Fig. 11
KTI4 does not inhibit SlVPE3 proteolytic activity. a Determination of vacuolar processing enzyme (VPE) activity in tobacco (N. benthamiana). Proteins were extracted from N. benthamiana leaves expressing an empty vector (Vec), SlVPE3 alone (SlVPE3 + Vec), SlVPE3 and KTI4 (SlVPE3 + KTI4), and mutated SlVPE3 (SlVPE3 C69G/C208G) and subjected to a VPE activity assay with a VPE-specific fluorescent substrate. The mutated form of SlVPE3 (SlVPE3C69G/C208G) was generated by site-directed mutagenesis. The VPE inhibitor biotin-YVAD-fmk was added. Values are shown as the means ± standard deviation (SD). b The recombinant KTI4 protein from E. coli showed inhibitory activity against trypsin. Soybean trypsin inhibitor was used as a positive control. Trypsin activity was measured with the Nα-benzoyl-L-arginine ethyl ester substrate. Error bars represent the SD of three independent experiments. c VPE activity assay with recombinant KTI4 protein (His-KTI4). Extracts from N. benthamiana expressing an empty vector (Vec), intact SlVPE3, or a mutated form of SlVPE3 (SlVPE3 C69G/C208G) were incubated with recombinant KTI4 protein purified from E. coli, and then subjected to a VPE activity assay. Values are shown as the means ± SD