Plant Molecular Biology Reporter

, Volume 24, Issue 2, pp 185–196

Genetic transformation of Rangpur lime (Citrus limonia osbeck) with thebO (bacterio-opsin) genen and its initial evaluation forPhytophthora nicotianae resistance

  • F. A. Azevedo
  • F. A. A. Mourão Filho
  • B. M. J. Mendes
  • W. A. B. Almeida
  • E. H. Schinor
  • R. Pio
  • J. M. Barbosa
  • S Guidetti-Gonzalez
  • H. Carrer
  • E. Lam
Commentary

Abstract

Transgenic plants expressing the bacterio-opsin (bO) gene can spontaneously activate programmed cell death (ped) and may enhance broad-spectrum pathogen resistance by activating an intrinsic defense pathway in plant species such as tobacco and potato. In this work, we produced transgenic Rangpur lime plants with thebO gene, viaAgrobacterium tumefaciens-mediated transformation, and evaluated these plants forPhytophthora nicotianae resistance. Two transgenic lines were successfully regenerated and transformation was confirmed by GUS activity assay, PCR analysis, Southern, Northern and Western blot analyses, in addition to detecting the expressed bO protein by an immunological approach. Evaluation forPhytophthora nicotianae resistance was carried out by plant inoculations with the pathogen and quantification of the affected area. One of the two transgenic lines showed greater tolerance to the fungal pathogen as compared to the control, with significantly smaller stem lesions after pathogen challenge. This increase in pathogen tolerance is correlated with a significantly higher level of transgene expression in this line when compared with the other transgenic line. This is the first report of the introduction of a potentially important gene into Rangpur lime to provide novel pathogen tolerance.

Key words

Agrobacterium tumefaciens disease gummosis root rot rootstock transgenic 

Abbreviations

bO

bacterio-opsin

CTV

citrus tristeza virus

HR

hypersensitive response

SAR

systemic acquired resistance

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References

  1. Abad MS, Hakimi SM, Kaniewski WK, Rommens CMT, Shulaev V, Lam E, and Shah DM (1997) Characterization of acquired resistance in lesion-mimic transgenic potato expressing bacterio-opsin. Mol Plant-Microbe Interact 10: 635–645.PubMedCrossRefGoogle Scholar
  2. Almeida WAB, Mourão-Filho FAA, Mendes BMJ, Pavan A, and Rodriguez APM (2003)Agrobacterium-mediated transformation ofCitrus sinensis andCitrus limonia epicotyl segments. Sci Agric 60: 23–29.Google Scholar
  3. Brasileiro ACM, and Dusi DMA (1999) Transformação genética de plantas. In: Cultura de tecidos e transformação genética de plantas (Eds Torres AC, Caldas LS, Buso JB) 863p (EMBRAPA-SPI/EMBRAPA-CNPH, Brasília).Google Scholar
  4. Broadbent P (1997) rootstock tolerance to biotic stress, Proc of Int Soc of Citriculture, pp. 1255–1257.Google Scholar
  5. Dellaporta SL, Wood J, and Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 4: 19–21.CrossRefGoogle Scholar
  6. Domíngues A, Guerri J, Cambra M, Navarro L, Moreno P, and Peña L, (2000) Efficient production of transgenic citrus plants expressing the coat protein gene citrus tristeza virus. Plant Cell Rep 19: 427–433.CrossRefGoogle Scholar
  7. Erwin DC, and Ribeiro OK (1996) Phytophtora Diseases Worldwide, 562p. (APS Press, London).Google Scholar
  8. Fagoaga C, Rodrigo I, Conjero V, Hinarejos C, Tuset JJ, Arnau J, Pina JA, Navarro L, and Peña L (2001) Increased tolerance toPhytophtora citrophthora in transgenic orange plants constitutively expressing a tomato pathogenesis related protein PR-5. Mol Breeding 7: 175–185.CrossRefGoogle Scholar
  9. FAO, Production Yearbook, 2006. (http://apps.fao.org).Google Scholar
  10. Febres VJ, Niblett CL, Lee RF, and Moore GA (2003) Characterization of grapefruit plants (Citrus paradisi Macf.) transformed with citrus tristeza closterovirus genes. Plant Cell Reports 21: 421–428.PubMedGoogle Scholar
  11. Feichtenberger E (2001) Doenças incitadas porPhytophthora em citros In: Doenças causadas porPhytophthora no Brasil (Eds Edmn L, Santos AF, Matsuoka K, and Bezerra JR) pp. 283–342 (Livraria e Editora Campinas, Campinas).Google Scholar
  12. Feichtenberger E (2000) Manejo integrado das principais doenças dos citros no Brasil Seminário internacional de citros: produção integrada 6: 176–216.Google Scholar
  13. Ghorbel R, López C, Fagoaga C, Moreno P, Navarro L, Flores R, and Peña L (2001) Transgenic citrus plants expressing the citrus tristeza virus p23 protein exhibit viral-like symptoms. Mol Plant Path 2: 27–36.CrossRefGoogle Scholar
  14. Graham JH, and Menge JA (2000) Phytophthora-induced diseases In: Compendium of Citrus Diseases (Eds, Timmer LW, Garnsey SM, and Graham JH) pp. 12–15 (St. Paul, MN, APS Press).Google Scholar
  15. Grosser, JW, Gmitter, FG (1990) Protoplast fusion and citrus improvement. Plant Breed Rev 8, 339–374.Google Scholar
  16. Gutiérrez-E MA, Luth D, and Moore GA (1997) Factors affecting Agrobacterium-mediated transformation inCitrus and production of sour orange (Citrus aurantium L.) plants expressing the coat protein gene of citrus tristeza virus. Plant Cell Rep 16: 745–753.CrossRefGoogle Scholar
  17. Jefferson RA (1987) Assaying chimeric genes in, plants: the GUS gene fusion system. Plant Mol Biol Rep 5: 387–405.CrossRefGoogle Scholar
  18. Kaosiri T, Zentmyer GA, and Erwin DC (1978) Stalk length as a taxonomic criterion forPhytophthora palmivora isolates from cacao. Can J Bot 56: 1730–1738.CrossRefGoogle Scholar
  19. Lam E (2004) Controlled cell death, plant survival and development. Nat Rev Mol Cell Biol 5: 305–315.PubMedCrossRefGoogle Scholar
  20. Medina Filho HP, Bordignon R, Siqueira WJ, Feichtenberger E, and Carvalho MRT (2004) Tolerância de híbridos e clones de porta-enxertos de citros à infecção de raízes porPhytophthora nicotiana. Fitopatol Bras 28: 534–540.Google Scholar
  21. Mendes BMJ, Boscariol RL, and Mourão Filho FAA, Almeida WAB (2002)Agrobacterium-mediated transformation of citrus Hamlin cultivar (Citrus sinensis L. Osbeck) epicotyl segments. Pesquisa Agropec Bras 37: 955–961.Google Scholar
  22. Mittler R, Lam E (1996) Sacrifice in the face of foes: pathogen-induced programmed cell death in higher plants. Trends Microbiol 4: 10–15.PubMedCrossRefGoogle Scholar
  23. Mittler R, Shulaev V, and Lam E (1995) Coordinated activation of programmed cell death and defense mechanisms in transgenic tobacco plants expressing a bacterial proton pump. Plant Cell 7: 29–42.PubMedCrossRefGoogle Scholar
  24. Murashige T, and Tucker DPH (1969) Growth factor requirement of citrus tissue culture. Proc First Int Citrus Symp 3: 1155–1169.Google Scholar
  25. Olivares-Fuster O, Fleming GH, Albiach-Marti MR, Gowda S, Dawson WO, Grosser JW (2003) Citrus tristeza virus (CTV) resistance in transgenic citrus based on virus challenge of protoplasts. In Vitro Cell Dev Biol Plant 39: 567–572.CrossRefGoogle Scholar
  26. Peña L, Cervera N, Ghorbel R, Domínguez A, Fagoaga C, Juárez J, Pina JA, and Navarro L (2003) Transgenic citrus. In; Plant genetic engineering, (Eds Jaiwal PK, Singh, RP) pp. 261–282 (Tx: Sci-Tech Publish, Houston).Google Scholar
  27. Pontier D, Mittler R, and Lam E (2002) Mechanism of cell death and disease resistance induction by transgenic expression of bacterio-opsin, Plant J 30: 499–509.PubMedCrossRefGoogle Scholar
  28. Siviero A, Furtado EL, Boava LP, Barbasso DV, Machado MA (2002) Avaliação de métodos de inoculação dePhytophthora parasitica em plântulas e plantas jovens de citros. Fitopatol bras 27: 574–580.CrossRefGoogle Scholar
  29. Timmer LW, Garnsey SM, and Graham JM (1993) Compedium of citrus diseases. 80 p. (APS Press: St. Paul).Google Scholar
  30. Widmer TL, Graham JH, and Mitchell DJ (1998) Histological comparasion of fibrous root infection of disease- tolerant and susceptible citrus hosts byPhytophthora nicotianae andP. Palmivora. Phytopath 88: 389–395.CrossRefGoogle Scholar
  31. Yalpani N, Silverman P, Wilson TMA, Kleier DA, and Raskin I (1991) Salicylic acid is a systemic signal and an inducer of pathogenesis-related proteins in virus-infected tobacco. Plant Cell 3: 809–818.PubMedCrossRefGoogle Scholar

Copyright information

© Springer 2006

Authors and Affiliations

  • F. A. Azevedo
    • 1
  • F. A. A. Mourão Filho
    • 2
  • B. M. J. Mendes
    • 3
  • W. A. B. Almeida
    • 4
  • E. H. Schinor
    • 2
  • R. Pio
    • 5
  • J. M. Barbosa
    • 3
  • S Guidetti-Gonzalez
    • 6
  • H. Carrer
    • 6
  • E. Lam
    • 7
  1. 1.Centro APTA Citros Sylvio Moreira-Instituto Agronômico de CampinasCordeirópolisBrazil
  2. 2.Departmento de Produção VegetalUSP/ESALQPiracicabaBrazil
  3. 3.Laboratório de Biotecnologia VegetalUSP/CENAPiracicabaBrazil
  4. 4.Escola de Agronomia da UFBACampus UniversitárioCruz das AlmasBrazil
  5. 5.Centro APTA FrutasInstituto Agronômico de Campinas-IACJundiaíBrazil
  6. 6.Depto Ciências BiológicasUSP/ESALQPiracicabaBrazil
  7. 7.Biotechnology Center, RutgersThe State University of New Jersey, Cook CollegeNew BrunswickUSA

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