Transgenic Research

, Volume 27, Issue 4, pp 379–396 | Cite as

Expression of disease resistance in genetically modified grapevines correlates with the contents of viral sequences in the T-DNA and global genome methylation

  • Daniela Dal Bosco
  • Iraci Sinski
  • Patrícia S. Ritschel
  • Umberto A. Camargo
  • Thor V. M. Fajardo
  • Ricardo Harakava
  • Vera QueciniEmail author
Original Paper


Increased tolerance to pathogens is an important goal in conventional and biotechnology-assisted grapevine breeding programs worldwide. Fungal and viral pathogens cause direct losses in berry production, but also affect the quality of the final products. Precision breeding strategies allow the introduction of resistance characters in elite cultivars, although the factors determining the plant’s overall performance are not fully characterized. Grapevine plants expressing defense proteins, from fungal or plant origins, or of the coat protein gene of grapevine leafroll-associated virus 3 (GLRaV-3) were generated by Agrobacterium-mediated transformation of somatic embryos and shoot apical meristems. The responses of the transformed lines to pathogen challenges were investigated by biochemical, phytopathological and molecular methods. The expression of a Metarhizium anisopliae chitinase gene delayed pathogenesis and disease progression against the necrotrophic pathogen Botrytis cinerea. Modified lines expressing a Solanum nigrum osmotin-like protein also exhibited slower disease progression, but to a smaller extent. Grapevine lines carrying two hairpin-inducing constructs had lower GLRaV-3 titers when challenged by grafting, although disease symptoms and viral multiplication were detected. The levels of global genome methylation were determined for the genetically engineered lines, and correlation analyses demonstrated the association between higher levels of methylated DNA and larger portions of virus-derived sequences. Resistance expression was also negatively correlated with the contents of introduced viral sequences and genome methylation, indicating that the effectiveness of resistance strategies employing sequences of viral origin is subject to epigenetic regulation in grapevine.


Chitinase Epigenetics Fungus Grapevine leafroll-associated virus 3 Pathogenesis related protein 5 Vitis 



The authors would like to thank the following for kindly sharing their expertise and excellent assistance: Renata Gava for fungus growth and aid with pathogenesis analyses, and Heitor Corbelini for grafting and conducting the plants in the greenhouse. The work was financed by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Grant No. 381107/97-3)/BioEX and Sistema Embrapa de Gestão (SEG) (Grant No. to PSR, TVMF, UAC and VQ.

Author contributions

DDB, IS and VQ performed genetic transformation experiments, plant selection and culture, fungal pathogen challenges, and insertion/expression molecular analyses. PSR and UAC designed the strategies for fungal disease resistance, and selected the grapevine cultivars. RH and TVMF designed and constructed the virus hairpin-inducing constructs and performed viral pathogen challenges. PSR, UAC, TVMF and VQ conceived the work, analyzed the results and drafted the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11248_2018_82_MOESM1_ESM.docx (19 kb)
Supplementary material 1 (DOCX 19 kb)
11248_2018_82_MOESM2_ESM.tif (2.7 mb)
Supplementary material 2 (TIFF 2734 kb)
11248_2018_82_MOESM3_ESM.tif (3.6 mb)
Supplementary material 3 (TIFF 3679 kb)
11248_2018_82_MOESM4_ESM.tif (3.7 mb)
Supplementary material 4 (TIFF 3753 kb)
11248_2018_82_MOESM5_ESM.tif (4.9 mb)
Supplementary material 5 (TIFF 5032 kb)


  1. Baránek M, Křižan B, Ondrušiková E, Pidra M (2010) DNA-methylation changes in grapevine somaclones following in vitro culture and thermotherapy. Plant Cell Tissue Organ Cult 101:11–22. CrossRefGoogle Scholar
  2. Baránek M, Čechová J, Raddová J, Holleinová V, Ondrušíková E, Pidra M (2015) Dynamics and reversibility of the DNA methylation landscape of grapevine plants (Vitis vinifera) stressed by in vitro cultivation and thermotherapy. PLoS ONE 10(5):e0126638. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Bogo MR, Rota CA, Pinto H Jr, Ocampos M, Correa CT, Vainstein MH, Schrank A (1998) A chitinase encoding gene (chit1 gene) from the entomopathogen Metarhizium anisopliae: isolation and characterisation of genomic and full-length cDNA. Curr Microbiol 73:221–225. CrossRefGoogle Scholar
  4. Bolar JP, Norelli JL, Harman GE, Brown SK, Aldwinckle HS (2001) Synergistic activity of endochitinase and exochitinase from Trichoderma atroviride (T. harzianum) against the pathogenic fungus (Venturia inaequalis) in transgenic apple plants. Transgenic Res 10(6):533–543. CrossRefPubMedGoogle Scholar
  5. Bornhoff BA, Harst M, Zyprian E, Topfer R (2005) Transgenic plants of Vitis vinifera ‘Seyval Blanc’. Plant Cell Rep 24:433–438. CrossRefPubMedGoogle Scholar
  6. Breen S, Solomon PS, Bedon F, Vincent D (2015) Surveying the potential of secreted antimicrobial peptides to enhance plant disease resistance. Front Plant Sci 6:900. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Burger JT, Maree HJ, Gouveia P, Naidu RA (2017) Grapevine leafroll-associated virus 3. In: Meng B, Martelli GP, Golino DA, Fuchs M (eds) Grapevine viruses: molecular biology, diagnostics and management. Springer, Cham, pp 167–195. CrossRefGoogle Scholar
  8. Camargo UA, Nachtigal JC, Maia JDG, Oliveira PRD, Protas JFS (2003) BRS Clara: nova cultivar de uva branca de mesa sem semente. Bento Gonçalves: Embrapa Uva e Vinho (Comunicado Técnico, 46). Accessed 12 May 2015
  9. Campos Ma, Silva MS, Magalhães CP, Ribeiro SG, Sarto RP, Vieira EA, Grossi de Sá MF (2008) Expression in Escherichia coli, purification, refolding and antifungal activity of an osmotin from Solanum nigrum. Microb Cell Fact 7:7. CrossRefPubMedCentralGoogle Scholar
  10. Carstens M, Vivier MA, Pretorius IS (2003) The Saccharomyces cerevisiae chitinase, encoded by the CTS12 gene, confers antifungal activity against Botrytis cinerea to transgenic tobacco. Transgenic Res 12:497–508. CrossRefPubMedGoogle Scholar
  11. Choi DS, Hong JK, Hwang BK (2013) Pepper osmotin-like protein 1 (CaOSM1) is an essential component for defense response, cell death, and oxidative burst in plants. Planta 238(6):1113–1124. CrossRefGoogle Scholar
  12. Chong J, Le Henanff G, Bertsch C, Walter B (2008) Identification, expression analysis and characterization of defense and signaling genes in Vitis vinifera. Plant Physiol Biochem 46:469–481. CrossRefPubMedGoogle Scholar
  13. Chowdhury S, Basu A, Kundu S (2015) Cloning, characterization, and bacterial over-expression of an osmotin-like protein gene from Solanum nigrum L. with antifungal activity against three necrotrophic fungi. Mol Biotechnol 57(4):371–381. CrossRefPubMedGoogle Scholar
  14. Chowdhury S, Basu A, Kundu S (2017) Overexpression of a new osmotin-like protein gene (SindOLP) confers tolerance against biotic and abiotic stresses in sesame. Front Plant Sci 8:410. PubMedPubMedCentralCrossRefGoogle Scholar
  15. Das M, Chauhan H, Chhibbar A, Rizwanul Haq QM, Khurana P (2011) High-efficiency transformation and selective tolerance against biotic and abiotic stress in mulberry, Morus indica cv. K2, by constitutive and inducible expression of tobacco osmotin. Transgenic Res 20(2):231–246. CrossRefPubMedGoogle Scholar
  16. Dhekney S, Li Z, Gray DJ (2011) Grapevines engineered to express cisgenic Vitis vinifera thaumatin-like protein exhibit fungal disease resistance. In Vitro Cell Dev Biol Plant 47:458–466. CrossRefGoogle Scholar
  17. Dutt M, Li ZT, Dhekney SA, Gray DJ (2007) Transgenic plants from shoot apical meristems of Vitis vinifera L. “Thompson Seedless” via Agrobacterium-mediated transformation. Plant Cell Rep 26(12):2101–2110. CrossRefPubMedGoogle Scholar
  18. Fajardo TVM, Dianese EC, Eiras M, Cerqueira DM, Lopes DB, Ferreira MASV, Martins CRF (2007) Variability of the coat protein gene of Grapevine leafroll-associated virus 3 in Brazil. Fitopatol Bras 32(4):335–340. CrossRefGoogle Scholar
  19. Fox J, Weisberg S (2011) An R companion to applied regression. Sage, Thousand Oaks. Google Scholar
  20. Gadoury DM, Seem RC, Wilcox WF, Henick-Kling T, Conterno L, Day A, Ficke A (2007) Effects of diffuse colonization of grape berries by Uncinula necator on bunch rots, berry microflora, and juice and wine quality. Phytopathology 97(10):1356–13565. CrossRefPubMedGoogle Scholar
  21. Gambino G, Gribaudo I, Leopold S, Schartl A, Laimer M (2005) Molecular characterization of grapevine plants transformed with GFLV resistance genes: I. Plant Cell Rep 24:655–662. CrossRefPubMedGoogle Scholar
  22. Gambino G, Perrone I, Gribaudo I (2008) A rapid and effective method for RNA extraction from different tissues of grapevine and other woody plants. Phytochem Anal 19(6):520–525. CrossRefPubMedGoogle Scholar
  23. 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(1):17–27. CrossRefPubMedGoogle Scholar
  24. Gamborg O, Miller RA, Ojima K (1968) Nutrient requirement of suspensions cultures of soybean root cells. Exp Cell Res 50:151–155CrossRefPubMedGoogle Scholar
  25. Gray DJ, Li ZT, Dhekney SA (2014) Precision breeding of grapevine (Vitis vinifera L.) for improved traits. Plant Sci 228:3–10. CrossRefPubMedGoogle Scholar
  26. Haile ZM, Pilati S, Sonego P, Malacarne G, Vrhovsek U, Engelen K, Tudzynski P, Zottini M, Baraldi E, Moser C (2017) Molecular analysis of the early interaction between the grapevine flower and Botrytis cinerea reveals that prompt activation of specific host pathways leads to fungus quiescence. Plant Cell Environ 40(8):1409–1428. CrossRefPubMedGoogle Scholar
  27. Harrell Jr FE, With contributions from Charles Dupont and many others (2017) Hmisc: harrell miscellaneous. R package version 4.0-3. Accessed 22 Dec 2017
  28. Hassan F, Meens J, Jacobsen H, Kiesecker H (2009) A family 19 chitinase (Chit30) from Streptomyces olivaceoviridis ATCC 11238 expressed in transgenic pea affects the development of T. harzianum in vitro. J Biotechnol 143:302–330. CrossRefPubMedGoogle Scholar
  29. He R, Wu J, Zhang Y, Agüero CB, Li X, Liu S, Wang C, Walker MA, Lu J (2017) Overexpression of a thaumatin-like protein gene from Vitis amurensis improves downy mildew resistance in Vitis vinifera grapevine. Protoplasma 254(4):1579–1589. CrossRefPubMedGoogle Scholar
  30. Hewezi T, Pantalone V, Bennett M, Neal Stewart C, Burch-Smith TM Jr (2017) Phytopathogen-induced changes to plant methylomes. Plant Cell Rep. CrossRefPubMedGoogle Scholar
  31. Iocco P, Franks T, Thomas MR (2001) Genetic transformation of major wine grape cultivars of Vitis vinifera L. Transgenic Res 10(2):105–112. CrossRefPubMedGoogle Scholar
  32. Jardak-Jamoussi R, Winterhagen P, Bouamama B, Dubois C, Mliki A, Wetzel T, Ghorbel A, Reustle GM (2009) Development and evaluation of GFLV inverted repeat construct for genetic transformation of grapevine. Plant Cell Tissue Organ Cult 97:187–196. CrossRefGoogle Scholar
  33. Jelly NS, Schellenbaum P, Walter B, Maillo P (2012) Transient expression of artificial microRNAs targeting Grapevine fanleaf virus and evidence for RNA silencing in grapevine somatic embryos. Transgenic Res 21(6):1319–1327. CrossRefPubMedGoogle Scholar
  34. Langner T, Göhre V (2016) Fungal chitinases: function, regulation, and potential roles in plant/pathogen interactions. Curr Genet 62(2):243–254. CrossRefPubMedGoogle Scholar
  35. Lê Cao K-A, Rohart F, Gonzalez I, Dejean S with Key Contributors Gautier B, Bartolo F, Contributions from Monget P, Coquery J, Yao F, Liquet B (2016). mixOmics: omics data integration project. R package version 6.1.1. Accessed 16 Feb 2017
  36. Lewsey MG, Hardcastle TJ, Melnyk CW, Molnar A, Valli A, Urich MA, Nery JR, Baulcombe DC, Ecker JR (2016) Mobile small RNAs regulate genome-wide DNA methylation. Proc Natl Acad Sci USA 113(6):E801–E810. CrossRefPubMedGoogle Scholar
  37. Li ZT, Hopkins DL, Gray DJ (2015) Overexpression of antimicrobial lytic peptides protects grapevine from Pierce’s disease under greenhouse but not field conditions. Transgenic Res 24(5):821–836. CrossRefPubMedGoogle Scholar
  38. Li ZT, Dhekney S, Dutt M, Van Aman M, Tattersall J, Kelley KT, Gray DJ (2006) Optimizing Agrobacterium-mediated transformation of grapevine. In Vitro Cell Dev Biol—Plant 42(3):220–227. CrossRefGoogle Scholar
  39. Liu G, Kennedy R, Greenshields DL, Peng G, Forseille L, Selvaraj G, Wei Y (2007) Detached and attached Arabidopsis leaf assays reveal distinctive defense responses against hemibiotrophic Colletotrichum spp. Mol Plant Microbe Interact 20(10):1308–1319. CrossRefPubMedGoogle Scholar
  40. Liu JJ, Sturrock R, Ekramoddoullah AK (2010) The superfamily of thaumatin-like proteins: its origin, evolution, and expression towards biological function. Plant Cell Rep 29(5):419–436. CrossRefPubMedGoogle Scholar
  41. Lodhi MA, Ye G-N, Weeden NF, Reisch BI (1994) A simple and efficient method for DNA extraction from grapevine cultivars, Vitis species and Ampelopsis. Plant Mol Biol Rep 12(1):6–13. CrossRefGoogle Scholar
  42. López C, Cervera M, Fagoaga C, Moreno P, Navarro L, Flores R, Peña L (2010) Accumulation of transgene-derived siRNAs is not sufficient for RNAi-mediated protection against Citrus tristeza virus in transgenic Mexican lime. Mol Plant Pathol 11(1):33–41. CrossRefPubMedGoogle Scholar
  43. Maliogka VI, Martelli GP, Fuchs M, Katis NI (2015) Control of viruses infecting grapevine. Adv Virus Res 91:175–227. CrossRefPubMedGoogle Scholar
  44. Marcato R, Sella L, Lucchetta M, Vincenzi S, Odorizzi S, Curioni A, Favaron F (2017) Necrotrophic fungal plant pathogens display different mechanisms to counteract grape chitinase and thaumatin-like protein. Physiol Mol Plant Pathol 99:7–15. CrossRefGoogle Scholar
  45. Matzke MA, Kanno T, Matzke AJ (2015) RNA-directed DNA methylation: the evolution of a complex epigenetic pathway in flowering plants. Annu Rev Plant Biol 66:243–267. CrossRefPubMedGoogle Scholar
  46. Mauro MC, Toutain A, Walter B, Pinck L, Otten L, Coutos-Thevenot P, Deloire A, Barbier P (1995) High efficiency regeneration of grapevine plants transformed with the GFLV coat protein gene. Plant Sci 112:97–106. CrossRefGoogle Scholar
  47. Miyao A, Nakagome M, Ohnuma T, Yamagata H, Kanamori H, Katayose Y, Takahashi A, Matsumoto T, Hirochika H (2012) Molecular spectrum of somaclonal variation in regenerated rice revealed by whole-genome sequencing. Plant Cell Physiol 53(1):256–264. CrossRefPubMedGoogle Scholar
  48. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497CrossRefGoogle Scholar
  49. Núñez de Cáceres González FF, Davey MR, Cancho Sanchez E, Wilson ZA (2015) Conferred resistance to Botrytis cinerea in Lilium by overexpression of the RCH10 chitinase gene. Plant Cell Rep 34(7):1201–1209. CrossRefPubMedGoogle Scholar
  50. Pooggin MM (2017) RNAi-mediated resistance to viruses: a critical assessment of methodologies. Curr Opin Virol 26:28–35. CrossRefPubMedGoogle Scholar
  51. R Core Team (2015) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna.
  52. Raham SK, Rinaldi S, Ikuo N, Masahiro M (2008) Production of transgenic potato exhibiting enhanced resistance to fungal infections and herbicide applications. Plant Biotechnol Rep 2:13–20CrossRefGoogle Scholar
  53. Ribeiro SG, Lohuis H, Goldbach R, Prins M (2007) Tomato chlorotic mottle virus is a target of RNA silencing but the presence of specific short interfering RNAs does not guarantee resistance in transgenic plants. J Virol 81(4):1563–1573. CrossRefPubMedGoogle Scholar
  54. Robert N, Roche K, Lebeau Y, Breda C, Boulay M, Esnault R, Buffard D (2002) Expression of grapevine chitinase genes in berries and leaves infected by fungal or bacterial pathogens. Plant Sci 162:389–400. CrossRefGoogle Scholar
  55. Rubio J, Montes C, Castro Á, Álvarez C, Olmedo B, Muñoz M, Tapia E, Reyes F, Ortega M, Sánchez E, Miccono M, Dalla Costa L, Martinelli L, Malnoy M, Prieto H (2015) Genetically engineered Thompson Seedless grapevine plants designed for fungal tolerance: selection and characterization of the best performing individuals in a field trial. Transgenic Res 24(1):43–60. CrossRefPubMedGoogle Scholar
  56. Schellenbaum P, Mohler V, Wenzel G, Walter B (2008) Variation in DNA methylation patterns of grapevine somaclones (Vitis vinifera L.). BMC Plant Biol 15(8):78. CrossRefGoogle Scholar
  57. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675. CrossRefPubMedPubMedCentralGoogle Scholar
  58. Seymour DK, Becker C (2017) The causes and consequences of DNA methylome variation in plants. Curr Opin Plant Biol 36:56–63. CrossRefPubMedGoogle Scholar
  59. Singh NK, Nelson DE, Kuhn D, Hasegawa PM, Bressan PA (1989) Molecular cloning of osmotin and regulation of its expression by ABA and adaptation to low water potential. Plant Physiol 90:1096–1101. CrossRefPubMedPubMedCentralGoogle Scholar
  60. Smith NA, Singh SP, Wang MB, Stoutjesdijk PA, Green AG, Waterhouse PM (2000) Total silencing by intron-spliced hairpin RNAs. Nature 407(6802):319–320. CrossRefPubMedGoogle Scholar
  61. Terakawa T, Takaya N, Horiuchi H, Koike M, Takagi M (1997) A fungal chitinase gene from Rhizopus oligosporus confers antifungal activity to transgenic tobacco. Plant Cell Rep 16:439–443. CrossRefGoogle Scholar
  62. Valat L, Fuchs M, Burrus M (2006) Transgenic grapevine rootstock clones expressing the coat protein or movement protein genes of Grapevine fanleaf virus: characterization and reaction to virus infection upon protoplast electroporation. Plant Sci 170:739–747. CrossRefGoogle Scholar
  63. Vigne E, Komar V, Fuchs M (2004) Field safety assessment of recombination in transgenic grapevines. Transgenic Res 13:165–179. CrossRefPubMedGoogle Scholar
  64. Wan R, Hou X, Wang X, Qu J, Singer SD, Wang Y, Wang X (2015) Resistance evaluation of Chinese wild Vitis genotypes against Botrytis cinerea and different responses of resistant and susceptible hosts to the infection. Front Plant Sci 6:854. PubMedPubMedCentralCrossRefGoogle Scholar
  65. Weber RL, Wiebke-Strohm B, Bredemeier C, Margis-Pinheiro M, de Brito GG, Rechenmacher C, Bertagnolli PF, de Sá ME, Campos MA, de Amorim RM, Beneventi MA, Margis R, Grossi-de-Sa MF, Bodanese-Zanettini MH (2014) Expression of an osmotin-like protein from Solanum nigrum confers drought tolerance in transgenic soybean. BMC Plant Biol 14:343. CrossRefPubMedPubMedCentralGoogle Scholar
  66. Wright K (2017) Corrgram: plot a correlogram. R package version 1.12. Accessed 8 June 2017
  67. Yamamoto T, Iketani H, Ieki H, Nishizawa Y, Notsuka K, Hibi T, Hayashi T, Matsuta N (2000) Transgenic grapevine plants expressing a rice chitinase with enhanced resistance to fungal pathogens. Plant Cell Rep 19:639–646. CrossRefGoogle Scholar
  68. Zhang W, Corwin JA, Copeland D, Feusier J, Eshbaugh R, Chen F, Atwell S, Kliebenstein DJ (2017) Plastic transcriptomes stabilize immunity to pathogen diversity: the jasmonic acid and salicylic acid networks within the Arabidopsis/Botrytis pathosystem. Plant Cell 29(11):2727–2752. CrossRefPubMedPubMedCentralGoogle Scholar
  69. Zhu B, Chen TH, Li PH (1995) Activation of two osmotin-like protein genes by abiotic stimuli and fungal pathogen in transgenic potato plants. Plant Physiol 108(3):929–937. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Embrapa Uva e VinhoBento GonçalvesBrazil
  2. 2.Instituto Biológico, Secretaria da Agricultura e AbastecimentoAgência Paulista de Tecnologia dos Agronegocios (APTA)São PauloBrazil
  3. 3.CNPUV (National Center for Grapevine and Wine Research)Embrapa (Brazilian Agricultural Corporation)Bento GonçalvesBrazil

Personalised recommendations