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. 2019 Jul 3;213(1):195–211. doi: 10.1534/genetics.119.302363

Figure 7.

Figure 7

PEF1-GFP localizes to the septal plug after injury and during programmed cell death. (A) PEF1-GFP recruitment to the septal pore after cutting of the hypha with a laser (strain GN3-17, Pccg-1-pef-1-gfp). Arrow: injured, empty compartment. Asterisk indicates the septal plug. The cell wall was stained with the chitin-binding dye calcofluor white (CFW, 100 µg/ml) (false colored in blue). Images from the left: (1) DIC; (2) GFP fluorescence; (3) CFW; (4) merged image of GFP and CFW; (5) merged image of 1–3. (B) Co-localization of PEF1-GFP and the Woronin body, which is visualized by dsRED-HEX-1, in a heterokaryon of strains S10 (Pccg-1-pef-1-gfp) and S20 (Pccg-1-dsred-hex-1). The asterisk indicates the septal plug, the arrow the injured compartment. Images from the left: (1) DIC; (2) GFP fluorescence; (3) dsRED fluorescence; (4) merged image of GFP and dsRED; (5) merged image of 1–3. (C) Recruitment of PEF1-GFP to the septal pore in the ∆hex-1 strain (S48, Pccg-1-pef-1-gfp), which does not possess Woronin bodies, after cutting the hypha with a laser. The asterisk indicates the septal plug. Left: DIC; center: GFP; right: merged image of DIC and GFP. (D) PEF1-GFP recruitment to septal plugs in hyphae undergoing programmed cell death induced by heterokaryon incompatibility [forced heterokaryon of C9-15 and GN3-36 (Pccg-1-pef-1-gfp)]. Arrows indicate compartments undergoing cell death. Asterisks indicate septal plugs. Note the absence of GFP fluorescence in the dead compartments. Left: DIC; right: GFP. Bar, 10 µm (A–D). (E) Quantification of PEF1-GFP recruitment to the septal plug after cutting with a laser on MM and MM + 200 mM EGTA (strain GN3-17, Pccg-1-pef-1-gfp). Error bars indicate the SD calculated from three independent experiments (n = 100 each). (F) Comparison of the time required for complete sealing of the septal pore in the wild type (FGSC 988) and the ∆pef1 strain (FGSC 15890) on MM and MM + 200 mM EGTA. Two hundred hyphae of each strain were cut with a laser and the time until cytoplasmic leakage through the septal pore ceased was measured.