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Protocols in ecological and environmental plant physiology

 

 Just Accepted

This article has been peer reviewed and accepted for publication. It is in production and has not been edited, so may differ from the final published form.


Functional characterization of a WRKY Transcription Factor of wheat and its expression analysis during leaf rust pathogenesis

Dhananjay Kumar, Anjali Kapoor, Dharmendra Singh, Lopamudra Satapathy, Ashwini Singh, Manish Kumar, Kumble Prabhu, Kunal Mukhopadhyay

Abstract

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 to 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 characterized using high-resolution atomic force microscopic imaging. The results contribute to an understanding of the molecular structure and function of a previously uncharacterized WRKY transcription factor in wheat that can be manipulated to improve biotic stress tolerance.

FP14077  Accepted 04 June 2014
 
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