Fig. 3.

Sequence analysis and functional validation revealed that the signal peptides of seven MoCDIPs were required for cell death-inducing activity in N. benthaminana. a Structural analysis of the eight MoCDIPs. b Functional validation of the predicted signal peptides of seven MoCDIPs based on a yeast secretion assay system. pYST-2, yeast colony transformed with an empty vector containing a truncated yeast invertase Suc2 gene lacking its original signal peptide sequence. FL-MoCDIPs-Suc2, yeast colonies transformed with full length MoCDIPs fused in frame with Suc2. NS-MoCDIPs-Suc2, truncated version lacking the predicted signal peptide sequence of MoCDIPs fused in-frame with Suc2. Yeast colonies grown on SD/−Leu medium were replica-plated onto sucrose selection medium for about 3 days. Strains unable to secrete invertase were inhibited on sucrose medium (white color), whereas strains able to secrete invertase could grow on sucrose medium (red color). c Transient expression of truncated non-signal peptide version of MoCDIPs did not cause cell death in N. benthamiana leaves. EV, an empty vector pGD. H₂O₂ accumulations in N. benthamiana leaves were shown by DAB staining. d RT-PCR analysis of MoCDIPs mRNA expression in agroinfiltrated N. benthamiana leaves. Total RNAs were extracted from infiltrated leaf tissues at 36 h post infiltration. EV, RT-PCR results from leaf tissues infiltrated with the empty vector pGD. FL, RT-PCR results from leaf tissues infiltrated with FL-MoCDIPs. NS, RT-PCR results from leaf tissues infiltrated with NS-MoCDIPs