Advertisement

European Journal of Plant Pathology

, Volume 155, Issue 4, pp 1345–1352 | Cite as

Topical application of double stranded RNA molecules deriving from Sesbania mosaic virus (SeMV) CP and MP genes protects Sesbania plants against SeMV

  • Naga Charan Konakalla
  • Athanasios Kaldis
  • Hema MasarapuEmail author
  • Andreas E. VoloudakisEmail author
Article
  • 153 Downloads

Abstract

RNA interference (RNAi) is a sequence-specific, gene silencing mechanism, induced by double-stranded RNA (dsRNA). It is a defense mechanism that protects eukaryotic cells from invasive nucleic acids such as viruses and transposons. In this study, we used a non-transgenic strategy in order to activate the antiviral RNAi mechanism against Sesbania mosaic virus (SeMV) in its natural host Sesbania grandiflora. DsRNA molecules from SeMV coat protein (CP) and movement protein (MP) (sobemovirus MP is considered a silencing suppressor) genes were produced by a two-step PCR approach followed by in vitro transcription and exogenously applied on sesbania plants along with SeMV. DsRNA for CP and MP conferred 53% and 64% resistance against SeMV, respectively based on the disease incidence data. The effect of dsRNA molecules against SeMV infection on sesbania plants was confirmed by direct antigen coating ELISA (DAC-ELISA). The strategy employed demonstrated the applicability of the RNA-based vaccination method, for the first time, to control sobemoviruses in a simple, highly specific and environmentally safe way.

Keywords

Coat protein DAC-ELISA Double-stranded RNA Movement protein RNA interference RNA-based vaccination Silencing suppressor 

Notes

Acknowledgments

Ramamurthy Prakash and Vaishnavi Pidugu are acknowledged for maintaining the sesbania plants and assisting in the virus inoculations at the Department of Virology, Sri Venkateswara University, India.

Author contributions

HM and AV conceived and designed the study. NK and AK perfomed the exepriments. NK, AK, HM, and AV contributed to the manuscript writing.

Funding

This work was financially supported by an Erasmus Mundus action BRAVE scholarship sponsored by European Union (2013–2536) and N.T.R Videshi Vidyadarana scholarship from the State Government of Andhra Pradesh, India to Naga Charan Konakalla.

Compliance with ethical standards

Conflict of interest

No conflict of interest exits in the submission of this manuscript.

Human studies and participants

There was no involvement of human participants and/or animals in the present study.

References

  1. Aalto, A. P., Sarin, L. P., van Dijk, A. A., Saarma, M., Poranen, M. M., Arumäe, U., & Bamford, D. H. (2007). Large-scale production of dsRNA for siRNA pools for RNA interference utilizing bacteriophage phi6 RNA-dependent RNA polymerase. RNA, 13, 422–429.PubMedPubMedCentralGoogle Scholar
  2. Beachy, R. N. (1997). Mechanisms and applications of pathogen-derived resistance in transgenic plants. Current Opinion in Biotechnology, 8, 215–220.PubMedGoogle Scholar
  3. Brodersen, P., & Voinnet, O. (2006). The diversity of RNA silencing pathways in plants. Trends in Genetics, 22, 268–280.PubMedGoogle Scholar
  4. Brugidou, C., Holt, C., Yassi, M. N., Zhang, S., Beachy, R., & Fauquet, C. (1995). Synthesis of an infectious full-length cDNA clone of rice yellow mottle virus and mutagenesis of the coat protein. Virology, 206, 108–115.PubMedGoogle Scholar
  5. Chowdhury, S. R., & Savithri, H. S. (2011a). Interaction of Sesbania mosaic virus movement protein with the coat protein–implications for viral spread. FEBS Journal, 278, 257–272.PubMedGoogle Scholar
  6. Chowdhury, S. R., & Savithri, H. S. (2011b). Interaction of Sesbania mosaic virus movement protein with VPg and P10: Implication to specificity of genome recognition. PLoS One, 6, e15609.Google Scholar
  7. Clark, M. F., & Adams, A. N. (1977). Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant viruses. Journal of General Virology, 34, 475–483.PubMedGoogle Scholar
  8. Dalakouras, A., Wassenegger, M., McMillan, J. N., Cardoza, V., Maegele, I., Dadami, E., Runne, M., Krczal, G., & Wassenegger, M. (2016). Induction of silencing in plants by high-pressure spraying of in vitro-synthesized small RNAs. Frontiers in Plant Science, 7, 1327.PubMedPubMedCentralGoogle Scholar
  9. Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., & Mello, C. C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 391, 806–811.Google Scholar
  10. Gan, D., Zhang, J., Jiang, H., Jiang, T., Zhu, S., & Cheng, B. (2010). Bacterially expressed dsRNA protects maize against SCMV infection. Plant Cell Reports, 29, 1261–1268.PubMedGoogle Scholar
  11. Gogoi, A., Sarmah, N., Kaldis, A., Perdikis, D., & Voloudakis, A. (2017). Plant insects and mites uptake double-stranded RNA upon its exogenous application on tomato leaves. Planta, 246, 1233–1241.PubMedGoogle Scholar
  12. Govind, K., Mäkinen, K., & Savithri, H. S. (2012). Sesbania mosaic virus (SeMV) infectious clone: Possible mechanism of 3′ and 5′ end repair and role of polyprotein processing in viral replication. PLoS One, 7, e31190.PubMedPubMedCentralGoogle Scholar
  13. Hammond, S. M., Boettcher, S., Caudy, A. A., Kobayashi, R., & Hannon, G. J. (2001). Argonaute2 a link between genetic and biochemical analyses of RNAi. Science, 293, 1146–1150.PubMedGoogle Scholar
  14. Hobbs, H. A., Reddy, D. V. R., Rajeshwari, R., & Reddy, A. S. (1987). Use of direct antigen coating and protein a coating ELISA procedures for detection of three peanut viruses. Plant Disease, 71, 747–749.Google Scholar
  15. Holeva, M. C., Sclavounos, A. P., Milla, S. P., Kyriakopoulou, P. E., & Voloudakis, A. E. (2007). External application of dsRNA of the capsid protein (CP) or 2b gene of CMV reduces the severity of CMV-infection in tobacco. ΧΙΙΙ international congress on molecular plant-microbe interactions, 21–27 July 2007, Sorrento, Italy.Google Scholar
  16. Ioannidou, D., Pinel, A., Brugidou, C., Albar, L., Ahmadi, N., Ghesquiere, A., Nicole, M., & Fargette, D. (2003). Characterization of the effects of a major QTL of the partial resistance to Rice yellow mottle virus using a near-isogenic–line approach. Physiological and Molecular Plant Pathology, 63, 213–221.Google Scholar
  17. Kaldis, A., Berbati, M., Melita, O., Reppa, C., Holeva, M., Otten, P., & Voloudakis, A. (2018). Exogenously applied dsRNA molecules deriving from Zucchini yellow mosaic virus (ZYMV) genome move systemically and protect cucurbits against ZYMV. Molecular Plant Pathology, 19, 883–895.PubMedGoogle Scholar
  18. Ketting, R. F., Fischer, S. E., Bernstein, E., Sijen, T., Hannon, G. J., & Plasterk, R. H. (2001). Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes & Development, 15, 2654–2659.Google Scholar
  19. Koch, A., Beidenkopf, D., Furch, A., Weber, L., Rossbach, O., Abdellatef, E., Linicus, L., Johannsmeier, J., Jelonek, L., Goesmann, A., Cardoza, V., McMillan, J., Mentzel, T., & Kogel, K. H. (2016). An RNAi-based control of Fusarium graminearum infections through spraying of long dsRNAs involves a plant passage and is controlled by the fungal silencing machinery. PLoS Pathogens, 12, e1005901.PubMedPubMedCentralGoogle Scholar
  20. Konakalla, N. C., Kaldis, A., Berbati, M., Masarapu, H., & Voloudakis, A. E. (2016). Exogenous application of double-stranded RNA molecules from TMV p126 and CP genes confers resistance against TMV in tobacco. Planta, 244, 961–969.PubMedGoogle Scholar
  21. Lacombe, S., Bangratz, M., Vignols, F., & Brugidou, C. (2010). The rice yellow mottle virus P1 protein exhibits dual functions to suppress and activate gene silencing. Plant Journal, 61, 371–382.PubMedGoogle Scholar
  22. Lindbo, J. A. (2012). A historical overview of RNAi in plants. Methods in Molecular Biology, 894, 1–16.PubMedGoogle Scholar
  23. Liu, J., Carmell, M. A., Rivas, F. V., Marsden, C. G., Thomson, J. M., Song, J. J., Hammond, S. M., Joshua-Tor, L., & Hannon, G. J. (2004). Argonaute2 is the catalytic engine of mammalian RNAi. Science, 305, 1437–1441.PubMedGoogle Scholar
  24. Liu, H. M., Zhu, C. X., Zhu, X. P., Guo, X. Q., Song, Y. Z., & Wen, F. J. (2007). A link between PVYN CP gene-mediated virus resistance and transgene arrangement. Journal of Phytopathology, 155, 676–682.Google Scholar
  25. Lokesh, G. L., Gopinath, K., Satheshkumar, P. S., & Savithri, H. S. (2001). Complete nucleotide sequence of Sesbania mosaic virus: A new virus species of the genus Sobemovirus. Archives of Virology, 146, 209–223.PubMedGoogle Scholar
  26. Meier, M., Paves, H., Olspert, A., Tamm, T., & Truve, E. (2006). P1 protein of cocksfoot mottle virus is indispensable for the systemic spread of the virus. Virus Genes, 32, 321–326.PubMedGoogle Scholar
  27. Mendoza, V. B. (1980). Katurai: A plant of many uses. Canopy, 12–13.Google Scholar
  28. Mitter, N., Worrall, E. A., Robinson, K. E., Li, P., Jain, R. G., Taochy, C., Fletcher, S. J., Carroll, B. J., Lu, G. Q., & Xu, Z. P. (2017). Clay nanosheets for topical delivery of RNAi for sustained protection against plant viruses. Nature Plants, 3, 16207.PubMedGoogle Scholar
  29. Namgial, T., Kaldis, A., Chakraborty, S., Voloudakis, A. (2019). Topical application of double-stranded RNA molecules containing sequences of Tomato leaf curl virus and Cucumber mosaic virus confers protection against the cognate viruses. Physiological and Molecular Plant Pathology, 101432.Google Scholar
  30. Ndjiondjop, M. N., Albar, L., Fargette, D., Fauquet, C., & Ghesquière, A. (1999). The genetic basis of high resistance to rice yellow mottle virus (RYMV) in cultivars of two cultivated rice species. Plant Disease, 83, 931–935.PubMedGoogle Scholar
  31. Numata, K., Ohtani, M., Yoshizumi, T., Demura, T., & Kodama, Y. (2014). Local gene silencing in plants via synthetic dsRNA and carrier peptide. Plant Biotechnology Journal, 12, 1027–1034.PubMedGoogle Scholar
  32. Pinto, Y. M., Kok, R. A., & Baulcombe, D. C. (1999). Resistance to rice yellow mottle virus (RYMV) in cultivated African rice varieties containing RYMV transgenes. Nature Biotechnology, 17, 702–707.PubMedGoogle Scholar
  33. Sarmiento, C., Gomez, E., Meier, M., Kavanagh, T. A., & Truve, E. (2007). Cocksfoot mottle virus P1 suppresses RNA silencing in Nicotiana benthamiana and Nicotiana tabacum. Virus Research, 123, 95–99.PubMedGoogle Scholar
  34. Sivakumaran, K., Fowler, B. C., & Hacker, D. L. (1998). Identification of viral genes required for cell-to-cell movement of southern bean mosaic virus. Virology, 252, 376–386.PubMedGoogle Scholar
  35. 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.PubMedGoogle Scholar
  36. Sreenivasalu, P., & Nayudu, M. V. (1982). Purification and partial characterization of sesbania mosaic virus. Current Science, 51, 86–87.Google Scholar
  37. Sun, Y., Qiao, X., & Mindich, L. (2004). Construction of carrier state viruses with partial genomes of the segmented dsRNA bacteriophages. Virology, 319, 274–279.PubMedGoogle Scholar
  38. Tabara, H., Sarkissian, M., Kelly, W. G., Fleenor, J., Grishok, A., Timmons, L., Fire, A., & Mello, C. C. (1999). The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell, 99, 123–132.PubMedPubMedCentralGoogle Scholar
  39. Tamm, T., & Truve, E. (2000). Sobemoviruses. Journal of Virology, 74, 6231–6241.PubMedPubMedCentralGoogle Scholar
  40. Tenllado, F., & Díaz-Ruíz, J. R. (2001). Double-stranded RNA-mediated interference with plant virus infection. Journal of Virology, 75, 12288–12297.PubMedPubMedCentralGoogle Scholar
  41. Tenllado, F., Llave, C., & Díaz-Ruíz, J. R. (2004). RNA interference as a new biotechnological tool for the control of virus diseases in plants. Virus Research, 102, 85–96.PubMedGoogle Scholar
  42. Tepfer, M. (2002). Risk assessment of virus-resistant transgenic plants. Annual Review of Phytopathology, 40, 467–491.PubMedGoogle Scholar
  43. Timmons, L. (2006). Construction of plasmids for RNA interference and in vitro transcription of double-stranded RNA. In C. elegans (pp. 109–117). Humana Press, New York.Google Scholar
  44. Turner, C. T., Davy, M. W., MacDiarmid, R. M., Plummer, K. M., Birch, N. P., & Newcomb, R. D. (2006). RNA interference in the light brown apple moth Epiphyas postvittana (Walker) induced by double-stranded RNA feeding. Insect Molecular Biology, 15, 383–391.PubMedGoogle Scholar
  45. Voinnet, O., Pinto, Y. M., & Baulcombe, D. C. (1999). Suppression of gene silencing: A general strategy used by diverse DNA and RNA viruses of plants. Proceedings of the National Academy of Sciences, 96, 14147–14152.Google Scholar
  46. Voloudakis, A. E., Holeva, M. C., Sarin, L. P., Bamford, D. H., Vargas, M., Poranen, M. M., & Tenllado, F. (2015). Efficient double-stranded RNA production methods for utilization in plant virus control. Methods in Molecular Biology, 1236, 255–274.PubMedGoogle Scholar
  47. Wang, M. B., & Metzlaff, M. (2005). RNA silencing and antiviral defense in plants. Current Opinion in Plant Biology, 8, 216–222.PubMedGoogle Scholar
  48. Yin, G., Sun, Z., Liu, N., Zhang, L., Song, Y., Zhu, C., & Wen, F. (2009). Production of double-stranded RNA for interference with TMV infection utilizing a bacterial prokaryotic expression system. Applied Microbiology and Biotechnology, 84, 323–333.PubMedGoogle Scholar
  49. Zhu, C. X., Song, Y. Z., Yin, G. H., & Wen, F. J. (2009). Induction of RNA-mediated multiple viral resistance to Potato Virus Y, Tobacco mosaic virus and Cucumber mosaic virus. Journal of Phytopathology, 157, 101–107.Google Scholar
  50. Ηoleva, M., Sclavounos, A. P., Kyriakopoulou, P. E., & Voloudakis, A. E. (2006). In vitro produced dsRNA induces resistance against a severe Hellenic CMV isolate in tobacco and tomato. 8th international congress of plant molecular biology, 20–25 august 2006, Adelaide, Australia.Google Scholar

Copyright information

© Koninklijke Nederlandse Planteziektenkundige Vereniging 2019

Authors and Affiliations

  1. 1.Laboratory of Plant Breeding and BiometryAgricultural University of AthensAthensGreece
  2. 2.Department of VirologySri Venkateswara UniversityTirupatiIndia

Personalised recommendations