Figure 8.

PsbO1 and PsbO2 Interact with the Thioredoxin-Like Domain of LTO1 in a Yeast Two-Hybrid Assay.

The thioredoxin-like domain of wild-type (WCSHC) or mutant (WCSHS) LTO1 constitutes the bait and is expressed as a fusion with the GAL4 DNA binding domain from the TRP1-based pGBKT7 vector. Arabidopsis PsbO1 or PsbO2 constitute the prey and are expressed as a fusion with the GAL4 activation domain from the LEU2-based pGADT7 vector. The yeast Y2HGold reporter strain was cotransformed with various combinations of two-hybrid plasmids as indicated on the top right side of the figure (“Empty” refers to the pGBKT7 or pGADT7 vectors). The yeast transformants were tested for adenine/His prototrophies and resistance to aureobasidin A, which depend on reconstitution of an active GAL4. One representative transformant for each combination was plated on solid medium lacking Leu and Trp (SD-L-W), incubated for 3 d at 28°C, and replicated on medium lacking Leu, Trp, adenine, and His (SD-L-W-A-H) or lacking Leu, Trp, adenine, and His with aureobasidin A (SD-L-W-A-H+AbA) and incubated at 28°C for 5 d. GAL4-dependent adenine and His prototrophies and resistance to aureobasidin A indicate interaction between PsbO2 and wild-type LTO1 and also between PsbO1 and mutant LTO1.