Roles for rice membrane dynamics and plasmodesmata during biotrophic invasion by the blast fungus

Abstract

Rice blast disease is caused by the hemibiotrophic fungus Magnaporthe oryzae, which invades living plant cells using intracellular invasive hyphae (IH) that grow from one cell to the next. The cellular and molecular processes by which this occurs are not understood. We applied live-cell imaging to characterize the spatial and temporal development of IH and plant responses inside successively invaded rice (Oryza sativa) cells. Loading experiments with the endocytotic tracker FM4-64 showed dynamic plant membranes around IH. IH were sealed in a plant membrane, termed the extra-invasive hyphal membrane (EIHM), which showed multiple connections to peripheral rice cell membranes. The IH switched between pseudohyphal and filamentous growth. Successive cell invasions were biotrophic, although each invaded cell appeared to have lost viability when the fungus moved into adjacent cells. EIHM formed distinct membrane caps at the tips of IH that initially grew in neighboring cells. Time-lapse imaging showed IH scanning plant cell walls before crossing, and transmission electron microscopy showed IH preferentially contacting or crossing cell walls at pit fields. This and additional evidence strongly suggest that IH co-opt plasmodesmata for cell-to-cell movement. Analysis of biotrophic blast invasion will significantly contribute to our understanding of normal plant processes and allow the characterization of secreted fungal effectors that affect these processes.

Figure 1.

Live-Cell Imaging of M. oryzae IH and Rice Cell Membranes. (A) and (B) Differential staining patterns with RB and FM4-64 in rice sheath epidermal cells invaded by EYFP-labeled fungal strain KV1 (green). Arrows indicate sites where appressoria had penetrated into host cells. (A) RB dye (purple) stained the endoplasmic reticulum inside rice cells and fungal IH at 36 hpi. Bar = 10 μm. (B) PM and endocytotic membranes in rice cells were stained to saturation with FM4-64 (red). Narrow primary hyphae (P) extending from the penetration site differentiated into bulbous IH inside two invaded rice cells at 27 hpi. This image is a three-dimensional projection of 20 optical sections acquired with a z-interval of 0.44 μm. Bar = 10 μm. (C) to (E) FM4-64 outlines IH but is not internalized by them. These images show separate and merged fluorescence channels for the upper rice cell in Figure 1B. Shown are EYFP fluorescence (C), FM4-64 fluorescence (white in this image) (D), and merged channels (E). Bars = 5 μm. (F) to (H) Invasive-like hyphae formed in vitro on dialysis membrane internalize FM4-64. Shown are EYFP fluorescence (F), FM4-64 fluorescence (red) (G), and merged channels (H). Bars = 10 μm. (I) The membrane encasing the IH had an FM4-64–stained connection (arrow) to rice membrane at the cell periphery. This is an enlarged view of a single optical section from the infection site in (C) to (E). The bright-field channel is included in this view (gray scale). Bar = 5 μm. (J) and (K) TEM images show EIHM surrounding an IH inside an epidermal cell. (J) Transverse section of an IH at 26 hpi. The arrow indicates a fibrillar inclusion inside the generally close-fitting EIHM. RC, rice cell. Bar = 500 nm. (K) High-magnification view of the IH–host interface from the cell in (J). FCW, fungal cell wall; FPM, fungal plasma membrane. Bar = 150 nm.

Figure 2.

Rice Membrane Dynamics and Fungal Nuclear Movement. (A) and (B) Numerous membrane tubules occur around an IH (green) in an epidermal cell during early stages of FM4-64 (white) uptake. Arrows indicate a membrane tubule (A) that appeared to be rounding up 4 min later (B). Note that (A) is imaged at a slightly lower magnification than (B). Each image is a projection of four optical sections taken at 0.5-μm z-intervals. Complete z-series are shown in Supplemental Movies 1 and 2 online. Bars = 10 μm. (C) and (D) Shifting of internal rice membranes (white) around an IH (green) after loading to saturation with FM4-64. Arrows indicate a shift in the rice vacuolar membrane position from (C) to (D) 90 min later. Numerous connections are seen between the EIHM and peripheral rice membranes. Both images are projections of four optical sections taken at 0.5-μm z-intervals. Complete z-series are shown in Supplemental Movies 3 and 4 online. Bars = 10 μm. (E) Endoplasmic reticulum (purple) in an RB-stained epidermal cell aggregated around the IH (green) at 36 hpi. Bar = 5 μm. (F) TEM image of a complex aggregation (white arrow) of endoplasmic reticulum–like membrane and vesicles internalized between the EIHM (black arrowhead) and the IH cell wall. Bar = 150 nm. (G) TEM image of EIHM elaborations containing electron-transparent material between the membrane and the IH wall. Bar = 75 nm. (H) to (K) Fungal nuclear movement to IH growing in neighboring cells. Infected cells were visualized using differential interference contrast (DIC) optics ([H] and [J]). IH nuclei were visualized by fluorescence from a histone-GFP fusion protein expressed by the fungus ([I] and [K]). Ten minutes elapsed between (H)/(I) and (J)/(K). Arrows mark equivalent cellular positions for the localization of nuclear fluorescence relative to the developing IH. The lower arrows indicate fading nuclear fluorescence in (K) relative to (I), and the upper arrows indicate the appearance of nuclear fluorescence in (K) relative to (I). Bars = 10 μm.

Figure 3.

Biotrophic Invasion Continues in Neighboring Rice Cells. (A) An IH was surrounded by the shrinking protoplast (arrow) after plasmolysis in 0.75 M sucrose solution. These cells were visualized at 27 hpi using DIC optics. Bar = 5 μm. (B) A rice cell at 48 hpi still plasmolyzes even though seven IH have invaded it from the first-invaded cell. This image shows the green hyphal fluorescence merged with the bright field (gray scale) of the plasmolyzed plant PM (arrow). Note that all IH expressed strong EYFP fluorescence. Some IH in this view do not appear green because they were below the focal plane. Bar = 5 μm. (C) Quantification of plasmolysis in successively invaded rice cells. Percentage of plasmolysis was measured in first-invaded cells at 26.5 hpi, in second-invaded cells at 38.5 hpi, and in third- or fourth-invaded cells at 49 hpi. This experiment was performed independently from the experiment in Table 1. (D) and (E) Filamentous IH growing in rice epidermal cells were sheathed in EIHM with prominent membrane caps at their tips (arrows). The membrane caps visible by DIC microscopy (D) stained with FM4-64 dye, shown as purple (E). Bars = 5 μm.

Figure 4.

Live-Cell Imaging Suggested That Cell-to-Cell Movement Involves Plasmodesmata. (A) to (D) IH seek out specific locations to cross rice cell walls. Four still images from Supplemental Movie 5 online show IH growing in epidermal cells. Image (A) was obtained at 36 hpi. During the 2.5-h period recorded, an IH (arrow) reached the cell wall and swelled slightly before crossing. The arrowhead indicates a fixed point in the cell and shows an IH developing by pseudohyphal budding and moving over time. Stars indicate IH that had each moved along the rice cell wall for ∼5 μm before swelling and crossing. The IH in the top right corner was not visible in (A). Other examples can be seen in the movie, in addition to IH growing along the cell wall and not crossing. Bars = 5 μm. (E) IH at 32 hpi exhibit extreme constriction (arrows) as they cross the rice cell wall. Only EYFP fluorescence is shown. This same image with merged EYFP and FM4-64 channels (see Supplemental Figure 1 online) indicates the locations of rice cell walls. Bar = 5 μm.

Figure 5.

TEM Images Show IH Associated with Plasmodesmata. (A) to (C) IH associated with rice cell walls (RCW) at pit fields. Arrows indicate plasmodesmata. Semithick sections (250 nm) were used to visualize fine connections between IH and pit fields. Therefore, the resolution of individual plasmodesma was reduced. (A) and (B) Two views of IH with fibrillar extensions toward pit fields. Note that the section just grazed the tip of the hypha in (A). Bars = 300 nm in (A) and 800 nm in (B). (C) An IH pressed against the cell wall at a pit field. Bar = 500 nm. (D) Ultrathin section (80 nm) of an IH that had traversed the host cell wall beside a plasmodesma (arrow). Bar = 1 μm.

Figure 6.

Images Relating to Plasmodesmata and Cell-to-Cell Movement. (A) IH (green) packed into a subsidiary cell failed to invade the neighboring guard cell. Plant membranes were stained with FM4-64 at 48 hpi. This image is a projection of 10 optical sections, each 1 μm thick. GC, guard cell. Bar = 5 μm. (B) to (D) Colocalization of GFP-labeled TMV-MP and FM4-64 spots identify pit fields (arrows) in epidermal walls. Shown are FM4-64 fluorescence (purple) (B), MP-GFP fluorescence (green) (C), and merged channels (D). Bars = 10 μm. (E) FM4-64 staining (white) of an IH at 36 hpi showed EIHM that appeared continuous with the membranes in pit field regions. EYFP and bright-field channels are not shown to highlight the FM4-64 pattern. The left arrow indicates an IH with two adjacent membrane connections, and the right arrow indicates an example in which the EIHM appeared continuous with membrane in the adjacent cell. Bar = 5 μm. (F) and (G) An alb⁻ mutant that fails to produce the high pressure needed for appressorial penetration crosses internal walls normally. DIC images show infection at 38 hpi (F) and 42 hpi (G). The arrow indicates swelling before movement. Bars = 10 μm.

Figure 7.

Viability and Morphology of IH in Invaded Rice Sheath Cells. Propidium iodide staining (red) identifies dead IH (arrows) among EYFP-expressing IH (green) in infected leaf sheath cells at 55 hpi. Bar = 10 μm.