Fig. 4

Workflow and CLSM analysis of purified SpyTag-labelled organelles isolated with magnetic SpyCatcher-beads via SpyTag/SpyCatcher interaction. a Transiently transformed tobacco leaves expressing organelle-specific SpyTag fusion proteins were harvested homogenized, filtered through miracloth and mixed with SpyCatcher-coated magnetic beads. After incubation, beads with covalently bound organelles were washed and further analysed. b Washed beads after incubation with wild type filtrate (negative control). c Washed beads after incubation with filtrate from plants expressing Plastid-SpyTag show co-localization of beads (dark dots) and chloroplasts (chloroplast autofluorescence is shown in red). d Washed beads after incubation with filtrate from plants expressing Mito-SpyTag, show co-localization of beads (dark dots) and mitochondria (red) indicated by white arrows. Mitochondria were stained with MitoTracker Orange CM-H2TMRos. Scale bars represent 10 μm and 5 µm in the zoomed images