Detached Maize Sheaths for Live-Cell Imaging of Infection by Fungal Foliar Maize Pathogens

Renata Belisário, Maria F. Torres, Ester A.S. Buiate, Katia V. Xavier, Etta M. Nuckles, Lisa J. Vaillancourt

Research output: Contribution to journalArticlepeer-review

Abstract

We have optimized a protocol to inoculate maize leaf sheaths with hemibiotrophic and necrotrophic foliar pathogenic fungi. The method is modified from one originally applied to rice leaf sheaths and allows direct microscopic observation of fungal growth and development in living plant cells. Leaf sheaths collected from maize seedlings with two fully emerged leaf collars are inoculated with 20 µL drops of 5 x 105 spores/ mL fungal spore suspensions and incubated in humidity chambers at 23 °C under continuous fluorescent light. After 24-72 h, excess tissue is removed with a razor blade to leave a single layer of epidermal cells, an optically clear sample that can be imaged directly without the necessity for chemical fixation or clearing. Plant and fungal cells remain alive for the duration of the experiment and interactions can be visualized in real-time. Sheaths can be stained or subjected to plasmolysis to study the developmental cytology and viability of host and pathogen cells during infection and colonization. Fungal strains transformed to express fluorescent proteins can be inoculated or co-inoculated on the sheaths for increased resolution and to facilitate the evaluation of competitive or synergistic interactions. Fungal strains expressing fluorescent fusion proteins can be used to track and quantify the production and targeting of these individual proteins in planta. Inoculated sheath tissues can be extracted to characterize nucleic acids, proteins, or metabolites. The use of these sheath assays has greatly advanced the detailed studies of the mechanisms of fungal pathogenicity in maize and also of fungal protein effectors and secondary metabolites contributing to pathogenicity.

Original languageEnglish
Article numbere65755
JournalJournal of Visualized Experiments
Volume2023
Issue number199
DOIs
StatePublished - Sep 2023

Bibliographical note

Publisher Copyright:
© 2023 JoVE Journal of Visualized Experiments.

ASJC Scopus subject areas

  • General Neuroscience
  • General Chemical Engineering
  • General Biochemistry, Genetics and Molecular Biology
  • General Immunology and Microbiology

Fingerprint

Dive into the research topics of 'Detached Maize Sheaths for Live-Cell Imaging of Infection by Fungal Foliar Maize Pathogens'. Together they form a unique fingerprint.

Cite this