Skip to main content

Advertisement

SpringerLink
Log in

RNAi-Mediated Simultaneous Resistance Against Three RNA Viruses in Potato

  • Original Paper
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

RNA interference (RNAi) technology has been successfully applied in stacking resistance against viruses in numerous crop plants. During RNAi, the production of small interfering RNAs (siRNAs) from template double-standard RNA (dsRNA) derived from expression constructs provides an on-switch for triggering homology-based targeting of cognate viral transcripts, hence generating a pre-programmed immunity in transgenic plants prior to virus infection. In the current study, transgenic potato lines (Solanum tuberosum cv. Desiree) were generated, expressing fused viral coat protein coding sequences from Potato virus X (PVX), Potato virus Y (PVY), and Potato virus S (PVS) as a 600-bp inverted repeat expressed from a constitutive 35S promoter. The expression cassette (designated Ec1/p5941) was designed to generate dsRNAs having a hairpin loop configuration. The transgene insertions were confirmed by glufosinate resistance, gene-specific PCR, and Southern blotting. Regenerated lines were further assayed for resistance to virus inoculation for up to two consecutive crop seasons. Nearly 100% resistance against PVX, PVY, and PVS infection was observed in transgenic lines when compared with untransformed controls, which developed severe viral disease symptoms. These results establish the efficacy of RNAi using the coat protein gene as a potential target for the successful induction of stable antiviral immunity in potatoes.

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.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Jeffries, C., Barker, H., & Khurana, S. (2006). Potato viruses (and viroids) and their management. In S. M. Khurana (Ed.), Handbook of Potato production, improvement and post-harvest management (Gopal J. New York: The Haworth’s Food Products Press.

    Google Scholar 

  2. Chaube, H. S., & Pundhir, V. S. (2005). Crop diseases and their management. New Delhi: PHI Learning Pvt. Ltd.

    Google Scholar 

  3. Hameed, A., Iqbal, Z., Asad, S., & Mansoor, S. (2014). Detection of multiple potato viruses in the field suggests synergistic interactions among potato viruses in Pakistan. The Plant Pathology Journal, 30, 407–415.

    Article  Google Scholar 

  4. Fletcher, J. (2012). A virus survey of New Zealand fresh, process and seed potato crops during 2010–11. New Zealand Plant Protection, 65, 197–203.

    Google Scholar 

  5. Tollenaere, C., Susi, H., & Laine, A. L. (2016). Evolutionary and epidemiological implications of multiple infection in plants. Trends in Plant Science, 21, 80–90.

    Article  CAS  Google Scholar 

  6. Vance, V. B. (1991). Replication of potato virus X RNA is altered in coinfections with potato virus Y. Virology, 182, 486–496.

    Article  CAS  Google Scholar 

  7. Gonzalez-Jara, P., Tenllado, F., Martinez-Garcia, B., Atencio, F., Barajas, D., Vargas, M., et al. (2004). Host-dependent differences during synergistic infection by Potyviruses with Potato virus X. Molecular Plant Pathology, 5, 29–35.

    Article  CAS  Google Scholar 

  8. Syller, J. (2012). Facilitative and antagonistic interactions between plant viruses in mixed infections. Molecular Plant Pathology, 13, 204–216.

    Article  Google Scholar 

  9. Hosseini, A., Massumi, H., Heydarnejad, J., Hosseini Pour, A., & Varsani, A. (2011). Characterisation of potato virus Y isolates from Iran. Virus Genes, 42, 128–140.

    Article  CAS  Google Scholar 

  10. Chung, B. N., Yoon, J. Y., & Palukaitis, P. (2013). Engineered resistance in potato against Potato leafroll virus, Potato virus A and Potato virus Y. Virus Genes, 47, 86–92.

  11. Beachy, R. N., Loesch-Fries, S., & Tumer, N. E. (1990). Coat protein-mediated resistance against virus infection. Annual review of Phytopathology, 28, 451–472.

    Article  CAS  Google Scholar 

  12. Lawson, C., Kaniewski, W., Haley, L., Rozman, R., Newell, C., Sanders, P., et al. (1990). Engineering resistance to mixed virus infection in a commercial potato cultivar: Resistance to Potato virus X and Potato virus Y in transgenic Russet Burbank. Nature Biotechnology, 8, 127–134.

    Article  CAS  Google Scholar 

  13. Wilson, T. (1993). Strategies to protect crop plants against viruses: Pathogen-derived resistance blossoms. Proceedings of the National Academy of Sciences, 90, 3134–3141.

    Article  CAS  Google Scholar 

  14. Goldbach, R., Bucher, E., & Prins, M. (2003). Resistance mechanisms to plant viruses: An overview. Virus Research, 92, 207–212.

    Article  CAS  Google Scholar 

  15. Mansoor, S., Amin, I., Hussain, M., Zafar, Y., & Briddon, R. W. (2006). Engineering novel traits in plants through RNA interference. Trends in Plant Science, 11, 559–565.

    Article  CAS  Google Scholar 

  16. Grishok, A., Pasquinelli, A. E., Conte, D., Li, N., Parrish, S., Ha, I., et al. (2001). Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing. Cell, 106, 23–34.

    Article  CAS  Google Scholar 

  17. Holoch, D., & Moazed, D. (2015). RNA-mediated epigenetic regulation of gene expression. Nature Reviews Genetics, 16, 71–84.

    Article  CAS  Google Scholar 

  18. Ruiz-Ferrer, V., & Voinnet, O. (2009). Roles of plant small RNAs in biotic stress responses. Annual Review of Plant Biology, 60, 485–510.

    Article  CAS  Google Scholar 

  19. Saurabh, S., Vidyarthi, A. S., & Prasad, D. (2014). RNA interference: Concept to reality in crop improvement. Planta, 239, 543–564.

  20. Smith, N. A., Singh, S. P., Wang, M. B., Stoutjesdijk, P. A., Green, A. G., & Waterhouse, P. M. (2000). Total silencing by intron-spliced hairpin RNAs. Nature, 407, 319–320.

    Article  CAS  Google Scholar 

  21. Arif, M., Azhar, U., Arshad, M., Zafar, Y., Mansoor, S., & Asad, S. (2011). Engineering broad-spectrum resistance against RNA viruses in potato. Transgenic Research, 21, 303–311.

    Article  Google Scholar 

  22. Sambrook, J., Frisch, E. F., & Maniatis, T. (1989). Molecular cloning: A laboratory manual. New York: Cold Spring Harbor Laboratory Press.

    Google Scholar 

  23. Van Eck, J., Conlin, B., Garvin, D., Mason, H., Navarre, D., & Brown, C. (2007). Enhancing beta-carotene content in potato by RNAi-mediated silencing of the beta-carotene hydroxylase gene. American Journal of Potato Research, 84, 331–342.

    Article  Google Scholar 

  24. Ausubel, F. M. (2002). Short protocols in molecular biology: A compendium of methods from current protocols in molecular biology. 2. New York: Wiley.

    Google Scholar 

  25. Khatoon, S., Kumar, A., Sarin, N. B., & Khan, J. A. (2016). RNAi-mediated resistance against Cotton leaf curl disease in elite Indian cotton (Gossypium hirsutum) cultivar Narasimha. Virus Genes, 52, 530–537.

  26. Ammara, U., Mansoor, S., Saeed, M., Amin, I., Briddon, R. W., & Al-Sadi, A. M. (2015). RNA interference-based resistance in transgenic tomato plants against Tomato yellow leaf curl virus-Oman (TYLCV-OM) and its associated betasatellite. Virology Journal, 12, 38.

    Article  Google Scholar 

  27. Missiou, A., Kalantidis, K., Boutla, A., Tzortzakaki, S., Tabler, M., & Tsagris, M. (2004). Generation of transgenic potato plants highly resistant to Potato virus Y (PVY) through RNA silencing. Molecular Breeding, 14, 185–197.

    Article  CAS  Google Scholar 

  28. Wesley, S. V., Helliwell, C., Smith, N. A., Wang, M. B., Rouse, D., Liu, Q., et al. (2001). Constructs for efficient, effective and high throughput gene silencing in plants. The Plant Journal, 27, 581–590.

    Article  CAS  Google Scholar 

  29. Rojas, M. R., Zerbini, F. M., Allison, R. F., Gilbertson, R. L., & Lucas, W. J. (1997). Capsid protein and helper component-proteinase function as potyvirus cell-to-cell movement proteins. Virology, 237, 283–295.

    Article  CAS  Google Scholar 

  30. Bendahmane, M., Chen, I., Asurmendi, S., Bazzini, A. A., Szecsi, J., & Beachy, R. N. (2007). Coat protein-mediated resistance to TMV infection of Nicotiana tabacum involves multiple modes of interference by coat protein. Virology, 366, 107–116.

    Article  CAS  Google Scholar 

  31. Seternes, T., Tonheim, T. C., Myhr, A. I., & Dalmo, R. A. (2016). A plant 35S CaMV promoter induces long-term expression of luciferase in Atlantic salmon. Scientific Reports, 6, 25096. doi:10.1038/srep25096.

    Article  CAS  Google Scholar 

  32. Bucher, E., Lohuis, D., Van Poppel, P. M., Geerts-Dimitriadou, C., Goldbach, R., & Prins, M. (2006). Multiple virus resistance at a high frequency using a single transgene construct. Journal of General Virology, 87, 3697–3701.

    Article  CAS  Google Scholar 

  33. Rezk, A., Aboul-Ata, A., & Mazyad, H. (2008). siRNA silencing of PVX coat protein gene affects accumulation of viral RNA in potato and tobacco plants. International Journal of Virology, 4, 14–25.

    Article  Google Scholar 

  34. Casacuberta, J. M., Devos, Y., Du Jardin, P., Ramon, M., Vaucheret, H., & Nogue, F. (2015). Biotechnological uses of RNAi in plants: Risk assessment considerations. Trends in Biotechnology, 33, 145–147.

    Article  CAS  Google Scholar 

  35. Jackson, A. L., & Linsley, P. S. (2010). Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application. Nature Reviews Drug Discovery, 9, 57–67.

    Article  CAS  Google Scholar 

  36. Sharma, V. K., Basu, S., & Chakraborty, S. (2015). RNAi mediated broad-spectrum transgenic resistance in Nicotiana benthamiana to chilli-infecting begomoviruses. Plant Cell Reports, 34, 1389–1399.

    Article  CAS  Google Scholar 

  37. Halpin, C. (2005). Gene stacking in transgenic plants—The challenge for 21st century plant biotechnology. Plant Biotechnology Journal, 3, 141–155.

    Article  CAS  Google Scholar 

  38. Hamilton, A. J., Voinnet, O., Chappell, L., & Baulcombe, D. (2002). Two classes of short interfering RNA in RNA silencing. EMBO Journal, 21, 4671–4679.

    Article  CAS  Google Scholar 

  39. Bazzini, A., Asurmendi, S., Hopp, H., & Beachy, R. (2006). Tobacco mosaic virus (TMV) and Potato virus X (PVX) coat proteins confer heterologous interference to PVX and TMV infection, respectively. Journal of General Virology, 87, 1005–1012.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was funded by Higher Education Commission (HEC), Government of Pakistan, under the indigenous PhD fellowship and research initiative program (IRSIP), and United States Department of Agriculture award 2014-67013-21659 to GJ. The authors would like to thank Ms. Kerry Swartwood for the assistance in tissue culture work conducted at the Boyce Thomason Institute for Plant Research.

Author information

Authors and Affiliations

  1. Molecular Virology and Gene Silencing Group, National Institute for Biotechnology and Genetic Engineering, Faisalabad, 38000, Pakistan

    Amir Hameed, Muhammad Nouman Tahir, Shaheen Asad, Rakhshanda Bilal & Shahid Mansoor

  2. Department of Bioinformatics and Biotechnology, Government College University, Allama Iqbal Road, Faisalabad, Pakistan

    Amir Hameed

  3. Boyce Thompson Institute for Plant Research, 533 Tower Road, Ithaca, NY, 14853, USA

    Joyce Van Eck & Georg Jander

Authors
  1. Amir Hameed
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Muhammad Nouman Tahir
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shaheen Asad
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rakhshanda Bilal
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Joyce Van Eck
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Georg Jander
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Shahid Mansoor
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Shahid Mansoor.

Ethics declarations

Conflict of interest

The authors declare no financial or commercial conflict of interest.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 13 kb)

Supplementary material 2 (DOCX 13 kb)

Supplementary material 3 (DOCX 12 kb)

Supplementary Fig. 1

Resistance analysis of transgenic plants (T0 generation, second-year crop). a Photograph taken 10 days of postemergence of transgenic plants shows healthy appearance with no disease symptoms anywhere. b Untransformed control plant showing viral disease symptoms of leaf thickening and greening at 10 days postemergence. c Transgenic plant showing healthy growth at 30 days postemergence. d Untransformed control plant showing severe viral disease symptoms of leaf curling, shrinking, mosaic and stunted growth at 30 days postemergence. (JPEG 1599 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hameed, A., Tahir, M.N., Asad, S. et al. RNAi-Mediated Simultaneous Resistance Against Three RNA Viruses in Potato. Mol Biotechnol 59, 73–83 (2017). https://doi.org/10.1007/s12033-017-9995-9

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-017-9995-9

Keywords

Search

Navigation