Fig. 7

Confocal microscopy imaging of N. benthamiana leaves infiltrated with C-terminal BKN:YFP fusion proteins. a-b. BKN1 is predominantly localized to the plasma membrane (PM) with some nuclear localization (N) for both At-BKN1 (a) and Al-BKN1 (b). The BKN1 cDNA from the Hh-0 ecotype was used for expressing the At-BKN1 protein (see text). c-d. BKN2 is predominantly localized to the plasma membrane (PM) for both At-BKN2 (c) and Al-BKN1 (d).e-g. Plasma membrane localization is disrupted for Al-BKN2 versions mutated at the myristoylation (G2A) and palmitoylation (C4A) sites. Images are shown for the single G2A mutant (e), single C4A mutant (f) and double G2A C4A mutant (g) versions of Al-BKN2. Localization is seen in the cytoplasm (CS = cytoplasmic strands) and nucleus (N). h. A. thaliana CAM4:YFP was used to compare the localization of YFP fluorescence in the cytoplasm (CS = cytoplasmic strands) and nucleus (N). i. Cells infiltrated with Al-BKN2 were plasmolysed using 0.8 M mannitol. Localization in plasmolysed cells remains predominantly at the cell periphery. There is also some localization to Hechtian strands in the apoplastic space (A). Hechtian strands are plasma membrane tubules connected to the cell wall [80]. YFP fusion constructs were infiltrated at OD600 = 0.5, and images were taken 24 h post-inoculation. Scale bars = 30 μm