title: 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.
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.
authors.affiliation: Department of Studies in Biotechnology, University of Mysore, Manasagangotri, Mysore, 570 006, Karnataka, India.
authors.forename: Sreedhara Ashok
authors.initials: SA
authors.lastname: Prabhu
authors.forename: Martin
authors.initials: M
authors.lastname: Wagenknecht
authors.forename: Prasad
authors.initials: P
authors.lastname: Melvin
authors.forename: Belur Shivappa
authors.initials: BS
authors.lastname: Gnanesh Kumar
authors.forename: Mariswamy
authors.initials: M
authors.lastname: Veena
authors.forename: Sekhar
authors.initials: S
authors.lastname: Shailasree
authors.forename: Bruno Maria
authors.initials: BM
authors.lastname: Moerschbacher
authors.forename: Kukkundoor Ramachandra
authors.initials: KR
authors.lastname: Kini
mesh_terms: D000595:Amino Acid Sequence; D060467:Disease Resistance / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D004791:Enzyme Inhibitors / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D005656:Fungal Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006031:Glycosylation; D054884:Host-Pathogen Interactions / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009868:Oomycetes / Q000187:drug effects / Q000502:physiology; D031782:Pennisetum / Q000235:genetics / Q000378:metabolism* / Q000382:microbiology; D010935:Plant Diseases / Q000235:genetics / Q000382:microbiology; D019441:Plant Epidermis / Q000235:genetics / Q000378:metabolism / Q000382:microbiology; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D058526:Plant Vascular Bundle / Q000235:genetics / Q000378:metabolism / Q000382:microbiology; D011096:Polygalacturonase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D055550:Protein Stability; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
chemical_list: D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D010940:Plant Proteins; D011096:Polygalacturonase