Figure 3.

Interaction between TRAB1 and VP1 in rice protoplast cells. (A) Two-hybrid interactions in the plant cell. Ten micrograms each of bait plasmid [p35S-ShΔ-stop (control), p35S-ShΔ-GBD (GBD alone), or p35S-ShΔ-GBD∷VP1-N (GBD∷VP1-N)], prey plasmid [p35S-ShΔ-stop (control) or p35S-ShΔ-GAD∷TRAB1 (GAD∷TRAB1)], and the UAS-TATA-GUS reporter plasmid were cotransfected into protoplasts of rice suspension-cultured cells by electroporation. The electroporated cells were cultured for 40 h. The control plasmid (p35S-ShΔ-stop) consisted of CaMV 35S promoter, Sh1 first intron with an internal deletion, and NOS terminator, but no coding region. Each transfection also included 5 μg of a ubiquitin promoter∷luciferase plasmid as an internal standard. GUS activities were normalized by luciferase activity. The effects of GAD∷TRAB1 (prey) fusion on the expression of the reporter gene are expressed by fold activation of normalized GUS activities relative to that obtained with a combination of indicated bait and control plasmids. Each value is the mean of four replicate electroporations with standard error. (B) Interaction between VP1 and GBD∷TRAB1 and the capacity of GBD∷TRAB1 fusion to confer ABA responsiveness to a promoter carrying GAL4-binding sites. Ten micrograms each of bait (GBD fusion) plasmid [p35S-ShΔ-stop (control), p35S-ShΔ-GBD (GBD alone), or p35S-ShΔ-GBD∷TRAB1 (GBD∷TRAB1], prey plasmid [p35-S-ShΔ-stop (control) or p35-S-Sh-OSVP1 (OSVP1)], and the UAS-TATA-GUS reporter plasmid were cotransfected into protoplasts of rice suspension-cultured cells by electroporation, and the cells were cultured for 40 h in the absence (−) or presence (+) of 5 × 10⁻⁵ M ABA. The effects of OSVP1 and ABA on the expression of the reporter gene are expressed by fold activation of normalized GUS activities relative to that obtained with a combination of indicated GBD (bait) fusion and control plasmid in the absence of ABA. Other details are as described for A.