Fig. 2

Analysis of the secretion and subcellular localization of the CFEM-containing  proteins VdSCP76 and VdSCP77 from Verticillium dahliae. A Functional validation of the putative N-terminal signal peptide of VdSCP76 and VdSCP77 by a yeast signal trap assay. The sequence of the putative VdSCP76 or VdSCP77 signal peptide was fused in-frame to the invertase sequence in the vector pSUC2, and then transformed into the yeast strain YTK12. The untransformed YTK12 and the YTK12 carrying the empty pSUC2 vector were used as negative controls. The signal peptide of the oomycete effector Avr1b was used as a positive control. B Functional analysis of the signal peptides for the cell death suppression activities of VdSCP76 and VdSCP77 against VdEG1 in Nicotiana benthamiana leaves. Deletion of the signal peptide of VdSCP76 or VdSCP77 resulted in no suppression against the cell death induced by VdEG1 in 4-week-old plants at 6 days after infiltration. GFP was used as control. C Subcellular localization of C-terminal mCherry tagged VdSCP76 and VdSCP77 when transiently expressed in N. benthamiana leaves. The mCherry fluorescence was scanned by a Leica TCS SP8 confocal microscopy system with an excitation wave length of 580 nm and emission of 610 nm. Bars = 50 μm. D The secretion and localization analysis of VdSCP76 and VdSCP77. The conidial suspension from EC::VdSCP76-GFP or EC::VdSCP77-GFP was co-incubated with onion epidermal cells for 4 days. Wild-type Vd991-GFP was used as control. The fluorescence was scanned by a Leica TCS SP8 confocal microscopy system with an excitation wavelength of 488 nm and emission of 510 nm for GFP, and excitation of 543 nm and emission at 562 nm for FM4-64 dye, respectively. Bars = 100 μm