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. 2013 Jan 18;161(3):1433–1444. doi: 10.1104/pp.112.211011

Figure 2.

Figure 2.

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.