Purification and Characterization of a Polygalacturonase-Inhibiting Protein from <i>Phaseolus vulgaris</i> L.

To invade a plant tissue, phytopathogenic fungi produce several cell wall-degrading enzymes; among them, endopolygalacturonase (PG) catalyzes the fragmentation and solubilization of homogalacturonan. Polygalacturonase-inhibiting proteins (PGIPs), found in the cell wall of many plants, counteract fungal PGs by forming specific complexes with them. We report the crystal structure at 1.73 Å resolution of PG from the phytopathogenic fungus Fusarium moniliforme (FmPG). The structure of FmPG was useful to study the mode of interaction of the enzyme with PGIP-2 from Phaseolus vulgaris. Several amino acids of FmPG were mutated, and their contribution to the formation of the complex with PGIP-2 was investigated by surface plasmon resonance. The residues Lys-269 and Arg-267, located inside the active site cleft, and His-188, at the edge of the active site cleft, are critical for the formation of the complex, which is consistent with the observed competitive inhibition of the enzyme played by PGIP-2. The replacement of His-188 with a proline or the insertion of a tryptophan after position 270, variations that both occur in plant PGs, interferes with the formation of the complex. We suggest that these variations are important structural requirements of plant PGs to prevent PGIP binding.

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“…PGIP-2 was purified from PVX-infected tissues of Nicotiana benthamiana as previously described (21).…”

Section: Methodsmentioning

confidence: 99%

Structural requirements of endo polygalacturonase for the interaction with PGIP (polygalacturonase-inhibiting protein)

Federici

Caprari

Mattei

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

135

129

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“…During microbial infections, OGs are expected to be released through the action of pathogen-encoded enzymes, such as polygalacturonases (PGs) (18). In vitro, the generation of elicitor-active OGs is promoted by plant-encoded PG-inhibiting proteins (PGIPs), key components of the plant defense response, which block the complete hydrolysis of homogalacturonan to galacturonic acid (19,20). However, the hypothesis that PGIPs are responsible for the production of OGs in vivo and, in turn, that OGs act as endogenous DAMPs during infection, has never been proven directly and relies on evidence based on the exogenous application of elicitors obtained from commercial sources of pectin.…”

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confidence: 99%

Plant immunity triggered by engineered in vivo release of oligogalacturonides, damage-associated molecular patterns

Benedetti

Pontiggia

Raggi

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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177

165

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Oligogalacturonides (OGs) are fragments of pectin that activate plant innate immunity by functioning as damage-associated molecular patterns (DAMPs). We set out to test the hypothesis that OGs are generated in planta by partial inhibition of pathogen-encoded polygalacturonases (PGs). A gene encoding a fungal PG was fused with a gene encoding a plant polygalacturonase-inhibiting protein (PGIP) and expressed in transgenic Arabidopsis plants. We show that expression of the PGIP-PG chimera results in the in vivo production of OGs that can be detected by mass spectrometric analysis. Transgenic plants expressing the chimera under control of a pathogen-inducible promoter are more resistant to the phytopathogens Botrytis cinerea, Pectobacterium carotovorum, and Pseudomonas syringae. These data provide strong evidence for the hypothesis that OGs released in vivo act as a DAMP signal to trigger plant immunity and suggest that controlled release of these molecules upon infection may be a valuable tool to protect plants against infectious diseases. On the other hand, elevated levels of expression of the chimera cause the accumulation of salicylic acid, reduced growth, and eventually lead to plant death, consistent with the current notion that trade-off occurs between growth and defense.plant immunity | DAMPs | polygalacturonase | PGIP | oligogalacturonides

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“…The major components of primary cell walls of higher plants are complex polysaccharides; increasing evidence indicates that these molecules contribute to disease resistance not just as mechanical barriers but also as sensors for incoming infections (Vorwerk et al, 2004). For example, partial degradation of homogalacturonan (HGA) by fungal endopolygalacturonases (PGs) releases oligogalacturonides (OGs) with a degree of polymerization between 10 and 15 that show elicitor activity (Cervone et al, 1987a(Cervone et al, , 1987b(Cervone et al, , 1989. Treatment with OGs causes accumulation of reactive oxygen species, biosynthesis of phytoalexins (Hahn et al, 1981), and expression of pathogenesis-related (PR) proteins (Davis and Hahlbrock, 1987;Broekaert and Pneumas, 1988) in several plant species.…”

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confidence: 99%

Transgenic Expression of a Fungal endo-Polygalacturonase Increases Plant Resistance to Pathogens and Reduces Auxin Sensitivity

et al. 2007

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Polygalacturonases (PGs), enzymes that hydrolyze the homogalacturonan of the plant cell wall, are virulence factors of several phytopathogenic fungi and bacteria. On the other hand, PGs may activate defense responses by releasing oligogalacturonides (OGs) perceived by the plant cell as host-associated molecular patterns. Tobacco (Nicotiana tabacum) and Arabidopsis (Arabidopsis thaliana) plants expressing a fungal PG (PG plants) have a reduced content of homogalacturonan. Here, we show that PG plants are more resistant to microbial pathogens and have constitutively activated defense responses. Interestingly, either in tobacco PG or wild-type plants treated with OGs, resistance to fungal infection is suppressed by exogenous auxin, whereas sensitivity to auxin of PG plants is reduced in different bioassays. The altered plant defense responses and auxin sensitivity in PG plants may reflect an increased accumulation of OGs and subsequent antagonism of auxin action. Alternatively, it may be a consequence of perturbations of cellular physiology and elevated defense status as a result of altered cell wall architecture.

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Purification and Characterization of a Polygalacturonase-Inhibiting Protein from Phaseolus vulgaris L.

Cited by 131 publications

References 30 publications

Structural requirements of endo polygalacturonase for the interaction with PGIP (polygalacturonase-inhibiting protein)

Structural requirements of endo polygalacturonase for the interaction with PGIP (polygalacturonase-inhibiting protein)

Plant immunity triggered by engineered in vivo release of oligogalacturonides, damage-associated molecular patterns

Transgenic Expression of a Fungal endo-Polygalacturonase Increases Plant Resistance to Pathogens and Reduces Auxin Sensitivity

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