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A Physiologically Regulated Multidomain Cystatin of Wheat Shows Stage-Dependent Immunity Against Karnal Bunt (Tilletia indica)

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Abstract

To identify novel components of basal resistance against the Tellitia indica of wheat, breeding for disease resistance was carried out on resistant and susceptible genotype of Karnal Bunt. The different members of wheat cystatin gene families were cloned, and their role in triggering differential resistance through co-expression was analyzed in our lab. The multidomain wheat cystatin (WCM) is a proteinase inhibitor characterized by cloning the gene from susceptible (WH542) and resistant genotype (HD 29). A WCM cDNA was isolated from both genotypes and sequenced. The WCM had a highly conserved N-terminal cystatin domain and a long C-terminal extension containing a second region, which exhibited similarity to the cystatin domain. The expression level was significantly (P > 0.001) higher in resistant compared to susceptible genotype at all the physiological stages of wheat spikes. In order to characterize the biochemical properties of WCM, the coding sequence was expressed in Escherichia coli using pET expression vector. The recombinant WCM was purified from soluble fraction of the cell extract by using affinity chromatography. WCM, with 23 KDa molecular mass, showed cysteine proteinase inhibitory activity against papain (Ki 3.039 × 10−7 M) as determined by using BAPNA as substrate. Furthermore, it was able to arrest the fungal mycelial growth of T. indica. Hyphae growth was inhibited, and morphological changes such as swelling and fragmentation of the fungus were observed. Overall, these observations suggest an endogenous high expression of cystatin, possibly associated with the resistance of wheat against Karnal bunt.

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Acknowledgments

I am highly thankful to the Department of Science and Technology for the financial support to complete this project. I am also thankful to Dr. Avinash Pandey, Coordinator, Nanotechnology Application Center, Allahabad University, Allahabad for providing the Scanning Electron Microscope facility.

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Authors and Affiliations

  1. Center for Biotechnology, Nehru Science Center, University of Allahabad, Allahabad, India

    Shalini Purwar & Shanthy Sundaram

  2. National Institute of Plant Genome Research, New Delhi, India

    Praveen Verma

  3. Jawaharlal Nehru University, New Delhi, India

    Shaili Srivastava

  4. Department of Molecular Biology & Genetic Engineering, GBPUA & T, Pantnagar, India

    Anil Kumar

  5. Center for Biotechnology, University of Allahabad, Allahabad, UP, India

    Shalini Purwar

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  1. Shalini Purwar
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Correspondence to Shalini Purwar.

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Purwar, S., Sundaram, S., Verma, P. et al. A Physiologically Regulated Multidomain Cystatin of Wheat Shows Stage-Dependent Immunity Against Karnal Bunt (Tilletia indica). Appl Biochem Biotechnol 168, 2344–2357 (2012). https://doi.org/10.1007/s12010-012-9941-z

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