Fig. 9

SlVPE3 interacts with KTI4 in vacuoles. a Interactions between KTI4 and SlVPE3 in a Y2H analysis. The ORF of KTI4 and the cDNA fragment encoding the mature protein of SlVPE3 were cloned into the pGBKT7 (BD) and pGADT7 (AD) vectors, respectively, resulting in the KTI4-BD and SlVPE3-AD plasmids, which were co-transformed into yeast. As negative controls, KTI4-BD and AD or SlVPE3-AD and BD were co-transformed into yeast. The transformants were streaked on SD/-Leu/-Trp medium (SD/-2). Protein–protein interactions were assessed by examining growth on SD/-Leu/-Trp/-His/-Ade medium (SD/-4) and further confirmed by monitoring β-galactosidase activity (blue coloration). b Subcellular localization of SlVPE3 and KTI4 visualized by monomeric red fluorescent protein (mRFP) analysis. The constructs used for transformation are indicated (left): mRFP alone, control showing the signals throughout the cell except in the vacuolar lumen; SlVPE3-mRFP, signals from the SlVPE3-mRFP fusion protein; KTI4-mRFP, signals from the KTI4-mRFP fusion protein. Protoplasts of tobacco (Nicotiana benthamiana) leaves transiently expressing the mRFP-alone control, SlVPE3-mRFP, or KTI4-mRFP were isolated and observed under a Leica confocal microscope (Leica DMI600CS). c Subcellular colocalization of SlVPE3 and KTI4 determined using N. benthamiana leaf protoplasts co-expressing SlVPE3-mRFP and KTI4-PRpHluorin. The constructs used for transformation are indicated (left): mRFP + PRpHluorin, control showing the signals throughout the cell, except in the vacuolar lumen; SlVPE3-mRFP + KTI4-PRpHluorin, signals from SlVPE3-mRFP and KTI4-PRpHluorin fusion proteins. Colocalization is shown by merging mRFP and PRpHluorin images (Merged). Scale bars, 25 μm