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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.

SUBMITTER: Prabhu SA

PROVIDER: S-EPMC4144779 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Experimental and bioinformatic characterization of a recombinant polygalacturonase-inhibitor protein from pearl millet and its interaction with fungal polygalacturonases.

Prabhu S Ashok SA Singh Ratna R Kolkenbrock Stephan S Sujeeth Neerakkal N El Gueddari Nour Eddine NE Moerschbacher Bruno M BM Kini Ramachandra K RK Wagenknecht Martin M

Journal of experimental botany 20140630 17

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 ...[more]

PMID: 24980909

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Project description:The phenomenon of heterosis has fascinated plant breeders ever since it was first described by Charles Darwin in 1876 in the vegetable kingdom and later elaborated by George H Shull and Edward M East in maize during 1908. Heterosis is the phenotypic and functional superiority manifested in the F1 crosses over the parents. Various classical complementation mechanisms gave way to the study of the underlying potential cellular and molecular mechanisms responsible for heterosis. In cereals, such as maize, heterosis has been exploited very well, with the development of many single-cross hybrids that revolutionized the yield and productivity enhancements. Pearl millet (Pennisetum glaucum (L.) R. Br.) is one of the important cereal crops with nutritious grains and lower water and energy footprints in addition to the capability of growing in some of the harshest and most marginal environments of the world. In this highly cross-pollinating crop, heterosis was exploited by the development of a commercially viable cytoplasmic male-sterility (CMS) system involving a three-lines breeding system (A-, B- and R-lines). The first set of male-sterile lines, i.e., Tift 23A and Tift18A, were developed in the early 1960s in Tifton, Georgia, USA. These provided a breakthrough in the development of hybrids worldwide, e.g., Tift 23A was extensively used by Punjab Agricultural University (PAU), Ludhiana, India, for the development of the first single-cross pearl millet hybrid, named Hybrid Bajra 1 (HB 1), in 1965. Over the past five decades, the pearl millet community has shown tremendous improvement in terms of cytoplasmic and nuclear diversification of the hybrid parental lines, which led to a progressive increase in the yield and adaptability of the hybrids that were developed, resulting in significant genetic gains. Lately, the whole genome sequencing of Tift 23D2B1 and re-sequencing of circa 1000 genomes by a consortium led by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) has been a significant milestone in the development of cutting-edge genetic and genomic resources in pearl millet. Recently, the application of genomics and molecular technologies has provided better insights into genetic architecture and patterns of heterotic gene pools. Development of whole-genome prediction models incorporating heterotic gene pool models, mapped traits and markers have the potential to take heterosis breeding to a new level in pearl millet. This review discusses advances and prospects in various fronts of heterosis for pearl millet.

Project description:Erwinia chrysanthemi 3937 secretes an arsenal of pectinolytic enzymes, including at least eight endo-pectate lyases encoded by pel genes, which play a major role in the soft-rot disease caused by this bacterium on various plants. E. chrysanthemi also produces some hydrolases that cleave pectin. Three adjacent hydrolase genes, pehV, pehW, and pehX, encoding exo-poly-alpha-D-galacturonosidases, have been characterized. These enzymes liberate digalacturonides from the nonreducing end of pectin. We report the identification of a novel gene, named pehN, encoding a protein homologous to the glycosyl hydrolases of family 28, which includes mainly polygalacturonases. PehN has a low hydrolase activity on polygalacturonate and on various pectins. PehN action favors the activity of the secreted endo-pectate lyases, mainly PelB and PelC, and that of the periplasmic exo-pectate lyase PelX. However, removal of the pehN gene does not significantly alter the virulence of E. chrysanthemi. Regulation of pehN transcription was analyzed by using gene fusions. Like other pectinase genes, pehN transcription is dependent on several environmental conditions. It is induced by pectic catabolic products and is affected by growth phase, catabolite repression, osmolarity, anaerobiosis, nitrogen starvation, and the presence of calcium ions. The transcription of pehN is modulated by the repressor KdgR, which controls almost all the steps of pectin catabolism, and by cyclic AMP receptor protein (CRP), the global activator of sugar catabolism. The regulator PecS, which represses the transcription of the pel genes but activates that of pehV, pehW, and pehX, also activates transcription of pehN. The three regulators KdgR, PecS, and CRP act by direct interaction with the pehN promoter region. The sequences involved in the binding of these three regulators and of RNA polymerase have been precisely defined. Analysis of the simultaneous binding of these proteins indicates that CRP and RNA polymerase bind cooperatively and that the binding of KdgR could prevent pehN transcription. In contrast, the activator effect of PecS is not linked to competition with KdgR or to cooperation with CRP or RNA polymerase. This effect probably results from competition between PecS and an unidentified repressor involved in peh regulation.

Dataset Information

Experimental and bioinformatic characterization of a recombinant polygalacturonase-inhibitor protein from pearl millet and its interaction with fungal polygalacturonases.

Publications

Experimental and bioinformatic characterization of a recombinant polygalacturonase-inhibitor protein from pearl millet and its interaction with fungal polygalacturonases.

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