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

  • Genetic Transformation and Hybridization
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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.

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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

References

  • 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

    Article  Google Scholar 

  • 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 79:1531–1537

    PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Beachy RN, Loesch-Fries S, Tumer NE (1990) Coat protein-mediated resistance against virus infection. Annu Rev Phytopathol 28:451–474

    Article  CAS  Google Scholar 

  • 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 17:269–277

    Article  CAS  Google Scholar 

  • 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 159–164

  • Cervera M (2005) Histochemical and fluorometric assays for uidA (GUS) gene detection. Methods Mol Biol 286:203–214

    PubMed  CAS  Google Scholar 

  • 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 265–277

  • 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

    Article  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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 215

  • 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

    Article  CAS  Google Scholar 

  • 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 75:3585–3590

    Article  PubMed  Google Scholar 

  • 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 174–179

  • 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 63–67

  • 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 2:27–36

    Article  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  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 resistence to the virus. Mol Plant Microbe Interact 15:826–833

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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, Mexico

  • 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 82:1223–1229

    Article  CAS  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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 8:85–91

    Google Scholar 

  • 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 81:1231–1235

    Article  CAS  Google Scholar 

  • 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 115–126

  • 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 139–154

  • 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

  • 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 83:1777–1781

    Google Scholar 

  • Sanford JC, Johnston SA (1985) The concept of parasite-derived resistance—deriving resistance genes from the parasite’s own genome. J Theor Biol 113:395–405

    Article  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • 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 8

  • 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 220:245–250

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

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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.

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Authors and Affiliations

  1. Facultad de Ciencias Exactas, Instituto de Bioquímica y Biología Molecular (IBBM), U.N.L.P., Calles 47 y 115, 1900, La Plata, Argentina

    María Cecilia Zanek, Carina Andrea Reyes, Eduardo José Peña, Karelia Velázquez, Oscar Grau & María Laura García

  2. Estación Experimental Agropecuaria, INTA, Concordia, Argentina

    Norma Costa & Maria Inés Plata

  3. Dpto de Protección Vegetal y Biotecnologia, Instituto Valenciano de Investigaciones Agrarias (IVIA), Moncada, Spain

    Magdalena Cervera & Leandro Peña

Authors
  1. María Cecilia Zanek
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  2. Carina Andrea Reyes
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  3. Magdalena Cervera
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  4. Eduardo José Peña
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  5. Karelia Velázquez
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  6. Norma Costa
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  7. Maria Inés Plata
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  8. Oscar Grau
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  9. Leandro Peña
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  10. María Laura García
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Correspondence to María Cecilia Zanek.

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Communicated by W. Harwood.

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Zanek, M.C., Reyes, C.A., Cervera, M. et al. Genetic transformation of sweet orange with the coat protein gene of Citrus psorosis virus and evaluation of resistance against the virus. Plant Cell Rep 27, 57–66 (2008). https://doi.org/10.1007/s00299-007-0422-8

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  • DOI: https://doi.org/10.1007/s00299-007-0422-8

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