Bio-Protocol (Mar 2016)

EdU Based DNA Synthesis and Cell Proliferation Assay in Maize Infected by the Smut Fungus Ustilago maydis

  • Amey Redkar,
  • Gunther Doehlemann

DOI
https://doi.org/10.21769/BioProtoc.1761
Journal volume & issue
Vol. 6, no. 6

Abstract

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The basidiomycetous smut fungus Ustilago maydis (U. maydis) infects all aerial parts of its host plant maize (Zea mays L.). Infection is seen in the form of prominent tumorous symptoms after the establishment of a biotrophic interaction with the host, usually around 5-6 days after infection. The fungus colonizes the various developmentally distinct aerial organs at different stages of development. Formation of tumors is coupled with the induction of host cell division. Activation of cell division can be understood as a measure of DNA synthesis which is triggered to induce rapid divisions in host cell. This developed protocol helps in tracking tumor induction in U. maydis by monitoring of DNA synthesis in planta. Infected leaves were treated with 5-ethynyl-2-deoxyuridine (EdU) at several stages of infection in the seedling leaves and labeled. EdU incorporation in the S phase cells, was visualized by attaching a fluorescent tag and non-dividing maize nuclei were stained with propidium iodide (PI). This protocol helped to understand the tumor development in U. maydis by confocal laser scanning microscopy (Kelliher and Walbot, 2011; Redkar et al., 2015)