The grapevine polygalacturonase-inhibiting protein (VvPGIP1) reduces Botrytis cinerea susceptibility in transgenic tobacco and differentially inhibits fungal polygalacturonases

Polygalacturonase-inhibiting proteins (PGIPs) selectively inhibit polygalacturonases (PGs) secreted by invading plant pathogenic fungi. PGIPs display differential inhibition towards PGs from different fungi, also towards different isoforms of PGs originating from a specific pathogen. Recently, a PGIP-encoding gene from Vitis vinifera (Vvpgip1) was isolated and characterised. PGIP purified from grapevine was shown to inhibit crude polygalacturonase extracts from Botrytis cinerea, but this inhibitory activity has not yet been linked conclusively to the activity of the Vvpgip1 gene product. Here we use a transgenic over-expression approach to show that the PGIP encoded by the Vvpgip1 gene is active against PGs of B. cinerea and that over-expression of this gene in transgenic tobacco confers a reduced susceptibility to infection by this pathogen. A calculated reduction in disease susceptibility of 47–69% was observed for a homogeneous group of transgenic lines that was statistically clearly separated from untransformed control plants following infection with Botrytis over a 15-day-period. VvPGIP1 was subsequently purified from transgenic tobacco and used to study the specific inhibition profile of individual PGs from Botrytis and Aspergillus. The heterologously expressed and purified VvPGIP1 selectively inhibited PGs from both A. niger and B.␣cinerea, including BcPG1, a PG from B. cinerea that has previously been shown to be essential for virulence and symptom development. Altogether our data confirm the antifungal nature of the VvPGIP1, and the in vitro inhibition data suggest at least in part, that the VvPGIP1 contributed to the observed reduction in disease symptoms by inhibiting the macerating action of certain Botrytis PGs in planta. The ability to correlate inhibition profiles to individual PGs provides a more comprehensive analysis of PGIPs as antifungal genes with biotechnological potential, and adds to our understanding of the importance of PGIP:PG interactions during disease and symptom development in plants.Dirk A. Joubert and Ana R. Slaughter contributed equally to this work.     

A Single Amino-Acid Substitution Allows Endo-Polygalacturonase of Fusarium verticillioides to Acquire Recognition by PGIP2 from Phaseolus vulgaris

Polygalacturonases (PGs) are secreted by phytopathogenic fungi to degrade the plant cell wall homogalacturonan during plant infection. To counteract Pgs, plants have evolved polygalacturonase-inhibiting proteins (PGIPs) that slow down fungal infection and defend cell wall integrity. PGIPs favour the accumulation of oligogalacturonides, which are homogalacturonan fragments that act as endogenous elicitors of plant defence responses. We have previously shown that PGIP2 from Phaseolus vulgaris (PvPGIP2) forms a complex with PG from Fusarium phyllophilum (FpPG), hindering the enzyme active site cleft from substrate. Here we analyse by small angle X-ray scattering (SAXS) the interaction between PvPGIP2 and a PG from Colletotrichum lupini (CluPG1). We show a different shape of the PG-PGIP complex, which allows substrate entry and provides a structural explanation for the different inhibition kinetics exhibited by PvPGIP2 towards the two isoenzymes. The analysis of SAXS structures allowed us to investigate the basis of the inability of PG from Fusarium verticilloides (FvPG) to be inhibited by PvPGIP2 or by any other known PGIP. FvPG is 92.5% identical to FpPG, and we show here, by both loss- and gain-of-function mutations, that a single amino acid site acts as a switch for FvPG recognition by PvPGIP2.     

Antisense expression of the Arabidopsis thaliana AtPGIP1 gene reduces polygalacturonase-inhibiting protein accumulation and enhances susceptibility to Botrytis cinerea

Polygalacturonases (PGs) hydrolyze the homogalacturonan of plant cell-wall pectin and are important virulence factors of several phytopathogenic fungi. In response to abiotic and biotic stress, plants accumulate PG-inhibiting proteins (PGIPs) that reduce the activity of fungal PGs. In Arabidopsis thaliana, PGIPs with comparable activity against BcPG1, an important pathogenicity factor of the necrotrophic fungus Botrytis cinerea, are encoded by two genes, AtPGIP1 and AtPGIP2. Both genes are induced by fungal infection through different signaling pathways. We show here that transgenic Arabidopsis plants expressing an antisense AtPGIP1 gene have reduced AtPGIP1 inhibitory activity and are more susceptible to B. cinerea infection. These results indicate that PGIP contributes to basal resistance to this pathogen and strongly support the vision that this protein plays a role in Arabidopsis innate immunity.     

Plants use identical inhibitors to protect their cell wall pectin against microbes and insects

As fundamentally different as phytopathogenic microbes and herbivorous insects are, they enjoy plant‐based diets. Hence, they encounter similar challenges to acquire nutrients. Both microbes and beetles possess polygalacturonases (PGs) that hydrolyze the plant cell wall polysaccharide pectin. Countering these threats, plant proteins inhibit PGs of microbes, thereby lowering their infection rate. Whether PG‐inhibiting proteins (PGIPs) play a role in defense against herbivorous beetles is unknown. To investigate the significance of PGIPs in insect–plant interactions, feeding assays with the leaf beetle Phaedon cochleariae on Arabidopsis thaliana pgip mutants were performed. Fitness was increased when larvae were fed on mutant plants compared to wild‐type plants. Moreover, PG activity was higher, although PG genes were downregulated in larvae fed on PGIP‐deficient plants, strongly suggesting that PGIPs impair PG activity. As low PG activity resulted in delayed larval growth, our data provide the first in vivo correlative evidence that PGIPs act as defense against insects.     

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.

     

Polygalacturonase-inhibiting proteins in defense against phytopathogenic fungi

Polygalacturonase-inhibiting proteins (PGIPs) are ubiquitous plant cell wall proteins that are directed against fungal polygalacturonases (PGs), which are important pathogenicity factors. The inhibiting activity of PGIPs directly reduces the aggressive potential of PGs. In addition, it causes PGs to form more long-chain oligogalacturonides that are able to induce defense responses, thereby indirectly contributing to the plant defense. Recent evidence demonstrates that PGIPs are efficient defense proteins and limit fungal invasion. PGIPs and the products of many plant resistance genes share a leucine-rich repeat (LRR) structure, which provides specific recognition of pathogen-derived molecules. The high level of polymorphism of both PGIPs and polygalacturonases is an invaluable tool for deciphering the structure, function and evolution of plant LRR proteins and their ligands. Furthermore, information about PGIP structure and evolution paves the way to the development of efficient strategies for crop protection.     

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.     

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.     

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.     

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.     

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.     

Apple polygalacturonase inhibiting protein1 expressed in transgenic tobacco inhibits polygalacturonases from fungal pathogens of apple and the anthracnose pathogen of lupins

Extracts from apple fruit (cultivar "Granny Smith") inhibited the cell-wall degrading polygalacturonase (PG) activity of Colletotrichum lupini, the causal agent of anthracnose on lupins, as well as Aspergillus niger PG. Southern blot analysis indicated that this cultivar of apple has a small gene family of polygalacturonase inhibiting proteins (pgips), and therefore heterologous expression in transgenic tobacco was used to identify the specific gene product responsible for the inhibitory activity. A previously isolated pgip gene, termed Mdpgip1, was introduced into tobacco (Nicotiana tabacum) by Agrobacterium-mediated transformation. The mature MdPGIP1 protein was purified to apparent homogeneity from tobacco leaves by high salt extraction, clarification by DEAE-Sepharose and cation exchange HPLC. Purified MdPGIP1 inhibited PGs from C. lupini and PGs from two economically important pathogens of apple trees, Botryosphaeria obtusa and Diaporthe ambigua. It did not inhibit the A. niger PG, which was in contrast to the apple fruit extract used in this study. We conclude that there are at least two active PGIPs expressed in apple, which differ in their charge properties and ability to inhibit A. niger PG.     

Perspectives in the biological function and the technological application of polygalacturonases

Polygalacturonases (PG) have evolved in the past years from a pectinase “simply” being used for food processing to an important parameter in plant–fungal interaction. PG-inhibiting proteins (PGIP) that are synthesised in plants as a specific response to PGs of pathogenic fungi, have become a focus as a possible target in resistance breeding, and PGIPs are also a concern as an inhibiting factor in food processing. Plant PGs have been identified as a major factor in fruit ripening, and PG-deficient transgenic plants have been bred. Mainly fungal PGs are used in industrial processes for juice clarification and the range of enzymes is being extended through new recombinant and non-recombinant fungal strains. Finally, novel fields of application can be envisaged for PGs in the production of oligogalacturonides as functional food components. Here we aim to highlight the various fields where PGs are encountered and where they are of biological or technological importance. Received: 22 June 1999 / Received revision: 4 October 1999 / Accepted: 10 October 1999     

Molecular cloning, functional analysis and localization of a novel gene encoding polygalacturonase-inhibiting protein in Chorispora bungeana

Polygalacturonase-inhibiting proteins (PGIPs) are plant defense proteins. To date, no spatial distribution of PGIPs and interaction between PGIPs and nitric oxide (NO) in plant were described. Here, we first reported the full-length cDNA sequence of PGIP of Chorispora bungeana (CbPGIP1). Notably, immunofluorescence localization showed that the CbPGIP was evenly distributed in leaves but it was mainly localized in epidermis and vascular bundle in stems and roots. Further studies indicated that CbPGIP had higher abundance in roots than in stems and leaves. Conversely, the bulk PGIP of C. bungeana showed a higher activity in leaves than in stems and roots. In addition, quantitative real-time polymerase chain reaction demonstrated that CbPGIP1 expression was induced by Stemphylium solani, salicylic acid (SA), 4, ?4°C and NO. This is a first report attempting to predict if NO can induce the PGIP expression. Taken together, these findings showed that the gene was spatially regulated and NO and SA might take part in CbPGIP1 expression induced by biotic and abiotic stresses. This study highlighted the potential importance of CbPGIP1 and NO in plant resistance.     

Prokaryotic expression and protein function of Brassica napus PGIP2 and its genetic transformation

Sclerotinia rot is a fungal disease caused by Sclerotinia sclerotiorum (Lib.) de Bary, which has severely reduced rapeseed production worldwide. Polygalacturonase-inhibiting proteins (PIGPs) inhibit the activity of polygalacturonases, which are secreted during fungal infection in plants. This study investigated the function of the polygalacturonase-inhibitor gene 2 (PGIP2) in sclerotinia rot resistance. The PGIP2 was successfully expressed in a prokaryotic system, and recombinant PGIP2 protein, purified after enterokinase treatment to remove tag peptide, inhibited S. sclerotiorum PG activity in vitro. PGIP2 was overexpressed in the susceptible Brassica napus cultivar 98c40 via Agrobacterium-mediated transformation. After inoculation with S. sclerotiorum mycelia, the transgenic rapeseed demonstrated greatly reduced leaf damage, as compared with their non-transgenic plants. Therefore, the PGIP2 encodes a functional protein and would be a candidate gene for enhancing Sclerotinia rot resistance.