Transgenic Research

, Volume 19, Issue 6, pp 949–958 | Cite as

Resistance to crown gall disease in transgenic grapevine rootstocks containing truncated virE2 of Agrobacterium

  • Stoyanka V. Krastanova
  • Vasudevan Balaji
  • Michele R. Holden
  • Mary Sekiya
  • Baodi Xue
  • Esengul A. Momol
  • Thomas J. BurrEmail author
Original Paper


A truncated form of the Ti-plasmid virE2 gene from Agrobacterium tumefaciens strains C58 and A6, and A. vitis strain CG450 was transferred and expressed in somatic embryos of grapevine rootstocks 110 Richter (Vitis rupestris × V. berlandieri), 3309 Couderc (V. rupestris × V. riparia) and Teleki 5C (V. berlandieri × V. riparia) via Agrobacterium-mediated transformation to confer resistance to crown gall disease. Transformation was confirmed in 98% of the 322 lines by enzyme-linked immunosorbent assay for the neomycin phosphotransferase II protein and 97% of 295 lines by polymerase chain reaction for the truncated virE2 transgene. Southern blot analysis revealed the insertion of truncated virE2 at one to three loci in a subset of seven transgenic 110 Richter lines. In vitro resistance screening assays based on inoculations of shoot internode sections showed reduced tumorigenicity and very small galls in 23 of 154 transgenic lines. Non-transformed controls had a 100% tumorigenicity rate with very large galls. Disease resistance assay at the whole plant level in the greenhouse revealed seven transgenic lines (3 lines of 110 Richter, 2 lines of 3309 Couderc and 2 lines of Teleki 5C) were resistant to A. tumefaciens strain C58 and A. vitis strains TM4 and CG450 with a substantially reduced percentage of inoculation sites showing gall as compared to controls. No association was found between the level of resistance to crown gall disease and the source Agrobacterium strain of virE2. Taken together, our data showed that resistance to crown gall disease can be achieved by expressing a truncated form of virE2 in grapevines.


Truncated virE2 Crown gall disease Grapevine Agrobacterium tumefaciens Agrobacterium vitis 



We are grateful to Dr. Marc Fuchs for critically reading the manuscript.

Supplementary material

11248_2010_9373_MOESM1_ESM.docx (109 kb)
(DOCX 108 kb)


  1. Anand A, Krichevsky A, Schornack S, Lahaye T, Tzfira T, Tang Y, Citovsky V, Mysore KS (2007) Arabidopsis VirE2 interacting protein2 is required for Agrobacterium T-DNA integration in plants. Plant Cell 19:1695–1708CrossRefPubMedGoogle Scholar
  2. Anand A, Uppalapati SR, Ryu CM, Allen SN, Kang L, Tang Y, Mysore KS (2008) Salicylic acid and systemic acquired resistance play a role in attenuating crown gall disease caused by Agrobacterium tumefaciens. Plant Physiol 146:703–715CrossRefPubMedGoogle Scholar
  3. Bevan M (1984) Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res 12:8711–8721CrossRefPubMedGoogle Scholar
  4. Boodley JW, Sheldrake R (1982) Cornell peat lite mixes for commercial plant growing. Cornell Univ Coop Ext Ithaca NY. Info Bul 43Google Scholar
  5. Bouquet A, Piganeau AM, Lamaison AM (1982) Influence du genotype sur la production de cals d’embryoides et de plants entières. CR Acad Sci Paris 295:574–596Google Scholar
  6. Burr TJ, Katz BH (1984) Grapevine cuttings as potential sites of survival and means of dissemination of Agrobacterium tumefaciens and A. radiobacter biovar 3. Phytopathology 77:1424–1427CrossRefGoogle Scholar
  7. Burr TJ, Otten L (1999) Crown gall of grape: biology and disease management. Annu Rev Phytopathol 37:53–80CrossRefPubMedGoogle Scholar
  8. Burr TJ, Norelli J, Rats B, Bishop A (1990) Use of Ti plasmid DNA probes for determining tumorgenicity of Agrobacterium strains. Appl Environ Microbiol 6:1782–1785Google Scholar
  9. Burr TJ, Bazzi C, Sule S, Otten L (1998) Biology of Agrobacterium vitis and the development of disease control strategies. Plant Dis 82:1288–1297CrossRefGoogle Scholar
  10. Citovsky V, Zupan J, Warnick D, Zambryski P (1992) Nuclear localization of Agrobacterium VirE2 protein in plant cells. Science 256:1802–1805CrossRefPubMedGoogle Scholar
  11. Citovsky V, Warnick D, Zambryski P (1994) Nuclear import of Agrobacterium VirD2 and VirE2 proteins in maize and tobacco. Proc Natl Acad Sci USA 91:3210–3214CrossRefPubMedGoogle Scholar
  12. Colova Tsolova V, Perl A, Krastanova S, Tsvetkov I, Atanassov A (2001) Molecular biology and biotechnology of the grapevine. Roubelakis-Angelakis KA (ed) Kluwer, pp 411–443Google Scholar
  13. Escobar MA, Civerolo EL, Summerfelt KR, Dandekar AM (2001) RNAi-mediated oncogene silencing confers resistance to crown gall tumorigenesis. Proc Natl Acad Sci USA 98:13437–13442CrossRefPubMedGoogle Scholar
  14. Escobar MA, Lesli CA, McGranahan GH, Dandekar AM (2002) Silencing crown gall disease in walnut (Juglans regia L.). Plant Sci 163:591–597CrossRefGoogle Scholar
  15. Escobar MA, Civerolo EL, Polito VS, Pinney KA, Dandekar AM (2003) Characterization of oncogene-silenced transgenic plants: implication for Agrobacterium biology and post-transcriptional gene silencing. Mol Plant Pathol 4:57–65CrossRefPubMedGoogle Scholar
  16. Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:1515–1518CrossRefGoogle Scholar
  17. Hiroka T, Rogowsky PM, Kado CI (1987) Characterization of the virE locus of Agrobacterium tumefaciens plasmid pTiC58. J Bacteriol 169:1529–1536Google Scholar
  18. Holden M, Krastanova S, Xue B, Pang S, Sekiya M, Momol EA, Gonsalves D, Burr TJ (2003) Genetic engineering of grape for resistance to crown gall. Acta Hortic 603:481–484Google Scholar
  19. Krastanova S, Walter B, Perrin M, Bardonnet N, Pinck L, Otten L (1993) Transfer and expression of the coat protein gene of grapevine fanleaf virus in grapevine. Extended abstracts of the 11th meeting ICVG, Montreux, Switzerland, pp 79–80Google Scholar
  20. Krastanova S, Perrin M, Barbier P, Demangeat G, Cornuet P, Bardonnet N, Otten L, Pink L, Walter B (1995) Transformation of grapevine rootstock with the coat protein gene of grapevine fanleaf nepovirus. Plant Cell Rep 14:550–554CrossRefGoogle Scholar
  21. Krastanova S, Ling K, Zhu H, Xue B, Burr T, Gonsalves D (2000) Development of transgenic grapevine rootstocks with genes from grapevine fanleaf virus and grapevine leafroll associated closteroviruses 2 and 3. Acta Hortic 528:367–372Google Scholar
  22. Ling KS, Zhu HY, Alvizo H, Hu JS, Drong RF, Gonsalves D (1997) The coat protein gene of grapevine leafroll associated closterovirus-3: cloning, nucleotide sequencing and expression in transgenic plants. Arch Virol 142:1101–1116CrossRefPubMedGoogle Scholar
  23. Lodhi MA, Ye GN, Weeden NF, Reisch BI (1994) A simple and efficient method for DNA extraction from grapevine cultivars and Vitis species. Plant Mol Biol Rep 12:6–13CrossRefGoogle Scholar
  24. McCown BH, Lloyd G (1981) Woody plant medium a mineral nutrient formulation for microculture of woody plant species. HortScience 3:453Google Scholar
  25. Momol EA, Burr TJ, Reid CL, Momol SH, Otten L (1998) Genetic diversity of Agrobacterium vitis as determined by DNA fingerprinting of the 5′-end of the 23S rRNA gene and random amplified polymorphic DNA. J Appl Microbiol 85:685–692CrossRefGoogle Scholar
  26. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  27. Otten L, Ruffray R, Momol EA, Momol MT, Burr TJ (1996) Phylogenetic relationships between Agrobacterium vitis isolates and their Ti plasmids. Mol Plant Microbe Interact 9:782–786Google Scholar
  28. Pearson R, Goheen AC (1988) Compendium of grape diseases. APS Press, St. PaulGoogle Scholar
  29. Pu X, Goodman RN (1992) Induction of necrogenesis by Agrobacterium tumefaciens on grape explants. Physiol Mol Plant Pathol 1:241–254CrossRefGoogle Scholar
  30. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  31. Schroth MN, McCain AH, Foottt JH, Huisman OC (1988) Reduction in yield and vigor of grapevine caused by crown gall disease. Plant Dis 72:241–246CrossRefGoogle Scholar
  32. Stover EW, Swartz HJ, Burr TJ (1997) Crown gall formation in a diverse collection of Vitis genotypes inoculated with Agrobacterium vitis. Am J Enol Vitic 48:26–32Google Scholar
  33. Sule S, Burr TJ (1998) The effect of resistance of rootstocks to crown gall (Agrobacterium ssp.) on the susceptibility of scions in grapevine cultivars. Plant Pathol 47:84–88CrossRefGoogle Scholar
  34. Tzfira T, Vaidya M, Citovsky V (2001) VIP1, an Arabidopsis protein that interacts with Agrobacterium VirE2, is involved in VirE2 nuclear import and Agrobacterium infectivity. EMBO J 20:3596–3607CrossRefPubMedGoogle Scholar
  35. Vidal J, Kikkert J, Malnoy M, Wallace P, Barnard J, Reisch B (2006) Evaluation of transgenic “Chardonnay” (Vitis vinifera) containing magainin genes for resistance to crown gall and powdery mildew. Transgenic Res 15:69–82CrossRefPubMedGoogle Scholar
  36. Viss WJ, Pitrak J, Humann MC, Driver J, Ream W (2003) Crown gall resistant transgenic apple trees that silence Agrobacterium tumefaciens oncogenes. Mol Breed 4:283–295CrossRefGoogle Scholar
  37. Ward DV, Zupan JK, Zambryski PC (2002) Agrobacterium VirE2 gets the VIP1 treatment in plant nuclear import. Trends Plant Sci 7:1–3CrossRefPubMedGoogle Scholar
  38. Winans SC, Allenza P, Stachel SE, McBride KE, Nester EW (1987) Characterization of the virE operon of the Agrobacterium Ti plasmid pTiA6. Nucleic Acids Res 5:825–837CrossRefGoogle Scholar
  39. Xue B, Ling KS, Reid CL, Krastanova S, Sekiya M, Momol EA, Sule S, Mozsar J, Gonsalves D, Burr TJ (1999) Transformation of five rootstocks with plant virus genes and a virE2 gene from Agrobacterium tumefaciens. In Vitro Cell Dev Biol Plant 35:226–231CrossRefGoogle Scholar
  40. Zhu Y, Nam J, Humara JM, Mysore KS (2003) Identification of Arabidopsis rat mutants. Plant Physiol 132:494–505CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Stoyanka V. Krastanova
    • 1
  • Vasudevan Balaji
    • 1
  • Michele R. Holden
    • 1
  • Mary Sekiya
    • 1
  • Baodi Xue
    • 1
  • Esengul A. Momol
    • 1
  • Thomas J. Burr
    • 1
    Email author
  1. 1.Department of Plant Pathology and Plant-Microbe BiologyCornell University, New York State Agricultural Experiment StationGenevaUSA

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