Figure 4.

Localization of the GFP-tagged callose synthase PMR4 in epidermal leaf cells. Three-week-old 35S::PMR4-GFP lines were inoculated with the virulent powdery mildew Gc (B–E), and unchallenged wild-type and 35S::PMR4-GFP plants served as controls (A). All tests were conducted with rosette leaves. Micrographs were taken by confocal laser-scanning microscopy. Green color was assigned to GFP-emitted fluorescence, red color to FM 4-64 membrane stain, and blue color to aniline blue-stained callose. A, Localization of the GFP-tagged callose synthase PMR4 at the FM 4-64-stained plasma membrane of unchallenged epidermal leaf cells of 35S::PMR4-GFP lines. No GFP-based fluorescence was seen in wild-type cells. Bars = 10 µm. B, Shadow 3D projection of germinated Gc conidium on a 35S::PMR4-GFP leaf surface at 6 hpi to visualize the position of an attempted fungal penetration site in subsequent microscopic analysis (C–E). The blue frame indicates the plane of the in silico cross section in C. Bars = 5 µm. C, In silico cross section at the site of attempted fungal penetration indicating PMR4-GFP accumulation in the plasma membrane and callose deposition at this site. Bars = 5 µm. D, Maximum-intensity 3D reconstruction at the site of attempted penetration. Shown is the view from the cytosol to the plasma membrane of the epidermal cell. Bars = 5 µm. E, Surface rendering at the site of attempted penetration. Bars = 1 µm. apt, Appressorial germ tube; c, conidium; pm, plant plasma membrane.