FIGURE 1.

Both the repeat region and its flanking sequences are necessary for PB biogenesis. A–D, confocal images of tobacco leaf epidermal cells at 4 dpi using constructs Zera-ECFP (A), SP-ECFP (B), R8-ECFP (C), and ΔR8-ECFP (D). Only the wild type Zera (A) sequence induced PBs in transformed cells. SP-ECFP (B) was used as a control of protein secretion. The truncated Zera protein fusions (C and D) were secreted. The inset in C indicates fluorescence in the apoplast after cell plasmolysis. A schematic diagram of each of the expressed constructs is shown above the corresponding confocal image. SP, γ-zein signal peptide; N-, first 10 amino acids of Zera sequence; PX, Pro-Xaa sequence. ECFP was used as reporter. The upper vertical lines in the diagrams represent Cys residues. Bar, 10 μm (A) or 20 μm (B–D). E and F, immunoblots of total protein extracts from tobacco leaves transformed with Zera-ECFP (lanes 1), ΔR8-ECFP (lanes 2), and R8-ECFP (lanes 3), using anti-GFP (E) and anti-R8 (F) antibodies.