Transgenic Research

, Volume 26, Issue 2, pp 225–235 | Cite as

Citrus psorosis virus coat protein-derived hairpin construct confers stable transgenic resistance in citrus against psorosis A and B syndromes

Original Paper
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Abstract

Citrus psorosis virus (CPsV) is the causal agent of psorosis, a serious and widespread citrus disease. Two syndromes of psorosis, PsA and PsB, have been described. PsB is the most aggressive and rampant form. Previously, we obtained Pineapple sweet orange plants transformed with a hairpin construct derived from the CPsV coat protein gene (ihpCP). Some of these plants were resistant to CPsV 90-1-1, a PsA isolate homologous to the transgene. In this study, we found that expression of the ihpCP transgene and siRNA production in lines ihpCP-10 and -15 were stable with time and propagation. In particular, line ihpCP-15 has been resistant for more than 2 years, even after re-inoculation. The ihpCP plants were also resistant against a heterologous CPsV isolate that causes severe PsB syndrome. Line ihpCP-15 manifested complete resistance while line ihpCP-10 was tolerant to the virus, although with variable behaviour, showing delay and attenuation in PsB symptoms. These lines are promising for a biotech product aimed at eradicating psorosis.

Keywords

Citrus psorosis virus Hairpin-RNA Psorosis A Psorosis B Transgenic sweet orange Virus resistance 

Supplementary material

11248_2016_1_MOESM1_ESM.docx (24 kb)
Supplementary material 1 (DOCX 24 kb)
11248_2016_1_MOESM2_ESM.docx (29 kb)
Supplementary material 2 (DOCX 28 kb)

References

  1. Alioto D, Gangemi M, Deaglio S, Sposato S, Noris E, Luisoni E, Milne RG (1999) Improved detection of Citrus psorosis virus using polyclonal and monoclonal antibodies. Plant Pathol 48:735–741. doi:10.1023/B:EJPP.0000041570.28825.29 CrossRefGoogle Scholar
  2. Anderson C (2000) Presentation on the Argentinean certification program. In: Proceedings of global citrus germplasm network 7–8 December 2000Google Scholar
  3. Axtell MJ (2013) Classification and comparison of small RNAs from plants. Annu Rev Plant Biol 64:137–159. doi:10.1146/annurev-arplant-050312-120043 CrossRefPubMedGoogle Scholar
  4. Baulcombe D (2004) RNA silencing in plants. Nature 437:356–363. doi:10.1038/nature02874 CrossRefGoogle Scholar
  5. Bond DM, Baulcombe DC (2014) Small RNAs and heritable epigenetic variation in plants. Trends Cell Biol 24:100–107. doi:10.1016/j.tcb.2013.08.001 CrossRefPubMedGoogle Scholar
  6. Brosnan CA, Voinnet O (2011) Cell-to-cell and long-distance siRNA movement in plants: mechanisms and biological implications. Curr Opin Plant Biol 14:580–587. doi:10.1016/j.pbi.2011.07.011 CrossRefPubMedGoogle Scholar
  7. Catoni M, Lucioli A, Doblas-Ibáñez P, Accotto GP, Vaira AM (2013) From immunity to susceptibility: virus resistance induced in tomato by a silenced transgene is lost as TGS overcomes PTGS. Plant J 75:941–953. doi:10.1111/tpj.12253 CrossRefPubMedGoogle Scholar
  8. Danós E (1990) La psorosis de los cítricos: la epidemia en curso en Argentina y el desafío de su control. International Foundation for Science (IFS) e Instituto Nacional de Tecnología Agropecuaria (INTA). Revista de Investigaciones Agropecuaria 22:265–277Google Scholar
  9. De Francesco A, Costa N, Plata MI, García ML (2015) Improved detection of Citrus psorosis virus and coat protein-derived transgenes in citrus plants: comparison between RT-qPCR and TAS-ELISA. J Phytopathol 163:915–925. doi:10.1111/jph.12392 CrossRefGoogle Scholar
  10. Di Nicola-Negri E, Tavazza M, Salandri L, Ilardi V (2010) Silencing of Plum pox virus 5′UTR/P1 sequence confers resistance to a wide range of PPV strains. Plant Cell Rep 29:1435–1444. doi:10.1007/s00299-010-0933-6 CrossRefPubMedGoogle Scholar
  11. Fawcett HS, Klotz LJ (1938) Types and symptoms of psorosis and psorosis-like diseases of citrus. Phytopathol 32:22Google Scholar
  12. Fire A (1999) RNA-triggered gene silencing. Trends Genet 15:358–363. doi:10.1016/S0168-9525(99)01818-1 CrossRefPubMedGoogle Scholar
  13. Folimonova SY, Harper SJ, Leonard MT, Triplett EW, Shilts T (2014) Superinfection exclusion by Citrus tristeza virus does not correlate with the production of viral small RNAs. Virol J. doi:10.1016/j.virol.2014.08.031 Google Scholar
  14. Frizzi A, Huang S (2010) Tapping RNA silencing pathways for plant biotechnology. Plant Biotechnol J 8:655–677. doi:10.1111/j.1467-7652.2010.00505.x CrossRefPubMedGoogle Scholar
  15. Gambino G, Perrone I, Carra A, Chitarra W, Boccacci P, Torello Marinoni D, Barberis M, Maghuly F, Laimer M, Gribaudo I (2010) Transgene silencing in grapevines transformed with GFLV resistance genes: analysis of variable expression of transgene, siRNAs production and cytosine methylation. Transgenic Res 19:17–27. doi:10.1007/s11248-009-9289-5 CrossRefPubMedGoogle Scholar
  16. García ML, Dal Bo E, Grau O, Milne RG (1994) The closely related citrus ringspot and Citrus psorosis viruses have particles of novel filamentous morphology. J Gen Virol 75:3585–3590. doi:10.1099/0022-1317-75-12-3585 CrossRefPubMedGoogle Scholar
  17. Hily JM, Scorza R, Webb K, Ravelonandro M (2005) Accumulation of the long class of siRNA is associated with resistance to Plum pox virus in a transgenic woody perennial plum tree. Mol Plant Microbe Interact 18:794–799. doi:10.1094/MPMI-18-0794 CrossRefPubMedGoogle Scholar
  18. Martín S, Alioto D, Milne RG, Garnsey SM, García ML, Grau O, Guerri Moreno P (2004) Detection of Citrus psorosis virus by ELISA, molecular hybridization, RT-PCR and immunosorbent electron microscopy and its association with citrus psorosis disease. Eur J Plant Pathol 110:747–757. doi:10.1023/B:EJPP.0000041570.28825.29 CrossRefGoogle Scholar
  19. Milne RG, García ML, Grau O (2000) Genus Ophiovirus. In: van Regenmortel MVH, Fauquet CM, Bishop DHL, Carstens EB, Estes MK, Lemon SM, Maniloff J, Mayo MA, Mc Geoch DJ, Pringle CR, Wickner RB (eds) Virus taxonomy. Sevent Report of the International Committee on Taxonomy of Virus. Academic Press, New York, pp 627–631Google Scholar
  20. Molnar A, Melnyk CW, Bassett A, Hardcastle TJ, Dunn R, Baulcombe DC (2010) Small silencing RNAs in plants are mobile and direct epigenetic modification in recipient cells. Science 328:872–875. doi:10.1126/science.1187959 CrossRefPubMedGoogle Scholar
  21. Moreno P, Guerri J, García ML (2015) The psorosis disease of citrus: a pale light at the end of the tunnel. J Citrus PatholGoogle Scholar
  22. Naum-Ongania G, Gago-Zachert S, Peña E, Grau O, Garcia ML (2003) Citrus psorosis virus RNA 1 is of negative polarity and potentially encodes in its complementary strand a 24 K protein of unknown function and 280 K putative RNA dependent RNA polymerase. Virus Res 96:49–61. doi:10.1016/S0168-1702(03)00172-2 CrossRefPubMedGoogle Scholar
  23. Pall GS, Hamilton AJ (2008) Improved northern blot method for enhanced detection of small RNA. Nat Protoc 3:1077–1084. doi:10.1038/nprot.2008.67 CrossRefPubMedGoogle Scholar
  24. Reyes CA, De Francesco A, Peña EJ, Costa N, Plata MI, Sendin L, Castagnaro AP, García ML (2011) Resistance to Citrus psorosis virus in transgenic sweet orange plants is triggered by coat protein–RNA silencing. J Biotechnol 151:151–158. doi:10.1016/j.jbiotec.2010.11.007 CrossRefPubMedGoogle Scholar
  25. Reyes CA, Ocolotobiche EE, Marmisollé FE, Robles Luna G, Borniego MB, Bazzini AA, Asurmendi S, García ML (2015) Citrus psorosis virus 24 K protein interacts with citrus miRNA precursors, affects their processing and subsequent miRNA accumulation and target expression. Mol Plant Pathol. doi:10.1111/mpp.12282 PubMedGoogle Scholar
  26. Reyes CA, De Francesco A, Ocolotobiche EE, Costa N, García ML (2016) Uncontrolled Citrus psorosis virus infection in Citrus sinensis transgenic plants expressing a viral 24 K-derived hairpin that does not trigger RNA silencing. Physiol Mol Plant Pathol. doi:10.1016/j.pmpp.2016.05.001 Google Scholar
  27. Robles Luna G, Peña EJ, Borniego MB, Heinlein M, García ML (2013) Ophioviruses CPsV and MiLBVV movement protein is encoded in RNA 2 and interacts with the coat protein. Virol J 441:152–161. doi:10.1016/j.virol.2013.03.019 CrossRefGoogle Scholar
  28. Roistacher CN (1991) Psorosis complex: psorosis-A, psorosis-B and ringspot. In: International Organization of Citrus Virologists (ed) Graft-transmissible diseases of citrus: Handbook for detection and diagnosis. Food and Agriculture Organization of the United Nations, Rome, pp 115–126Google Scholar
  29. Roistacher CN (1993) Psorosis—a review. In: Moreno P, da Graca JV, Timmer LW (eds) Proceedings of 12th conference of the international organization of citrus virologists, 23–27 November 1992, Riverside, CA, pp 139–154Google Scholar
  30. Sánchez de la Torre ME, Riva O, Zandomeni R, Grau O, García ML (1998) The top component of Citrus psorosis virus contains two ssRNAs, the smaller encodes the coat protein. Mol Plant Pathol. http://www.bspp.org.uk/mppol/1998/1019sanchez
  31. Smith NA, Singh SP, Wang MB, Stoutjesdijk PA, Green AG, Waterhouse PM (2000) Total silencing by intron-spliced hairpin RNAs. Nature 407:319–320. doi:10.1038/35030305 CrossRefPubMedGoogle Scholar
  32. Velázquez K, Renovell A, Comellas M, Serra P, García ML, Pina JA, Navarro L, Moreno P, Guerri J (2010) Effect of temperature on RNA silencing of a negative-stranded RNA plant virus: Citrus psorosis virus. Plant Pathol 59:982–990. doi:10.1111/j.1365-3059.2010.02315.x CrossRefGoogle Scholar
  33. Velázquez K, Pina JA, Navarro L, Moreno P, Guerri J (2012) Association of citrus psorosis B symptoms with a sequence variant of the Citrus psorosis virus RNA 2. Plant Pathol 61:448–456. doi:10.1111/j.1365-3059.2011.02525.x CrossRefGoogle Scholar
  34. Yadav RK, Chattopadhyay D (2011) Enhanced viral intergenic region-specific short interfering RNA accumulation and DNA methylation correlates with resistance against a geminivirus. Mol Plant Microbe Interact 24(10):1189–1197. doi:10.1094/MPMI-03-11-0075 CrossRefPubMedGoogle Scholar
  35. Zanek MC, Peña E, Reyes CA, Figueroa J, Stein B, Grau O, García ML (2006) Detection of Citrus psorosis virus in the northwestern citrus production area of Argentina by using an improved TAS-ELISA. J Virol Methods 137:245–251. doi:10.1016/j.jviromet.2006.06.021 CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2016

Authors and Affiliations

  1. 1.Instituto de Biotecnología y Biología Molecular, CCT-La PlataCONICET - UNLPLa PlataArgentina
  2. 2.Estación Experimental AgropecuariaINTA ConcordiaConcordiaArgentina

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