Skip to main content

Advertisement

SpringerLink
Log in

In Vivo Gene Silencing of Potato Virus X by Small Interference RNAs in Transgenic Potato

  • Published:
Potato Research Aims and scope Submit manuscript

Abstract

RNA silencing is an important antiviral mechanism in plants. Small interfering RNAs (siRNA) or short hairpin RNAs (shRNA) are key stimulators for RNA silencing by acting as executors of viral restriction. Here, we have utilized RNAi technology to suppress potato virus X (PVX) in a transgenic potato cultivar. A stable shRNA of 107 bp directed against a conserved region in the coat protein (CP) gene of PVX was designed with stem and loop sequences derived from a microRNA (miR403; an active regulatory miRNA in potato). The shRNA transgene was directionally cloned in a plant binary vector under the influence of the cauliflower mosaic virus 35S (CaMV35S) constitutive promoter. The pre-shRNA construct was introduced into the potato cultivar Sante through Agrobacterium and transgene insertion was confirmed by testing using PCR (polymerase chain reaction). Upon artificial inoculation of transgenic and non-transgenic potato lines with PVX, variable resistance was revealed through qRT-PCR among the transgenic potato lines. Compared to non-transgenic potato plants, the transgenic potato lines—D5, P3, P9, P14, P21, S11 and S21—showed undetectable levels of CP-PVX mRNA. However, the transgenic lines D4 and P16 exhibited 70% and 75%, respectively, reducing mRNA expression of CP-PVX. The transgenic potato lines remained healthy, with no detectable disease symptoms as compared to the control plants which showed chlorosis and mosaic symptoms. Hence, the expression of virus specific shRNAs is a novel, effective and predictable approach to engineer resistance against PVX in transgenic plants.

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

Similar content being viewed by others

References

  • Abbas MF, ud-Din A, Ghani A, Qadir A, Ahmed R (2013) Major potato viruses in potato crop of Pakistan: a brief review. Int J Biol Biotechnol 10(3):425–430

    Google Scholar 

  • Andika IB, Kondo H, Tamada T (2005) Evidence that RNA silencing-mediated resistance to Beet necrotic yellow vein virus is less effective in roots than in leaves. Mol Plant Microbe In 18:194–204

    Article  CAS  Google Scholar 

  • Antignus Y, Vunsh R, Lachman O, Pearlsman M, Maslenin L, Hananya U, Rosner A (2004) Truncated Rep gene originated from Tomato yellow leaf curl virus Israel [Mild] confers strain-specific resistance in transgenic tomato. Ann Appl Biol 144:39–44

    Article  Google Scholar 

  • Aslam U, Tabassum B, Nasir IA, Khan A, Husnain T (2018) A virus-derived short hairpin RNA confers resistance against sugarcane mosaic virus in transgenic sugarcane. Transgenic Res 27:203–210

    Article  CAS  PubMed  Google Scholar 

  • Bai Y, Guo Z, Wang X, Bai D, Zhang W (2009) Generation of double-virus-resistant marker-free transgenic potato plants. Prog Nat Sci 19:543–548

    Article  CAS  Google Scholar 

  • Beachy RN, Loesch-Fries S, Tumer NE (1990) Coat protein-mediated resistance against virus infection. Annu Rev Phytopathol 28:451–472

    Article  CAS  Google Scholar 

  • Bruening G (1998) Plant gene silencing regularized. Proc Natl Acad Sci 95:13349–13351

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bucher E, Lohuis D, van Poppel PM, Geerts-Dimitriadou C, Goldbach R, Prins M (2006) Multiple virus resistance at a high frequency using a single transgene construct. J Gen Virol 87:3697–3701

    Article  CAS  PubMed  Google Scholar 

  • Bukovinszki Á, Divéki Z, Csányi M, Palkovics L, Balázs E (2007) Engineering resistance to PVY in different potato cultivars in a marker-free transformation system using a ‘shooter mutant’ A. tumefaciens. Plant Cell Rep 26:459–465

    Article  CAS  PubMed  Google Scholar 

  • Chen Y-K, Lohuis D, Goldbach R, Prins M (2004) High frequency induction of RNA-mediated resistance against Cucumber mosaic virus using inverted repeat constructs. Mol Breed 14:215–226

    Article  Google Scholar 

  • Clark MF, Adams A (1977) Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J Gen Virol 34:475–483

    Article  CAS  PubMed  Google Scholar 

  • Di Nicola-Negri E, Brunetti A, Tavazza M, Ilardi V (2005) Hairpin RNA-mediated silencing of Plum pox virus P1 and HC-Pro genes for efficient and predictable resistance to the virus. Transgenic Res 14:989–994

    Article  PubMed  CAS  Google Scholar 

  • Ding S-W, Voinnet O (2007) Antiviral immunity directed by small RNAs. Cell 130:413–426

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fedorkin O, Solovyev A, Yelina N, Zamyatnin A Jr, Zinovkin R, Mäkinen K, Schiemann J, Morozov SY (2001) Cell-to-cell movement of potato virus X involves distinct functions of the coat protein. J Gen Virol 82:449–458

    Article  CAS  PubMed  Google Scholar 

  • Food and Agriculture Organization of the United Nations (2005). Potato fact sheet. http://www.fao.org/3/i1127e/i1127e03.pdf

  • Gargouri-Bouzid R, Jaoua L, Rouis S, Saïdi MN, Bouaziz D, Ellouz R (2006) PVY-resistant transgenic potato plants expressing an anti-NIa protein scFv antibody. Mol Biotechnol 33:133–140

    Article  CAS  PubMed  Google Scholar 

  • Golemboski DB, Lomonossoff GP, Zaitlin M (1990) Plants transformed with a tobacco mosaic virus nonstructural gene sequence are resistant to the virus. Proc Natl Acad Sci 87:6311–6315

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hameed A, Tahir MN, Asad S, Bilal R, Eck JV, Jander G, Mansoor S (2017) RNAi-mediated simultaneous resistance against three RNA viruses in potato. Mol Biotechnol 59:73–83

    Article  CAS  PubMed  Google Scholar 

  • Hily J-M, Ravelonandro M, Damsteegt V, Bassett C, Petri C, Liu Z, Scorza R (2007) Plum pox virus coat protein gene Intron-hairpin-RNA (ihpRNA) constructs provide resistance to plum pox virus in Nicotiana benthamiana and Prunus domestica. J Am Soc Hortic Sci 132:850–858

    Article  CAS  Google Scholar 

  • Hirai S, Oka S-i, Adachi E, Kodama H (2007) The effects of spacer sequences on silencing efficiency of plant RNAi vectors. Plant Cell Rep 26:651–659

    Article  CAS  PubMed  Google Scholar 

  • Jeffries C, Barker H, Khurana S (2005) Potato viruses (and viroids) and their management. Potato production, improvement and post-harvest management. The Haworth’s Food Products Press, New York

  • Kalantidis K, Psaradakis S, Tabler M, Tsagris M (2002) The occurrence of CMV-specific short RNAs in transgenic tobacco expressing virus-derived double-stranded RNA is indicative of resistance to the virus. Mol Plant Microbe In 15:826–833

    Article  CAS  Google Scholar 

  • Kamachi S, Mochizuki A, Nishiguchi M, Tabei Y (2007) Transgenic Nicotiana benthamiana plants resistant to cucumber green mottle mosaic virus based on RNA silencing. Plant Cell Rep 26:1283–1288

    Article  CAS  PubMed  Google Scholar 

  • King JC, Slavin JL (2013) White potatoes, human health, and dietary guidance. Adv Nutr 4:393S–401S

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Knowles N, Hovi T, Hyypiä T, King A, Lindberg AM, Pallansch M, Palmenberg A, Simmonds P, Skern T, Stanway G (2012) Virus taxonomy, Ninth Report of the International Committee on Taxonomy of Viruses: Picornaviridae

  • Lapidot M, Gafny R, Ding B, Wolf S, Lucas WJ, Beachy RN (1993) A dysfunctional movement protein of tobacco mosaic virus that partially modifies the plasmodesmata and limits virus spread in transgenic plants. Plant J 4:959–970

    Article  CAS  Google Scholar 

  • Lee NS, Dohjima T, Bauer G, Li H, Li M-J, Ehsani A, Salvaterra P, Rossi J (2002) Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat Biotechnol 20:500–505

    Article  CAS  PubMed  Google Scholar 

  • Lomonossoff GP (1995) Pathogen-derived resistance to plant viruses. Annu Rev Phytopathol 33:323–343

    Article  CAS  PubMed  Google Scholar 

  • Love S, Pavek J (2008) Positioning the potato as a primary food source of vitamin C. Am J Potato Res 85:277–285

    Article  CAS  Google Scholar 

  • 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. Mol Breed 14:185–197

    Article  CAS  Google Scholar 

  • Miyagishi M, Taira K (2002) U6 promoter–driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells. Nat Biotechnol 20:497–500

    Article  CAS  PubMed  Google Scholar 

  • Mubin M, Briddon R, Mansoor S (2009) Complete nucleotide sequence of chili leaf curl virus and its associated satellites naturally infecting potato in Pakistan. Arch Virol 154:365–368

    Article  CAS  PubMed  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Paul CP, Good PD, Winer I, Engelke DR (2002) Effective expression of small interfering RNA in human cells. Nat Biotechnol 20:505–508

    Article  CAS  PubMed  Google Scholar 

  • Samac DA, Tesfaye M, Dornbusch M, Saruul P, Temple SJ (2004) A comparison of constitutive promoters for expression of transgenes in alfalfa (Medicago sativa). Transgenic Res 13:349–361

    Article  CAS  PubMed  Google Scholar 

  • Smith NA, Singh SP, Wang M-B, Stoutjesdijk PA, Green AG, Waterhouse PM (2000) Gene expression: total silencing by intron-spliced hairpin RNAs. Nature 407:319–320

    Article  CAS  PubMed  Google Scholar 

  • Sui G, Soohoo C, Affar EB, Gay F, Shi Y, Forrester WC, Shi Y (2002) A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci 99:5515–5520

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Qamar N, Khan MA, Rashid A (2003) Screening of potato germplasm against potato virus X (PVX) and potato virus Y (PVY). Pak J Phytopath 15:41–45

    Google Scholar 

  • Tabassum B, Nasir IA, Khan A, Aslam U, Tariq M, Shahid N, Husnain T (2016) Short hairpin RNA engineering: in planta gene silencing of potato virus Y. Crop Prot 86:1–8

    Article  CAS  Google Scholar 

  • Taylor SH BL, Rey MEC (2004) The development of SACMV resistant cassava lines using an antisense RNA. Paper presented at the 4th International geminivirus symposium and 2nd International ssDNA comparative virology workshop, Cape Town, South Africa

  • Tian J, Chen J, Ye X, Chen S (2016) Health benefits of the potato affected by domestic cooking: a review. Food Chem 202:165–175

    Article  CAS  PubMed  Google Scholar 

  • Vance VB (1991) Replication of potato virus X RNA is altered in coinfections with potato virus Y. Virology 182:486–494. https://doi.org/10.1016/0042-6822(91)90589-4

    Article  CAS  PubMed  Google Scholar 

  • Voinnet O (2005) Induction and suppression of RNA silencing: insights from viral infections. Nat Rev Genet 6:206–220

    Article  CAS  PubMed  Google Scholar 

  • Wang MB, Abbott DC, Waterhouse PM (2000) A single copy of a virus-derived transgene encoding hairpin RNA gives immunity to barley yellow dwarf virus. Mol Plant Pathol 1:347–356

    Article  CAS  PubMed  Google Scholar 

  • Waterhouse PM, Graham MW, Wang M-B (1998) Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc Natl Acad Sci 95:13959–13964

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Watson JM, Fusaro AF, Wang M, Waterhouse PM (2005) RNA silencing platforms in plants. FEBS Lett 579:5982–5987

    Article  CAS  PubMed  Google Scholar 

  • Wolfe M, Baresel J, Desclaux D, Goldringer I, Hoad S, Kovacs G, Löschenberger F, Miedaner T, Østergård H, Van Bueren EL (2008) Developments in breeding cereals for organic agriculture. Euphytica 163:323

    Article  Google Scholar 

  • Yu J-Y, DeRuiter SL, Turner DL (2002) RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci 99:6047–6052

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang R, Marshall D, Bryan GJ, Hornyik C (2013) Identification and characterization of miRNA transcriptome in potato by high-throughput sequencing. PLoS One 8:e57233

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhu Y, Qian W, Hua J (2010) Temperature modulates plant defense responses through NB-LRR proteins. PLoS Pathog 6:e1000844

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Centre of Excellence in Molecular Biology, University of the Punjab, Lahore, Pakistan

    Imtiaz Ahmad Sajid, Bushra Tabassum, Iqra Yousaf, Olawale Samuel Adeyinka, Naila Shahid, Idrees Ahmad Nasir & Tayyab Husnain

  2. Department of Microbiology, BUITEMS, Quetta, Pakistan

    Anwar Khan

Authors
  1. Imtiaz Ahmad Sajid
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bushra Tabassum
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Iqra Yousaf
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Anwar Khan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Olawale Samuel Adeyinka
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Naila Shahid
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Idrees Ahmad Nasir
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Tayyab Husnain
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Bushra Tabassum.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sajid, I.A., Tabassum, B., Yousaf, I. et al. In Vivo Gene Silencing of Potato Virus X by Small Interference RNAs in Transgenic Potato. Potato Res. 63, 143–155 (2020). https://doi.org/10.1007/s11540-019-09433-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11540-019-09433-0

Keywords

Search

Navigation