Fig. 3: The N+-BDP mechanosensor provides direct evidence for melanin-dependent appressorium turgor generation in M. oryzae.
a, FLIM image of an alb1- melanin-deficient mutant at 24 h germinated on glass coverslips and stained with the mechanosensory rotor probe N+-BDP. b, FLIM image of a buf1- melanin-deficient mutant at 24 h germinated on glass coverslips and stained with N+-BDP. c, FLIM image of Guy11 appressoria at 24 h germinated on glass coverslips and stained with N+-BDP. d, FLIM image of tricyclazole-treated appressoria of Guy11 at 24 h germinated on glass coverslips and stained with N+-BDP. The corresponding black and white images highlight membrane spatial heterogeneity/lack of spatial heterogeneity at 24 h. e, Dot plot showing the average fluorescence lifetime for alb1-, buf1-, Guy11+tricyclazole and Guy11 control appressoria imaged at 24 h. Each dot corresponds to the average fluorescence lifetime obtained for an ROI drawn around the membrane of an individual appressorium in a 2D FLIM image. Total observations n = 108 appressoria examined in three biological replicates; typical range 23–31 appressoria; each biological replicate is colour coded (R1, replicate 1; R2, replicate 2; R3, replicate 3); data are presented as mean ± s.e.m. ****P < 0.0001, as determined by one-way analysis (ANOVA) with Dunnett’s multiple comparisons test. Images are representative of n = 3 independent repeats of the experiment. Scale bars, 1 μm.
