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 Received: 11 July 2014 / Accepted: 9 January 2015 Ó Springer Science+Business Media Dordrecht 2015 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 char- acterize a native PGIP from pearl millet and understand its role in resistance against downy mildew. A native gly- cosylated 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 vas- cular 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 Á Confocal immunolocalization Á Peptide mass fingerprinting Á Plant–pathogen interaction Á Quantitative real-time PCR Á Two-dimensional gel electrophoresis Introduction The plant cell wall (CW) which acts as both a rigid structural embankment as well as a flexible layer during cell expansion has evolved into a complex network of polysaccharides such as cellulose, hemicellulose, pectin and proteins [1]. Due to their strategic position, CWs are crucial in plant–microbe interactions, including defense against phytopathogens [2]. Homogalacturonides (HGs) are linear, a-1,4-linked-D-galactopyranosyluronic acid chains, with a significant portion of the residues methyl esterified and/or acetylated, and constitute the ‘‘smooth region’’ of the complex pectin [3]. Polygalacturonases (PGs) are Electronic supplementary material The online version of this article (doi:10.1007/s11033-015-3850-5) contains supplementary material, which is available to authorized users. S. A. Prabhu Á P. Melvin Á M. Veena Á K. R. Kini (&) Department of Studies in Biotechnology, University of Mysore, Manasagangotri, Mysore 570 006, Karnataka, India e-mail: krk@appbot.uni-mysore.ac.in M. Wagenknecht Á B. M. Moerschbacher Institut fu ¨r Biologie und Biotechnologie der Pflanzen, Westfa ¨lische Wilhelms-Universita ¨t Mu ¨nster, Schlossplatz 8, 48143 Mu ¨nster, Germany B. S. Gnanesh Kumar Protein Biochemistry and Glycobiology Laboratory, Department of Biochemistry, University of Hyderabad, Hyderabad 500046, Andhra Pradesh, India S. Shailasree Institution of Excellence, Vijnana Bhavan, University of Mysore, Manasagangotri, Mysore 570 006, Karnataka, India 123 Mol Biol Rep DOI 10.1007/s11033-015-3850-5