Fig. 4

Functional evaluation of Fg62 signal peptide. a Cell death detection on Nicotiana benthamiana leaves after transient expression of Fg62, Fg62-nsp, GFP, and PsXEG1. Representative images were taken at 7 days post-inoculation (dpi). b Detection of electrolyte leakage in N. benthamiana leaves transiently expressing Fg62, Fg62-nsp, GFP, and PsXEG1 at 7 dpi. Different letters above the bars indicate significant differences (one-way ANOVA, Tukey’s test; P < 0.05). c Representative immunoblots showing the protein levels of Fg62 and Fg62-nsp, all fused with HA tags and transiently expressed in N. benthamiana leaves. Ponceau S staining indicates equal loading. d Functional validation of Fg62 signal peptide. The indicated strains were grown on YPDA, CMD-W, or YPRAA medium for 2 days. The invertase enzymatic activity was detected by reducing 2,3,5-triphenyl tetrazolium chloride (TTC) to insoluble red-colored 1,3,5-triphenyl formazan. e These full-length Fg62 and Fg62-nsp tagged with GFP and GFP alone were transiently expressed in N. benthamiana. Plant cells were treated with 70 mM NaCl for plasmolysis and analyzed by confocal microscopy at 36 h after Agrobacterium infiltration. The asterisk indicates the apoplast region (bars = 50 μm)