Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Supplemental Figure 1. AM-induced PPA formation in the epidermis of M. truncatularoots expressing GFP-HDEL and co-localization with in vivo FM 4-64 staining (A-D)Time course showing ER structure prior to and during fungal infection. (A, B) A hollow tube of ER (green fluorescence; arrow) organizes within the cytoplasmic column, directly underneath the appressorium contact site (arrowhead), shown by comparing the z-stack projection (A) with the orthogonal x-z section (B). Fungal autofluorescence (red) shows that cell penetration has not yet occurred. (C, D) Five hours later the ER tube density has decreased (arrow) and the fungus (red) has penetrated (double arrowhead) the cell within the ER tube. n = position of nucleus. Bar = 20 μm. (E-G) FM 4-64 labeling co-localizes with the PPA and precedes fungal entry of the epidermal cell. (E) Z-axis projection of several optical sections across the cell body of a trichoblast (the root hair, RH, is visible on the left as a circular section). The GFP-HDEL labeling is in green and the FM 4-64 staining in red. (F-G) 3D reconstructed side views of the cell volume enclosed between the dashed lines marked in (E). (F) Image with same color-coding as(E) showing FM 4-64 staining (appearing as yellow/orange, grey arrow) within the PPA ER tube (green, white arrow). (G). Same as (F), except that the ER fluorescence has been removed and the FM 4-64 staining is now color-coded in green (grey arrow). This image shows clearly that the hypha (red) has not yet entered the cell. Arrowheads indicate the appressorium contact site and in all images the AM fungal autofluorescence is in red. n = position of nucleus. Bar = 20 μm.
• Supplemental Video 1 - Supplemental Video 1. AM hyphal penetration into a root epidermal cell following the path of the PPA and showing progressive widening of the fluorescent-labeled ER surrounding the apoplastic compartment. Root cells express a GFP-HDEL tag which labels the ER (green), and the fungus is visualized in red. Timing is shown in the top right-hand corner, and hyphal penetration takes place between 4-7 hours after initial appressorium formation on the epidermal cell surface. Images are z-axis projections of serial optical sections. Stills of this video are presented in Figure 1P to 1R. Image width = 120 μm.
• Supplemental Video 2 - Supplemental Video 2. Animated model showing intracellular dynamics within the host epidermis prior to and during AM fungal infection. See text for details of the intracellular processes depicted in the animation. Four stills from the movie are shown in Figure 2J to 2M. The different cellular components are color-coded as follows: cell wall in brown; plasma membrane in yellow; actin cables in red; microtubules in green; endoplasmic reticulum in cyan; nucleus in orange; AM fungus in magenta. The animation does not respect the real timing of the various cellular events, which take place over a total of approximately 7 hours.