Background

PS externalization constitutes one of the hallmarks of apoptosis-like PCD that can be detected very early (Martin et al., 1995). Hence the appearance of PS on the outer leaflet of the plasma membrane marks the onset of an apoptotic cell death phenomenon. Ustilago maydis is a biotrophic plant pathogen and infects host plant Zea mays. The lifecycle of U. maydis has been demonstrated to comprise of primarily two morphological forms namely the non-pathogenic haploid sporidial form and the pathogenic diploid filamentous form. The transition from the haploid to the diploid takes place through the mating of the compatible haploid strains on plant surfaces (Kahmann and Kamper, 2004). This leads to the generation of an infectious structure called appressoria that further penetrates the host plant as the filamentous form of the pathogen. Within the plant cells, the filamentous form of U. maydis again undergoes several transitions between morphologically distinct phases leading to sporulation. PCD has been demonstrated to play a significant role in the morphological transformations of different cell types primarily in higher eukaryotes (Buss et al., 2006; Suzanne and Steller, 2013). Also in some phytopathogenic fungi, PCD has been evidenced to be absolutely essential for the generation of appressoria (Veneault-Fourrey et al., 2006). Besides aiding in the switching between distinct morphological forms, PCD is also an end result of a harsh environmental stress response (Phillips et al., 2003). During penetration of the host plant the pathogens are exposed to a number of host defense response derived stress conditions. Among them, exposure to an increasingly oxidative environment is the most common. The primary reason behind this is the increased production of reactive oxygen species by the host in response to pathogen invasion (Torres et al., 2006). Assaying PCD in the fungal pathogen under each of these conditions mentioned can give significant insights into the pathogenic development as well as stress response of U. maydis. This protocol described the steps in detail to stain U. maydis sporidia under axenic culture conditions with annexinV-FITC to detect onset of any apoptosis-like cell death event upon exposure to adverse environmental conditions. However, it doesn’t include the staining of filamentous hyphae of U. maydis during its growth in-planta. Therefore this protocol is only applicable to the U. maydis sporidial cell suspension.