Plant Cell Reports

, Volume 37, Issue 5, pp 819–832 | Cite as

Overexpression of VpPR10.1 by an efficient transformation method enhances downy mildew resistance in V. vinifera

  • Hang Su
  • Yun-Tong Jiao
  • Fang-Fang Wang
  • Yue-E Liu
  • Wei-Li Niu
  • Guo-Tian Liu
  • Yan Xu
Original Article


Key message

Putrescine and spermidine increase the transformation efficiency of Vitis vinifera L. cv. Thompson seedless. Accumulation of VpPR10.1 in transgenic V. vinifera Thompson seedless, likely increases its resistance to downy mildew.


A more efficient method is described for facilitating Agrobacterium-mediated transformation of Vitis vinifera L. cv. Thompson Seedless somatic embryogenesis using polyamines (PAs). The efficacies of putrescine, spermidine and spermine are identified at a range of concentrations (10 µM, 100 µM and 1 mM) added to the culture medium during somatic embryo growth. Putrescine (PUT) and spermidine (SPD) promote the recovery of proembryonic masses (PEM) and the development of somatic embryos (SE) after co-cultivation. Judging from the importance of the time-frame in genetic transformation, PAs added at the co-cultivation stage have a stronger effect than delayed selection treatments, which are superior to antibiotic treatments in the selection stage. Best embryogenic responses are with 1 mM PUT and 100 µM SPD added to the co-culture medium. Using the above method, a pathogenesis-related gene (VpPR10.1) from Chinese wild Vitis pseudoreticulata was transferred into Thompson Seedless for functional evaluation. The transgenic line, confirmed by western blot analysis, was inoculated with Plasmopara viticola to test for downy mildew resistance. Based on observed restrictions of hyphal growth and increases in H2O2 accumulation in the transgenic plants, the accumulation of VpPR10.1 likely enhanced the transgenic plants resistance to downy mildew.


Grapevine Polyamine Transformation Downy mildew VpPR10.1 Resistance 



This work was supported by the National Natural Science Foundation of China (Grant Nos. 31471844, 31601716, 31272125). Support was also received from the “948” Program, Ministry of Agriculture, China (Grant No. 2016-X11). The authors thank Dr. Alexander (Sandy) Lang from RESCRIPT Co. (New Zealand) for useful comments and language editing which have greatly improved the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Adie BA, Pérez-Pérez J, Pérez-Pérez MM, Godoy M, Sánchez-Serrano J-J, Schmelz EA, Solano R (2007) ABA is an essential signal for plant resistance to pathogens affecting JA biosynthesis and the activation of defenses in Arabidopsis. Plant Cell 19:1665–1681CrossRefPubMedGoogle Scholar
  2. Agüero CB, Meredith CP (2006) Genetic transformation of Vitis vinifera L. cvs Thompson seedless and chardonnay with the pear PGIP and GFP encoding genes. Vitis 45:1–8Google Scholar
  3. Alexander D et al (1993) Increased tolerance to two oomycete pathogens in transgenic tobacco expressing pathogenesis-related protein 1a. Proc Natl Acad Sci USA 90:7327–7331CrossRefPubMedPubMedCentralGoogle Scholar
  4. Alvarez I, Tomaro ML, Benavides MP (2003) Changes in polyamines, proline and ethylene in sunflower calluses treated with NaCl. Plant Cell Tissue Organ Cult 74:51–59CrossRefGoogle Scholar
  5. Arun M et al (2016) Involvement of exogenous polyamines enhances regeneration and Agrobacterium. 3 Biotech 6:1–12Google Scholar
  6. Asselbergh B, Curvers K, França SC, Audenaert K, Vuylsteke M, Van Breusegem F, Höfte M (2007) Resistance to Botrytis cinerea in sitiens, an abscisic acid-deficient tomato mutant, involves timely production of hydrogen peroxide and cell wall modifications in the epidermis. Plant Physiol 144:1863–1877CrossRefPubMedPubMedCentralGoogle Scholar
  7. Bais HP, Ravishankar G (2002) Role of polyamines in the ontogeny of plants and their biotechnological applications. Plant Cell Tissue Organ Culture 69:1–34CrossRefGoogle Scholar
  8. Bornhoff B-A, Harst M, Zyprian E, Töpfer R (2005) Transgenic plants of Vitis vinifera cv. Seyval blanc. Plant Cell Rep 24:433–438CrossRefPubMedGoogle Scholar
  9. Bouquet A, Torregrosa L, Iocco P, Thomas MR (2008) Grapes. In: Kole C, Hall TC (eds) Compendium of transgenic crop plants: transgenic temperate fruits and nuts. Wiley-Blackwell, Oxford, pp 189–232CrossRefGoogle Scholar
  10. Breiteneder H et al (1989) The gene coding for the major birch pollen allergen Betv1, is highly homologous to a pea disease resistance response gene. EMBO J 8:1935Google Scholar
  11. Castro A, Vidal S, de León IP (2016) Moss pathogenesis-related-10 Protein enhances resistance to Pythium irregulare in Physcomitrella patens and Arabidopsis thaliana. Front Plant Sci 7:580PubMedPubMedCentralGoogle Scholar
  12. Cavalier-smith T (1998) A revised six-kingdom system of life. Biol Rev 73:203–266CrossRefPubMedGoogle Scholar
  13. Chong-Pérez B, Reyes M, Rojas L, Ocaña B, Pérez B, Kosky RG, Angenon G (2012) Establishment of embryogenic cell suspension cultures and Agrobacterium-mediated transformation in banana cv.‘Dwarf Cavendish’(Musa AAA): effect of spermidine on transformation efficiency Plant Cell. Tissue Organ Culture (PCTOC) 111:79–90CrossRefGoogle Scholar
  14. Christensen AB et al (2002) The molecular characterization of two barley proteins establishes the novel PR-17 family of pathogenesis-related proteins. Mol Plant Pathol 3:135–144CrossRefPubMedGoogle Scholar
  15. Dai L et al (2015) Establishment of a picloram-induced somatic embryogenesis system in Vitis vinifera cv. chardonnay and genetic transformation of a stilbene synthase gene from wild-growing Vitis species. Plant Cell Tissue Organ Cult (PCTOC) 121:397–412CrossRefGoogle Scholar
  16. Dai L et al. (2016) The novel gene VPPR4-1 from Vitis pseudoreticulata increases powdery mildew resistance in transgenic Vitis vinifera L. Front Plant Sci 7:695PubMedPubMedCentralGoogle Scholar
  17. Dhekney SA, Li ZT, Zimmerman TW, Gray DJ (2009) Factors influencing genetic transformation and plant regeneration of Vitis American. J Enol Viticult 60:285–292Google Scholar
  18. Díez-Navajas AM, Greif C, Poutaraud A, Merdinoglu D (2007) Two simplified fluorescent staining techniques to observe infection structures of the oomycete Plasmopara viticola in grapevine leaf tissues. Micron 38:680–683CrossRefPubMedGoogle Scholar
  19. Ebadi A, Aalifar M, Farajpour M, Fatahi Moghaddam M (2016) Investigating the most effective factors in the embryo rescue technique for use with ‘Flame Seedless’ grapevine (Vitis vinifera). J Horticult Sci Biotechnol 91:441–447CrossRefGoogle Scholar
  20. Fagoaga C et al (2001) Increased tolerance to Phytophthora citrophthora in transgenic orange plants constitutively expressing a tomato pathogenesis related protein PR-5. Mol Breed 7:175–185CrossRefGoogle Scholar
  21. Fernandes H, Michalska K, Sikorski M, Jaskolski M (2013) Structural and functional aspects of PR-10 proteins. Febs J 280:1169–1199CrossRefPubMedGoogle Scholar
  22. Franks T, Gang He D, Thomas M (1998) Regeneration of transgenic shape Vitis vinifera L. Sultana plants: genotypic phenotypic analysis. Mol Breed 4:321–333CrossRefGoogle Scholar
  23. Galambos A, Zok A, Kuczmog A, Oláh R, Putnoky P, Ream W, Szegedi E (2013) Silencing Agrobacterium oncogenes in transgenic grapevine results in strain-specific crown gall resistance. Plant Cell Rep 32:1751–1757CrossRefPubMedGoogle Scholar
  24. Gambino G, Ruffa P, Vallania R, Gribaudo I (2007) Somatic embryogenesis from whole flowers, anthers and ovaries of grapevine (Vitis spp.). Plant Cell Tissue Organ Cult 90:79–83CrossRefGoogle Scholar
  25. Gill SS, Tuteja N (2010) Polyamines and abiotic stress tolerance in plants. Plant Signal Behav 5:26–33CrossRefPubMedPubMedCentralGoogle Scholar
  26. Gindro K, Spring J, Pezet R, Richter H, Viret O (2006) Histological and biochemical criteria for objective and early selection of grapevine cultivars resistant to Plasmopara viticola. Vitis 45:191–196Google Scholar
  27. He P (1999) Viticulture. China agriculture press, BeijingGoogle Scholar
  28. He M et al (2013) Subcellular localization and functional analyses of a PR10 protein gene from Vitis pseudoreticulata in response to Plasmopara viticola infection. Protoplasma 250:129–140CrossRefPubMedGoogle Scholar
  29. He R, Wu J, Zhang Y et al (2017) Overexpression of a thaumatin-like protein gene from Vitis amurensis improves downy mildew resistance in Vitis vinifera grapevine. Protoplasma 254:1579–1589CrossRefPubMedGoogle Scholar
  30. Hood M, Shew H (1996) Applications of KOH-aniline blue fluorescence in the study of plant-fungal interactions. Phytopathology 86:704–708CrossRefGoogle Scholar
  31. Hussain SS, Ali M, Ahmad M, Siddique KH (2011) Polyamines: natural and engineered abiotic and biotic stress tolerance in plants. Biotechnol Adv 29:300–311CrossRefPubMedGoogle Scholar
  32. Ingram DS (1981) Physiology and biochemistry of host–parasite interaction. In: Spencer DM (ed) The downy mildews, pp 143–161Google Scholar
  33. Jain S, Kumar D, Jain M, Chaudhary P, Deswal R, Sarin NB (2012) Ectopic overexpression of a salt stress-induced pathogenesis-related class 10protein (PR10) gene from peanut (Arachis hypogaea L.) affords broad spectrum abiotic stress tolerance in transgenic tobacco Plant Cell. Tissue Organ Cult (PCTOC) 109:19–31CrossRefGoogle Scholar
  34. Kiefer B, Riemann M, Büche C, Kassemeyer H-H, Nick P (2002) The host guides morphogenesis and stomatal targeting in the grapevine pathogen Plasmopara viticola. Planta 215:387–393CrossRefPubMedGoogle Scholar
  35. Koistinen KM et al (2002) Birch PR-10c is induced by factors causing oxidative stress but appears not to confer tolerance to these agents. New Phytol 155:381–391CrossRefGoogle Scholar
  36. Kuehn GD, Phillips GC (2005) Role of polyamines in apoptosis and other recent advances in plant polyamines. Crit Rev Plant Sci 24:123–130CrossRefGoogle Scholar
  37. Kumar SV, Rajam M (2005) Polyamines enhance Agrobacterium tumefaciens vir gene induction and T-DNA transfer. Plant Sci 168:475–480CrossRefGoogle Scholar
  38. Kumria R, Rajam M (2002) Alteration in polyamine titres during Agrobacterium-mediated transformation of indica rice with ornithine decarboxylase gene affects plant regeneration potential. Plant Sci 162:769–777CrossRefGoogle Scholar
  39. Lafon R, Bulit J (1981) Downy mildew of the vine. In: Spencer DM (ed) The downy mildews, pp 601–614Google Scholar
  40. Li Z, Dhekney S, Dutt M, Van Aman M, Tattersall J, Kelley K, Gray D (2006) Optimizing Agrobacterium-mediated transformation of grapevine. Vitro Cell Dev Biol Plant 42:220–227CrossRefGoogle Scholar
  41. Li H, Li F-l, Du J-c, Lu H, He Z-q (2008a) Somatic embryogenesis and histological analysis from zygotic embryos in Vitis vinifera L.‘Moldova’. For Stud China 10:253CrossRefGoogle Scholar
  42. Li ZT, Dhekney S, Dutt M, Gray D (2008b) An improved protocol for Agrobacterium-mediated transformation of grapevine (Vitis vinifera L.). Plant Cell Tissue Organ Cult 93:311–321CrossRefGoogle Scholar
  43. Lindow S, Newman K, Chatterjee S, Baccari C, Iavarone AT, Ionescu M (2014) Production of Xylella fastidiosa diffusible signal factor in transgenic grape causes pathogen confusion and reduction in severity of Pierce’s disease Molecular. Plant Microbe Interact 27:244–254CrossRefGoogle Scholar
  44. Liu J-J (2004) Characterization, expression and evolution of two novel subfamilies of Pinus monticola cDNAs encoding pathogenesis-related (PR)-10 proteins. Tree Physiol 24:1377–1385CrossRefPubMedGoogle Scholar
  45. Liu D, Raghothama KG, Hasegawa PM, Bressan RA (1994) Osmotin overexpression in potato delays development of disease symptoms. Proc Natl Acad Sci 91:1888–1892CrossRefPubMedPubMedCentralGoogle Scholar
  46. Liu J-J, Ekramoddoullah AK, Piggott N, Zamani A (2005) Molecular cloning of a pathogen/wound-inducible PR10 promoter from Pinus monticola and characterization in transgenic Arabidopsis plants. Planta 221:159–169CrossRefPubMedGoogle Scholar
  47. Liu J-J, Ekramoddoullah AK, Hawkins B, Shah S (2013) Overexpression of a western white pine PR10 protein enhances cold tolerance in transgenic Arabidopsis. Plant Cell Tissue Organ Cult (PCTOC) 114:217–223CrossRefGoogle Scholar
  48. Liu R, Wang L, Zhu J, Chen T, Wang Y, Xu Y (2015) Histological responses to downy mildew in resistant and susceptible grapevines. Protoplasma 252:259–270CrossRefPubMedGoogle Scholar
  49. Méchin V, Damerval C, Zivy M (2007) Total protein extraction with TCA-acetone. In: Thiellement H, Zivy M, Damerval C, Méchin V (eds) Plant proteomics. Springer, New York, pp 1–8. Google Scholar
  50. Moons A, Prinsen E, Bauw G, Van Montagu M (1997) Antagonistic effects of abscisic acid and jasmonates on salt stress-inducible transcripts in rice roots. Plant Cell 9:2243–2259CrossRefPubMedPubMedCentralGoogle Scholar
  51. Nakano M, Hoshino Y, Mii M (1994) Regeneration of transgenic plants of grapevine (Vitis vinifera L.) via Agrobacterium rhizogenesmediated transformation of embryogenic calli. J Exp Bot 45:649–656CrossRefGoogle Scholar
  52. Oliver JP, Castro A, Gaggero C, Cascón T, Schmelz EA, Castresana C, De León IP (2009) Pythium infection activates conserved plant defense responses in mosses. Planta 230:569–579CrossRefPubMedGoogle Scholar
  53. Palomo-Ríos E, Barceló-Muñoz A, Mercado JA, Pliego-Alfaro F (2012) Evaluation of key factors influencing Agrobacterium-mediated transformation of somatic embryos of avocado (Persea americana Mill.). Plant Cell Tissue Organ Cult (PCTOC) 109:201–211CrossRefGoogle Scholar
  54. Park CJ, Kim KJ, Shin R, Park JM, Shin YC, Paek KH (2004) Pathogenesis-related protein 10 isolated from hot pepper functions as a ribonuclease in an antiviral pathway. Plant J 37:186–198CrossRefPubMedGoogle Scholar
  55. Petri C, Alburquerque N, Pérez-Tornero O, Burgos L (2005) Auxin pulses and a synergistic interaction between polyamines and ethylene inhibitors improve adventitious regeneration from apricot leaves and Agrobacterium-mediated transformation of leaf tissues. Plant Cell Tissue Organ Cult 82:105–111CrossRefGoogle Scholar
  56. Pnueli L, Hallak-Herr E, Rozenberg M, Cohen M, Goloubinoff P, Kaplan A, Mittler R (2002) Molecular and biochemical mechanisms associated with dormancy and drought tolerance in the desert legume Retama raetam. Plant J 31:319–330CrossRefPubMedGoogle Scholar
  57. Satish L et al (2016) Influence of plant growth regulators and spermidine on somatic embryogenesis and plant regeneration in four Indian genotypes of finger millet (Eleusine coracana (L.) Gaertn). Plant Cell Tissue Organ Cult (PCTOC) 124:15–31CrossRefGoogle Scholar
  58. Sels J, Mathys J, De Coninck BM, Cammue BP, De Bolle MF (2008) Plant pathogenesis-related (PR) proteins: a focus on PR peptides. Plant Physiol Biochem 46:941–950CrossRefPubMedGoogle Scholar
  59. Sikorski MM, Biesiadka J, Kasperska AE, Kopcińska J, Łotocka B, Golinowski W, Legocki AB (1999) Expression of genes encoding PR10 class pathogenesis-related proteins is inhibited in yellow lupine root nodules. Plant Sci 149:125–137CrossRefGoogle Scholar
  60. Takeuchi K et al (2016) Overexpression of RSOsPR10, a root-specific rice PR10 gene, confers tolerance against drought stress in rice and drought and salt stresses in bentgrass. Plant Cell Tissue Organ Cult (PCTOC) 127:35–46CrossRefGoogle Scholar
  61. Thomas M, Matsumoto S, Cain P, Scott N (1993) Repetitive DNA of grapevine: classes present and sequences suitable for cultivar identification. Theor Appl Genet 86:173–180CrossRefPubMedGoogle Scholar
  62. Thordal-Christensen H, Zhang Z, Wei Y, Collinge DB (1997) Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley—powdery mildew interaction. Plant J 11:1187–1194CrossRefGoogle Scholar
  63. Tillett RL, Wheatley MD, Tattersall EA, Schlauch KA, Cramer GR, Cushman JC (2012) The Vitis vinifera C-repeat binding protein 4 (VvCBF4) transcriptional factor enhances freezing tolerance in wine grape. Plant Biotechnol J 10:105–124CrossRefPubMedGoogle Scholar
  64. Torregrosa L, Iocco P, Thomas M (2002) Influence of Agrobacterium strain, culture medium, and cultivar on the transformation efficiency of Vitis vinifera L. American. J Enol Viticult 53:183–190Google Scholar
  65. Trouvelot S et al (2008) A β-1, 3 glucan sulfate induces resistance in grapevine against Plasmopara viticola through priming of defense responses, including HR-like cell death Molecular. Plant-Microbe Interact 21:232–243CrossRefGoogle Scholar
  66. Ukaji N, Kuwabara C, Takezawa D, Arakawa K, Fujikawa S (2004) Accumulation of pathogenesis-related (PR) 10/Bet v 1 protein homologues in mulberry (Morus bombycis Koidz.) tree during winter. Plant Cell Environ 27:1112–1121CrossRefGoogle Scholar
  67. Van Loon L, Van Strien E (1999) The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type proteins. Physiol Mol Plant Pathol 55:85–97CrossRefGoogle Scholar
  68. Van Loon L, Pierpoint W, Boller T, Conejero V (1994) Recommendations for naming plant pathogenesis-related proteins. Plant Mol Biol Rep 12:245–264CrossRefGoogle Scholar
  69. Vidal J, Kikkert J, Wallace P, Reisch B (2003) High-efficiency biolistic co-transformation and regeneration of’Chardonnay‘ (Vitis vinifera L.) containing npt-II and antimicrobial peptide genes. Plant Cell Rep 22:252–260CrossRefPubMedGoogle Scholar
  70. Vidal J, Gomez C, Cutanda M, Shrestha B, Bouquet A, Thomas M, Torregrosa L (2010) Use of gene transfer technology for functional studies in grapevine Australian. J Grape Wine Res 16:138–151CrossRefGoogle Scholar
  71. Wan Y, Schwaninger H, He P, Wang Y (2007) Comparison of resistance to powdery mildew and downy mildew in Chinese wild grapes. Vitis 46:132–136Google Scholar
  72. Wang Y, He P (1997) Study on inheritance of leaves’ resistance to powdery mildew in Chinese native wild. Vitis 30:19–25Google Scholar
  73. Wang Y, Liu Y, He P, Chen J, Lamikanra O, Lu J (1995) Evaluation of foliar resistance to Uncinula necator in Chinese wild Vitis species. Vitis 34:159–164Google Scholar
  74. Wang Q et al (2005) Improvement of Agrobacterium-mediated transformation efficiency and transgenic plant regeneration of Vitis vinifera L. by optimizing selection regimes and utilizing cryopreserved cell suspensions. Plant Sci 168:565–571CrossRefGoogle Scholar
  75. Wang L, Wei J, Zou Y, Xu K, Wang Y, Cui L, Xu Y (2014) Molecular Characteristics and biochemical functions of VpPR10s from Vitis pseudoreticulata associated with biotic and abiotic stresses. Int J Mol Sci 15:19162–19182CrossRefPubMedPubMedCentralGoogle Scholar
  76. Xie X, Agüero CB, Wang Y, Walker MA (2016) Genetic transformation of grape varieties and rootstocks via organogenesis. Plant Cell Tissue Organ Cult (PCTOC) 126:541–552CrossRefGoogle Scholar
  77. Xu Y, Yu H, He M, Yang Y, Wang Y (2010) Isolation and expression analysis of a novel pathogenesis-related protein 10 gene from Chinese wild Vitis pseudoreticulata induced by Uncinula necator. Biologia 65:653–659CrossRefGoogle Scholar
  78. Xu T-F, Zhao X-C, Jiao Y-T, Wei J-Y, Wang L, Xu Y (2014) A pathogenesis related protein, VpPR-10.1, from Vitis pseudoreticulata: an insight of its mode of antifungal activity. Plos One 9:e95102CrossRefPubMedPubMedCentralGoogle Scholar
  79. Yamamoto M, Torikai S, Oeda K (1997) A major root protein of carrots with high homology to intracellular pathogenesis-related (PR) proteins and pollen allergens. Plant Cell Physiol 38:1080–1086CrossRefPubMedGoogle Scholar
  80. Yamamoto T et al (2000) Transgenic grapevine plants expressing a rice chitinase with enhanced resistance to fungal pathogens. Plant Cell Rep 19:639–646CrossRefGoogle Scholar
  81. Yang Y, Jittayasothorn Y, Chronis D, Wang X, Cousins P, Zhong G-Y (2013) Molecular characteristics and efficacy of 16D10 siRNAs in inhibiting root-knot nematode infection in transgenic grape hairy roots. Plos One 8:e69463CrossRefPubMedPubMedCentralGoogle Scholar
  82. Zhang H et al (2011) Histological and molecular studies of the non-host interaction between wheat and Uromyces fabae. Planta 234:979CrossRefPubMedGoogle Scholar
  83. Zhang W-J, Dewey RE, Boss W, Phillippy BQ, Qu R (2013) Enhanced Agrobacterium-mediated transformation efficiencies in monocot cells is associated with attenuated defense responses. Plant Mol Biol 81:273–286CrossRefPubMedGoogle Scholar
  84. Zhou Q, Dai L, Cheng S, He J, Wang D, Zhang J, Wang Y (2014) A circulatory system useful both for long-term somatic embryogenesis and genetic transformation in Vitis vinifera L. cv. Thompson seedless. Plant Cell Tissue Organ Cult (PCTOC) 118:157–168CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.State Key Laboratory of Crop Stress Biology in Arid Areas, College of HorticultureNorthwest A&F UniversityYanglingPeople’s Republic of China
  2. 2.Key Laboratory of Horticultural Plant Biology and Germplasm Innovation in Northwest ChinaMinistry of AgricultureYanglingPeople’s Republic of China

Personalised recommendations