Manipulation of Auxin and Cytokinin Balance During the Plasmodiophora brassicaeArabidopsis thaliana Interaction

  • Jutta Ludwig-MüllerEmail author
  • Susann Auer
  • Sabine Jülke
  • Sabine Marschollek
Part of the Methods in Molecular Biology book series (MIMB, volume 1569)


The symptoms of the clubroot disease on Brassica species caused by the obligate biotrophic protist Plasmodiophora brassicae relies, among other factors, on the modulation of plant hormones. Signaling, transport as well as biosynthesis and metabolism are key features how the levels of auxins and cytokinins are controlled. We here describe (a) how to inoculate the model plant Arabidopsis thaliana with P. brassicae, (b) qualitative and quantitative methods to evaluate disease severity in auxin and cytokinin mutants, (c) molecular methods to monitor changes in plant and pathogen transcripts, (d) prerequisites for the establishment of transgenic lines manipulated in an auxin or cytokinin pathway, and (e) methods for β-glucuronidase staining in root galls and sections of infected roots to determine auxin and cytokinin responsive promoter activities.

Key words

Arabidopsis thaliana Auxin Clubroot Cytokinins β-Glucuronidase Hormone responsive promoter Plasmodiophora brassicae 


  1. 1.
    Siemens J, Nagel M, Ludwig-Müller J, Sacristán MD (2002) The interaction of Plasmodiophora brassicae and Arabidopsis thaliana: parameters for disease quantification and screening of mutant lines. J Phytopathol 150:592–605CrossRefGoogle Scholar
  2. 2.
    Kageyama K, Asano T (2009) Life cycle of Plasmodiophora brassicae. J Plant Growth Regul 28:203–211CrossRefGoogle Scholar
  3. 3.
    Jahn L, Mucha S, Bergmann S, Horn C, Siemens J, Staswick P, Steffens B, Ludwig-Müller J (2013) The clubroot pathogen (Plasmodiophora brassicae) influences auxin signaling to regulate auxin homeostasis. Plants 2:726–749CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Siemens J, Keller I, Sarx J, Kunz S, Schuller A, Nagel W, Schmülling T, Parniske M, Ludwig-Müller J (2006) Transcriptome analysis of Arabidopsis clubroots indicate a key role for cytokinins in disease development. Mol Plant Microbe Interact 19:480–494CrossRefPubMedGoogle Scholar
  5. 5.
    Schuller A, Kehr J, Ludwig-Müller J (2014) Laser microdissection coupled to transcriptional profiling of Arabidopsis roots inoculated by Plasmodiophora brassicae indicates a role for brassinosteroids in clubroot formation. Plant Cell Physiol 55:392–411CrossRefPubMedGoogle Scholar
  6. 6.
    Grsic-Rausch S, Kobelt P, Siemens J, Bischoff M, Ludwig-Müller J (2000) Expression and localization of nitrilase during symptom development of the clubroot disease in Arabidopsis thaliana. Plant Physiol 122:369–378CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Päsold S, Siegel I, Seidel C, Ludwig-Müller J (2010) Flavonoid accumulation in Arabidopsis thaliana root galls caused by the obligate biotrophic pathogen Plasmodiophora brassicae. Mol Plant Pathol 11:545–562CrossRefPubMedGoogle Scholar
  8. 8.
    Devos S, Laukens K, Deckers P, Van Der Straeten D, Beeckman T, Inzé D, Van Onckelen H, Witters E, Prinsen E (2006) A hormone and proteome approach to picturing the initial metabolic events during Plasmodiophora brassicae infection on Arabidopsis. Mol Plant Microbe Interact 19:1431–1443CrossRefPubMedGoogle Scholar
  9. 9.
    Malinowski R, Smith JA, Fleming AJ, Scholes JD, Rolfe SA (2012) Gall formation in clubroot-infected Arabidopsis results from an increase in existing meristematic activities of the host but is not essential for the completion of the pathogen life cycle. Plant J 71:226–238CrossRefPubMedGoogle Scholar
  10. 10.
    Knaust A, Ludwig-Müller J (2013) The ethylene signaling pathway is needed to restrict root gall growth in Arabidopsis after infection with the obligate biotrophic protist Plasmodiophora brassicae. J Plant Growth Regul 32:9–21CrossRefGoogle Scholar
  11. 11.
    Siemens J, Gonzales M-C, Wolf S, Hofmann C, Greiner S, Du Y, Rausch T, Roitsch T, Ludwig-Müller J (2011) Extracellular invertase is involved in the regulation of the clubroot disease in Arabidopsis thaliana. Mol Plant Pathol 12:247–262CrossRefPubMedGoogle Scholar
  12. 12.
    Faggian R, Strelkov SE (2009) Detection and measurement of Plasmodiophora brassicae. J Plant Growth Regul 28:282–288CrossRefGoogle Scholar
  13. 13.
    Schwelm A, Fogelqvist J, Knaust A, Jülke S, Lilja T, Bonilla-Rosso G, Karlsson M, Shevchenko A, Choi SR, Dhandapani V, Kim HG, Park JY, Lim YP, Ludwig-Müller J, Dixelius C (2015) The Plasmodiophora brassicae genome reveals insights in its life cycle and ancestry of chitin synthases. Sci Rep 5:11153. doi: 10.1038/srep11153 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Staswick PE, Serban B, Rowe M, Tiryaki I, Maldonado MT, Maldonado MC, Suza W (2005) Characterization of an Arabidopsis enzyme family that conjugates amino acids to indole-3-acetic acid. Plant Cell 17:616–627CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Feng J, Hwang SF, Strelkov SE (2013) Genetic transformation of the obligate parasite Plasmodiophora brassicae. Phytopathology 103:1052–1057CrossRefPubMedGoogle Scholar
  16. 16.
    Fähling M, Graf H, Siemens J (2003) Pathotype-separation of Plasmodiophora brassicae by the host plant. J Phytopathol 151:425–430CrossRefGoogle Scholar
  17. 17.
    Graf H, Fähling M, Siemens J (2004) Chromosome polymorphism of the obligate biotrophic parasite Plasmodiophora brassicae. J Phytopathol 152:86–91CrossRefGoogle Scholar
  18. 18.
    Puzio PS, Newe M, Grymaszewska G, Ludwig-Müller J, Grundler FMW (2000) Plasmodiophora brassicae-induced expression of pyk20, an Arabidopsis thaliana gene with glutamine-rich domain. Physiol Mol Plant Pathol 56:79–84CrossRefGoogle Scholar
  19. 19.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−ΔΔCt) method. Methods 2001(25):402–408CrossRefGoogle Scholar
  20. 20.
    Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743CrossRefPubMedGoogle Scholar
  21. 21.
    Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622CrossRefPubMedGoogle Scholar
  22. 22.
    Agarwal A, Kaul V, Faggian R, Rookes JE, Ludwig-Müller J, Cahill DM (2011) Analysis of global host gene expression during the primary phase of the Arabidopsis thaliana-Plasmodiophora brassicae interaction. Funct Plant Biol 38:1–16CrossRefGoogle Scholar
  23. 23.
    Hruz T, Laule O, Szabo G, Wessendorp F, Bleuler S, Oertle L, Widmayer P, Gruissem W, Zimmermann P (2008) Genevestigator V3: a reference expression database for the meta-analysis of transcriptomes. Adv Bioinformatics 2008, article ID 420747. doi: 10.1155/2008/420747
  24. 24.
    Winter D, Vinegar B, Nahal H, Ammar R, Wilson G, Provart N (2007) An “Electronic Fluorescent Pictograph” browser for exploring and analyzing large-scale biological data sets. PLoS One. doi: 10.1371/journal.pone.0000718 Google Scholar
  25. 25.
    Nitz I, Berkefeld H, Puzio P, Grundler FMW (2001) Pyk10, a seedling and root specific gene and promoter from Arabidopsis thaliana. Plant Sci 161:337–346CrossRefPubMedGoogle Scholar
  26. 26.
    Mollier P, Hoffmann B, Orsel M, Pelletier G (2000) Tagging of a cryptic promoter that confers root-specific gus expression in Arabidopsis thaliana. Plant Cell Rep 19:1076–1083CrossRefGoogle Scholar
  27. 27.
    Ludwig‐Müller J, Jülke S, Geiß K, Richter F, Mithöfer A, Šola I, Rusak G, Keenan S, Bulman S (2015) A novel methyltransferase from the intracellular pathogen Plasmodiophora brassicae methylates salicylic acid. Mol Plant Pathol 16:349–364CrossRefPubMedGoogle Scholar
  28. 28.
    Yu P, Lor P, Ludwig-Müller J, Hegeman AD, Cohen JD (2015) Quantitative evaluation of IAA conjugate pools in Arabidopsis thaliana. Planta 241:539–548CrossRefPubMedGoogle Scholar
  29. 29.
    Buczacki ST, Moxham SE (1979) A triple stain for differentiating resin-embedded sections of Plasmodiophora brassicae in host tissues under the light microscope. Trans Br Mycol Soc 72:311–347CrossRefGoogle Scholar
  30. 30.
    Humphrey CD, Pittman A (1974) Simple methylene blue—azure II—basic fuchsin stain for epoxy-embedded tissue sections. Stain Technol 40(1):9–14CrossRefGoogle Scholar
  31. 31.
    Siemens J, Glawischnig E, Ludwig-Müller J (2008) Indole glucosinolates and camalexin do not influence the development of the clubroot disease in Arabidopsis thaliana. J Phytopathol 156:332–337CrossRefGoogle Scholar
  32. 32.
    Ludwig-Müller J, Pieper K, Ruppel M, Cohen JD, Epstein E, Kiddle G, Bennett R (1999) Indole glucosinolate and auxin biosynthesis in Arabidopsis thaliana L. glucosinolate mutants and the development of the clubroot disease. Planta 208:409–419CrossRefPubMedGoogle Scholar
  33. 33.
    Neuhaus K, Grsic-Rausch S, Sauerteig S, Ludwig-Müller J (2000) Arabidopsis plants transformed with nitrilase 1 or 2 in antisense direction are delayed in clubroot development. J Plant Physiol 156:756–761CrossRefGoogle Scholar
  34. 34.
    Alix K, Lariagon C, Delourme R, Manzanares-Dauleux M (2007) Exploiting natural genetic diversity and mutant resources of Arabidopsis thaliana to study the A. thalianaPlasmodiophora brassicae interaction. Plant Breed 126:218–221CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Jutta Ludwig-Müller
    • 1
    Email author
  • Susann Auer
    • 1
  • Sabine Jülke
    • 1
  • Sabine Marschollek
    • 1
  1. 1.Institut für BotanikTechnische Universität DresdenDresdenGermany

Personalised recommendations