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Comparative evaluation of resistance to potato virus Y (PVY) in three different RNAi-based transgenic potato plants

Abstract

Small interfering RNAs (siRNAs) produced from template double-stranded RNAs (dsRNAs) can activate the immune system in transgenic plants by detecting virus transcripts to degrade. In the present study, an RNA interference (RNAi) gene silencing mechanism was used for the development of transgenic potato plants resistant to potato virus Y (PVY), the most harmful viral disease. Three RNAi gene constructs were designed based on the coat protein (CP) and the untranslated region parts of the PVY genome, being highly conserved among all strains of the PVY viruses. Transgenic potato plants were generated using Agrobacterium containing pCAMRNAiCP, pCAMRNAiUR, and pCAMRNAiCP-UR constructs. The transgene insertions were confirmed by molecular analysis containing polymerase chain reaction (PCR) and southern blotting. The resistance of transgenic plants to PVY virus was determined using bioassay and evaluating the amount of viral RNA in plants by RT-PCR, dot blotting of PVY coating protein, and enzyme-linked immunosorbent assay (ELISA). Bioassay analysis revealed that more than 67% of transgenic potato plants were resistant to PVY compared with the non-transgenic plants, which showed viral disease symptoms. No phenotypic abnormalities were observed in transgenic plants. Out of six lines in southern blot analysis, four lines had one copy of the transgene and two lines had two copies of the target genes. No correlation was detected between the copy number of the genes and the resistance level of the plant to PVY. Transgenic lines obtained from all three constructs indicated more or less similar levels of resistance against viral infection; however, CP-UR lines exhibited relatively high resistance followed by CP and UR expressing lines, respectively. Meanwhile, some lines showed a delay in symptoms 35 days after infection which were classified as susceptible.

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References

  • Ammara U, Mansoor S, Saeed M, Amin I, Briddon RW, Al-Sadi AM (2015) RNA interference-based resistance in transgenic tomato plants against Tomato yellow leaf curl virus-Oman (TYLCV-OM) and its associated betasatellite. Virol J 12:38

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  • Arif M, Azhar U, Arshad M, Zafar Y, Mansoor S, Asad S (2012) Engineering broad-spectrum resistance against RNA viruses in potato. Transgenic Res 21:303–311

    CAS  PubMed  Article  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

    CAS  Article  Google Scholar 

  • 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. J Gen Virol 87:1005–1012

    CAS  PubMed  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  • Betts S et al (2019) Uniform expression and relatively small position effects characterize sister transformants in maize and soybean. Front Plant Sci 10:1209

    PubMed  PubMed Central  Article  Google Scholar 

  • Casacuberta JM, Devos Y, Du Jardin P, Ramon M, Vaucheret H, Nogue F (2015) Biotechnological uses of RNAi in plants: risk assessment considerations. Trends Biotechnol 33:145–147

    CAS  PubMed  Article  Google Scholar 

  • Chen X, Liu J, Xu L, Jiang F, Xie X, Zhu C, Wen F (2010) Inhibiting virus infection by RNA interference of the eight functional genes of the potato virus Y genome. J Phytopathol 158:776–784

    CAS  Article  Google Scholar 

  • Chung BN, 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

    CAS  PubMed  Article  Google Scholar 

  • Cillo F, Palukaitis P (2014) Transgenic resistance. In: Adv Virus Res, vol 90. Elsevier, pp 35–146

  • Dalakouras A, Wassenegger M, Dadami E, Ganopoulos I, Pappas ML, Papadopoulou K (2020) Genetically modified organism-free RNA interference: exogenous application of RNA molecules in plants. Plant Physiol 182:38–50

    CAS  PubMed  Article  Google Scholar 

  • Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1:19–21

    CAS  Article  Google Scholar 

  • Dong OX, Ronald PC (2019) Genetic engineering for disease resistance in plants: recent progress and future perspectives Plant Physiol: pp 01224–02018

  • Eamens A, Wang M-B, Smith NA, Waterhouse PM (2008) RNA silencing in plants: yesterday, today, and tomorrow. Plant Physiol 147:456–468

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • 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

    CAS  PubMed  Article  Google Scholar 

  • Ghanbari Jahromi M, Rahnama H, Mousavi A, Safarnejad MR, Kalatejari S, Soheilivand S (2015) Transient expression of coding and non-coding regions of PVY confer resistance to virus infection. Prog Biol Sci 5:19–31

    Google Scholar 

  • Goldbach R, Bucher E, Prins M (2003) Resistance mechanisms to plant viruses: an overview. Virus Res 92:207–212

    CAS  PubMed  Article  Google Scholar 

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

    CAS  PubMed  Article  Google Scholar 

  • Holoch D, Moazed D (2015) RNA-mediated epigenetic regulation of gene expression. Nat Rev Genet 16:71–84

    CAS  PubMed  PubMed Central  Article  Google Scholar 

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

    CAS  PubMed  Article  Google Scholar 

  • Hung Y-H, Slotkin RK (2021) The initiation of RNA interference (RNAi) in plants. Curr Opin Plant Biol 61:102014

    CAS  PubMed  Article  Google Scholar 

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

  • Jiang F, Wu B, Zhang C, Song Y, An H, Zhu C, Wen F (2011) Special origin of stem sequence influence the resistance of hairpin expressing plants against PVY. Biol Plant 55:528–535

    CAS  Article  Google Scholar 

  • 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 Interact 15:826–833

    CAS  PubMed  Article  Google Scholar 

  • Kaldis A, Berbati M, Melita O, Reppa C, Holeva M, Otten P, Voloudakis A (2018) Exogenously applied dsRNA molecules deriving from the Zucchini yellow mosaic virus (ZYMV) genome move systemically and protect cucurbits against ZYMV. Mol Plant Pathol 19:883–895

    CAS  PubMed  Article  Google Scholar 

  • Khatoon S, Kumar A, Sarin NB, Khan JA (2016) RNAi-mediated resistance against cotton leaf curl disease in elite Indian cotton (Gossypium hirsutum) cultivar Narasimha. Virus Genes 52:530–537

    CAS  PubMed  Article  Google Scholar 

  • Konakalla NC, Kaldis A, Berbati M, Masarapu H, Voloudakis AE (2016) Exogenous application of double-stranded RNA molecules from TMV p126 and CP genes confers resistance against TMV in tobacco. Planta 244:961–969

    CAS  PubMed  Article  Google Scholar 

  • Kreuze J, Souza-Dias J, Jeevalatha A, Figueira A, Valkonen J, Jones R (2020) Viral diseases in potato. The potato crop. Springer, Cham, pp 389–430

    Chapter  Google Scholar 

  • Lawson C, Kaniewski W, Haley L, Rozman R, Newell C, Sanders P, Tumer NE (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. Bio/technol 8:127–134

    CAS  Google Scholar 

  • Leibman D, Ortega-Parra N, Wolf D, Shterkman M, Hanssen I, Gal-On A (2021) A transgenic RNAi approach for developing tomato plants immune to Pepino mosaic virus. Plant Pathol 70:1003–1012

    CAS  Article  Google Scholar 

  • Lenka B, Satapathy SN, Senapati M (2020) Engineering plant virus resistance: gene silencing to genome editing. Int J Curr Microbiol App Sci 9:3086–3096

    CAS  Article  Google Scholar 

  • Luan H, Liao W, Song Y, Niu H, Hu T, Zhi H (2020) Transgenic plant generated by RNAi-mediated knocking down of soybean Vma12 and soybean mosaic virus resistance evaluation. AMB Exp 10:1–10. https://doi.org/10.1186/s13568-020-00997-6

    Article  Google Scholar 

  • Mansoor S, Amin I, Hussain M, Zafar Y, Briddon RW (2006) Engineering novel traits in plants through RNA interference. Trends Plant Sci 11:559–565

    CAS  PubMed  Article  Google Scholar 

  • McCue KF, Ponciano G, Rockhold DR, Whitworth JL, Gray SM, Fofanov Y, Belknap WR (2012) Generation of PVY coat protein siRNAs in transgenic potatoes resistant to PVY Am J. Potato Res 89:374–383

    CAS  Article  Google Scholar 

  • Miroshnichenko D, Timerbaev V, Okuneva A, Klementyeva A, Sidorova T, Pushin A, Dolgov S (2020) Enhancement of resistance to PVY in intragenic marker-free potato plants by RNAi-mediated silencing of eIF4E translation initiation factors. Plant Cell Tiss Org Cult 140:691–705

    CAS  Article  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

    CAS  Article  Google Scholar 

  • Mitter N et al (2017a) Clay nanosheets for topical delivery of RNAi for sustained protection against plant viruses. Nat Plants 3:1–10

    Article  CAS  Google Scholar 

  • Mitter N, Worrall EA, Robinson KE, Xu ZP, Carroll BJ (2017b) Induction of virus resistance by exogenous application of double-stranded RNA. Curr Opin Virol 26:49–55

    CAS  PubMed  Article  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

    CAS  Article  Google Scholar 

  • Niraula PN, Fondong VN (2021) Development and adoption of genetically engineered plants for virus resistance: advances opportunities and challenges. Plants 10:2339. https://doi.org/10.3390/plants10112339

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Ntui VO, Kynet K, Azadi P, Khan RS, Chin DP, Nakamura I, Mii M (2013) Transgenic accumulation of a defective cucumber mosaic virus (CMV) replicase derived double stranded RNA modulates plant defence against CMV strains O and Y in potato. Transgenic Res 22:1191–1205

    CAS  PubMed  Article  Google Scholar 

  • Oerke E-C, Dehne H-W (2004) Safeguarding production—losses in major crops and the role of crop protection. Crop Prot 23:275–285

    Article  Google Scholar 

  • Patil BL et al (2011) RNAi-mediated resistance to diverse isolates belonging to two virus species involved in Cassava brown streak disease. Mol Plant Pathol 12:31–41

    CAS  PubMed  Article  Google Scholar 

  • Petrov N, Stoyanova M, Andonova R, Teneva A (2015) Induction of resistance to potato virus Y strain NTN in potato plants through RNAi. Biotechnol Biotechnol Equip 29:21–26

    CAS  PubMed  Article  Google Scholar 

  • Pooggin MM (2017) Rnai-mediated resistance to viruses: a critical assessment of methodologies. Curr Opin Virol 26:28–35

    CAS  PubMed  Article  Google Scholar 

  • Rosa C, Kuo Y-W, Wuriyanghan H, Falk BW (2018) RNA interference mechanisms and applications in plant pathology. Annu Rev Phytopathol 56:581–610

    CAS  PubMed  Article  Google Scholar 

  • Sambrook J (2001) Molecular cloning: a laboratory manual/Joseph Sambrook, David W. Russell. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory

  • Schaefer LK et al (2020) Cross-kingdom RNAi of pathogen effectors leads to quantitative adult plant resistance in wheat. Front Plant Sci 11:253

    PubMed  PubMed Central  Article  Google Scholar 

  • Smith NA, Eamens AL (2012) Isolation and detection of small RNAs from plant tissues. Antiviral resistance in plants. Springer, Berlin, pp 155–172

    Chapter  Google Scholar 

  • Souza MT, Tennant PF, Gonsalves D (2005) Influence of coat protein transgene copy number on resistance in transgenic line 63–1 against Papaya ringspot virus isolates. HortScience 40:2083–2087

    Article  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

    CAS  Article  Google Scholar 

  • Vadlamudi T, Patil BL, Kaldis A, Gopal DVRS, Mishra R, Berbati M, Voloudakis A (2020) DsRNA-mediated protection against two isolates of Papaya ringspot virus through topical application of dsRNA in papaya. J Virol Methods 275:113750

    CAS  PubMed  Article  Google Scholar 

  • van Esse HP, Reuber TL, van der Does D (2020) Genetic modification to improve disease resistance in crops. New Phytol 225:70–86

    PubMed  Article  Google Scholar 

  • Visser JC, Bellstedt DU, Pirie MD (2012) The recent recombinant evolution of a major crop pathogen, Potato virus Y. PLoS One 7:e50631

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Wang J, Meng F, Chen R, Liu J, Nie X, Nie B (2016) RT-PCR differentiation, molecular and pathological characterization of Andean and ordinary strains of Potato virus S in potatoes in China. Plant Dis 100:1580–1585

    PubMed  Article  Google Scholar 

  • Wilson TM (1993) Strategies to protect crop plants against viruses: pathogen-derived resistance blossoms. PNAS 90:3134–3141

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Worrall EA, Bravo-Cazar A, Nilon AT, Fletcher SJ, Robinson KE, Carr JP, Mitter N (2019) Exogenous application of RNAi-inducing double-stranded RNA inhibits aphid-mediated transmission of a plant virus. Front Plant Sci 10:265

    PubMed  PubMed Central  Article  Google Scholar 

  • Yadav JS et al (2011) RNAi-mediated resistance to Cassava brown streak Uganda virus in transgenic cassava. Mol Plant Pathol 12:677–687

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  • Zhan X et al (2019) Generation of virus-resistant potato plants by RNA genome targeting. Plant Biotechnol J 17:1814–1822

    CAS  PubMed  PubMed Central  Article  Google Scholar 

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Agricultural Biotechnology Research Institute of Iran.

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Correspondence to Hassan Rahnama.

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Jahromi, M.G., Rahnama, H., Mousavi, A. et al. Comparative evaluation of resistance to potato virus Y (PVY) in three different RNAi-based transgenic potato plants. Transgenic Res 31, 313–323 (2022). https://doi.org/10.1007/s11248-022-00302-0

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  • DOI: https://doi.org/10.1007/s11248-022-00302-0

Keywords

  • Coat protein (CP)
  • Potato virus Y (PVY)
  • RNAi
  • Transgenic
  • Untranslated region (UTR)