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Protocols in ecological and environmental plant physiology

 

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Functional characterisation of a WRKY transcription factor of wheat and its expression analysis during leaf rust pathogenesis

Dhananjay Kumar A , Anjali Kapoor A , Dharmendra Singh A , Lopamudra Satapathy A , Ashwini Kumar Singh B , Manish Kumar A , Kumble Vinod Prabhu C and Kunal Mukhopadhyay A D

A Department of Biotechnology, Birla Institute of Technology, Mesra, Ranchi 835215, India.
B Central Instrumentation Facility, Birla Institute of Technology, Mesra, Ranchi 835215, India.
C National Phytotron Facility, Indian Agricultural Research Institute, New Delhi 110012, India.
D Corresponding author. Email: kmukhopadhyay@bitmesra.ac.in

Functional Plant Biology - http://dx.doi.org/10.1071/FP14077
Submitted: 7 March 2014  Accepted: 4 June 2014   Published online: 5 August 2014


 
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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 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.

Additional keywords: atomic force microscopy, gene expression, in silico modeling and docking, in silico modelling and docking, leaf-rust of wheat, WRKY transcription factors.


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