Three aspartic acid residues of polygalacturonase-inhibiting protein (PGIP) from Phaseolus vulgaris are critical for inhibition of Fusarium phyllophilum PG

Polygalacturonase-inhibiting proteins (PGIPs) are plant cell wall proteins that specifically inhibit the activity of endo polygalacturonases (PGs) produced by fungi during the infection process. The interaction with PGIPs limits the destructive potential of PGs and may trigger plant defence responses through the release of elicitor active oligogalacturonides. In order to pinpoint the residues of PvPGIP2 from Phaseolus vulgaris involved in the interaction with PGs, we used site-directed mutagenesis to mutate the residues D131, D157 and D203, and tested for the inhibitory activity of the mutant proteins expressed in Pichia pastoris against Fusarium phyllophilum and Aspergillus niger PGs. Here, we report that mutation of these residues affects the inhibition capacity of PvPGIP2 against F. phyllophilum PG.     

The polygalacturonase-inhibiting protein PGIP2 of Phaseolus vulgaris has evolved a mixed mode of inhibition of endopolygalacturonase PG1 of Botrytis cinerea

Botrytis cinerea is a phytopathogenic fungus that causes gray mold in >1,000 plant species. During infection, it secretes several endopolygalacturonases (PGs) to degrade cell wall pectin, and among them, BcPG1 is constitutively expressed and is an important virulence factor. To counteract the action of PGs, plants express polygalacturonase-inhibiting proteins (PGIPs) that have been shown to inhibit a variety of PGs with different inhibition kinetics, both competitive and noncompetitive. The PG-PGIP interaction promotes the accumulation of oligogalacturonides, fragments of the plant cell wall that are general elicitors of plant defense responses. Here, we characterize the enzymatic activity of BcPG1 and investigate its interaction with PGIP isoform 2 from Phaseolus vulgaris (PvPGIP2) by means of inhibition assays, homology modeling, and molecular docking simulations. Our results indicate a mixed mode of inhibition. This is compatible with a model for the interaction where PvPGIP2 binds the N-terminal portion of BcPG1, partially covering its active site and decreasing the enzyme affinity for the substrate. The structural framework provided by the docking model is confirmed by site-directed mutagenesis of the residues that distinguish PvPGIP2 from the isoform PvPGIP1. The finding that PvPGIP2 inhibits BcPG1 with a mixed-type kinetics further indicates the versatility of PGIPs to evolve different recognition specificities.     

The expression of a bean PGIP in transgenic wheat confers increased resistance to the fungal pathogen Bipolaris sorokiniana

A possible strategy to control plant pathogens is the improvement of natural plant defense mechanisms against the tools that pathogens commonly use to penetrate and colonize the host tissue. One of these mechanisms is represented by the host plant's ability to inhibit the pathogen's capacity to degrade plant cell wall polysaccharides. Polygalacturonase-inhibiting proteins (PGIP) are plant defense cell wall glycoproteins that inhibit the activity of fungal endopolygalacturonases (endo-PGs). To assess the effectiveness of these proteins in protecting wheat from fungal pathogens, we produced a number of transgenic wheat lines expressing a bean PGIP (PvPGIP2) having a wide spectrum of specificities against fungal PGs. Three independent transgenic lines were characterized in detail, including determination of the levels of PvPGIP2 accumulation and its subcellular localization and inhibitory activity. Results show that the transgene-encoded protein is correctly secreted into the apoplast, maintains its characteristic recognition specificities, and endows the transgenic wheat with new PG recognition capabilities. As a consequence, transgenic wheat tissue showed increased resistance to digestion by the PG of Fusarium moniliforme. These new properties also were confirmed at the plant level during interactions with the fungal pathogen Bipolaris sorokiniana. All three lines showed significant reductions in symptom progression (46 to 50%) through the leaves following infection with this pathogen. Our results illustrate the feasibility of improving wheat's defenses against pathogens by expression of proteins with new capabilities to counteract those produced by the pathogens.     

The interaction between endopolygalacturonase from Fusarium moniliforme and PGIP from Phaseolus Vulgaris studied by surface plasmon resonance and mass spectrometry

A combination of surface plasmon resonance (SPR) and matrix-assisted laser-desorptionionization- time-of-flight mass spectrometry (MALDI-TOF-MS) was used to study the interaction between endopolygalacturonase (PG) from Fusarium moniliforme and a polygalacturonase-inhibiting protein (PGIP) from Phaseolus vulgaris. PG hydrolyses the homogalacturonan of the plant cell wall and is considered an important pathogenicity factor of many fungi. PGIP is a specific inhibitor of fungal PGs and is thought to be involved in plant defence against phytopathogenic fungi. SPR was used either to study the effect of the PG glycosylation on the formation of the complex with PGIP, and as a sensitive affinity capture of an interacting peptide from a mixture of PG fragments obtained by limited proteolysis. Mass spectrometry allowed to characterise the interacting peptide eluted from the sensor surface.     

Transgenic expression of polygalacturonase-inhibiting proteins in Arabidopsis and wheat increases resistance to the flower pathogen Fusarium graminearum

Fusarium head blight (FHB), caused by Fusarium graminearum, is one of the most important diseases of wheat worldwide, resulting in yield losses and mycotoxin contamination. The molecular mechanisms regulating Fusarium penetration and infection are poorly understood. Beside mycotoxin production, cell wall degradation may play a role in the development of FHB. Many fungal pathogens secrete polygalacturonases (PGs) during the early stages of infection, and plants have evolved polygalacturonase-inhibiting proteins (PGIPs) to restrict pectin degradation during fungal infection. To investigate the role of plant PGIPs in restricting the development of FHB symptoms, we first used Arabidopsis thaliana, whose genome encodes two PGIPs (AtPGIP1 and AtPGIP2). Arabidopsis transgenic plants expressing either of these PGIPs under control of the CaMV 35S promoter accumulate inhibitory activity against F.?graminearum PG in their inflorescences, and show increased resistance to FHB. Second, transgenic wheat plants expressing the bean PvPGIP2 in their flowers also had a significant reduction of symptoms when infected with F.?graminearum. Our data suggest that PGs likely play a role in F.?graminearum infection of floral tissues, and that PGIPs incorporated into wheat may be important for increased resistance to FHB.     

Polygalacturonase-inhibiting protein is a structural component of plant cell wall

It is generally believed that plants "evolved a strategy of defending themselves from a phytopathogen attack" during evolution. This metaphor is used frequently, but it does not facilitate understanding of the mechanisms providing plant resistance to the invasion of foreign organisms and to other unfavorable external factors, as well as the role of these mechanisms in plant growth and development. Information on processes involving one of the plant resistance factors--polygalacturonase-inhibiting protein (PGIP)--is considered in this review. The data presented here indicate that PGIP, being an extracellular leucine-rich repeat-containing protein, performs important functions in the structure of plant cell wall. Amino acid residues participating in PGIP binding to homogalacturonan in the cell wall have been determined. The degree of methylation and the mode of distribution of homogalacturonan methyl groups are responsible for the formation of a complex structure, which perhaps determines the specificity of PGIP binding to pectin. PGIP is apparently one of the components of plant cell wall determining some of its mechanical properties; it is involved in biochemical processes related to growth, expansion, and maceration, and it influences plant morphology. Polygalacturonase (PG) is present within practically all plant tissues, but the manifestation of its activity varies significantly depending on physiological conditions in the tissue. Apparently, the regulation of PG functioning in apoplast significantly affects the development of processes associated with the modification of the structure of plant cell wall. PGIP can regulate PG activity through binding to homogalacturonan. The genetically determined structure of PGIP in plants determines the mode of its interaction with an invader and perhaps is one of the factors responsible for the set of pathogens causing diseases in a given plant species.     

Cloning and functional analysis of three genes encoding polygalacturonase-inhibiting proteins from Capsicum annuum and transgenic CaPGIP1 in tobacco in relation to increased resistance to two fungal pathogens

Polygalacturonase-inhibiting proteins (PGIPs) are plant cell wall glycoproteins that can inhibit fungal endopolygalacturonases (PGs). The PGIPs directly reduce the aggressive potential of PGs. Here, we isolated and functionally characterized three members of the pepper (Capsicum annuum) PGIP gene family. Each was up-regulated at a different time following stimulation of the pepper leaves by Phytophthora capcisi and abiotic stresses including salicylic acid, methyl jasmonate, abscisic acid, wounding and cold treatment. Purified recombinant proteins individually inhibited activity of PGs produced by Alternaria alternata and Colletotrichum nicotianae, respectively, and virus-induced gene silencing in pepper conferred enhanced susceptibility to P. capsici. Because three PGIP genes acted similarily in conferring resistance to infection by P. capsici, and because individually purified proteins showed consistent inhibition against PG activity of both pathogens, CaPGIP1 was selected for manipulating transgenic tobacco. The crude proteins from transgenic tobacco exhibited distinct enhanced resistance to PG activity of both fungi. Moreover, the transgenic tobacco showed effective resistance to infection and a significant reduction in the number of infection sites, number of lesions and average size of lesions in the leaves. All results suggest that CaPGIPs may be involved in plant defense response and play an important role in a plant’s resistance to disease.     

Polygalacturonase-inhibiting protein interacts with pectin through a binding site formed by four clustered residues of arginine and lysine

Polygalacturonase-inhibiting protein (PGIP) is a cell wall protein that inhibits fungal polygalacturonases (PGs) and retards the invasion of plant tissues by phytopathogenic fungi. Here, we report the interaction of two PGIP isoforms from Phaseolus vulgaris (PvPGIP1 and PvPGIP2) with both polygalacturonic acid and cell wall fractions containing uronic acids. We identify in the three-dimensional structure of PvPGIP2 a motif of four clustered arginine and lysine residues (R183, R206, K230, and R252) responsible for this binding. The four residues were mutated and the protein variants were expressed in Pichia pastoris. The ability of both wild-type and mutated proteins to bind pectins was investigated by affinity chromatography. Single mutations impaired the binding and double mutations abolished the interaction, thus indicating that the four clustered residues form the pectin-binding site. Remarkably, the binding of PGIP to pectin is displaced in vitro by PGs, suggesting that PGIP interacts with pectin and PGs through overlapping although not identical regions. The specific interaction of PGIP with polygalacturonic acid may be strategic to protect pectins from the degrading activity of fungal PGs.     

Polygalacturonase inhibitor protein from fruits of anthracnose resistant and susceptible varieties of Chilli (Capsicum annuum L)

Chilli fruit is highly susceptible to anthracnose infection at the stage of harvest maturity, due to which the fruit yield in the leading commercial variety Byadgi is severely affected. Field studies on screening of several varieties for resistance to anthracnose have shown that a variety of chilli AR-4/99K is resistant to anthracnose infection. In many crops, resistance to fungal attack has been correlated with PGIP activity in developing fruits based on which transgenic varieties have been developed with resistance to fungi. The present study was carried out to determine whether anthracnose resistance in AR-4/99K was due to the increased levels of PGIP alone and/ or due to differences, if any, in the properties of PGIP. Hence, a comparative study of the properties of polygalacturonase inhibitor protein (PGIP) isolated from fruits of anthracnose resistant chilli var AR-4/99K and a susceptible variety Byadgi was conducted with the objective of utilizing the information in genetic transformation studies. Both the PGIPs from anthracnose resistant and susceptible varieties of chilli exhibited similarities in the elution pattern on Sephadex gel, DEAE cellulose, PAGE and SDS-PAGE. The two PGIPs were active over a wide range of pH and temperature. Both PGIPs showed differential inhibitory activity against polygalacturonase (PG) secreted by Colletotrichum gleosporoides, C. capsici, C. lindemuthianum, Fusarium moniliforme and Sclerotium rolfsii. The inhibitory activity of PGIP from both resistant and susceptible varieties was the highest (82% and 76%, respectively) against the PG from Colletotrichum capsici, a pathogen causing anthracnose rot of chilli, while the activity was lower (1.27 to 12.3%) on the other fungal PGs. Although PGIP activity decreased with fruit maturation in both the varieties, the resistant variety maintained a higher activity at 45 days after flowering (DAF) as compared to the susceptible variety which helped it to overcome the infection by anthracnose as against the susceptible variety (Byadgi) in which PGIP activity was drastically reduced at maturity. The molecular mass of PGIP as determined by SDS-PAGE was found to be 37 kDa. N-terminal sequence analysis of the PGIP showed the first six amino acid residues from N-terminal end were Asp-Thr-His-Lys-Ser-Glu (DTHKSE), respectively. The similarities in properties of the two PGIPs support the earlier findings that resistance of AR-4/99K to anthracnose fungus is a result of its higher PGIP activity at maturity.     

Polygalacturonase inhibiting protein in plant cell walls

Polygalacturonase inhibiting protein (PGIP) is localized in plant cell walls and plays an important role both in pectic substance metabolism and in prevention of the penetration of phytopathogenic microorganisms. Apparently, PGIP is responsible for the specificity of cell--cell interactions during pollination or inoculation by fungi nonpathogenic for the particular plant. PGIPs from different plants share a basic common structure. They are rather thermostable glycoproteins enriched with leucine and contain about 20% carbohydrates; the molecular weight varies between 37-54 kD. The synthesis of PGIP is encoded by one gene, and its expression is stimulated by injury and fungal infection. The resistance of plant tissues to infection frequently correlates with PGIP expression and with inhibiting action on fungal PG. Thus, PGIP is believed to be useful for gene engineering to obtain transgenic plants resistant to fungal infection or retaining commercial value during storage.     

Study of Q224K,V152G double mutation in bean PGIP2, an LRR protein for plant defense—An in silico approach

Polygalacturonase inhibiting proteins (PGIPs) are leucine‐rich repeat (LRR) proteins from plants that are organized into multigene families. They act as specific inhibitors against Polygalacturonases (PGs) from phytopathogens and share high sequence identity within species. We performed in silico mutation (Q224K and V152G) in PGIP2 from Phaseolus vulgaris to corresponding residues of another member, PGIP1. This mutation is known to cause 100% loss of inhibition against the PG of fungus Fusarium phyllophilum (Fp). A comparative analysis between PGIP2 and the double mutant, using 50 ns molecular dynamics simulations explored structural difference affecting PG binding properties. Simulations revealed that the mutation at 224, strains this residue which acts as a lock for the PGIP‐PG complex through main chain H‐bond. Changes in secondary structural elements and strain in the bend region along the convex face of the solenoidal protein affected the flexibility of the mutant protein. At the concave interacting face of the mutant, subtle changes in the sidechain behavior of the PG‐binding residues occurred in a concerted manner revealing flipping of aromatic rings to be crucial to avoid steric clash with FpPG in PGIP2. Docking PGIP2 and the mutant protein individually to FpPG illustrated the inability of the latter to inhibit FpPG leaving its active site free. Our study demonstrates that the effect of mutation affects the flexibility of the protein along the convex face, while binding specificity is altered through the concave face imparting minimal change in the typical structure supported by the LRRs. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Directed mutagenesis confirms the functional importance of positively selected sites in polygalacturonase inhibitor protein

Polygalacturonase inhibitor proteins (PGIPs) protect plants against invasion by diverse microbial and invertebrate enemies that use polygalacturonase (PG) to breach the plant cell wall. Directed mutagenesis has identified specific natural mutations conferring novel defensive capability in green bean PGIP against a specific fungal PG. These same sites are identified as positively selected by phylogenetic codon-substitution models, demonstrating the utility of such models for connecting retrospective comparative analyses with contemporary, ecologically relevant variation.     

A single amino acid substitution in highly similar endo-PGs from Fusarium verticillioides and related Fusarium species affects PGIP inhibition.

Endo-polygalacturonase (PG) may be a critical virulence factor secreted by several fungi upon plant invasion. The single-copy gene encoding PG in Fusarium verticillioides and in eight other species of the Gibberella fujikuroi complex (F. sacchari, F. fujikuroi, F. proliferatum, F. subglutinans, F. thapsinum, F. nygamai, F. circinatum, and F. anthophilum) was functionally analyzed in this paper. Both the nucleotide and amino acid sequences were highly similar among the 12 strains of F. verticillioides analyzed, as well as among those from the G. fujikuroi complex. The PGs were not inhibited by the polygalacturonase-inhibiting proteins (PGIPs) from the monocot asparagus and leek plants, but were inhibited to variable extents by bean PGIP. PGs from F. verticillioides, F. nygamai and one strain of F. proliferatum were barely inhibited. Residue 97 within PG was demonstrated to contribute to the different levels of inhibition. Together these findings provide new insights into the structural and functional relationships between the PG from the species of the G. fujikuroi complex and the plant PGIP.     

A single amino acid substitution in highly similar endo-PGs from Fusarium verticillioides and related Fusarium species affects PGIP inhibition

Endo-polygalacturonase (PG) may be a critical virulence factor secreted by several fungi upon plant invasion. The single-copy gene encoding PG in Fusarium verticillioides and in eight other species of the Gibberella fujikuroi complex (F. sacchari, F. fujikuroi, F. proliferatum, F. subglutinans, F. thapsinum, F. nygamai, F. circinatum, and F. anthophilum) was functionally analyzed in this paper. Both the nucleotide and amino acid sequences were highly similar among the 12 strains of F. verticillioides analyzed, as well as among those from the G. fujikuroi complex. The PGs were not inhibited by the polygalacturonase-inhibiting proteins (PGIPs) from the monocot asparagus and leek plants, but were inhibited to variable extents by bean PGIP. PGs from F. verticillioides, F. nygamai and one strain of F. proliferatum were barely inhibited. Residue 97 within PG was demonstrated to contribute to the different levels of inhibition. Together these findings provide new insights into the structural and functional relationships between the PG from the species of the G. fujikuroi complex and the plant PGIP.     

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

Homogeneous endo-polygalacturonase (PG) was covalently bound to cyanogen-bromide-activated Sepharose, and the resulting PG-Sepharose conjugate was utilized to purify, by affinity chromatography, a protein from Phaseolus vulgaris hypocotyls that binds to and inhibits PG. Isoelectric focusing of the purified PG-inhibiting protein (PGIP) showed a major protein band that coincided with PG-inhibiting activity. PGIP formed a complex with PG at pH 5.0 and at low salt concentrations. The complex dissociated in 0.5 m Na-acetate and pH values lower than 4.5 or higher than 6.0. Formation of the PG-PGIP complex resulted in complete inhibition of PG activity. PG activity was restored upon dissociation of the complex. The protein exhibited inhibitory activity toward PGs from Colletotrichum lindemuthianum, Fusarium moniliforme and Aspergillus niger. The possible role of PGIP in regulating the activity of fungal PG's and their ability to elicit plant defense reactions are discussed.     

Characterization and functional analysis of a novel PGIP gene from Pyrus pyrifolia Nakai cv Huobali

Polygalacturonase-inhibiting proteins (PGIPs) are multifunctional proteins related to plant autoimmunity and belong to the plant extracellular leucine-rich repeat (eLRR) protein superfamily. PGIPs play a role in host defense in many plants. In the present study, a novel PGIP gene, PpPGIP was isolated from Pyrus pyrifolia Nakai cv Huobali. The nucleotide sequence of PpPGIP was highly homologous with PGIPs from other plant species and the protein encoded by PpPGIP has several conserved LRR domains. The putative protein PpPGIP was closely clustered with several PGIPs from horticultural plants on the phylogenetic tree. The constructed homology model of PpPGIP indicated that the main-chain conformation and the folding patterns of PpPGIP were highly similar to structural features of PvPGIP2 from Phaseolus vulgaris. The expression levels of PpPGIP in healthy tissue and organ of ‘Huobali’ were analyzed with RT-PCR, and PpPGIP accumulated a little in young leaves, but PpPGIP was expressed abundantly in the pericarp of ‘Huobali’ fruits. Furthermore, in order to verify the function of PpPGIP, the constitutive plant expression vector of PpPGIP was constructed and transferred into tobacco (Nicotiana tabacum L. cv Xanthi). The Southern blot and real-time PCR analyses demonstrated that the PpPGIP gene was integrated into the genome of the tobacco transformants and highly expressed in the transgenic lines. The antifungal activity of PpPGIP was detected in vitro plates, and the crude protein extract of transgenic tobacco plants inhibited the hyphal growth of Phomopsis sp., Alternaria sp., Penicillium sp., and Aspergillus niger in different degrees.     

Suppression of Fusarium oxysporum with recombinant polygalacturonase inhibiting proteins (BvPGIPs) extracted from sugar beet roots

Soil-borne fungus Fusarium oxysporum f. sp. betae (Fob) is the causative agent of Fusarium yellows in sugar beet. Leaf interveinal yellowing and root vascular discoloration significantly reduce root yield as well as sucrose content and juice purity. Fob, like other fungal pathogens, initiates disease development by secreting polygalacturonase (PG) enzymes to break down plant cell walls during early stages of infection. To protect themselves, plants produce polygalacturonase-inhibiting proteins (PGIPs). In our study of sugar beet root defense responses, several PGIP genes (BvPGIPs) were identified. To determine if BvPGIPs inhibit Fob PGs, genes BvPGIP1, BvPGIP2 and Bv(FC607)PGIP1 were fused with the CaMV 35S promoter and each was expressed individually in sugar beet hairy roots. We demonstrate that all three recombinant BvPGIP proteins inhibited Fob and F. oxysporum f. sp. gladioli (Fog) PGs. A comparable level of BvPGIP activity was observed against Fob PGs, while BvPGIP2 showed higher activity against Fog PGs. Similar results were obtained when recombinant PGIPs were used to bioassay effects on Fob and Fog spore germination and hyphal growth. This is a first report that documents F. oxysporum inhibition by overexpressing BvPGIPs that may lead to improved Fusarium yellows resistance in sugar beet.