Molecular Biology Reports

, Volume 42, Issue 6, pp 1123–1138 | Cite as

Immuno-affinity purification of PglPGIP1, a polygalacturonase-inhibitor protein from pearl millet: studies on its inhibition of fungal polygalacturonases and role in resistance against the downy mildew pathogen

  • Sreedhara Ashok Prabhu
  • Martin Wagenknecht
  • Prasad Melvin
  • Belur Shivappa Gnanesh Kumar
  • Mariswamy Veena
  • Sekhar Shailasree
  • Bruno Maria Moerschbacher
  • Kukkundoor Ramachandra Kini
Article

Abstract

Polygalacturonase-inhibitor proteins (PGIPs) are important plant defense proteins which modulate the activity of microbial polygalacturonases (PGs) leading to elicitor accumulation. Very few studies have been carried out towards understanding the role of PGIPs in monocot host defense. Hence, present study was taken up to characterize a native PGIP from pearl millet and understand its role in resistance against downy mildew. A native glycosylated PGIP (PglPGIP1) of ~43 kDa and pI 5.9 was immunopurified from pearl millet. Comparative inhibition studies involving PglPGIP1 and its non-glycosylated form (rPglPGIP1; recombinant pearl millet PGIP produced in Escherichia coli) against two PGs, PG-II isoform from Aspergillus niger (AnPGII) and PG-III isoform from Fusarium moniliforme, showed both PGIPs to inhibit only AnPGII. The protein glycosylation was found to impact only the pH and temperature stability of PGIP, with the native form showing relatively higher stability to pH and temperature changes. Temporal accumulation of both PglPGIP1 protein (western blot and ELISA) and transcripts (real time PCR) in resistant and susceptible pearl millet cultivars showed significant Sclerospora graminicola-induced accumulation only in the incompatible interaction. Further, confocal PGIP immunolocalization results showed a very intense immuno-decoration with highest fluorescent intensities observed at the outer epidermal layer and vascular bundles in resistant cultivar only. This is the first native PGIP isolated from millets and the results indicate a role for PglPGIP1 in host defense. This could further be exploited in devising pearl millet cultivars with better pathogen resistance.

Keywords

Chemical deglycosylation Confocal immunolocalization Peptide mass fingerprinting Plant–pathogen interaction Quantitative real-time PCR Two-dimensional gel electrophoresis 

Supplementary material

11033_2015_3850_MOESM1_ESM.pdf (42 kb)
Supplementary material 1 (PDF 43 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Sreedhara Ashok Prabhu
    • 1
  • Martin Wagenknecht
    • 2
  • Prasad Melvin
    • 1
  • Belur Shivappa Gnanesh Kumar
    • 3
  • Mariswamy Veena
    • 1
  • Sekhar Shailasree
    • 4
  • Bruno Maria Moerschbacher
    • 2
  • Kukkundoor Ramachandra Kini
    • 1
  1. 1.Department of Studies in BiotechnologyUniversity of MysoreMysoreIndia
  2. 2.Institut für Biologie und Biotechnologie der PflanzenWestfälische Wilhelms-Universität MünsterMünsterGermany
  3. 3.Protein Biochemistry and Glycobiology Laboratory, Department of BiochemistryUniversity of HyderabadHyderabadIndia
  4. 4.Institution of Excellence, Vijnana BhavanUniversity of MysoreMysoreIndia

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