Fig 3. BbAFP1 depolarizes target membranes and results in the production of a reactive oxygen species (ROS) burst.

(A) Dynamic processes of BbAFP1 entering target fungal cells. Pre-germinated A. brassicae conidia were treated with BbAFP1^FITC and the protein internalization was observed (5–60 min). (B) Effects of BbAFP1 on membrane potential change in A. brassicae cells using DiSC3(5) staining. After treated germinated A. brassicae conidia with membrane potential-sensitive dye DiSC3(5), BbAFP1 (5–20 μM) was added to the cells and the fluorescence intensity was detected (2–20 min). (C) Effects of BbAFP1 on ROS burst using H₂DCFDA staining. (D) Propidium iodide (PI) uptake analysis. After treated A. brassicae cells with BbAFP1^FITC and PI solution for 10 min or 60 min, fluorescent signals were observed. In these experiments, A. brassicae cells were pre-cultured at 26°C for 5 h in PDB. All experiments were performed in triplicate with at least three independent biological samples. Error bars = SD.