Figure 3.

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.