Live-cell fluorescence imaging to investigate the dynamics of plant cell death during infection by the rice blast fungus Magnaporthe oryzae

Background: Plant cell death plays important roles during plant-pathogen interactions. To study pathogen-induced cell death, there is a need for cytological tools that allow determining not only host cell viability, but also cellular events leading to cell death with visualization of pathogen development. Here we describe a live cell imaging method to provide insights into the dynamics of cell death in rice (Oryza sativa). This method uses live-cell confocal microscopy of rice sheath cells mechanically damaged or invaded by fluorescently-tagged Magnaporthe oryzae together with fluorescent dyes fluorescein diacetate (FDA) and propidium iodide (PI). FDA stains the cytoplasm of live cells exclusively, thus also visualizing the vacuole, whereas PI stains nuclei of dead cells. Results: We first demonstrated that confocal microscopy of rice leaf sheaths stained with FDA and PI discriminated between live cells and mechanically-killed cells. FDA-derived fluorescein was confined to the cytoplasm of live cells, indicating the intact vacuolar and plasma membranes. We also observed previously unreported fluorescein patterns in mechanically damaged cells. These patterns include: (1) homogeneous distribution of fluorescein in the increased area of the cytoplasm due to the shrunken vacuole; (2) the increase of the fluorescein intensity; and (3) containment of the brighter fluorescein signal only in affected cells likely due to closure of plasmodesmata. We refer to these as novel fluorescein patterns in this study. Simultaneous imaging of fluorescently-tagged M. oryzae (red) and FDA staining (green) in rice cells revealed characteristic features of the hemibiotrophic interaction. That is, newly invaded cells are alive but subsequently become dead when the fungus spreads into neighbor cells, and biotrophic interfacial complexes are associated with the host cytoplasm. This also revealed novel fluorescein patterns in invaded cells. Time-lapse imaging suggested that the FDA staining pattern in the infected host cell progressed from typical cytoplasmic localization (live cell with the intact vacuole), to novel patterns (dying cell with closed plasmodesmata with the shrunken or ruptured vacuole), to lack of fluorescence (dead cell). Conclusion: We have developed a method to visualize cellular events leading to host cell death during rice blast disease. This method can be used to compare and contrast host cell death associated with disease resistance and susceptibility in rice-M. oryzae and other host-pathogen interactions.