title: Experimental and bioinformatic characterization of a recombinant polygalacturonase-inhibitor protein from pearl millet and its interaction with fungal polygalacturonases.
abstract: Polygalacturonases (PGs) are hydrolytic enzymes employed by several phytopathogens to weaken the plant cell wall by degrading homopolygalacturonan, a major constituent of pectin. Plants fight back by employing polygalacturonase-inhibitor proteins (PGIPs). The present study compared the inhibition potential of pearl millet PGIP (Pennisetum glaucum; PglPGIP1) with the known inhibition of Phaseolus vulgaris PGIP (PvPGIP2) against two PGs, the PG-II isoform from Aspergillus niger (AnPGII) and the PG-III isoform from Fusarium moniliforme (FmPGIII). The key rationale was to elucidate the relationship between the extent of sequence similarity of the PGIPs and the corresponding PG inhibition potential. First, a pearl millet pgip gene (Pglpgip1) was isolated and phylogenetically placed among monocot PGIPs alongside foxtail millet (Setaria italica). Upstream sequence analysis of Pglpgip1 identified important cis-elements responsive to light, plant stress hormones, and anoxic stress. PglPGIP1, heterologously produced in Escherichia coli, partially inhibited AnPGII non-competitively with a pH optimum between 4.0 and 4.5, and showed no inhibition against FmPGIII. Docking analysis showed that the concave surface of PglPGIP1 interacted strongly with the N-terminal region of AnPGII away from the active site, whereas it weakly interacted with the C-terminus of FmPGIII. Interestingly, PglPGIP1 and PvPGIP2 employed similar motif regions with few identical amino acids for interaction with AnPGII at non-substrate-binding sites; however, they engaged different regions of AnPGII. Computational mutagenesis predicted D126 (PglPGIP1)-K39 (AnPGII) to be the most significant binding contact in the PglPGIP1-AnPGII complex. Such protein-protein interaction studies are crucial in the future generation of designer host proteins for improved resistance against ever-evolving pathogen virulence factors.
authors: Department of Studies in Biotechnology, University of Mysore, Manasagangotri, Mysore-570 006, Karnataka, India Institut für Biologie und Biotechnologie der Pﬂanzen, Westfälische Wilhelms-Universität Münster, Schlossplatz 8, D-48143 Münster, Germany.
authors: S Ashok
authors: SA
authors: Prabhu
authors: Institut für Biologie und Biotechnologie der Pﬂanzen, Westfälische Wilhelms-Universität Münster, Schlossplatz 8, D-48143 Münster, Germany.
authors: Ratna
authors: R
authors: Singh
authors: Institut für Biologie und Biotechnologie der Pﬂanzen, Westfälische Wilhelms-Universität Münster, Schlossplatz 8, D-48143 Münster, Germany.
authors: Stephan
authors: S
authors: Kolkenbrock
authors: Molecular Biology of Plants, Groningen Biomolecular Sciences and Biotechnology Institute, Centre for Life Sciences, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands.
authors: Neerakkal
authors: N
authors: Sujeeth
authors: Institut für Biologie und Biotechnologie der Pﬂanzen, Westfälische Wilhelms-Universität Münster, Schlossplatz 8, D-48143 Münster, Germany.
authors: Nour Eddine
authors: NE
authors: El Gueddari
authors: Institut für Biologie und Biotechnologie der Pﬂanzen, Westfälische Wilhelms-Universität Münster, Schlossplatz 8, D-48143 Münster, Germany.
authors: Bruno M
authors: BM
authors: Moerschbacher
authors: Department of Studies in Biotechnology, University of Mysore, Manasagangotri, Mysore-570 006, Karnataka, India krk@appbot.uni-mysore.ac.in.
authors: Ramachandra K
authors: RK
authors: Kini
authors: Institut für Biologie und Biotechnologie der Pﬂanzen, Westfälische Wilhelms-Universität Münster, Schlossplatz 8, D-48143 Münster, Germany.
authors: Martin
authors: M
authors: Wagenknecht
mesh_terms: D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000378:metabolism; D001483:Base Sequence; D005656:Fungal Proteins / Q000378:metabolism*; D005670:Fusarium / Q000378:metabolism; D008969:Molecular Sequence Data; D031782:Pennisetum / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D011096:Polygalacturonase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D005656:Fungal Proteins; C081479:PGIP protein, plant; D010940:Plant Proteins; D011994:Recombinant Proteins; D011096:Polygalacturonase
keywords: Computational mutagenesis; PGIPs; PGs; Phaseolus vulgaris; electrostatic surface potential; inhibition studies; pearl millet; protein modelling and docking.