Yeast one-hybrid analyses of the factors binding to W-box elements. A. Scheme of reporter and effector vectors. Fragments of the TaeIF5A1 promoter were inserted into the upstream of the His3 reporter gene. Promoter fragments of 461 bp and 165 bp containing the W-box motif (construct R1, R2) and promoter fragment of 165 bp containing the mutated sequence (construct mR2) were tested. The W-box and mutated W-boxes were respectively cloned into the upstream of the His3 reporter gene: W-box (construct R3); the mutants (constructs R4–R6). The effector vectors, E1: pGADT7-Rec2 harboring TaRAV; E2: pGADT7-Rec2 harboring TaWRKY. B. TaRAV and TaWRKY interaction with W-box (R3) or mutants (R4–R6) sequences. C. Determination of TaRAV and TaWRKY binding to the promoter fragments containing W-box or mutated motif (R1, R2, mR2). The effector and the reporter constructs were co-transformed into yeast strain Y187. Positive transformants were determined by spotting serial dilutions (1:1, 1:10, 1:100, 1:1000) of yeast onto SD/-His/-Leu/-Trp plates with 3-AT. Negative controls: N1, p53HIS2 + E1 (AD-TaRAV); N2, p53HIS2 + E2 (AD-TaWRKY); Positive control: P, p53HIS2 + pGAD-Rec2-53. D. TaRAV and TaWRKY binding to the W-box and the promoter fragment containing the W-box motif in tobacco leaves. (a) Construction of reporter and effector plasmids for transient trans-activation assays; (b) GUS staining of tobacco leaves co-transformed with reporter and effector plasmids; (c) GUS activity assay of the co-expression of effector and reporter plasmids. The data represent mean values of three independent experiments.