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WRKY proteins are a large family of plant-specific transcription factors associated with regulation of biotic and abiotic stress responses, but how they respond to cereal rust pathogens has never been explored at the molecular level. Full-length cDNA of TaWRKY1B was obtained from a wheat cultivar HD2329 derivative containing leaf rust resistance gene Lr28 based on domain characteristics. The unique feature of this WRKY transcription factor gene was the close proximity of the DNA-binding domain and consensus DNA element W-Box within the open reading frame. Infection with a virulent race of leaf rust fungus resulted in 146-fold induction of the gene in resistant plants, but only 12-fold in the susceptible plants as compared with mock-inoculated controls. Docking models of 74 amino acids DNA-binding domain and 26 bp W-Box element showed that the WRKY domain, located on the β1 strand, only interacts with the W-Box at positions corresponding to W125, R126, K127 and Y128 amino acids. A truncated recombinant protein of 9.0 kD, encompassing the DNA-binding domain also showed binding specificity to the 32 bp W-Box element in electrophoretic mobility shift assays. The protein-DNA ensemble was also characterised using high-resolution atomic force microscopic imaging. The results contribute to an understanding of the molecular structure and function of a previously uncharacterised WRKY transcription factor in wheat that can be manipulated to improve biotic stress tolerance. The WRKY TFs has been a subject of intense research; however, their functional analysis in crops like wheat is still lacking. The molecular structure of a WRKY gene JX028549 was revealed and its spatio-temporal expression showed upregulation in response to the leaf rust pathogenesis. The results contribute towards understanding the structure and function of a wheat WRKY TF that can be used as a candidate gene to improve biotic stress tolerance. [ABSTRACT FROM AUTHOR]