Advertisement

Plant Cell Reports

, Volume 27, Issue 1, pp 57–66 | Cite as

Genetic transformation of sweet orange with the coat protein gene of Citrus psorosis virus and evaluation of resistance against the virus

  • María Cecilia ZanekEmail author
  • Carina Andrea Reyes
  • Magdalena Cervera
  • Eduardo José Peña
  • Karelia Velázquez
  • Norma Costa
  • Maria Inés Plata
  • Oscar Grau
  • Leandro Peña
  • María Laura García
Genetic Transformation and Hybridization

Abstract

Citrus psorosis is a serious viral disease affecting citrus trees in many countries. Its causal agent is Citrus psorosis virus (CPsV), the type member of genus Ophiovirus. CPsV infects most important citrus varieties, including oranges, mandarins and grapefruits, as well as hybrids and citrus relatives used as rootstocks. Certification programs have not been sufficient to control the disease and no sources of natural resistance have been found. Pathogen-derived resistance (PDR) can provide an efficient alternative to control viral diseases in their hosts. For this purpose, we have produced 21 independent lines of sweet orange expressing the coat protein gene of CPsV and five of them were challenged with the homologous CPV 4 isolate. Two different viral loads were evaluated to challenge the transgenic plants, but so far, no resistance or tolerance has been found in any line after 1 year of observations. In contrast, after inoculation all lines showed characteristic symptoms of psorosis in the greenhouse. The transgenic lines expressed low and variable amounts of the cp gene and no correlation was found between copy number and transgene expression. One line contained three copies of the cp gene, expressed low amounts of the mRNA and no coat protein. The ORF was cytosine methylated suggesting a PTGS mechanism, although the transformant failed to protect against the viral load used. Possible causes for the failed protection against the CPsV are discussed.

Keywords

Citrus psorosis virus PDR Post-transcriptional gene silencing Citrus Transgenic trees 

Abbreviations

CPsV

Citrus psorosis virus

CP

Coat protein

TAS-ELISA

Triple sandwich immunoassay

PDR

Pathogen-derived resistance

CaMV

Cauliflower mosaic virus

AMV

Alfalfa mosaic virus

nos

Nopaline synthase gene

OD

Optical density

dpi

Days post inoculation

C-I

Infected control

C-NI

Non-infected control

PTGS

Post-transcriptional gene silencing

CTV

Citrus tristeza virus

GFLV

Grapevine fan-leaf virus

TSWV

Tomato spotted wilt virus

Notes

Acknowledgments

We thank Dr. L.W. Timmer for helpful discussion and manuscript revision, and Ing. G. Chiarrone and Pto. Agr. Fabián Ramos for greenhouse work. M. L. García, Eduardo J. Peña, Carina A. Reyes and M. C. Zanek belong to the staff of Facultad de Ciencias Exactas, UNLP, O.G. is recipient of the research career award from CICBA, and MLG from CONICET. Lic. Zanek and Reyes are fellows of CONICET. Lic. E.J. Peña is fellows of ANPCyT. This work was supported by grants from BID802 OC-AR PICT 6198 SECyT-CONICET, AECI, CICBA and INTA EEA Concordia.

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: 10.1046/j.1365-3059.1999.00410.x CrossRefGoogle Scholar
  2. Barthe GA, Ceccardi TL, Manjunath KL, Derrick KS (1998) Citrus psorosis virus: nucleotide sequencing of the coat protein gene and detection by hybridisation and RT-PCR. J Gen Virol 79:1531–1537PubMedGoogle Scholar
  3. Baulcombe DC (1996) Mechanisms of pathogen-derived resistance to viruses in transgenic plants. Plant Cell 8:1833–1844. doi: 10.1105/tpc.8.10.1833 PubMedCrossRefGoogle Scholar
  4. Beachy RN, Loesch-Fries S, Tumer NE (1990) Coat protein-mediated resistance against virus infection. Annu Rev Phytopathol 28:451–474CrossRefGoogle Scholar
  5. Bekesiova I, Nap J-P, Mlynarova L (1999) Isolation of high Quality DNA and RNA from leaves of the carnivorous plant Drosera rotundifolia. Plant Mol Biol Rep 17:269–277CrossRefGoogle Scholar
  6. Beñatena HN, Portillo MM (1984) Natural spread of psorosis in sweet orange seedlings. In: Garnsey SM, Timmer LW and Dodds JA (eds) Proceedings of the 9th conference of the international organization of citrus virologists, IOCV, Riverside, pp 159–164Google Scholar
  7. Cervera M (2005) Histochemical and fluorometric assays for uidA (GUS) gene detection. Methods Mol Biol 286:203–214PubMedGoogle Scholar
  8. Danós E (1990) La psorosis de los cítricos: la epidemia en curso en Argentina y el desafio de su control. In: International Foundation for Science (IFS) e Instituto Nacional de Tecnologia Agropecuaria (INTA) (eds) Revista de Investigaciones Agropecuarias, vol 22, pp 265–277Google Scholar
  9. Domínguez A, Guerri J, Cambra M, Navarro L, Moreno P, Peña L (2000) Efficient production of citrus transgenic plants expressing the coat protein gene of Citrus Tristeza Virus. Plant Cell Rep. 19:427–433. doi: 10.1007/s002990050751 CrossRefGoogle Scholar
  10. Domínguez A, Hermoso de Mendoza A, Guerri J, Cambra M, Navarro L, Moreno P, Peña L (2002) Pathogen-derived resistance to Citrus tristeza virus (CTV) in transgenic Mexican lime (Citrus aurantifolia (Christ. ) Swing.) plants expressing its p25 coat protein gene. Mol Breed 10:1–10. doi: 10.1023/A:1020347415333 CrossRefGoogle Scholar
  11. Fagoaga C, López C, Hermoso de Mendoza A, Moreno P, Navarro L, Flores R, Peña L (2006) Post-transcriptional gene silencing of the p23 silencing supresor of Citrus tristeza virus confers resistance to the virus in transgenic Mexican lime. Plant Mol Biol 60:153-165. doi: 10.1007/s11103-005-3129-7 PubMedCrossRefGoogle Scholar
  12. Futterer J, Gisel A, Iglesias V, Kloti A, Kost B, Mittelsten Sceid O, Neuhaus-Url G, Schrott M, Shillito R, Spangenber G, Wang ZY (1995) Standard molecular techniques for analysis of trangenic plants. In: Potrykus I, Spangenberg G (eds) Gene tranfer to plants, Springer Lab manual, vol 25, pp 215Google Scholar
  13. Gambino G, Gribaudo I, Stephan L, Scharti A, Laimer M (2005) Molecular characterization of grapevine plants transformed with GFLV resistance genes:I. Plant Cell Rep 24:665–662. doi: 10.1007/s00299-005-0006-4 CrossRefGoogle Scholar
  14. García ML, Dal Bo E, Grau O, Milne R (1994) The closely related citrus ringspot and citrus psorosis viruses have particles of novel filamentous morphology. J Gen Virol 75:3585–3590PubMedCrossRefGoogle Scholar
  15. Garnsey SM, Timmer LW (1980) Mechanical transmissibility of citrus ringspot virus isolates from Florida, Texas, and California. In: Calavan EC, Garnsey SM, Timmer LW (eds) Proceedings of the Eighth conference of the international organization of citrus virologists, IOCV, Riverside, pp 174–179Google Scholar
  16. Garnsey SM, Youtsey CO, Bridges GD, Burnett HC (1976) A necrotic ringspot-like virus found in a “Star Ruby” grapefruit tree imported without authorization from Texas. In: Proceedings of the Florida State Horticultural Society, vol 89, pp 63–67Google Scholar
  17. Ghorbel R, Lopez C, Fagoaga C, Moreno P, Navarro L, Flores R, Peña L (2001) Transgenic citrus plants expressing the citrus tristeza virus p23 protein exhibit viral-like symptoms. Mol Plant Pathol 2:27–36CrossRefGoogle Scholar
  18. Goldbach R, Bucher, Prins M (2003) Resistance mechanisms to plant viruses: an overview. Virus Res 92:207–212. doi: 10.1016/S0168-1702(02)00353-2 PubMedCrossRefGoogle Scholar
  19. Hily JH, Scorza R, Malinowski T, Zawadzka B, Ravelonandro M (2004) Stability of gene silencing-based resistance to Plum pox virus in transgenic plum (Prunus domestica L.) under field conditions. Transgenic Res 13:427–436. doi: 10.1007/s11248-004-8702-3 PubMedCrossRefGoogle Scholar
  20. 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 resistence to the virus. Mol Plant Microbe Interact 15:826–833PubMedCrossRefGoogle Scholar
  21. Kouassi NK, Chen L, Siré C, Bangratz-Reyser M, Beachy RN, Fauquet CM, Brugidou C (2006) Expression of rice yellow mottle virus coat protein enhances virus infection in transgenic plants. Arch Virol 151:2111–2122. doi: 10.1007/s00705-006-0802-3 PubMedCrossRefGoogle Scholar
  22. Levin JS, Thompson WF, Csinos AS, Stephenson MG, Weissinger AK (2005) Matrix attachment regions increase the efficiency and stability of RNA-mediated resistance to Tomato Spotted Wilt Virus in transgenic tobacco. Transgenic Res 14:193–206. doi: 10.1007/s11248-004-5413-8 PubMedCrossRefGoogle Scholar
  23. Maghuly F, Stephan L, da Câmara Machado A, Borroto Fernandez E, Mahmood AK, Gambino G, Gribaudo I, Schartl A, Laimer M (2006) Molecular characterization of grapevine plants transformed with GFLV esistance genes: II. Plant Cell Rep 25:546–553. doi: 10.1007/s00299-005-0087-0 PubMedCrossRefGoogle Scholar
  24. Milne RG, Garcia ML, Moreno P (2003) Citrus psorosis virus. Association of applied biologists (AAB) descriptions of plant viruses. http://www.dpvweb.net/dpv/showdpv.php?dpvno=401
  25. Naum-Ongania G, Gago-Zachert S, Pena 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 PubMedCrossRefGoogle Scholar
  26. Palle SR, Miao H, Seyran M, Louzada ES, da Graça JV, Skaria M (2004) Preliminary evidence for natural transmission of citrus psorosis virus by an olpidium-like fungus. In: 16th conference of the international organization of citrus virologists, IOCV, Abstract conference, MexicoGoogle Scholar
  27. Pang SZ, Nagpala P, Wang M, Slightom JL, Gonsalves D (1992) Resistance to heterologous isolates of tomato spotted wilt virus in transgenic tobacco expressing its nucleocapsid protein gene. Phytopathology 82:1223–1229CrossRefGoogle Scholar
  28. Peña L, Cervera M, Juárez J, Navarro A, Pina JA, Durán-Vila N, Navarro L (1995) Agrobacterium-mediated transformation of sweet orange and regeneration of transgenic plant. Plant Cell Rep 14:616–619. doi: 10.1007/BF00232724 CrossRefGoogle Scholar
  29. Powell-Abel P, Nelson RS, De B, Hoffmann N, Rogers SG, Fraley RT, Beachy RN (1986) Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene. Science 232:738–743. doi: 10.1126/science.3457472 CrossRefGoogle Scholar
  30. Prins M de Haan P, Luyten R, van Veller M, van Grinsven MQ, Goldbach R (1995) Broad resistance to tospoviruses in transgenic tobacco plants expressing three tospoviral nucleoprotein gene sequences. Mol Plant Microbe Interact 8:85–91Google Scholar
  31. Ravelonandro M, Scorza R, Bachelier JC, Labonne G, Levy L, Damsteegt V (1997) Resistance of Prunus domestica L. to plum pox virus infection. Plant Dis 81:1231–1235CrossRefGoogle Scholar
  32. Roistacher CN (1991) Graft-transmissible diseases of citrus. In: Food and Agriculture Organization of the United Nations, FAO (ed) Handbook for detection and diagnosis, Rome, pp 115–126Google Scholar
  33. Roistacher CN (1993) Psorosis-A review. In: Moreno P, da Graça JV, Timmer LW (eds) Proceedings of the 12th conference of the international organization of citrus virologists, IOCV, Riverside, pp 139–154Google Scholar
  34. Sánchez de la Torre ME, Riva O, Zandomeni R, Grau O, García ML (1998) Mol Plant Pathol. Online http://www.bspp.org.uk/mppol/1998/1019sanchez
  35. Sánchez de la Torre ME, López C, Grau O, García ML (2002) RNA2 of citrus psorosis virus is of negative polarity and has a single open reading frame in its complementary strand. J Gen Virol 83:1777–1781Google Scholar
  36. Sanford JC, Johnston SA (1985) The concept of parasite-derived resistance—deriving resistance genes from the parasite’s own genome. J Theor Biol 113:395–405CrossRefGoogle Scholar
  37. Scorza R, Callahan A, Levy L, Damsteegt V, Webb K, Revelonandro M (2001) Post-transcriptional gene silencing in plum pox virus resistant transgenic European plum containing the plum potyvirus coat protein gene. Transgenic Res 10:201–209. doi: 10.1023/A:1016644823203 PubMedCrossRefGoogle Scholar
  38. Swingle WT, Webber HJ (1896) The principal diseases of citrus fruits in Florida. United States Department of Agriculture, Division of Vegetable Physiology and Pathology, Bulletin 8Google Scholar
  39. Vancanneyt G, Schmidt R, O´Connor-Sanchez L, Willmitzer L, Rocha-Sosa M (1990) Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation. Mol Gen Genet 220:245–250PubMedCrossRefGoogle Scholar
  40. Zanek MC, Peña EJ, 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: 101016/jjviromet200606021 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • María Cecilia Zanek
    • 1
    Email author
  • Carina Andrea Reyes
    • 1
  • Magdalena Cervera
    • 3
  • Eduardo José Peña
    • 1
  • Karelia Velázquez
    • 1
  • Norma Costa
    • 2
  • Maria Inés Plata
    • 2
  • Oscar Grau
    • 1
  • Leandro Peña
    • 3
  • María Laura García
    • 1
  1. 1.Facultad de Ciencias ExactasInstituto de Bioquímica y Biología Molecular (IBBM), U.N.L.P.La PlataArgentina
  2. 2.Estación Experimental Agropecuaria, INTAConcordiaArgentina
  3. 3.Dpto de Protección Vegetal y BiotecnologiaInstituto Valenciano de Investigaciones Agrarias (IVIA)MoncadaSpain

Personalised recommendations