A Standardized Method to Assess Infection Rates of Root-Knot and Cyst Nematodes in Arabidopsis thaliana Mutants with Alterations in Root Development Related to Auxin and Cytokinin Signaling

  • Rocío Olmo
  • Ana Cláudia Silva
  • Fernando E. Díaz-Manzano
  • Javier Cabrera
  • Carmen Fenoll
  • Carolina EscobarEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1569)


Plant parasitic nematodes cause a great impact in agricultural systems. The search for effective control methods is partly based on the understanding of underlying molecular mechanisms leading to the formation of nematode feeding sites. In this respect, crosstalk of hormones such as auxins and cytokinins (IAA, CK) between the plant and the nematode seems to be crucial. Thence, the study of loss of function or overexpressing lines with altered IAA and CK functioning is entailed. Those lines frequently show developmental defects in the number, position and/or length of the lateral roots what could generate a bias in the interpretation of the nematode infection parameters. Here we present a protocol to assess differences in nematode infectivity with the lowest interference of root architecture phenotypes in the results. Thus, tailored growth conditions and normalization parameters facilitate the standardized phenotyping of nematode infection.

Key words

Auxin Cytokinin Root-knot nematodes Cyst nematodes Lateral root Infection assay 



This work was supported by the Spanish Government (AGL2013-48787 to CE; and PCIN-2013-053 to CF) and by the Castilla-La Mancha Government (PEII-2014-020-P to CF). FEDM, RO, JC, and ACS acknowledge the European Social Fund, the University of Castilla La Mancha and the MINECO for cofinancing this work.

Supplementary material

332994_1_En_5_MOESM1_ESM.docx (603 kb)
Fig. S1 Plates within the growth chamber. (a) Plates with plants sown in a single row before covering them with the gauze; average light intensity received by the plants is 104 μmol/m2 s. (b) Plates covered with a gauze (plants) from fluorescent light exposure; average light intensity received by the plants is 48 μmol/m2 s. Scale bars: 10 cm (DOCX 602 kb)


  1. 1.
    Moreira S, Bishopp A, Carvalho H, Campilho A (2013) AHP6 inhibits cytokinin signaling to regulate the orientation of pericycle cell division during lateral root initiation. PLoS One 8(2):e56370CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Lavenus J, Goh T, Roberts I, Guyomarch S, Lucas M, De Smet I et al (2013) Lateral root development in Arabidopsis: fifty shades of auxin. Trends Plant Sci 18:450–458CrossRefPubMedGoogle Scholar
  3. 3.
    McCarter JP (2008) Molecular approaches toward resistance to plant-parasitic nematodes. In: Berg RH, Taylor CG (eds) Cell biology of plant nematode parasitism. Springer, Berlin, pp 239–267Google Scholar
  4. 4.
    Bohlmann H (2015) Introductory chapter on the basic biology of cyst nematodes. In: Escobar C, Fenoll C (eds) Advances in botanical research, vol 73. Elsevier Academic Press, Oxford, pp 33–59Google Scholar
  5. 5.
    Escobar C, Barcala M, Cabrera J, Fenoll C (2015) Overview of root-knot nematodes and giant cells. In: Escobar C, Fenoll C (eds) Advances in botanical research, vol 73. Elsevier Academic Press, Oxford, pp 1–32Google Scholar
  6. 6.
    Grunewald W, Van Noorden G, Van Isterdael G, Beeckman T, Gheysen G, Mathesius U (2009) Manipulation of auxin transport in plant roots during Rhizobium symbiosis and nematode parasitism. Plant Cell 21:2553–2562CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Cabrera J, Diaz-Manzano FE, Sanchez M, Rosso MN, Melillo T, Goh T et al (2014) A role for LATERAL ORGAN BOUNDARIES-DOMAIN 16 during the interaction Arabidopsis-Meloidogyne spp. provides a molecular link between lateral root and root-knot nematode feeding site development. New Phytol 203:632–645CrossRefPubMedGoogle Scholar
  8. 8.
    Cabrera J, Barcala M, Fenoll C, Escobar C (2015) The power of omics to identify plant susceptibility factors and to study resistance to root-knot nematodes. Curr Issues Mol Biol 19:53–72PubMedGoogle Scholar
  9. 9.
    Siddique S, Radakovic ZS, De La Torre CM, Chronis D, Novák O, Ramireddy E et al (2015) A parasitic nematode releases cytokinin that controls cell division and orchestrates feeding site formation in host plants. Proc Natl Acad Sci U S A 112:12669–12674CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Shanks CM, Rice JH, Zubo Y, Schaller GE, Hewezi T, Kieber JJ (2016) The role of cytokinin during infection of Arabidopsis thaliana by the cyst nematode Heterodera schachtii. Mol Plant Microbe Interact 29:57–68CrossRefPubMedGoogle Scholar
  11. 11.
    De Meutter J, Tytgat T, Witters E, Gheysen G, Van Onckelen H, Gheysen G (2003) Identification of cytokinins produced by the plant parasitic nematodes Heterodera schachtii and Meloidogyne incognita. Mol Plant Pathol 4:271–277CrossRefPubMedGoogle Scholar
  12. 12.
    De Meutter J, Tytgat T, Prinsen E, Gheysen G, Van Onckelen H, Gheysen G (2005) Production of auxin and related compounds by the plant parasitic nematodes Heterodera schachtii and Meloidogyne incognita. Commun Agric Appl Biol Sci 70:51–60PubMedGoogle Scholar
  13. 13.
    Grunewald W, Karimi M, Wieczorek K, Beeckman T, Gheysen G, Mathesius U (2008) A role for AtWRKY23 in feeding site establishment of plant-parasitic nematodes. Plant Physiol 148:358–368CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Cabrera J, Barcala M, García A, Rio-Machín A, Medina C, Jaubert-Possamai S et al (2016) Differentially expressed small RNAs in Arabidopsis galls formed by Meloidogyne javanica: a functional role for miR390 and its TAS3-derived tasiRNAs. New Phytol 209:1625–1640CrossRefPubMedGoogle Scholar
  15. 15.
    Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675CrossRefPubMedGoogle Scholar
  16. 16.
    Díaz-Manzano FE, Olmo R, Cabrera J, Barcala M, Escobar C, Fenoll C (2016) Long-term in vitro system for maintenance and amplification of root-knot nematodes in Cucumis sativus roots. Front Plant Sci 7:124PubMedPubMedCentralGoogle Scholar
  17. 17.
    Bohlmann H, Wieczorek K (2015) Infection assay of cyst nematodes on Arabidopsis roots. Bio-protocol 5(18):e1596CrossRefGoogle Scholar
  18. 18.
    Cabrera J, Díaz-Manzano FE, Barcala M, Arganda-Carreras I, de Almeida-Engler J, Engler G et al (2015) Phenotyping nematode feeding sites: three dimensional reconstruction and volumetric measurements of giant cells induced by root-knot nematodes in Arabidopsis. New Phytol 206:868–880CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Rocío Olmo
    • 1
  • Ana Cláudia Silva
    • 1
  • Fernando E. Díaz-Manzano
    • 1
  • Javier Cabrera
    • 1
  • Carmen Fenoll
    • 1
  • Carolina Escobar
    • 1
    Email author
  1. 1.Facultad de Ciencias Ambientales y BioquímicaUniversidad de Castilla-La ManchaToledoSpain

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