Abstact

WRKY transcription factors, a plant-specific family of transcriptional regulators, are classified into four groups (I-IV) and play pivotal roles in plant defense, development, and stress responses. These proteins are characterized by conserved WRKY domains that preferentially bind to the W-box cis-element C/TTGACC/T in target gene promoters. In Gossypium hirsutum (Gh; upland cotton), the group IId member GhWRKY17 regulates cotton fiber development by activating downstream target genes such as GhHOX3 through promoter W-box binding. However, the structural basis for its DNA recognition specificity remains elusive. Here, we present the 1.8 Å resolution crystal structure of the GhWRKY17 WRKY domain in complex with the GhHOX3 promoter DNA-the first structural characterization of a group IId WRKY protein. Structural analysis reveals that it consists of four antiparallel β-strands, with the β2-strand (harboring the conserved 249WRKYGQK255 motif) and β3-strand co-operatively engaging the DNA major groove. Key residues (R250, K251, Y252, Q254, K255, R264, Y266, Y267) form an intricate hydrogen-bonding network essential for recognizing the extended G/TTTGACC motif. Comparative structural analyses with group I/IIa/III WRKY-DNA complexes reveal that GhWRKY17's dual-strand engagement and extensively hydrogen bond-mediated specific interaction represent novel mechanistic features distinguishing group IId members from other WRKY subgroups, emphasizing the necessity for subgroup-specific investigations. These findings not only establish a structural paradigm for group IId WRKY function but also provide molecular insights for engineering cotton fiber traits through transcriptional regulation.