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

  • Imtiaz Ahmad Sajid
  • Bushra TabassumEmail author
  • Iqra Yousaf
  • Anwar Khan
  • Olawale Samuel Adeyinka
  • Naila Shahid
  • Idrees Ahmad Nasir
  • Tayyab Husnain


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.


Agrobacterium-mediated transformation Coat protein Post-transcriptional gene silencing PVX-resistant transgenic potato shRNA transgene 



  1. 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):425–430Google Scholar
  2. 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 18:194–204CrossRefGoogle Scholar
  3. 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 144:39–44CrossRefGoogle Scholar
  4. 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 27:203–210CrossRefGoogle Scholar
  5. Bai Y, Guo Z, Wang X, Bai D, Zhang W (2009) Generation of double-virus-resistant marker-free transgenic potato plants. Prog Nat Sci 19:543–548CrossRefGoogle Scholar
  6. Beachy RN, Loesch-Fries S, Tumer NE (1990) Coat protein-mediated resistance against virus infection. Annu Rev Phytopathol 28:451–472CrossRefGoogle Scholar
  7. Bruening G (1998) Plant gene silencing regularized. Proc Natl Acad Sci 95:13349–13351CrossRefGoogle Scholar
  8. 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 87:3697–3701CrossRefGoogle Scholar
  9. 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 26:459–465CrossRefGoogle Scholar
  10. 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 14:215–226CrossRefGoogle Scholar
  11. Clark MF, Adams A (1977) Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. J Gen Virol 34:475–483CrossRefGoogle Scholar
  12. 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 14:989–994CrossRefGoogle Scholar
  13. Ding S-W, Voinnet O (2007) Antiviral immunity directed by small RNAs. Cell 130:413–426CrossRefGoogle Scholar
  14. 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 82:449–458CrossRefGoogle Scholar
  15. Food and Agriculture Organization of the United Nations (2005). Potato fact sheet.
  16. 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 33:133–140CrossRefGoogle Scholar
  17. 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 87:6311–6315CrossRefGoogle Scholar
  18. 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 59:73–83CrossRefGoogle Scholar
  19. 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 132:850–858CrossRefGoogle Scholar
  20. 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 26:651–659CrossRefGoogle Scholar
  21. 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 YorkGoogle Scholar
  22. 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 15:826–833CrossRefGoogle Scholar
  23. 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 26:1283–1288CrossRefGoogle Scholar
  24. King JC, Slavin JL (2013) White potatoes, human health, and dietary guidance. Adv Nutr 4:393S–401SCrossRefGoogle Scholar
  25. 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: PicornaviridaeGoogle Scholar
  26. 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 4:959–970CrossRefGoogle Scholar
  27. 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 20:500–505CrossRefGoogle Scholar
  28. Lomonossoff GP (1995) Pathogen-derived resistance to plant viruses. Annu Rev Phytopathol 33:323–343CrossRefGoogle Scholar
  29. Love S, Pavek J (2008) Positioning the potato as a primary food source of vitamin C. Am J Potato Res 85:277–285CrossRefGoogle Scholar
  30. 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 14:185–197CrossRefGoogle Scholar
  31. Miyagishi M, Taira K (2002) U6 promoter–driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells. Nat Biotechnol 20:497–500CrossRefGoogle Scholar
  32. 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 154:365–368CrossRefGoogle Scholar
  33. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  34. Paul CP, Good PD, Winer I, Engelke DR (2002) Effective expression of small interfering RNA in human cells. Nat Biotechnol 20:505–508CrossRefGoogle Scholar
  35. 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 13:349–361CrossRefGoogle Scholar
  36. Smith NA, Singh SP, Wang M-B, Stoutjesdijk PA, Green AG, Waterhouse PM (2000) Gene expression: total silencing by intron-spliced hairpin RNAs. Nature 407:319–320CrossRefGoogle Scholar
  37. 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 99:5515–5520CrossRefGoogle Scholar
  38. Qamar N, Khan MA, Rashid A (2003) Screening of potato germplasm against potato virus X (PVX) and potato virus Y (PVY). Pak J Phytopath 15:41–45Google Scholar
  39. 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 86:1–8CrossRefGoogle Scholar
  40. 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 AfricaGoogle Scholar
  41. Tian J, Chen J, Ye X, Chen S (2016) Health benefits of the potato affected by domestic cooking: a review. Food Chem 202:165–175CrossRefGoogle Scholar
  42. Vance VB (1991) Replication of potato virus X RNA is altered in coinfections with potato virus Y. Virology 182:486–494. CrossRefGoogle Scholar
  43. Voinnet O (2005) Induction and suppression of RNA silencing: insights from viral infections. Nat Rev Genet 6:206–220CrossRefGoogle Scholar
  44. 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 1:347–356CrossRefGoogle Scholar
  45. 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 95:13959–13964CrossRefGoogle Scholar
  46. Watson JM, Fusaro AF, Wang M, Waterhouse PM (2005) RNA silencing platforms in plants. FEBS Lett 579:5982–5987CrossRefGoogle Scholar
  47. 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 163:323CrossRefGoogle Scholar
  48. 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 99:6047–6052CrossRefGoogle Scholar
  49. Zhang R, Marshall D, Bryan GJ, Hornyik C (2013) Identification and characterization of miRNA transcriptome in potato by high-throughput sequencing. PLoS One 8:e57233CrossRefGoogle Scholar
  50. Zhu Y, Qian W, Hua J (2010) Temperature modulates plant defense responses through NB-LRR proteins. PLoS Pathog 6:e1000844CrossRefGoogle Scholar

Copyright information

© European Association for Potato Research 2019

Authors and Affiliations

  1. 1.Centre of Excellence in Molecular BiologyUniversity of the PunjabLahorePakistan
  2. 2.Department of MicrobiologyBUITEMSQuettaPakistan

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