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Antimicrobial peptide (AMP) from Zingiber zerumbet rhizomes with inhibitory effect on Pythium myriotylum secretory proteases and zoospore viability

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Abstract

Protease mediated proteolysis has been widely implicated in virulence of necrotrophic fungal pathogens. This is counteracted in plants by evolving new and effective antimicrobial peptides (AMP) that constitute important components of innate immune system. Peptide extraction from rhizome of Zingiber zerumbet was optimized using ammonium sulphate (50–80% w/v) and acetone (60 and 100% v/v) with maximal protein recovery of 1.2 ± 0.4 mg/g obtained using 100% acetone. Evaluation of inhibitory potential of Z. zerumbet rhizome protein extract to prominent hydrolases of necrotrophic Pythium myriotylum revealed maximal inhibition of proteases (75.8%) compared to other hydrolytic enzymes. Protein was purified by Sephacryl S200HR resin resulting in twofold purification and protease inhibition of 84.4%. Non-reducing polyacrylamide gel electrophoresis (PAGE) of the fractions yielded two bands of 75 kDa and 25 kDa molecular size. Peptide mass fingerprint of the protein bands using matrix assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectroscopy (MS) and subsequent MASCOT searches revealed peptide match to methylesterase from Arabidopsis thaliana (15%) and to hypothetical protein from Oryza sativa (98%) respectively. Further centrifugal filter purification using Amicon Ultra (10,000 MW cut-off) filter, yielded a prominent band of 25 kDa size. Concentration dependent inhibition of zoospore viability by Z. zerumbet AMP designated as ZzAMP was observed with maximal inhibition of 89.5% at 4 µg protein and an IC50 value of 0.59 µg. Studies are of particular relevance in the context of identifying the molecules involved in imparting below ground defense in Z. zerumbet as well in development of AMPs as potential candidate molecules for control of necrotrophic pathogens of agricultural relevance.

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Acknowledgements

SR and PP acknowledges the Research Fellowship received from Ministry of Human Resource and Development (MHRD), Government of India. Authors are grateful to NITC and CUK for the research facilities extended.

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Authors and Affiliations

  1. School of Biotechnology, National Institute of Technology Calicut, Calicut, 673 601, India

    Sharmila Raj & Princy Peter

  2. Department of Biochemistry and Molecular Biology, Central University of Kerala (CUK), Kasaragod, 671 320, India

    R. Aswati Nair

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Correspondence to R. Aswati Nair.

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Raj, S., Aswati Nair, R. & Peter, P. Antimicrobial peptide (AMP) from Zingiber zerumbet rhizomes with inhibitory effect on Pythium myriotylum secretory proteases and zoospore viability. World J Microbiol Biotechnol 36, 77 (2020). https://doi.org/10.1007/s11274-020-02848-9

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