Disruption of the Interfacial Membrane Leads to Effector Re-location and Lifestyle Switch During Rice Blast Disease.

To cause the devastating rice blast disease, the hemibiotrophic fungus produces invasive hyphae (IH) that are enclosed in a plant-derived interfacial membrane, known as the extra-invasive hyphal membrane (EIHM), in living rice cells. Little is known about when the EIHM is disrupted and how the disruption contributes to blast disease. Here we show that the disruption of the EIHM correlates with the hyphal growth stage in first-invaded susceptible rice cells.

A strikingly-angled spindle mediates nuclear migration during colonization of rice cells infected by Magnaporthe oryzae.

To cause rice blast disease, Magnaporthe oryzae must properly organize microtubules and position nuclei during colonization of host cells. Live cell confocal imaging of fluorescently-tagged microtubules and nuclei of M. oryzae invasive hyphae reveals that microtubules form a cage-like arrangement around nuclei during interphase and that the mitotic spindle forms and mediates nuclear migration while integrity of the nuclear envelope is lost.

Visualizing the Movement of Magnaporthe oryzae Effector Proteins in Rice Cells During Infection.

During biotrophy, filamentous pathogens such as the rice blast fungus Magnaporthe oryzae deliver effector proteins into live host cells to facilitate colonization. We describe three complementary assays for visualizing M. oryzae effector translocation into the rice cytoplasm and cell-to-cell movement during infection.

Vacuole Dynamics in Rice Cells Invaded by the Blast Fungus Magnaporthe oryzae.

We describe a fluorescence imaging method to visualize the dynamics of the central vacuole in rice cells during invasion by the blast fungus Magnaporthe oryzae. This method utilizes the combination of confocal microscopy, rice sheath cells (optically transparent), fluorescently tagged M. oryzae (red fluorescence), and fluorescein diacetate staining (green fluorescence; visualizing vacuole dynamics).

Nuclear and structural dynamics during the establishment of a specialized effector-secreting cell by Magnaporthe oryzae in living rice cells.

Background: To cause an economically important blast disease on rice, the filamentous fungus Magnaporthe oryzae forms a specialized infection structure, called an appressorium, to penetrate host cells. Once inside host cells, the fungus produces a filamentous primary hypha that differentiates into multicellular bulbous invasive hyphae (IH), which are surrounded by a host-derived membrane. These hyphae secrete cytoplasmic effectors that enter host cells presumably via the biotrophic interfacial complex (BIC).

The appressorium of the rice blast fungus Magnaporthe oryzae remains mitotically active during post-penetration hyphal growth.

To investigate the mitotic dynamics of an appressorium, we used live-cell confocal imaging of a fluorescence-based mitotic reporter strain of Magnaporthe oryzae. We present evidence that the M. oryzae appressorium remains viable and mitotically active well after host penetration.

Mitotic stopwatch for the blast fungus Magnaporthe oryzae during invasion of rice cells.

To study nuclear dynamics of Magnaporthe oryzae, we developed a novel mitotic reporter strain with GFP-NLS (localized in nuclei during interphase but in the cytoplasm during mitosis) and H1-tdTomato (localized in nuclei throughout the cell cycle). Time-lapse confocal microscopy of the reporter strain during host cell invasion provided several new insights into nuclear division and migration in M. oryzae: (i) mitosis lasts about 5min; (ii) mitosis is semi-closed; (iii) septal pores are closed during mitosis; and (iv) a nucleus exhibits extreme constriction (approximately from 2μm to 0.

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).