FIGURE 1.

PHR1 and PHL1 interact with the AtFER1 promoter region. A, structure of AtFer1 minimal promoter. The IDRS is involved in AtFer1 repression under −Fe conditions. Alignments of plant ferritin genes promoter regions allowed the identification of conserved elements (8). Element 2 sequence is indicated, and the putative P1BS is in capital letters. B, yeast one-hybrid revealed interaction between PHR1 and Element 2. The yeast strain contains the AUR1-C gene, conferring resistance to aureobasidin A, fused to GAL4 minimal promoter and a tetramer of elements 2 and 3 of AtFer1 promoter. The strain was transformed with pGAD T7 AD vector (empty) of pGAD T7 AD-PHR1 (PHR1) containing full-length PHR1 ORF cloned in-frame with the GAL4 activation domain. Yeasts were plated on medium containing (+AbA) or not (−AbA) aureobasidin A. C, PHR1 and PHL1 interact with Element 2. PHR1 and PHL1 were produced using the TNT system. A fragment of 160 bp, containing all elements presented in A was radiolabeled and used as a probe in EMSA. Competitions were performed with a 100-fold molar excess of unlabeled wild type or mutated in Element 2 (fragments 1 and 2, respectively). O indicates absence of competition. Fp: free probe, M: mock. A mock translation mixture was used as control.