Jump to ContentJump to Main Navigation
Show Summary Details
More options …

Vegetable Crops Research Bulletin

2 Issues per year

Open Access
Online
ISSN
1898-7761
See all formats and pricing
More options …

A Simple Dual Stain for Detailed Investigations of Plant-Fungal Pathogen Interactions

Marcin Nowicki / Małgorzata Lichocka
  • Institute of Biochemistry and Biophysics, Polish Academy of Sciences Pawińskiego 5a, 02-106 Warszawa, Poland
  • Other articles by this author:
  • De Gruyter OnlineGoogle Scholar
/ Marzena Nowakowska / Urszula Kłosińska / Elżbieta U. Kozik
Published Online: 2013-05-15 | DOI: https://doi.org/10.2478/v10032-012-0016-z

Summary

Dramatic increase in confocal microscopy observation output has been gained by optimization of a simple trypan blue and aniline blue dual-stain and its application to two model pathosystems: Pseudoperonospora cubensiscucumber and Phytophthora infestans-tomato. Comparison of two dual-stain methods for confocal microscopy studies of P. cubensis-challenged cucumber leaves indicated the 'mild' approach most successful. This methodology provides simultaneous detection of different pathogen structures layered with the plant defense reactions. Moreover, ImageJ-assisted quantification of plant defense responses renders this method useful for addressing the host plant resistance reactions, as well as investigating the given isolate's pathogenicity. Application of this method for the P. infestans-challenged tomato leaf samples resulted in detection of several fungal infection structures, along with plant defense responses. The dual-stain also enabled detection of a peculiar aniline blue-sensitive material in the pathogen cell walls at the area of its hyphae emerging through the leaf stomata. Results presented herein indicate this method is applicable for detailed (possibly quantitative) investigations of multiple plant-fungal pathosystems.

Streszczenie

Optymalizowano metodę podwójnego barwienia (błękit trypanowy i błękit anilinowy) eksplantatów zainokulowanych liści celem mikroskopowych analiz konfokalnych dwóch patosystemów: Pseudoperonospora cubensis - ogórek oraz Phytophthora infestans - pomidor. Wskutek przeprowadzonej optymalizacji, uzyskano olbrzymi wzrost wydajności analiz obydwu badanych patosystemów. Porównanie dwóch technik podwójnego barwienia, celem późniejszych analiz konfokalnych eksplantatów ogórka zainokulowanych P. cubensis, wskazuje na lepsze wyniki przy zastosowaniu „łagodnego” protokołu barwienia. Aplikacja tego protokołu pozwoliła na detekcję struktur infekcyjnych patogenów w obu badanych patosystemach, przy równoczesnych obserwacjach reakcji obronnych roślin. Ilościowe analizy obrazów reakcji odpornościowych ogórka po inokulacji P. cubensis przeprowadzone przy pomocy programu ImageJ wskazują, że metoda ta jest przydatna zarówno do charakterystyki reakcji obronnych roślin, jak i do określania poziomu patogeniczności danego izolatu. Dodatkowo, metoda ta umożliwiła detekcję specyficznego materiału o powinowactwie do błękitu anilinowego w ścianach komórkowych P. infestans, w obszarze sporulującej grzybni przerastającej aparaty szparkowe liści pomidora. Zaprezentowane wyniki wskazują na możliwość zastosowania tej metody do szczegółowych (ilościowych) analiz, również innych patosystemów

This article offers supplementary material which is provided at the end of the article.

Keywords: histochemical dual-stain; cucumber downy mildew; tomato late blight; pathogen infection structures; host plant resistance; resistance reaction quantification

  • Adhikari B.N., Savory E.A., Vaillancourt B., Childs K.L., Hamilton J.P., Day B., Buell R.C. 2012. Expression profiling of Cucumis sativus in response to infection by Pseudoperonosporacubensis. PLoS ONE 7(4): e34954. [DOI:10.1371/journal.pone.0034954]CrossrefGoogle Scholar

  • An Y., Kang S.C., Kim K.D., Hwang B.K., Jeun Y. 2010. Enhanced defense responses of tomato plants against late blight pathogen Phytophthora infestans by preinoculation with rhizobacteria. Crop Protection 29: 1406-1412. [DOI:10.1016/j.bbr.2011.03.031]CrossrefGoogle Scholar

  • Bhadauria V., Miraz P., Kennedy R., Banniza S., Wei Y. 2010. Dual trypan-aniline blue fluorescence staining methods for studying fungus-plant interactions. Biotechnic & Histochemistry 85: 99-105. [DOI: 10.3109/10520290903132196]CrossrefWeb of ScienceGoogle Scholar

  • Call A.D., Criswell A.D., Wehner T.C., Kłosińska U., Kozik E.U. 2012. Screening cucumber (Cucumissativus L.) for resistance to downy mildew caused by Pseudoperonosporacubensis (Berk. & Curt.). Crop Science 52: 577-592. [DOI:10.2135/cropsci2011.06.0296]Web of ScienceCrossrefGoogle Scholar

  • Chen Y., Halterman D.A. 2011. Phenotypic characterization of potato late blight resistance mediated by the broad-spectrum resistance gene RB. Phytopathology 101: 263-270. [DOI: 10.1094/ PHYTO-04-10-0119]CrossrefGoogle Scholar

  • Currier H.B. 1957. Callose substance in plant cells. American Journal of Botany 44: 478-488. [DOI:10.2307/2438916]CrossrefGoogle Scholar

  • Diez-Navajas A.M., 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: 680-683. [DOI: 10.1016/j.micron.2006.09.009]CrossrefWeb of ScienceGoogle Scholar

  • Foolad M.R. 2007. Genome mapping and molecular breeding of tomato. International Journal of Plant Genomics 2007:64358. [DOI:10.1155/2007/64358] CrossrefGoogle Scholar

  • Freytag S., Arabatzis N., Hahlbrock K., Schmelzer E. 1994. Reversible cytoplasmic rearrangements precede wall apposition, hypersensitive cell death and defense-related gene activation in potato Phytophthorainfestans interactions. Planta 194: 123-135. [DOI:10.1007/bf00201043]CrossrefGoogle Scholar

  • Ganeshan S., Sharma P., Chibbar R.N.2009. Functional genomics for crop improvement. In: Molecular Techniques in Crop Improvement. (eds. Mohan J.S. & Brar D.S.) Springer, New York: 63-95. [DOI:10.1007/978-90-481-2967-6_3]CrossrefGoogle Scholar

  • Grandillo S., Chetelat R., Knapp S., Spooner D., Peralta I., Cammareri M., et al. 2011. Solanum sect. Lycopersicon. In: Wild crop relatives: Genomic and breeding resources. Vegetables. (ed.: Kole C.). Springer-Verlag Berlin Heidelberg: 129-215. [DOI: 10.1007/978-3-642- 20450-0_9]CrossrefGoogle Scholar

  • Haas B.J., Kamoun S., Zody M.C., Jiang R.H.Y., Handsaker R.E., Cano L.M., et al. 2009. Genome sequence and analysis of the Irish potato famine pathogen Phytophthorainfestans. Nature 461: 393-398. [DOI: 10.1038/nature08358]CrossrefGoogle Scholar

  • Hardham A.R., Shan W. 2009. Cellular and molecular biology of Phytophthora plant interactions. In: The Mycota. (ed. Deising, H.B.). Springer Verlag Berlin Heidelberg,. 5: 4-27. [DOI: 10.1007/978-3-540- 87407-2_1]CrossrefGoogle Scholar

  • Hood M.E., Shew H.D. 1996. Applications of KOH-aniline blue fluorescence in the study of plantfungal interactions. Phytopathology 86: 704-708. [DOI: 10.1094/Phyto- 86-704]CrossrefGoogle Scholar

  • Huang S., Li R., Zhang Z., Li L., Gu X., Fan W., et al. 2009. The genome of the cucumber, Cucumis sativus L. Nature Genetics 41: 1275-1281. [DOI: 10.1038/ng.475]CrossrefWeb of SciencePubMedGoogle Scholar

  • Latgé J.-P. 2007. The cell wall: a carbohydrate armour for the fungal cell. Molecular Microbiology 66: 279-290.Web of ScienceCrossrefGoogle Scholar

  • Lebeda A., Cohen Y. 2011. Cucurbit downy mildew (Pseudoperonosporacubensis) biology, ecology, epidemiology, host-pathogen interaction and control. European Journal of Plant Pathology 129: 157-192. [DOI: 10.1111/j.1365-2958.2007.05872.x]Web of ScienceCrossrefGoogle Scholar

  • Mueller L.A., Lankhorst R.K., Tanksley S.D., Giovannoni J.J., White R., Vrebalov J., et al. 2009. A snapshot of the emerging tomato genome sequence. Plant Genetics. 2: 78-92. [DOI: 10.3835/plantgenome2008.08.0005]CrossrefGoogle Scholar

  • Nowicki M., Foolad M.R., Nowakowska M., Kozik E.U. 2012a. Potato and tomato late blight caused by Phytophthora infestans: An overview of pathology and resistance breeding. Plant Disease 96: 4-17. [DOI: 10.1094/PDIS-05-11-0458]Web of ScienceCrossrefGoogle Scholar

  • Nowicki M., Kozik E.U., Foolad M.R. 2012b. Late blight of tomato. In: Genomics applications in plant breeding (eds. Varshney R.K. & Tuberosa R.). Wiley-Blacwell Publishers, USA: accepted.Google Scholar

  • Raffaele S., Win J., Cano L.M., Kamoun S. 2010. Analyses of genome architecture and gene expression reveal novel candidate virulence factors in the secretome of Phytophthora infestans. BMC Genomics 11: Article No.: 637. [DOI: 10.1186/1471-2164-11-637]Web of ScienceCrossrefGoogle Scholar

  • Savory E.A., Granke L.L., Quesada- Ocampo L.M., Varbanova M., Hausbeck M.K., Day B. 2010. The cucurbit downy mildew pathogen Pseudoperonospora cubensis. Molecular Plant Pathology 12: 217-226. [DOI: 10.1111/j.1364-3703.2010.00670.x]PubMedWeb of ScienceCrossrefGoogle Scholar

  • Savory E.A., Adhikari B.N., Hamilton J.P., Vaillancourt B., Day B. 2012a. mRNA-Seq analysis of the Pseudoperonospora cubensis transcriptome during cucumber (Cucumis sativus L.) infection. PLoS ONE 7(4): e35796. [DOI:10.1371/journal.pone.0035796]CrossrefGoogle Scholar

  • Savory E.A., Zou C., Adhikari B.N., Hamilton J.P., Buell R.C., Shiu S.- H., Day B. 2012b. Alternative splicing of a multi-drug transporter from Pseudoperonospora cubensis generates an RXLR effector protein that elicits a rapid cell death. PLoS ONE 7(4): e34701. [DOI:10.1371/ journal. pone.0034701]CrossrefWeb of ScienceGoogle Scholar

  • Shibata Y., Kawakita K., Takemoto D. 2010. Age-related resistance of Nicotiana benthamiana against hemibiotrophic pathogen Phytophthorainfestans requires both ethylene- and salicylic acid-mediated signaling pathways. Molecular Plant - Microbe Interactions 23: 1130-1142. [DOI: 10.1094/MPMI-23-9- 1130]CrossrefGoogle Scholar

  • The Potato Genome Sequencing Consortium. 2011. Genome sequence and analysis of the tuber crop potato. Nature 475: 189-195. [DOI: 10.1038/nature10158]Web of ScienceCrossrefGoogle Scholar

  • Tian M., Win J., Savory E., Burkhardt A., Held M., Brandizzi F., Day B. 2011. 454 Genome Sequencing of Pseudoperonospora cubensis reveals effector proteins with a QXLR translocation motif. Molecular Plant- Microbe Interactions 24: 543-553. [DOI: 10.1094/MPMI-08-10-0185]CrossrefWeb of ScienceGoogle Scholar

  • Vleeshouwers V., Raffaele S., Vossen J., Champouret N., Oliva R., Segretin M.E., Rietman H., Cano L.M., Lokossou A., Kessel G. 2011. Understanding and exploiting late blight resistance in the age of effectors. Annual Review of Phytopathology. [DOI: 10.1146/ annurev-phyto-072910-095326]CrossrefWeb of ScienceGoogle Scholar

  • Wang Y.H., Joobeur T., Dean R.A., Staub J.E. 2007. Cucurbits. In: Genome mapping and molecular breeding in plants, Volume 5: Vegetables. (ed. C. Kole), Springer Verlag Berlin- Heidelberg: 315-329. [DOI:10.1007/ 978-3-540-34536-7_10]CrossrefGoogle Scholar

  • Woycicki R., Witkowicz J., Gawronski P., Dabrowska J., Lomsadze A., Pawelkowicz M., et al. 2011. The genome sequence of the North- European cucumber (Cucumissativus L.) Unravels Evolutionary Adaptation Mechanisms in Plants. PLoS ONE 6:e22728. [DOI: 10.1371/journal.pone.0022728] CrossrefWeb of ScienceGoogle Scholar

About the article

Published Online: 2013-05-15

Published in Print: 2012-12-01


Citation Information: Vegetable Crops Research Bulletin, ISSN (Online) 1898-7761, ISSN (Print) 1506-9427, DOI: https://doi.org/10.2478/v10032-012-0016-z.

Export Citation

This content is open access.

Citing Articles

Here you can find all Crossref-listed publications in which this article is cited. If you would like to receive automatic email messages as soon as this article is cited in other publications, simply activate the “Citation Alert” on the top of this page.

[1]
Marzena Nowakowska, Marcin Nowicki, Urszula Kłosińska, Robert Maciorowski, Elżbieta U. Kozik, and Mark Gijzen
PLoS ONE, 2014, Volume 9, Number 10, Page e109328

Comments (0)

Please log in or register to comment.
Log in