved viral sequences for obtaining wide spectrum of resistance. Viral derived small non coding RNA was used to silence viral gene. To have efficient processing of hairpins, microRNA backbone has been used to harbour viral derived siRNA. An artificial microRNA having miRNA159a backbone harbouring 21-nt siRNA sequences from RNAi suppressor (GBNV–NSs and PRSV–Hc-Pro) genes were constructed. To establish the efficacy of these amiRNA constructs, transient expression assay were performed and attenuation of viral symptoms were observed on expression of artificial microRNA construct in infected plants. Our data demonstrate that expression of virus-specific siRNAs using artificial-miRNA is an effective and predictable approach to silence Groundnut bud necrosis virus (GBNV) and Papaya ringspot virus (PRSV).

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Alvarez, J. P., Pekker, I., Goldsmidt, A., Blum, E., Amsellum, Z., & Eshed, Y. (2006). Endogenous and synthetic MicroRNAs stimulate simultaneous, efficient and localized regulation of multiple targets in diverse species. Plant Cell, 18, 1135–1151.

    Article  Google Scholar 

  • Bag, S., Agarwal, S., & Jain, R. K. (2007). Sequence diversity in the coat proteins of Papaya ringspot virus isolates originating from different locations in India. Indian Phyopathology, 602, 244–250.

    Google Scholar 

  • Balol, G., & Patil, M. S. (2014). Biological characterization and detection of Groundnut bud necrosis virus (GBNV) in different parts of tomato. Journal of Pure and Applied Microbiolgy, 8(1), 749–752.

    CAS  Google Scholar 

  • Bartel, D. P. (2004). MicroRNAs: genomics, biogenesis, mechanism, and function. Cell, 116, 281–297.

    Article  CAS  PubMed  Google Scholar 

  • Brummelkamp, T. R., Bernards, R., & Agami, R. (2002). A system for stable expression of short interfering RNAs in mammalian cells. Science, 296, 550–553.

    Article  CAS  PubMed  Google Scholar 

  • Capoor, S. P., & Varma, P. M. (1958). A mosaic disease of papaya in Bombay. Indian Journal of Agricultural Sciences, 29, 225–233.

    Google Scholar 

  • Chapman, E. J., & Carrington, J. C. (2007). Specialization and evolution of endogenous small RNA pathways. Nature Reviews Genetics, 8, 884–896.

    Article  CAS  PubMed  Google Scholar 

  • Dykes, I. M., & Emanueli, C. (2017). Transcriptional and post-transcriptional gene regulation by long non-coding RNA. Genomics Proteomics Bioinformatics, 15, 177–186.

    Article  PubMed  PubMed Central  Google Scholar 

  • Elbashir, S. M., Lendeckel, W., & Tuschl, T. (2001). RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes & Development, 15, 188–200.

    Article  CAS  Google Scholar 

  • Fauquet, C. M., Mayo, M. A., Maniloff, J., Desselberger, U., & Ball, L. A. (2005). Virus taxonomy: Eight report of the international committee on taxonomy of viruses. San Diego: Elsevier Academic Press.

    Google Scholar 

  • Gonsalves, D. (1998). Control of papaya ringspot virus in papaya: A case study. Annual review of Phytopathology, 36, 415–437.

    Article  CAS  PubMed  Google Scholar 

  • Gregory, R. I., Chendrimada, T. P., Cooch, N., & Shiekhattar, R. (2005). Human RISC couples microRNA biogenesis and posttranscriptional gene silencing. Cell, 123, 631–640.

    Article  CAS  PubMed  Google Scholar 

  • Hofgen, R., & Willmitzer, L. (1988). Storage of competent cells for Agrobacterium transformation. Nucleic Acids Research, 16, 9877.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hutvagner, G., & Simard, M. J. (2008). Argonaute proteins: key players in RNA silencing. Nature Reviews Molecular Cell Biology, 9, 22–32.

    Article  CAS  PubMed  Google Scholar 

  • Jain, R. K., Pappu, H. R., Pappu, S. S., Varma, A., & Ram, R. D. (1998). Molecular characterization of Papaya ringspot potyvirus isolates from India. Annals of Applied Biology, 132, 413–425.

    Article  CAS  Google Scholar 

  • Liu, S. R., Zhou, J. J., Hu, C. G., Wei, C. L., & Zhang, J. Z. (2017). MicroRNA-mediated gene silencing in plant defense and viral counter-defense. Frontiers in Microbiology, 8, 1801.

    Article  PubMed  PubMed Central  Google Scholar 

  • Mickiewicz, A., Rybarczyk, A., Sarzynska, J., Figlerowicz, M., & Blazewicz, J. (2016). AmiRNA Designer—new method of artificial miRNA design. Acta Biochimica Polonica, 63(1), 71–77.

    Article  CAS  PubMed  Google Scholar 

  • Nakanishi, K. (2016). Anatomy of RISC: how do small RNAs and chaperones activate Argonaute proteins? WIREs RNA, 7, 637–660. https://doi.org/10.1002/wrna.1356.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Niu, Q. W., Lin, S. S., Reyes, J. L., Chen, K. C., Wu, H. W., Yeh, S. D., et al. (2006). Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance. Nature Biotechnology, 24, 1420–1428.

    Article  CAS  PubMed  Google Scholar 

  • Parizotto, E. A., Dunoyer, P., Rahm, N., Himber, C., & Voinnet, O. (2004). In vivo investigation of the transcription, processing, endonucleolytic activity, and functional relevance of the spatial distribution of a plant miRNA. Genes & Development, 18, 2237–2242.

    Article  CAS  Google Scholar 

  • Pfaffl, M. W. (2001). A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research, 1, 29–45.

    Article  Google Scholar 

  • Pratt, A. J., & MacRae, I. J. (2009). The RNA-induced silencing complex: a versatile gene-silencing machine. Journal of Biological Chemistry, 284(27), 17897–901.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Praveen, S., Ramesh, S. V., Mishra, A. K., Koundal, V., & Palukaitis, P. (2010). Silencing potential of viral derived RNAi constructs in Tomato leaf curl virus-AC4 gene suppression in tomato. Transgenic Research, 19(1), 45–55.

    Article  CAS  PubMed  Google Scholar 

  • Qu, J., Ye, J., & Fang, R. (2007). Artificial MicroRNA-Mediated Virus Resistance in Plants. Journal of Virology, 81, 6690–6699.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Roy, G. G., Jain, R. K., Bhat, A. I., & Varma, A. (1999). Comparative host range and serological studies of papaya ring spot poty virus isolates. Indian Phytopathology, 52, 14–17.

    Google Scholar 

  • Sanford, J. C., & Johnston, S. A. (1985). The concept of parasite-derived resistance-deriving resistance genes from the parasite’s own genome. Journal of Theoretical Biology, 113(2), 395–405.

    Article  Google Scholar 

  • Schwab, R., Ossowski, S., Riester, M., Warthmann, N., & Weigel, D. (2006). Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell, 18, 1121–1133.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shephard, D. N., Martin, D. P., & Thomson, J. A. (2009). Transgenic strategies for developing crop resistant to geminiviruses. Plant Science, 176, 1–11.

    Article  Google Scholar 

  • Unver, T., & Budak, H. (2009). Virus-induced gene silencing, a post transcriptional gene silencing method. International Journal of Plant Genomics, 2009, 8.

    Google Scholar 

  • Vaucheret, H., Varquez, F., Crete, P., & Bartel, D. (2004). The action of ARGONAUTE1 in the miRNA pathway and its regulation by the miRNA pathway are crucial for plant development. Genes & Devlopment, 18, 1187–1197.

    Article  CAS  Google Scholar 

  • Waterhouse, P. M., Graham, M. W., & Wang, M. B. (1998). Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proceedings of the National Academy of Sciences of the United States of America, 96, 13959–13964.

    Article  Google Scholar 

  • Yeh, S. D., Jan, F. J., Chiang, C. H., Doong, T. J., Chen, M. C., Chung, P. H., et al. (1992). Complete nucleotide sequence and genetic organization of Papaya ringspot virus RNA. Journal of General Virology, 73, 2531–2541.

    Article  CAS  PubMed  Google Scholar 

  • Yiu, S. M., Wong, P. W., Lam, T. W., Mui, Y. C., Kung, H. F., Lin, M., et al. (2005). Filtering of ineffective siRNAs and improved siRNA design tool. Bioinformatics, 21, 144–151.

    Article  CAS  PubMed  Google Scholar 

  • Yogindran, S., Ghosh, A., & Rajam, M. V. (2015). Artificial miRNAs for specific gene silencing and engineering virus resistance in plants. Cell Development Biology, 4, e137.

    Google Scholar 

  • Zhang, B., & Wang, Q. (2015). MicroRNA-based biotechnology for plant improvement. Journal of Cellular Physiology, 230(1), 1–15.

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

The financial support received from Indian Agricultural Research Institute (IARI), New Delhi for the facilities and financial support provided.

Author information

Authors and Affiliations

  1. Division of Biochemistry, Indian Agricultural Research Institute, New Delhi, 110012, India

    Suneha Goswami, Ranjeet Ranjan Kumar & Shelly Praveen

  2. Division of Plant Physiology, Indian Agricultural Research Institute, New Delhi, 110012, India

    Viswanathan Chinnusamy

Authors
  1. Suneha Goswami
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ranjeet Ranjan Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Viswanathan Chinnusamy
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Shelly Praveen
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

SG, SP: conceived and designed the experiments. SG RRK: involved in designing of viral derived siRNAs, isolation of miR159a backbone from A. thaliana, construction of plant expression vectors, mobilization of constructs into Agrobacterium tumefaciens, transient expression assay, and virus bioassay. SG RRK VC SP: wrote the paper and edited the manuscript.

Corresponding author

Correspondence to Suneha Goswami.

Ethics declarations

Conflict of interest

The authors declare that they have no competing interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Goswami, S., Kumar, R.R., Chinnusamy, V. et al. In planta silencing of NSs and Hc-Pro through RNAi constructs: to develop durable resistance. Ind J Plant Physiol. 22, 577–586 (2017). https://doi.org/10.1007/s40502-017-0344-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s40502-017-0344-6

Keywords

Search

Navigation