From Sample to Data: Preparing, Obtaining, and Analyzing Images of Plant-Pathogen Interactions Using Confocal Microscopy

  • Helen N. FonesEmail author
  • George R. Littlejohn
Part of the Methods in Molecular Biology book series (MIMB, volume 1734)


This chapter describes the steps needed to inoculate host plants with a fungus of interest, and subsequently to visualize the infection using confocal microscopy. As an exemplar, we consider the interaction between wheat and the Septoria leaf blotch fungus, Zymoseptoria tritici. This method is easiest when a GFP- or other fluorophore-tagged strain of the studied fungus is available, but notes are also provided which describe possible staining techniques which may be employed if fluorescent fungus is unavailable in your system.

Key words

Plant pathogenic fungi Zymoseptoria tritici GFP Confocal microscopy Image analysis 


  1. 1.
    Gong X, Hurtado O, Wang B et al (2015) pFPL vectors for high-throughput protein localization in fungi: detecting cytoplasmic accumulation of putative effector proteins. Mol Plant-Microbe Interact 28:107–121CrossRefPubMedGoogle Scholar
  2. 2.
    Gupta YK, Dagdas YF, Martinez-Rocha AL et al (2015) Septin-dependent assembly of the exocyst is essential for plant infection by Magnaporthe oryzae. Plant Cell 27:3277–3289CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Kilaru S, Steinberg G (2015) Yeast recombination-based cloning as an efficient way of constructing vectors for Zymoseptoria tritici. Fungal Genet Biol 79:76–83CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Kilaru S, Schuster M, Latz M et al (2015) A gene locus for targeted ectopic gene integration in Zymoseptoriatritici. Fungal Genet Biol 79:118–124CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Kilaru S, Schuster M, Ma W, Steinberg G (2017) Fluorescent markers of various organelles in the wheat pathogen Zymoseptoria tritici. Fungal Genet Biol 105:16–27CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Abràmoff MD, Magalhães PJ, Ram SJ (2004) Image processing with ImageJ. Biophotonics Int 11:36–42Google Scholar
  7. 7.
    Schuster M, Kilaru S, Guo M et al (2015) Red fluorescent proteins for imaging Zymoseptoria tritici during invasion of wheat. Fungal Genet Biol 79:132–140CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Kilaru S, Schuster M, Studholme D et al (2015) A codon-optimized green fluorescent protein for live cell imaging in Zymoseptoria tritici. Fungal Genet Biol 79:125–131CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Fones HN, Steinberg G, Gurr SJ (2015) Measurement of virulence in Zymoseptoria tritici through low inoculum-density assays. Fungal Genet Biol 79:89–93CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Keon J, Antoniw J, Carzaniga R et al (2007) Transcriptional adaptation of Mycosphaerella graminicola to programmed cell death (PCD) of its susceptible wheat host. Mol Plant-Microbe Interact 20:178–193CrossRefPubMedGoogle Scholar
  11. 11.
    Littlejohn GR, Gouveia JD, Edner C et al (2010) Perfluorodecalin enhances in vivo confocal microscopy resolution of Arabidopsis thaliana mesophyll. New Phytol 186:1018–1025CrossRefPubMedGoogle Scholar
  12. 12.
    Littlejohn GR, Mansfield JC, Christmas JT et al (2014) An update: improvements in imaging perfluorocarbon-mounted plant leaves with implications for studies of plant pathology, physiology, development and cell biology. Front Plant Sci 5:140PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  1. 1.BiosciencesUniversity of ExeterExeterUK
  2. 2.School of Biological SciencesPlymouth UniversityPlymouthUK

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