Fig. 5: Subcellular localization of BcCrh1 during saprophytic growth and host infection.

a Intracellular localization of BcCrh1 during saprophytic growth. Spores of BcCrh1-GFP strain were cultured in liquid PDB medium for 12 h, vacuoles (top) and nuclei (bottom) were stained with CMAC and DAPI, respectively, and samples were visualized using a Confocal microscope. Scale bars =  5 μm. The graph shows fluorescent intensity profiles of GFP/CMAC signals (top) and GFP/DAPI signals (bottom) in transects (white arrowheads). Y axis, GFP and CMAC or DAPI fluorescence intensity; X axis, transect length (μm). b Differential distribution of BcCrh1 in mycelia and infection cushions in vitro. Spores were germinated on a glass slide in PDB medium. At 24 h the GFP signal accumulates in the entire mycelium and in initiating infection cushions (left, marked with red arrow), at 36 h the signal is detected only in the mature infection cushions (right, indicated with white arrows). Scale bar = 20 μm. c–d Onion epidermis infection assay with cytoplasmic GFP and BcCrh1-GFP strains. c Images showing secretion of the protein from hyphal tips at early time points (12, 21 hpi) and from infection cushions at 36 hpi. Scale bars = 50 μm at 12 hpi and 20 μm in all other images. d Intracellular localization of secreted BcCrh1-GFP protein in plasmolysed onion cells 45 hpi. Infection area is marked by red dashed line. BcCrh1-derived GFP signal is observed in the cytoplasmic space of plasmolysed onion cells outside the invasion area (marked with arrows). Scale bars = 20 μm. All the experiments were repeated three times with similar results.