Probing the contractile vacuole as Achilles’ heel of the biotrophic grapevine pathogen Plasmopara viticola

Abstract

The causative agent of Grapevine Downy Mildew, the oomycete Plasmopara viticola, poses a serious threat to viticulture. In the current work, the contractile vacuole of the zoospore is analysed as potential target for novel plant protection strategies. Using a combination of electron microscopy, spinning disc confocal microscopy, and video differential interference contrast microscopy, we have followed the genesis and dynamics of this vacuole required during the search for the stomata, when the non-walled zoospore is exposed to hypotonic conditions. This subcellular description was combined with a pharmacological study, where the functionality of the contractile vacuole was blocked by manipulation of actin, by Na, Cu, and Al ions or by inhibition of the NADPH oxidase. We further observe that RGD peptides (mimicking binding sites for integrins at the extracellular matrix) can inhibit the function of the contractile vacuole as well. Finally, we show that an extract from Chinese liquorice (Glycyrrhiza uralensis) proposed as biocontrol for Downy Mildews can efficiently induce zoospore burst and that this activity depends on the activity of NADPH oxidase. The effect of the extract can be phenocopied by its major compound, glycyrrhizin, suggesting a mode of action for this biologically safe alternative to copper products.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

References

  1. Allen RN, Newhook FJ (1973) Chemotaxis of zoospores of Phytophthora cinnamomi to ethanol in capillaries of soil pore dimensions. Transact Brit Mycol Soc 61:287–302

    CAS  Article  Google Scholar 

  2. Baluška F, Šamaj J, Wojtaszek P, Volkmann D, Menzel D (2003) Cytoskeleton plasma membrane-cell wall continuum in plants. Emerging links revisited. Plant Physiol 133:482–491

    Article  PubMed  PubMed Central  Google Scholar 

  3. Bartlett DW, McClough J, Godwin JR, Hall AA, Hamer M, Parr-Dobrzanski B (2002) Thestrobilurin fungicides. Pest Manag Sci 58:649–662

    CAS  Article  PubMed  Google Scholar 

  4. Beakes GW, Glocklin SL, Sekimoto S (2012) The evolutionary phylogeny of the oomycete “fungi”. Protoplasma 249:3–19

    Article  PubMed  Google Scholar 

  5. Becker B, Doan JM, Wustman B, Carpenter EJ, Chen L, Zhang Y, Wong GK-S, Melkonian M (2015) The origin and evolution of the plant cell surface: algal integrin-associated proteins and a new family of integrin-like cytoskeleton-ECM linker proteins. Genome Biol Evolution 7:1580–1589

    CAS  Article  Google Scholar 

  6. Bleyer G, Kassemeyer H-H, Breuer M, Krause R, Viret O, Dubuis P. H, Fabre A.L, Bloesch B, Siegfried W, Naef A, Huber M (2011) “VitiMeteo”—a future-oriented forecasting system for viticulture. IOBC/Wprs Bulletin 67:69–77

  7. Brun LA, Maillet J, Richarte J, Herrmann P, Remy JC (1998) Relationships between extractable copper, soil properties and copper uptake by wild plants in vineyard soils. Environ Pollut 102:151–161

    CAS  Article  Google Scholar 

  8. Canut H, Carrasco A, Galaud JP, Cassan C, Bouyssou H, Vita N, Ferrara P, Pont-Lezica R (1998) High affinity RGD-binding sites at the plasma membrane of Arabidopsis thaliana links the cell wall. Plant J 16:63–71

    CAS  Article  PubMed  Google Scholar 

  9. Chen HR, Sheu SJ (1993) Determination of glycyrrhizin and glycyrrhetinic acid in traditional Chinese medicinal preparations by capillary electrophoresis. J Chromatogr A 29:184–188

    Article  Google Scholar 

  10. Chen J, Dai G, Gu Z, Miao Y (2002) Inhibition effect of 58 plant extracts against grape downy mildew (Plasmopara viticola). Natural Product Res Development 14:9–13

    Google Scholar 

  11. Chen WJ, Delmotte F, Richard-Cervera S, Douence L, Greif C, Corio-Costet MF (2007) At least two origins of fungicide resistance in grapevine downy mildew populations. Appl Environm Microbiol 73:5162–5172

    CAS  Article  Google Scholar 

  12. Chitcholtan K, Garrill A (2005) A beta4 integrin-like protein co-localises with a phosphotyrosine containing protein in the oomycete Achlya bisexualis: inhibition of tyrosine phosphorylation slows tip growth. Fung Genet Biol 42:534–545

    CAS  Article  Google Scholar 

  13. Cho CW, Fuller MS (1989) Observations of the water expulsion vacuole of Phytophthora palmivora. Protoplasma 149:47–55

    Article  Google Scholar 

  14. Dagostin S, Formolo T, Giovannini O, Pertot I, Schmitt A (2010) Salvia officinalis extract can protect grapevine against Plasmopara viticola. Plant Dis 94:575–580

    Article  Google Scholar 

  15. Dagostin S, Schärer HJ, Pertot I, Tamm L (2011) Are there alternatives to copper for controlling grapevine downy mildew in organic viticulture? Crop Prot 30:776–788

    CAS  Article  Google Scholar 

  16. Dercks W, Buchenauer H (1987) Comparative studies on the mode of action of aluminium ethyl phosphite in four Phytophthora species. Crop Prot 6:82–89

    CAS  Article  Google Scholar 

  17. Eggenberger K, Sanyal P, Hundt S, Wadhwani P, Ulrich AS, Nick P (2017) Challenge integrity: the cell-permeating peptide BP100 interferes with the actin-auxin oscillator. Plant Cell Physiol 58:71–85

    PubMed  Google Scholar 

  18. Eibach R, Zyprian E, Welter L, Töpfer R (2007) The use of molecular markers for pyramiding resistance genes in grapevine breeding. Vitis 46:120–124

    CAS  Google Scholar 

  19. Gay JL, Greenwood AD, Heath IB (1971) The Formation and Behaviour of Vacuoles (Vesicles) during Oosphere Development and Zoospore Germination in Saprolegnia. Microbiology 65:233–241

  20. Gessler C, Pertot I, Perazzolli M (2011) Plasmopara viticola: a review of knowledge on downy mildew of grapevine and effective disease management. Phytopathol Mediterr 50:3–44

    Google Scholar 

  21. Giraud F, Molitor D, Bleunven M, Evers D (2013) Fungicide sensitivity profiles of the Plasmopara viticola populations in the Luxembourgian grape-growing region. J Plant Pathol S1:55–62

    Google Scholar 

  22. Gómez-Zeledón J, Zipper R, Spring O (2013) Assessment of phenotypic diversity of Plasmopara viticola on Vitis genotypes with different resistance. Crop Prot 54:221–228

    Article  Google Scholar 

  23. Hardham AR (2005) Phytophthora cinnamomi. Mol Plant Pathol 6:589–604

    CAS  Article  PubMed  Google Scholar 

  24. Heath IB, Harold RL (1992) Actin has multiple roles in the formation and architecture of zoospores of the oomycetes, Saprolegnia ferax and Achlya bisexualis. J Cell Science 102:611–627

    CAS  Google Scholar 

  25. Heumann HG (1992) Microwave-stimulated glutaraldehyde and osmium tetroxide fixation of plant tissue: ultrastructural preservation in seconds. Histochemistry 97:341–347

    CAS  Article  PubMed  Google Scholar 

  26. Hostetter MK (2000) RGD-mediated adhesion in fungal pathogens of humans, plants and insects. Current Op Microbiol 3:344–348

    CAS  Article  Google Scholar 

  27. Hyde GJ, Hardham AR (1993) Microtubules regulate the generation of polarity in zoospores of Phytophthora cinnamomi. Eur J Cell Biol 62:75–85

    CAS  PubMed  Google Scholar 

  28. Hyde GJ, Lancelle S, Hepler PK, Hardham AR (1991) Freeze substitution reveals a new model for sporangial cleavage in Phytophthora, a result with implications for cytokinesis in other eukaryotes. J Cell Sci 100:735–746

    PubMed  Google Scholar 

  29. Ismail A, Takeda S, Nick P (2014) Life and death under salt stress: same players, different timing? J Exp Bot 65:2963–2979

    CAS  Article  PubMed  Google Scholar 

  30. Jürges G, Kassemeyer H-H, Dürrenberger M, Düggelin M, Nick P (2009) The mode of interaction between Vitis and Plasmopara viticola Berk. & Curt. Ex de Bary depends on the host species. Plant Biol 11:886–898

    Article  PubMed  Google Scholar 

  31. Kaminskyj SG, Heath IB (1995) Integrin and spectrin homologues, and cytoplasm-wall adhesion in tip growth. J Cell Sci 108:849–856

    CAS  PubMed  Google Scholar 

  32. Kiefer B, Riemann M, Büche C, Kassemeyer HH, Nick P (2002) The host guides morphogenesis and stomatal targeting in the grapevine pathogen Plasmopara viticola. Planta 215:387–393

    CAS  Article  PubMed  Google Scholar 

  33. Kleinig H, Sitte P (1986) Zellbiologie—ein Lehrbuch. Gustav-Fischer, Stuttgart-New York

    Google Scholar 

  34. Koch E, Enders M, Ullrich C, Molitor D, Berkelmann-Löhnertz B (2013) Effect of Primula root and other plant extracts in infection structure formation of Phyllosticta ampelicida (asexual stage of Guignardia bidwellii) and on black rot disease of grapevine in the greenhouse. J Plant Diseases Protection 120:26–33

    Article  Google Scholar 

  35. Koning AJ, Lum PY, Williams JM, Wright R (1993) DiOC6 staining reveals organelle structure and dynamics in living yeast cells. Cell Mot Cytoskelet 25:111–128

    CAS  Article  Google Scholar 

  36. Kortekamp A (2003) Leaf surface topography does not mediate tactic response of Plasmopara-zoospores to stomata. J Applied Bot 77:41–46

    Google Scholar 

  37. Kutschera U, Hossfeld U (2012) Physiological phytopathology-origin and evolution of a scientific discipline. J Appl Bot 85:1–5

    Google Scholar 

  38. Liu Q, Qiao F, Ismail A, Chang X, Nick P (2013) The plant cytoskeleton controls regulatory volume increase. BBA Membranes 1828:2111–2120

    CAS  Article  Google Scholar 

  39. Mitchell HJ, Hardham AR (1999) Characterisation of the water expulsion vacuole in Phytophthora nicotianae zoospores. Protoplasma 206:118–130

    Article  Google Scholar 

  40. Mitchell HJ, Kovac KA, Hardham AR (2002) Characterisation of Phytophthora nicotianae zoospore and cyst membrane proteins. Mycol Res 106:1211–1223

    CAS  Article  Google Scholar 

  41. Müller K, Sleumer H (1934) Biologische Untersuchungen über die Peronosporakrankheit des Weinstockes mit besonderer Berücksichtigung ihrer Bekämpfung nach der Inkubationskalendermethode. Landwirtschaftl Jahrb Z Wissenschaftl Landwirtschaft 79:509–576

    Google Scholar 

  42. Nick P (2011) Mechanics of the cytoskeleton. In: Wojtaszek P (ed) Mechanical integration of plant cells and plants. Springer, Berlin-Heidelberg, pp 53–90

    Google Scholar 

  43. Patterson DJ (1980) Contractile vacuoles and associated structures: their organization and function. Biol Rev 55:1–46

    CAS  Article  Google Scholar 

  44. Pickard BG (2008) “Second extrinsic organizational mechanism” for orienting cellulose: modeling a role for the plasmalemmal reticulum. Protoplasma 233:7–29

    CAS  Article  PubMed  Google Scholar 

  45. Riemann M, Büche C, Kassemeyer HH, Nick P (2002) Microtubules and actin microfilaments guide the establishment of cell polarity during early development of the wine pathogen Plasmopara viticola. Protoplasma 219:13–22

    CAS  Article  PubMed  Google Scholar 

  46. 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

    Article  PubMed  Google Scholar 

  47. Ruoslahti E (1996) RGD and other recognition sequences for integrins. Annu Rev Cell Develop Biol 12:697–715

    CAS  Article  Google Scholar 

  48. Schaubschläger WM, Becker G, Mazur G, Gödde M (1994) Occupational sensitization to Plasmopara viticola. J Allergy Clinical Immunol 93:457–463

    Article  Google Scholar 

  49. Scherf A, Treutwein J, Kleeberg H, Schmitt A (2012) Efficacy of leaf extract fractions of Glycyrrhiza glabra L. against downy mildew of cucumber (Pseudoperonospora cubensis). Eur J Plant Pathol 134:55–762

    Article  Google Scholar 

  50. Schuster C, Konstantinidou-Doltsinis S, Schmitt A (2010) Glycyrrhiza glabra extract protects plants against important phytopathogenic fungi. Commun Agric Appl Biol Sci 75:531–540

    CAS  PubMed  Google Scholar 

  51. Schützendübel A, Polle A (2002) Plant responses to abiotic stresses: heavy metal-induced oxidative stress and protection by mycorrhization. J Exp Bot 53:1351–1365

    PubMed  Google Scholar 

  52. Van Zwieten M, Stovold G, Van Zwieten L (2004) Literature review and inventory of alternatives to copper for disease control in the Australian organic industry. A report for the Rural Industries Research and Development Corporation. RIRDC Project DAN-208A. ISBN 0 7347 1590 0

  53. Williams MG, Magarey PA, Sivasithamparam K (2007) Effect of temperature and light intensity on early infection behaviour of a Western Australian isolate of Plasmopara viticola, the downy mildew pathogen of grapevine. Austral Plant Pathol 36:325–331

    Article  Google Scholar 

  54. Yokozawa T, Liu ZW, Chen CP (2000) Protective effects of Glycyrrhizae radix extract and its compounds in a renal hypoxia (ischemia)-reoxygenation (reperfusion) model. Phytomedicine 6:439–445

    CAS  Article  PubMed  Google Scholar 

  55. Yu ZL, Zhang JG, Wang XC, Chen J (2008) Excessive copper induces the production of reactive oxygen species, which is mediated by phospholipase D, nicotinamide adenine dinucleotide phosphate oxidase and antioxidant systems. J Integr Plant Biol 50:157–167

    CAS  Article  PubMed  Google Scholar 

  56. Zaban B, Maisch J, Nick P (2013) Dynamic actin controls polarity induction de novo in protoplasts. J Integr Plant Biol 55:142–159

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgements

We gratefully acknowledge Joachim Daumann and Kerstin Huber (Botanical Garden of the Karlsruhe Institute of Technology) for efficient support with the plant material. Also, we acknowledge Prof. Dr. Otmar Spring and Dr. Javier Goméz (University of Hohenheim) for kindly providing single-sporangia strains of P. viticola. This work was supported by the VITIFUTUR Interreg V Upper Rhine project co-financed by the European Union/European Regional Development Fund (ERDF) and the German Federal Agency for Agriculture (Programme for Sustainable Agriculture, BÖLN).

Author information

Affiliations

Authors

Corresponding author

Correspondence to Peter Nick.

Ethics declarations

Funding

This study was supported by funds from the BACCHUS Interreg IV Upper Rhine project co-financed by the European Union/European Regional Development Fund (ERDF) and the German Federal Agency for Agriculture (Programme for Sustainable Agriculture, BÖLN).

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

This study is dedicated to the memory of Peter Sitte, Albert-Ludwigs-University of Freiburg, who passed away in 2016.

Handling Editor: Uli Kutschera

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Tröster, V., Setzer, T., Hirth, T. et al. Probing the contractile vacuole as Achilles’ heel of the biotrophic grapevine pathogen Plasmopara viticola . Protoplasma 254, 1887–1901 (2017). https://doi.org/10.1007/s00709-017-1123-y

Download citation

Keywords

  • Contractile vacuole
  • Downy Mildew
  • Glycyrrhizin
  • Plasmopara viticola
  • RGD peptides