, Volume 256, Issue 4, pp 951–969 | Cite as

Two grapevine metacaspase genes mediate ETI-like cell death in grapevine defence against infection of Plasmopara viticola

  • Peijie GongEmail author
  • Michael Riemann
  • Duan Dong
  • Nadja Stoeffler
  • Bernadette Gross
  • Armin Markel
  • Peter Nick
Original Article


Metacaspase, as hypersensitive response (HR) executors, has been identified in many plant species. Previously, the entire gene family of metacaspase has been uncovered, but there are still questions that remain unclear regarding HR-regulating gene members. In this study, based on metacaspase expression during different grapevine genotypes interacting with Plasmopara viticola, we identified MC2 and MC5 as candidates involved in HR. We overexpressed both metacaspases as GFP fusions in tobacco BY-2 cells to address subcellular localization and cellular functions. We found MC2 located at the ER, while MC5 was nucleocytoplasmic. In these overexpressor lines, cell death elicited by the bacterial protein harpin, is significantly enhanced, indicating MC2 and MC5 mediated defence-related programmed cell death (PCD). This effect was mitigated, when the membrane-located NADPH oxidase was inhibited by the specific inhibitor diphenylene iodonium, or when cells were complemented with methyl jasmonate, a crucial signal of basal immunity. Both findings are consistent with a role of MC2 and MC5 in cell death-related immunity. Using a dual-luciferase reporter system in grapevine cells we demonstrated both MC2 and MC5 promoter alleles from V. rupestris were more responsive to harpin than those from V. vinifera cv ‘Müller-Thurgau’, while they were not induced by MeJA as signal linked with basal immunity. These findings support a model, where MC2 and MC5 act specifically as executors of the HR.


Metacaspase Programmed cell death (PCD) Plant immunity Vitis rupestris Hypersensitive response (HR) 


Funding information

This study was kindly supported by a fellowship from the Chinese Scholarship Council to Peijie Gong, as well as a project fund from the BACCHUS Interreg project IV Oberrhein/Rhin supérieur.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

709_2019_1353_MOESM1_ESM.docx (350 kb)
ESM 1 (DOCX 349 kb)
709_2019_1353_MOESM2_ESM.docx (3.6 mb)
ESM 2 (DOCX 3.60 mb)
709_2019_1353_MOESM3_ESM.docx (3.6 mb)
ESM 3 (DOCX 3.60 mb)
709_2019_1353_MOESM4_ESM.docx (3.8 mb)
ESM 4 (DOCX 3.83 mb)
709_2019_1353_MOESM5_ESM.docx (7.2 mb)
ESM 5 (DOCX 7.18 mb)
709_2019_1353_MOESM6_ESM.docx (6.5 mb)
ESM 6 (DOCX 6.47 mb)
709_2019_1353_MOESM7_ESM.docx (3.6 mb)
ESM 7 (DOCX 3.60 mb)
709_2019_1353_MOESM8_ESM.docx (298 kb)
ESM 8 (DOCX 297 kb)
709_2019_1353_MOESM9_ESM.docx (17 kb)
ESM 9 (DOCX 16.6 kb)
709_2019_1353_MOESM10_ESM.docx (16 kb)
ESM 10 (DOCX 15.9 kb)
709_2019_1353_MOESM11_ESM.docx (54 kb)
ESM 11 (DOCX 53.6 kb)


  1. Baker CJ, Orlandi EW, Mock NM (1993) Harpin, an elicitor of the hypersensitive response in tobacco caused by Erwinia amylovora, elicits active oxygen production in suspension cells. Plant Physiol 102:1341–1344CrossRefGoogle Scholar
  2. Boller T, He SY (2009) Innate immunity in plants: an arms race between pattern recognition receptors in plants and effectors in microbial pathogens. Science 324:742–744. CrossRefGoogle Scholar
  3. Bollhoner B, Zhang B, Stael S, Denance N, Overmyer K, Goffner D, Van Breusegem F, Tuominen H (2013) Post mortem function of AtMC9 in xylem vessel elements. New Phytol 200:498–510. CrossRefGoogle Scholar
  4. Bröker LE, Kruyt FAE, Giaccone G (2005) Cell death independent of caspases: a review clinical. Cancer Res 11:3155–3162. Google Scholar
  5. Buschmann H, Green P, Sambade A, Doonan JH, Lloyd CW (2011) Cytoskeletal dynamics in interphase, mitosis and cytokinesis analysed through Agrobacterium-mediated transient transformation of tobacco BY-2 cells. New Phytol 190:258–267. CrossRefGoogle Scholar
  6. Chang XL, Nick P (2012) Defence signalling triggered by flg22 and harpin is integrated into a different stilbene output in vitis cells. PLoS One 7(7):e40446. CrossRefGoogle Scholar
  7. Chang X, Heene E, Qiao F, Nick P (2011) The phytoalexin resveratrol regulates the initiation of hypersensitive cell death in Vitis cell. PLoS One 6(10):e26405. CrossRefGoogle Scholar
  8. Chang XL, Seo M, Takebayashi Y, Kamiya Y, Riemann M, Nick P (2017) Jasmonates are induced by the PAMP flg22 but not the cell death-inducing elicitor harpin in Vitis rupestris. Protoplasma 254:271–283. CrossRefGoogle Scholar
  9. Chaumont F, Tyerman SD (2014) Aquaporins: highly regulated channels controlling plant water relations. Plant Physiol 164:1600–1618. CrossRefGoogle Scholar
  10. Chen WQ, Provart NJ, Glazebrook J, Katagiri F, Chang HS, Eulgem T, Mauch F, Luan S, Zou GZ, Whitham SA, Budworth PR, Tao Y, Xie ZY, Chen X, Lam S, Kreps JA, Harper JF, Si-Ammour A, Mauch-Mani B, Heinlein M, Kobayashi K, Hohn T, Dangl JL, Wang X, Zhu T (2002) Expression profile matrix of Arabidopsis transcription factor genes suggests their putative functions in response to environmental stresses. Plant Cell 14:559–574. CrossRefGoogle Scholar
  11. Coll NS, Vercammen D, Smidler A, Clover C, Van Breusegem F, Dangl JL, Epple P (2010) Arabidopsis type I metacaspases control cell death. Science 330:1393–1397. CrossRefGoogle Scholar
  12. Coll NS, Epple P, Dangl JL (2011) Programmed cell death in the plant immune system. Cell Death Differ 18:1247–1256. CrossRefGoogle Scholar
  13. Czemmel S, Stracke R, Weisshaar B, Cordon N, Harris NN, Walker AR, Robinson SP, Bogs J (2009) The grapevine R2R3-MYB transcription factor VvMYBF1 regulates flavonol synthesis in developing grape berries. Plant Physiol 151:1513–1530. CrossRefGoogle Scholar
  14. del Pozo O, Lam E (1998) Caspases and programmed cell death in the hypersensitive response of plants to pathogens. Curr Biol 8:1129–1132. CrossRefGoogle Scholar
  15. Despres C (2003) The Arabidopsis NPR1 Disease Resistance Protein Is a Novel Cofactor That Confers Redox Regulation of DNA Binding Activity to the Basic Domain/Leucine Zipper Transcription Factor TGA1. Plant Cell Online 15(9):2181–2191CrossRefGoogle Scholar
  16. Diaz-De-Leon F, Klotz KL, Lagrimini LM (1993) Nucleotide-Sequence of the Tobacco (Nicotiana-Tabacum) Anionic Peroxidase Gene. Plant Physiol 101:1117–1118.
  17. Duan D, Halter D, Baltenweck R, Tisch C, Tröster V, Kortekamp A, Hugueney P, Nick P (2015) Genetic diversity of stilbene metabolism in Vitis sylvestris. J Exp Bot 66:3243–3257. CrossRefGoogle Scholar
  18. Duan D, Fischer S, Merz P, Bogs J, Riemann M, Nick P (2016) An ancestral allele of grapevine transcription factor MYB14 promotes plant defence. J Exp Bot 67:1795–1804. CrossRefGoogle Scholar
  19. Eibach R, Zyprian E, Welter L, Töpfer R (2007) The use of molecular markers for pyramiding resistance genes in grapevine breeding. Vitis 46:120–124Google Scholar
  20. Eulgem T, Rushton PJ, Robatzek S, Somssich IE (2000) The WRKY superfamily of plant transcription factors Trends. Plant Sci 5:199–206. CrossRefGoogle Scholar
  21. Fischer BM, Salakhutdinov I, Akkurt M, Eibach R, Edwards KJ, Topfer R, Zyprian EM (2004) Quantitative trait locus analysis of fungal disease resistance factors on a molecular map of grapevine TAG theoretical and applied genetics. Theoretische und angewandte Genetik 108:501–515. CrossRefGoogle Scholar
  22. Gaff DF, Okong'O-Ogola O (1971) The use of non-permeating pigments for testing the survival of cells. J Exp Bot 22:756–758. CrossRefGoogle Scholar
  23. Gao N, Wadhwani P, Muhlhauser P, Liu Q, Riemann M, Ulrich AS, Nick P (2016) An antifungal protein from Ginkgo biloba binds actin and can trigger cell death. Protoplasma 253:1159–1174. CrossRefGoogle Scholar
  24. Gomez-Zeledon J, Zipper R, Spring O (2013) Assessment of phenotypic diversity of Plasmopara viticola on Vitis genotypes with different resistance. Crop Prot 54:221–228. CrossRefGoogle Scholar
  25. Gasser SM, Amati BB, Cardenas ME, Hofmann JF (1989) Studies on scaffold attachment sites and their relation to genome function. Int Rev Cytol 119:57–96CrossRefGoogle Scholar
  26. Hatsugai N, Kuroyanagi M, Yamada K, Meshi T, Tsuda S, Kondo M, Nishimura M, Hara-Nishimura I (2004) A plant vacuolar protease, VPE, mediates virus-induced hypersensitive cell death. Science 305:855–858. CrossRefGoogle Scholar
  27. He R, Drury GE, Rotari VI, Gordon A, Willer M, Farzaneh T, Woltering EJ, Galloi P (2008) Metacaspase-8 modulates programmed cell death induced by ultraviolet light and H2O2 in Arabidopsis. J Biol Chem 283:774–783. CrossRefGoogle Scholar
  28. Holl J, Vannozzi A, Czemmel S, D'Onofrio C, Walker AR, Rausch T, Lucchin M, Boss PK, Dry IB, Bogs J (2013) The R2R3-MYB transcription factors MYB14 and MYB15 regulate stilbene biosynthesis in Vitis vinifera. Plant Cell 25:4135–4149. CrossRefGoogle Scholar
  29. Horstmann V, Huether CM, Jost W, Reski R, Decker EL (2004) Quantitative promoter analysis in Physcomitrella patens: a set of plant vectors activating gene expression within three orders of magnitude. BMC Biotechnol 4:13. CrossRefGoogle Scholar
  30. Huang L, Zhang H, Hong Y, Liu S, Li D, Song F (2015) Stress-responsive expression, subcellular localization and protein-protein interactions of the rice metacaspase family. Int J Mol Sci 16:16216–16241. CrossRefGoogle Scholar
  31. Ismail A, Riemann M, Nick P (2012) The jasmonate pathway mediates salt tolerance in grapevines. J Exp Bot 63:2127–2139. CrossRefGoogle Scholar
  32. Jones JD, Dangl JL (2006) The plant immune system. Nature 444:323–329. CrossRefGoogle Scholar
  33. Klemencic M, Funk C (2018) Structural and functional diversity of caspase homologues in non-metazoan organisms. Protoplasma 255:387–397. CrossRefGoogle Scholar
  34. Kumar GM, Mamidala P, Podile AR (2009) Regulation of Polygalacturonase-inhibitory proteins in plants is highly dependent on stress and light responsive elements. Plant Omics 2:238–249Google Scholar
  35. Laloi C (2004) The Arabidopsis Cytosolic Thioredoxin h5 Gene Induction by Oxidative Stress and Its W-Box-Mediated Response to Pathogen Elicitor. Plant Physiol 134(3):1006–1016CrossRefGoogle Scholar
  36. Lam E (2004) Controlled cell death, plant survival and development. Nat Rev Mol Cell Biol 5:305–315. CrossRefGoogle Scholar
  37. Lescot M, Dehais P, Thijs G, Marchal K, Moreau Y, Peer Y, Rouze 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 30:325–327CrossRefGoogle Scholar
  38. Luo H, Song F, Goodman RM, Zheng Z (2005) Up-regulation of OsBIHD1, a rice gene encoding BELL homeo-domain transcriptional factor, in disease resistance responses. Plant Biol 7:459–468. CrossRefGoogle Scholar
  39. Maleck K, Levine A, Eulgem T, Morgan A, Schmid J, Lawton KA, Dangl JL, Dietrich RA (2000) The transcriptome of Arabidopsis thaliana during systemic acquired resistance. Nat Genet 26(4):403–410CrossRefGoogle Scholar
  40. Nagata T, Nemoto Y, Hasezawa S (1992) Tobacco BY-2 Cell Line as the “HeLa” Cell in the Cell Biology of Higher Plants. Int Rev Cytol 132:1–30. CrossRefGoogle Scholar
  41. Nick P (2014) Schützen und nützen - von der Erhaltung zur Anwendung. Fallbeispiel Europäische Wildrebe. Handbuch Genbank WEL Hoppea Denkschr Regensb Bot Ges Sonderband:159–173Google Scholar
  42. Nocarova E, Fischer L (2009) Cloning of transgenic tobacco BY-2 cells; an efficient method to analyse and reduce high natural heterogeneity of transgene expression. BMC Plant Biol 9:44. CrossRefGoogle Scholar
  43. Park HC, Kim ML, Kang YH, Jeon JM, Yoo JH, Kim MC, Park CY, Jeong JC, Moon BC, Lee JH, Yoon HW, Lee SH, Chung WS, Lim CO, Lee SY, Hong JC, Cho MJ (2004) Pathogen- and NaCl-induced expression of the SCaM-4 promoter is mediated in part by a GT-1 box that interacts with a GT-1-like transcription factor. Plant Physiol 135:2150–2161. CrossRefGoogle Scholar
  44. Piszczek E, Gutman W (2007) Caspase-like proteases and their role in programmed cell death in plants. Acta Physiol Plant 29:391–398. CrossRefGoogle Scholar
  45. Pontier D, Balague C, Roby D (1998) The hypersensitive response. A programmed cell death associated with plant resistance. Cr Acad Sci Iii-Vie 321:721–734. CrossRefGoogle Scholar
  46. Rao S, El-Habbak M, Haudenshield JS, Zheng D, Hartman GL, Korban SS, Ghabrial SA (2010) Over-expression of the calmodulin gene SCaM-4 in soybean enhances resistance to Phytophthora sojae. Phytopathology 100:S107–S107Google Scholar
  47. Repka V, Fischerova I, Silharova K (2004) Methyl jasmonate is a potent elicitor of multiple defense responses in grapevine leaves and cell-suspension cultures. Biol Plant 48:273–283. CrossRefGoogle Scholar
  48. Rouxel M, Mestre P, Comont G, Lehman BL, Schilder A, Delmotte F (2013) Phylogenetic and experimental evidence for host-specialized cryptic species in a biotrophic oomycete. New Phytol 197:251–263. CrossRefGoogle Scholar
  49. Seibicke T (2002) Untersuchungen zur induzierten Resistenz a Vitis spec. PhD thesis University of FreiburgGoogle Scholar
  50. Suarez MF, Filonova LH, Smertenko A, Savenkov EI, Clapham DH, von Arnold S, Zhivotovsky B, Bozhkov PV (2004) Metacaspase-dependent is essential for PCD in plant embryogenesis. Curr Biol 14:339–340. CrossRefGoogle Scholar
  51. Svyatyna K, Jikumaru Y, Brendel R, Reichelt M, Mithofer A, Takano M, Kamiya Y, Nick P, Riemann M (2014) Light induces jasmonate-isoleucine conjugation via OsJAR1-dependent and -independent pathways in rice. Plant Cell Environ 37:827–839. CrossRefGoogle Scholar
  52. Takken FLW, Tameling WIL (2009) To nibble at plant resistance proteins. Science 324:744–746. CrossRefGoogle Scholar
  53. Thomma BP, Nurnberger T, Joosten MH (2011) Of PAMPs and effectors: the blurred PTI-ETI dichotomy. Plant Cell 23:4–15. CrossRefGoogle Scholar
  54. Trondle D, Schroder S, Kassemeyer HH, Kiefer C, Koch MA, Nick P (2010) Molecular phylogeny of the genus Vitis (Vitaceae) based on plastid markers. Am J Bot 97:1168–1178. CrossRefGoogle Scholar
  55. Tsuda K, Katagiri F (2010) Comparing signaling mechanisms engaged in pattern-triggered and effector-triggered immunity. Curr Opin Plant Biol 13:459–465. CrossRefGoogle Scholar
  56. Wang X, Feng H, Tang C, Bai P, Wei G, Huang L, Kang Z (2012) TaMCA4, a novel wheat metacaspase gene functions in programmed cell death induced by the fungal pathogen Puccinia striiformis f. sp. tritici. Molecular plant-microbe interactions. MPMI 25:755–764. CrossRefGoogle Scholar
  57. Watanabe N, Lam E (2011) Arabidopsis metacaspase 2d is a positive mediator of cell death induced during biotic and abiotic stresses. Plant J 66:969–982. CrossRefGoogle Scholar
  58. Xu SX, Liu GS, Chen RD (2006) Characterization of an anther- and tapetum-specific gene and its highly specific promoter isolated from tomato. Plant Cell Rep 25(3):231–240CrossRefGoogle Scholar
  59. Zhang CH, Gong PJ, Wei R, Li SX, Zhang XT, Yu YH, Wang YJ (2013) The metacaspase gene family of Vitis vinifera L.: characterization and differential expression during ovule abortion in stenospermocarpic seedless grapes. Gene 528:267–276. CrossRefGoogle Scholar
  60. Zhang X, Wu Q, Cui S, Ren J, Qian W, Yang Y, He S, Chu J, Sun X, Yan C, Yu X, An C (2015) Hijacking of the jasmonate pathway by the mycotoxin fumonisin B1 (FB1) to initiate programmed cell death in Arabidopsis is modulated by RGLG3 and RGLG4. J Exp Bot 66:2709–2721. CrossRefGoogle Scholar
  61. Zhou DX (1999) Regulatory mechanism of plant gene transcription by GT-elements and GT-factors. Trends Plant Sci 4:210–214CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Botanical Institute, Karlsruhe Institute of TechnologyKarlsruheGermany

Personalised recommendations