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Subcellular localization and functional analyses of a PR10 protein gene from Vitis pseudoreticulata in response to Plasmopara viticola infection

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Abstract

Downy mildew, caused by the oomycete Plasmopara viticola, is a serious fungal disease in the cultivated European grapevines (Vitis vinifera L.). The class 10 of pathogenesis-related (PR) genes in grapevine leaves was reported to be accumulated at mRNA level in response to P. viticola infection. To elucidate the functional roles of PR10 genes during plant–pathogen interactions, a PR10 gene from a fungal-resistant accession of Chinese wild Vitis pseudoreticulata (designated VpPR10.2) was isolated and showed high homology to PR10.2 from susceptible V. vinifera (designated VvPR10.2). Comparative analysis displayed that there were significant differences in the patterns of gene expression between the PR10 genes from the two host species. VpPR10.2 was induced with high level in leaves infected by P. viticola, while VvPR10.2 showed a low response to this inoculation. Recombinant VpPR10.2 protein showed DNase activity against host genomic DNA and RNase activity against yeast total RNA in vitro. Meanwhile, recombinant VpPR10.2 protein inhibited the growth of tobacco fungus Alternaria alternata and over-expression of VpPR10.2 in susceptible V. vinifera enhanced the host resistance to P. viticola. The results from subcellular localization analysis showed that VpPR10.2 proteins were distributed dynamically inside or outside of host cell. Moreover, they were found in haustorium of P. viticola and nucleus of host cell which was associated with a nucleus collapse at 10 days post-inoculation. Taken together, these results suggested that VpPR10.2 might play an important role in host plant defense against P. viticola infection.

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Abbreviations

PR:

Pathogenesis-related

VpPR10.2 :

Vitis pseudoreticulata PR10.2

qRT-PCR:

Quantitative reverse transcription polymerase chain reaction

EST:

Expressed sequence tag

GST:

Glutathione-S-transferase

IPTG:

Isopropyl-beta-d-thiogalactopyranoside

hpi:

Hours post-inoculation

dpi:

Days post-inoculation

References

  • Arnoys EJ, Wang JL (2007) Dual localization: proteins in extracellular and intracellular compartments. Acta Histochem 109(2):89–110

    Article  PubMed  CAS  Google Scholar 

  • Bellin D, Peressotti E, Merdinoglu D, Wiedemann-Merdinoglu S, Adam-Blondon A-F, Cipriani G, Morgante M, Testolin R, Di Gaspero G (2009) Resistance to Plasmopara viticola in grapevine ‘Bianca’ is controlled by a major dominant gene causing localised necrosis at the infection site. TAG Theor Appl Genet 120(1):163–176

    Article  Google Scholar 

  • Borie B, Jeandet P, Parize A, Bessis R, Adrian M (2004) Resveratrol and stilbene synthase mRNA production in grapevine leaves treated with biotic and abiotic phytoalexin elicitors. Am J Enol Vitic 55(1):60–64

    CAS  Google Scholar 

  • Cadle-Davidson L (2008) Variation within and between Vitis spp. for foliar resistance to the downy mildew pathogen Plasmopara viticola. Plant Dis 92(11):1577–1584

    Article  Google Scholar 

  • Castro AJ, Carapito C, Zorn N, Magne C, Leize E, Van Dorsselaer A, Clement C (2005) Proteomic analysis of grapevine (Vitis vinifera L.) tissues subjected to herbicide stress. J Exp Bot 56(421):2783–2795

    Article  PubMed  CAS  Google Scholar 

  • Chadha P, Das R (2006) A pathogenesis related protein, AhPR10 from peanut: an insight of its mode of antifungal activity. Planta 225:213–222

    Article  PubMed  CAS  Google Scholar 

  • Chang MM, Chiang CC, Martin MW, Hadwiger LA (1993) Expression of a pea disease resistance response gene in the potato cultivar shepody. Am Potato J 70(9):635–647

    Article  Google Scholar 

  • Constabel CP, Bertrand C, Brisson N (1993) Transgenic potato plants overexpressing the pathogenesis-related STH-2 gene show unaltered susceptibility to Phytophthora infestans and potato virus X. Plant Mol Biol 22(5):775–782

    Article  PubMed  CAS  Google Scholar 

  • Daraselia ND, Tarchevskaya S, Narita JO (1996) The promoter for tomato 3-hydroxy-3-methylglutaryl coenzyme A reductase gene 2 has unusual regulatory elements that direct high-level expression. Plant Physiol 112(2):727–733

    Article  PubMed  CAS  Google Scholar 

  • Denzer H, Staudt G, Schlosser E (1995) The behavior of Plasmopara viticola on resistant and susceptible grapevine varieties. Vitis 34(2):113–117

    Google Scholar 

  • Ekramoddoullah AKM (2004) Physiology and mrolecular biology of a family of pathogenesis-related PR-10 proteins in conifers. J Crop Improv 10(1):261–280

    Article  CAS  Google Scholar 

  • Fernandes H, Bujacz A, Bujacz G, Jelen F, Jasinski M, Kachlicki P, Otlewski J, Sikorski MM, Jaskolski M (2009) Cytokinin-induced structural adaptability of a Lupinus luteus PR-10 protein. FEBS J 276(6):1596–1609

    Article  PubMed  CAS  Google Scholar 

  • Fung RWM, Gonzalo M, Fekete C, Kovacs LG, He Y, Marsh E, McIntyre LM, Schachtman DP, Qiu W (2008) Powdery mildew induces defense-oriented reprogramming of the transcriptome in a susceptible but not in a resistant grapevine. Plant Physiol 146(1):236–249

    Article  PubMed  CAS  Google Scholar 

  • Gamm M, Heloir MC, Bligny R, Vaillant-Gaveau N, Trouvelot S, Alcaraz G, Frettinger P, Clement C, Pugin A, Wendehenne D, Adrian M (2011) Changes in carbohydrate metabolism in Plasmopara viticola-infected grapevine leaves. Mol Plant Microbe Interact 24(9):1061–1073

    Article  PubMed  CAS  Google Scholar 

  • Guan X, Zhao H, Xu Y, Wang Y (2011) Transient expression of glyoxal oxidase from the Chinese wild grape Vitis pseudoreticulata can suppress powdery mildew in a susceptible genotype. Protoplasma 248(2):415–423

    Article  PubMed  CAS  Google Scholar 

  • Gutha L, Casassa L, Harbertson J, Naidu R (2010) Modulation of flavonoid biosynthetic pathway genes and anthocyanins due to virus infection in grapevine (Vitis vinifera L.) leaves. BMC Plant Biol 10(1):187

    Article  PubMed  Google Scholar 

  • Jellouli N, Ben Jouira H, Skouri H, Ghorbel A, Gourgouri A, Mliki A (2008) Proteomic analysis of Tunisian grapevine cultivar Razegui under salt stress. J Plant Physiol 165(5):471–481

    Article  PubMed  CAS  Google Scholar 

  • Kang ZS, Huang L, Buchenauer B (2003) Subcellular localization of chitinase and β-1,3-glucanase in compatible and incompatible interactions between wheat and Puccinia striiformis f. sp. tritici. Allemand 110(2):170–183

    CAS  Google Scholar 

  • Kim S, Yu S, Kang Y, Kim S, Kim J-Y, Kim S-H, Kang K (2008) The rice pathogen-related protein 10 (JIOsPR10) is induced by abiotic and biotic stresses and exhibits ribonuclease activity. Plant Cell Rep 27(3):593–603

    Article  PubMed  CAS  Google Scholar 

  • Kim SG, Kim ST, Wang Y, Yu S, Choi IS, Kim YC, Kim WT, Agrawal GK, Rakwal R, Kang KY (2011) The RNase activity of rice probenazole-induced protein1 (PBZ1) plays a key role in cell death in plants. Mol Cells 31(1):25–31

    Article  PubMed  CAS  Google Scholar 

  • Kortekamp A (2006) Expression analysis of defence-related genes in grapevine leaves after inoculation with a host and a non-host pathogen. Plant Physiol Biochem 44(1):58–67

    Article  PubMed  CAS  Google Scholar 

  • Lebel S, Schellenbaum P, Walter B, Maillot P (2010) Characterisation of the Vitis vinifera PR10 multigene family. BMC Plant Biol 10(1):184

    Article  PubMed  Google Scholar 

  • Lescot M, Déhais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouzé P, Rombauts S (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res 30(1):325–327

    Article  PubMed  CAS  Google Scholar 

  • Liu J-J, Ekramoddoullah AKM (2006) The family 10 of plant pathogenesis-related proteins: their structure, regulation, and function in response to biotic and abiotic stresses. Physiol Mol Plant Pathol 68(1–3):3–13

    Article  CAS  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using realtime quantitative PCR and the 2-DDCT method. Methods 25:402–408

    Article  PubMed  CAS  Google Scholar 

  • Lodhi M, Ye G-N, Weeden N, Reisch B (1994) A simple and efficient method for DNA extraction from grapevine cultivars and Vitis species. Plant Mol Biol Rep 12(1):6–13

    Article  CAS  Google Scholar 

  • Mogensen JE, Wimmer R, Larsen JN, Spangfort MD, Otzen DE (2002) The major birch allergen, bet v 1, shows affinity for a broad spectrum of physiological ligands. J Biol Chem 277(26):23684–23692

    Article  PubMed  CAS  Google Scholar 

  • 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(2):186–198

    Article  PubMed  CAS  Google Scholar 

  • Polesani M, Bortesi L, Ferrarini A, Zamboni A, Fasoli M, Zadra C, Lovato A, Pezzotti M, Delledonne M, Polverari A (2010) General and species-specific transcriptional responses to downy mildew infection in a susceptible (Vitis vinifera) and a resistant (V. riparia) grapevine species. BMC Genomics 11:117

    Article  PubMed  Google Scholar 

  • Puehringer HM, Zinoecker I, Marzban G, Katinger H, Laimer M (2003) MdAP, a novel protein in apple, is associated with the major allergen Mal d 1. Gene 321:173–183

    Article  PubMed  CAS  Google Scholar 

  • Robert N, Ferran J, Breda C, Coutos-Thévenot P, Boulay M, Buffard D, Esnault R (2001) Molecular characterization of the incompatible interaction of Vitis vinifera leaves with Pseudomonas syringae Pv. Pisi: expression of genes coding for stilbene synthase and class 10 PR protein. Eur J Plant Pathol 107(2):249–261

    Article  CAS  Google Scholar 

  • Santos-Rosa M, Poutaraud A, Merdinoglu D, Mestre P (2008) Development of a transient expression system in grapevine via agro-infiltration. Plant Cell Rep 27(6):1053–1063

    Article  PubMed  CAS  Google Scholar 

  • Saraste M, Sibbald PR, Wittinghofer A (1990) The P-loop—a common motif in ATP- and GTP-binding proteins. Trends Biochem Sci 15(11):430–434

    Article  PubMed  Google Scholar 

  • Schmidlin L, Poutaraud A, Claudel P, Mestre P, Prado E, Santos-Rosa M, Wiedemann-Merdinoglu S, Karst F, Merdinoglu D, Hugueney P (2008) A stress-inducible resveratrol O-methyltransferase involved in the biosynthesis of pterostilbene in grapevine. Plant Physiol 148(3):1630–1639

    Article  PubMed  CAS  Google Scholar 

  • Scott MS, Calafell SJ, Thomas DY, Hallett MT (2005) Refining protein subcellular localization. PLoS Comput Biol 1(6):e66

    Article  PubMed  Google Scholar 

  • Somssich IE, Schmelzer E, Bollmann J, Hahlbrock K (1986) Rapid activation by fungal elicitor of genes encoding “pathogenesis-related” proteins in cultured parsley cells. Proc Natl Acad Sci USA 83(8):2427–2430

    Article  PubMed  CAS  Google Scholar 

  • Srivastava S, Emery RJN, Kurepin LV, Reid DM, Fristensky B, Kav NNV (2006) Pea PR 10.1 is a ribonuclease and its transgenic expression elevates cytokinin levels. Plant Growth Regul 49(1):17–25

    Article  CAS  Google Scholar 

  • Trifonova EA, Sapotsky MV, Komarova ML, Scherban AB, Shumny VK, Polyakova AM, Lapshina LA, Kochetov AV, Malinovsky VI (2007) Protection of transgenic tobacco plants expressing bovine pancreatic ribonuclease against tobacco mosaic virus. Plant Cell Rep 26(7):1121–1126

    Article  PubMed  CAS  Google Scholar 

  • van Loon LC, Rep M, Pieterse CM (2006) Significance of inducible defense-related proteins in infected plants. Annu Rev Phytopathol 44:135–162

    Article  PubMed  Google Scholar 

  • Wan Y, Schwaninger H, He P, Wang Y (2007) Comparison of resistance to powdery mildew and downy mildew in Chinese wild grapes. Vitis 46(3):132–136

    Google Scholar 

  • 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(3):159–164

    Google Scholar 

  • Wang YP, Nowak G, Culley D, Hadwiger LA, Fristensky B (1999) Constitutive expression of pea defense gene DRR206 confers resistance to blackleg (Leptosphaeria maculans) disease in transgenic canola (Brassica napus). Mol Plant-Microbe Interact 12(5):410–418

    Article  CAS  Google Scholar 

  • Wolf G, Fric F (1981) A rapid staining method for Erysiphe graminis f. sp. hordei in and on whole barley leaves with a protein-specific dye. Phytopathology 71:596–598

    Article  Google Scholar 

  • Xie Y-R, Chen Z-Y, Brown RL, Bhatnagar D (2010) Expression and functional characterization of two pathogenesis-related protein 10 genes from Zea mays. J Plant Physiol 167(2):121–130

    Article  PubMed  CAS  Google Scholar 

  • Xu Y, Zhu Z, Xiao Y, Wang Y (2009) Construction of a cDNA Library of Vitis pseudoreticulata native to China inoculated with Uncinula necator and the analysis of potential defence-related expressed sequence tags (ESTs). S Afr Soc Enol Vitic 30(1):65–71

    CAS  Google Scholar 

  • Xu Y, Yu H, He M, Yang Y, Wang Y (2010a) Isolation and expression analysis of a novel pathogenesis-related protein 10 gene from Chinese wild Vitis pseudoreticulata induced by Uncinula necator. Biologia 65(4):653–659

    Article  CAS  Google Scholar 

  • Xu W, Yu Y, Ding J, Hua Z, Wang Y (2010b) Characterization of a novel stilbene synthase promoter involved in pathogen- and stress-inducible expression from Chinese wild Vitis pseudoreticulata. Planta 231(2):475–487

    Article  PubMed  CAS  Google Scholar 

  • Yan Q, Qi X, Jiang Z, Yang S, Han L (2008) Characterization of a pathogenesis-related class 10 protein (PR-10) from Astragalus mongholicus with ribonuclease activity. Plant Physiol Biochem 46(1):93–99

    Article  PubMed  CAS  Google Scholar 

  • Zhang J, Wang Y, Wang X, Yang K, Yang J (2003) An improved method for rapidly extracting total RNA from Vitis. J Fruit Sci 20(3):178–181

    CAS  Google Scholar 

  • Zhang R, Wang Y, Liu G, Li H (2010) Cloning and characterization of a pathogenesis-related gene (ThPR10) from Tamarix hispida. Acta Biologica Cracoviensia Series Boanica 52(2):17–25

    Google Scholar 

  • Zhou X-J, Lu S, Xu Y-H, Wang J-W, Chen X-Y (2002) A cotton cDNA (GaPR-10) encoding a pathogenesis-related 10 protein with in vitro ribonuclease activity. Plant Sci 162(4):629–636

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors thank Qingmei Han and Guoyun Zhang (State Key Laboratory of Crop Stress Biology in Arid Areas, Shaanxi 712100, P. R. China) for technical assistance. This work was supported by the National Natural Science Foundation of China (Grant no. 30971972 and no. 30700547) and the program for New Century Excellent Talents in University (NCET-10-0692).

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Authors and Affiliations

  1. State Key Laboratory of Crop Stress Biology in Arid Areas, Northwest A&F University, Yangling, Shaanxi, 712100, People’s Republic of China

    Mingyang He, Yan Xu, Jiangling Cao, Ziguo Zhu, Yuntong Jiao, Yuejin Wang, Xin Guan, Yazhou Yang, Weirong Xu & Zhenfang Fu

  2. Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (Northwest Region), Ministry of Agriculture, Beijing, People’s Republic of China

    Mingyang He, Yan Xu, Jiangling Cao, Ziguo Zhu, Yuntong Jiao, Yuejin Wang, Xin Guan, Yazhou Yang, Weirong Xu & Zhenfang Fu

  3. College of Horticulture, Northwest A & F University, Yangling, Shaanxi, 712100, People’s Republic of China

    Mingyang He, Yan Xu, Jiangling Cao, Ziguo Zhu, Yuntong Jiao, Yuejin Wang, Xin Guan, Yazhou Yang, Weirong Xu & Zhenfang Fu

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  1. Mingyang He
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  2. Yan Xu
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Correspondence to Yuejin Wang.

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Handling Editor: Peter Nick

Mingyang He and Yan Xu contributed equally to this work.

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He, M., Xu, Y., Cao, J. et al. Subcellular localization and functional analyses of a PR10 protein gene from Vitis pseudoreticulata in response to Plasmopara viticola infection. Protoplasma 250, 129–140 (2013). https://doi.org/10.1007/s00709-012-0384-8

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