Elevated Early Callose Deposition Results in Complete Penetration Resistance to Powdery Mildew in Arabidopsis

Elevated callose deposition at early time points of infection prevents adapted powdery mildew penetration in 35S::PMR4-GFP plants. Three-week-old 35S::PMR4-GFP, pmr4, and wild-type plants were inoculated with the adapted powdery mildew Gc. All tests were conducted with rosette leaves. A and B, Micrographs showing callose deposition (blue fluorescence by aniline blue staining) at sites of attempted Gc penetration at 6, 12, and 24 hpi with fungal conidia present (A) and with conidia washed off to improve visualization of deposited callose (B). agt, Appressorial germ tube; c, conidium; cd, callose deposit; ht, haustorium; pp, penetration peg; sh, secondary hyphae. Bars = 10 µm. C, Diameter of the first and second pathogen-induced callose deposits in aniline blue-stained leaves. d, Diffuse deposit (determination of diameter not possible); nd, not detectable; pp, penetration peg. *P < 0.05, ****P < 0.0001 by Tukey’s test. Error bars represent se, and n = 100 of four independent leaves. D, Relative fluorescence intensity emitted by aniline blue-stained single callose deposits, discriminating between total and core area (only in 35S::PMR4-GFP) at 6 hpi. nd, Not detectable. ****P < 0.0001 by Tukey’s test. Error bars represent se, and n = 25 independent callose deposits. E, 3D surface plots of callose deposits at 6 hpi. F, Callose synthase activity of membrane fractions at 6, 12, and 24 hpi. Unchallenged leaf membrane fractions served as controls. Activity was determined in a fluorescence-based assay detecting produced callose via the emission of callose-bound aniline blue. Values of 35S::PMR4-GFP represent the means of lines 1 and 2 in biologically independent experiments. *P < 0.05, **P < 0.01, ****P < 0.0001 by Tukey’s test. Error bars represent se, and n = 5 of six independent leaves. G, Quantification of cell entry determined by haustorium formation per conidium. nd, Not detectable. ***P < 0.001 by Tukey’s test. Error bars represent se, and n = 50 of four independent leaves. H, Quantification of secondary hyphae formation at germinated conidia 24 hpi. **P < 0.01 by Tukey’s test. Error bars represent se, and n = 50 of four independent leaves.

Elevated callose deposition at early time points of infection prevents nonadapted powdery mildew penetration in 35S::PMR4-GFP plants. Three-week-old 35S::PMR4-GFP, pmr4, and wild-type plants were inoculated with the nonadapted powdery mildew Bgh. All tests were conducted with rosette leaves. A, Micrographs showing callose deposition (blue fluorescence after aniline blue staining) at sites of attempted Bgh penetration at 6, 12, and 24 hpi. Conidia were washed off in the wild-type, 24-hpi micrograph to improve visualization of the callose deposition and penetration peg. agt, Appressorial germ tube; c, conidium; cd, callose deposit; pgt, primary germ tube; pp, penetration peg. Bars = 10 µm. B, Diameter of the first (at primary germ tube) and second (at appressorial germ tube) callose deposits in aniline blue-stained leaves. nd, Not detectable; p, patch-like callose deposition of a whole cell (determination of diameter not possible); pp, penetration peg. ****P < 0.0001 by Tukey’s test. Error bars represent se, and n = 100 of four independent leaves. C, Callose synthase activity of membrane fractions at 6, 12, and 24 hpi. Unchallenged leaf membrane fractions served as controls. Activity was determined in a fluorescence-based assay detecting produced callose via the emission of callose-bound aniline blue. Values of 35S::PMR4-GFP represent the means of lines 1 and 2 in biologically independent experiments. ****P < 0.0001 by Tukey’s test. Error bars represent se, and n = 5 of six independent leaves. D, Quantification of nonhost cell entry determined by haustorium formation at germinated conidia. nd, Not detectable. ****P < 0.0001 by Tukey’s test. Error bars represent se, and n = 50 of four independent leaves.

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.

Expression profile of plant defense-related genes during powdery mildew infection of Arabidopsis. Three-week-old 35S::PMR4-GFP, pmr4, and wild-type plants were inoculated with the virulent powdery mildew Gc and the nonadapted powdery mildew Bgh. All tests were conducted with rosette leaves. Relative gene expression was determined by quantitative PCR. RNA was isolated from infected leaf tissue and used as template in complementary DNA generation. Gene expression at 0 hpi was used as a reference, and Actin2 expression was used for normalization. Values of 35S::PMR4-GFP represent the means of lines 1 and 2 in biologically independent experiments. *P < 0.05, **P < 0.01 by Tukey’s test. Error bars represent se, and n = 6. A, Relative expression of the salicylic acid-related gene ICS1. B, Relative expression of the salicylic acid-related gene EDS5. C, Relative expression of the jasmonate-related gene COI1. [See online article for color version of this figure.]