FIGURE 2.

Role of Cys residues in Zera-PB formation. A, diagram showing the positions of the cysteine residues in Zera fused to ECFP. B, total protein analysis of tobacco leaves transformed with Zera-Cys mutants fused to ECFP by SDS-PAGE/Coomassie Blue staining (top) and immunoblot using an anti-GFP antibody (bottom). Shown are untransformed tobacco protein extracts (lane 1), tobacco extracts of non-mutated Zera-ECFP (lane 2), and protein extracts of transformed tobacco with Zera Cys⁷ mutant (lane 3), Zera Cys⁹ (lane 4), Zera Cys⁶⁴ (lane 5), Zera Cys⁸² (lane 6), Zera Cys⁸⁴ (lane 7), Zera Cys⁹² (lane 8), Zera Cys⁷-Cys⁹ (lane 9), and Zera Cys⁸²-Cys⁸⁴-Cys⁹² (lane 10). The arrows indicate the electrophoretic bands of Zera-ECFP and Zera-ECFP Cys mutants, and the arrowhead shows an additional immunoreactive band. C–K, confocal images showing the fluorescence pattern of epidermal cells transformed with Zera-Cys mutants. Single Cys⁷ and Cys⁹ mutants were mainly secreted (C and D), but some cells were able to induce PBs (insets in C and D). E–H, single mutation of Cys⁶⁴, Cys⁸², Cys⁸⁴, or Cys⁹² had no effect on PB formation. I–K, Zera-ECFP containing multiple Cys mutations was not able to oligomerize and was secreted. Scale bars, 10 μm (C–H) or 20 μm (I–K).