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In plants, polygalacturonase-inhibiting proteins (PGIPs) play critical roles for resistance to fungal disease by inhibiting the pectin-depolymerizing activity of endopolygalacturonases (PGs), one type of enzyme secreted by pathogens that compromises plant cell walls and leaves the plant susceptible to disease. Here, the interactions between PGIPs from Phaseolus vulgaris (PvPGIP1 and PvPGIP2) and PGs from Aspergillus niger (AnPG2), Botrytis cinerea (BcPG1 and BcPG2), and Fusarium moniliforme (FmPG3) were reconstituted through a yeast two hybrid (Y2H) system to investigate the inhibition efficiency of various PvPGIP1 and 2 truncations and mutants. We found that tPvPGIP2_5-8, which contains LRR5 to LRR8 and is only one-third the size of the full length peptide, exhibits the same level of interactions with AnPG and BcPGs as the full length PvPGIP2 via Y2H. The inhibitory activities of tPvPGIP2_5-8 on the growth of A. niger and B. cinerea were then examined and confirmed on pectin agar. On pectin assays, application of both full length PvPGIP2 and tPvPGIP2_5-8 clearly slows down the growth of A. niger and B. cinerea. Investigation on the sequence-function relationships of PGIP utilizing a combination of site directed mutagenesis and a variety of peptide truncations suggests that LRR5 could have the most essential structural feature for the inhibitory activities, and may be a possible target for the future engineering of PGIP with enhanced activity. This study highlights the potential of plant-derived PGIPs as a candidate for future in planta evaluation as a pest control agent.

When nematodes invade and subsequently migrate within plant roots, they generate cell wall fragments (in the form of oligogalacturonides; OGs) that can act as damage-associated molecular patterns and activate host defence responses. However, the molecular mechanisms mediating damage responses in plant-nematode interactions remain unexplored. Here, we characterized the role of a group of cell wall receptor proteins in Arabidopsis, designated as polygalacturonase-inhibiting proteins (PGIPs), during infection with the cyst nematode Heterodera schachtii and the root-knot nematode Meloidogyne incognita. PGIPs are encoded by a family of two genes in Arabidopsis, and are involved in the formation of active OG elicitors. Our results show that PGIP gene expression is strongly induced in response to cyst nematode invasion of roots. Analyses of loss-of-function mutants and overexpression lines revealed that PGIP1 expression attenuates infection of host roots by cyst nematodes, but not root-knot nematodes. The PGIP1-mediated attenuation of cyst nematode infection involves the activation of plant camalexin and indole-glucosinolate pathways. These combined results provide new insights into the molecular mechanisms underlying plant damage perception and response pathways during infection by cyst and root-knot nematodes, and establishes the function of PGIP in plant resistance to cyst nematodes.

Polygalacturonase-inhibiting protein (PGIP) is an important plant biochemical anti-disease factor. PGIP has a leucine-rich repeat structure that can selectively bind and inhibit the activity of endo-polygalacturonase (endo-PG) in fungi, playing a key role in plant disease resistance. The regulation of PGIP in plant disease resistance has been well studied, and the effect of PGIP to increase disease resistance is clear. This review summarizes recent advances in understanding the PGIP protein structure, the PGIP mechanism of plant disease resistance, and anti-disease activity by PGIP gene transfer. This overview should contribute to a better understanding of PGIP function and can help guide resistance breeding of PGIP for anti-disease effects. [ABSTRACT FROM AUTHOR]

Abstract 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.

Hypericum perforatum L. (St. John's wort) is a well-known medicinal plant that possesses secondary metabolites with beneficial pharmacological properties. However, improvement in the production of secondary metabolites via genetic manipulation is a challenging task as H. perforatum remains recalcitrant to Agrobacterium tumefaciens-mediated transformation. Here, the transcripts of key genes involved in several plant defence responses (secondary metabolites, RNA silencing, reactive oxygen species (ROS) and specific defence genes) were investigated in H. perforatum suspension cells inoculated with A. tumefaciens by quantitative real-time PCR. Results indicated that key genes from the xanthone, hypericin and melatonin biosynthesis pathways, the ROS-detoxification enzyme HpAOX, as well as the defence genes Hyp-1 and HpPGIP, were all upregulated to rapidly respond to A. tumefaciens elicitation in H. perforatum. By contrast, expression levels of genes involved in hyperforin and flavonoid biosynthesis pathways were markedly downregulated upon A. tumefaciens elicitation. In addition, we compared the expression patterns of key genes in H. perforatum leaf tissues with and without dark glands, a major site of secondary metabolite production. Overall, we provide evidence for the upregulation of several phenylpropanoid pathway genes in response to elicitation by Agrobacterium, suggesting that production of secondary metabolites could modulate H. perforatum recalcitrance to A. tumefaciens-mediated transformation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Polygalacturonase-inhibiting protein (PGIP) is an important plant biochemical anti-disease factor. PGIP has a leucine-rich repeat structure that can selectively bind and inhibit the activity of endo-polygalacturonase (endo-PG) in fungi, playing a key role in plant disease resistance. The regulation of PGIP in plant disease resistance has been well studied, and the effect of PGIP to increase disease resistance is clear. This review summarizes recent advances in understanding the PGIP protein structure, the PGIP mechanism of plant disease resistance, and anti-disease activity by PGIP gene transfer. This overview should contribute to a better understanding of PGIP function and can help guide resistance breeding of PGIP for anti-disease effects.

• MYTH test screened 22 proteins interacted with PpVIN2 in peach fruit. • PpPGIP1 in peach fruit is sensitives to chilling temperature storage. • PpPGIP1 positively regulates VIN activity via an interaction with PpVIN2. • PpPGIP1 is related to the rapid decomposition of sucrose and CI in peach fruit. The vacuolar invertase (VIN) gene PpVIN2 is induced at chilling temperatures, and plays an important role in sucrose metabolism in peach fruit during cold storage. Using a dual membrane yeast two-hybrid system, we found 22 proteins interacted with PpVIN2. Of these, PpPGIP1 (polygalacturonase-inhibiting protein) is a leucine-rich repeat glycoprotein and sensitives to chilling temperature (5 °C), based on the expression level of which increased significantly accompanied with the chilling injury. Colocalization of PpPGIP1 and PpVIN2 was demonstrated by confocal microscopy and their interaction between was demonstrated by bimolecular fluorescence complementation (BiFC). Transient overexpression of PpPGIP1 in peach fruit increased VIN activity significantly. Transient virus-induced gene silencing of PpPGIP1 decreased VIN activity significantly, which led to decreases sucrose decomposition. PpPGIP1 recombinant protein was successfully expressed by constructing pMAL-c6T-PpPGIP1, and PpPGIP1 increased the VIN activity in vitro. Taken together, PpPGIP1 positively regulates VIN activity via an interaction with PpVIN2; this interaction results in accelerated sucrose decomposition in peaches under chilling temperature. These findings provide a new perspective for understanding the new function of PpPGIP1 related with the rapid decomposition of sucrose and the occurrence of chilling injury in peach fruit during cold storage. [ABSTRACT FROM AUTHOR]

Summary: Verticillium wilt, one of the most devastating diseases of cotton (Gossypium hirsutum), causes severe yield and quality losses. Given the effectiveness of plant polygalacturonase‐inhibiting proteins (PGIPs) in reducing fungal polygalacturonase (PG) activity, it is necessary to uncover the key functional amino acids to enhance cotton resistance to Verticillium dahliae. To identify novel antifungal proteins, the selectivity of key amino acids was investigated by screening against a panel of relevant PG‐binding residues. Based on the obtained results, homologous models of the mutants were established. The docking models showed that hydrogen bonds and structural changes in the convex face in the conserved portion of leucine‐rich repeats (LRRs) may be essential for enhanced recognition of PG. Additionally, we successfully constructed Cynanchum komarovii PGIP1 (CkPGIP1) mutants Asp176Val, Pro249Gln, and Asp176Val/Pro249Gln and G. hirsutum PGIP1 (GhPGIP1) mutants Glu169Val, Phe242Gln, and Glu169Val/Phe242Gln with site‐directed mutagenesis. The proteins of interest can effectively inhibit VdPG1 activity and V. dahliae mycelial growth in a dose‐dependent manner. Importantly, mutants that overproduced PGIP in Arabidopsis and cotton showed enhanced resistance to V. dahliae, with reduced Verticillium‐associated chlorosis and wilting. Furthermore, the lignin content was measured in mutant‐overexpressing plants, and the results showed enhanced lignification of the xylem, which blocked the spread of V. dahliae. Thus, using site‐directed mutagenesis assays, we showed that mutations in CkPGIP1 and GhPGIP1 give rise to PGIP versatility, which allows evolving recognition specificities for PG and is required to promote Verticillium resistance in cotton by restricting the growth of invasive fungal pathogens. Significance statement: By using site‐directed mutagenesis assays, demonstrated that mutations in CkPGIP1 and GhPGIP1 give rise to PGIP versatility, which allows evolving recognition specificities for PG and is required to promote Verticillium resistance in cotton by restricting the growth of invasive fungal pathogens. [ABSTRACT FROM AUTHOR]

Sclerotinia stem rot (SSR), caused by Sclerotinia sclerotiorum, is the most serious disease affecting the yield of the agriculturally and economically important crop Brassica napus (rapeseed). In this study, Oryza sativa polygalacturonase- inhibiting protein 2 (OsPGIP2) was found to effectively enhanced rapeseed immunity against S. sclerotiorum infection. Leaf extracts of B. napus plants overexpressing OsPGIP2 showed enhanced S. sclerotiorum resistance by delaying pathogen infection. The constitutive expression of OsPGIP2 in rapeseed plants provided a rapid and effective defense response, which included the production of reactive oxygen species, interactions with S. sclerotiorum polygalacturonases (SsPG3 and SsPG6), and effects on the expression of defense genes. RNA sequencing analysis revealed that the pathogen induced many differentially expressed genes associated with pathogen recognition, redox homeostasis, mitogen-activated protein kinase signaling cascades, hormone signaling pathways, pathogen-/defenserelated genes, and cell wall-related genes. The overexpression of OsPGIP2 also led to constitutively increased cell wall cellulose and hemicellulose contents in stems without compromising seed quality. The results demonstrate that OsPGIP2 plays a major role in rapeseed defense mechanisms, and we propose a model for OsPGIP2-conferred resistance to S. sclerotiorum in these plants. [ABSTRACT FROM AUTHOR]

Summary: Recognition of endogenous molecules acting as ‘damage‐associated molecular patterns’ (DAMPs) is a key feature of immunity in both animals and plants. Oligogalacturonides (OGs), i.e. fragments derived from the hydrolysis of homogalacturonan, a major component of pectin are a well known class of DAMPs that activate immunity and protect plants against several microbes. However, hyper‐accumulation of OGs severely affects growth, eventually leading to cell death and clearly pointing to OGs as players in the growth‐defence trade‐off. Here we report a mechanism that may control the homeostasis of OGs avoiding their deleterious hyper‐accumulation. By combining affinity chromatography on acrylamide‐trapped OGs and other procedures, an Arabidopsis thaliana enzyme that specifically oxidizes OGs was purified and identified. The enzyme was named OG OXIDASE 1 (OGOX1) and shown to be encoded by the gene At4g20830. As a typical flavo‐protein, OGOX1 is a sulphite‐sensitive H2O2‐producing enzyme that displays maximal activity on OGs with a degree of polymerization >4. OGOX1 belongs to a large gene family of mainly apoplastic putative FAD‐binding proteins [Berberine Bridge Enzyme‐like (BBE‐like); 27 members], whose biochemical and biological function is largely unexplored. We have found that at least four BBE‐like enzymes in Arabidopsis are OG oxidases (OGOX1–4). Oxidized OGs display a reduced capability of activating the immune responses and are less hydrolysable by fungal polygalacturonases. Plants overexpressing OGOX1 are more resistant to Botrytis cinerea, pointing to a crucial role of OGOX enzymes in plant immunity. [ABSTRACT FROM AUTHOR]

Brassica campestris male fertility 19 (BcMF19; GenBank accession number GQ902048.1), a gene that is specially expressed in tapetum cells and microspores during anther development in B. campestris ssp. chinensis, which is learned from the previous in situ hybridization study. In the present study, we constructed antisense-silenced plants of BcMF19 using B. campestris ssp. chinensis to validate this prediction. The morphology of the pistils, long anthers, and short anthers was significantly affected in 35sbcmf19 compared with the control samples. 4′-6-Diamidino-2-phenylindole staining revealed that two generative nuclei and one large vegetative nucleus were not affected in the mutant compared with control. Statistical analysis of Alexander's staining results showed that 96% of the control pollen grains had vitality, whereas only 86% of the mutant pollen grains did. Under scanning electron microscopy, the mutant demonstrated numerous abnormal pollen grains and resembled dried persimmon. The frequency of normal pollen grains was approximately 18%. Under transmission electron microscopy, the pollen intine during the binucleate and mature pollen stages in 35sbcmf19 exhibited abnormal thickening, especially at the germinal furrows, compared with control. In vitro pollen germination test showed that the tips of the mutant pollen tubes transformed into globular alveoli and stopped growing compared with control. On the other hand, in vivo pollen germination test suggested that BcMF19 affected the pollen tube extension in the pistil. These findings indicate that BcMF19 is essential to the pollen development and pollen tube extension of B. campestris ssp. chinensis.

Objectives: Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum is one of the major fungal diseases of canola. To develop resistance against this fungal disease, the chit42 from Trichoderma atroviride with chitin-binding domain and polygalacturonase-inhibiting protein 2 (PG1P2) of Phaseolus vulgaris were co-expressed in canola via Agrobacterium-mediated transformation. Results: Stable integration and expression of transgenes in T and T plants was confirmed by PCR, Southern blot and RT-PCR analyses. Chitinase activity and PGIP2 inhibition were detected by colorimetric and agarose diffusion assay in transgenic lines but not in untransformed plants. The crude proteins from single copy transformant leaves having high chitinase and PGIP2 activity (T16, T8 and T3), showed up to 44 % inhibition of S. sclerotiorum hyphal growth. The homozygous T plants, showing inheritance in Mendelian fashion (3:1), were further evaluated under greenhouse conditions for resistance to S. sclerotiorum. Intact plants contaminated with mycelia showed resistance through delayed onset of the disease and restricted size and expansion of lesions as compared to wild type plants. Conclusions: Combined expression of chimeric chit42 and pgip2 in Brassica napus L. provide subsequent protection against SSR disease and can be helpful in increasing the canola production in Iran. [ABSTRACT FROM AUTHOR]

Bruchids are stored-grain insect pests responsible for serious seed loss in legume crops. A previous study using an F 2 population (F2OA) derived from a cross between wild moth-bean (Vigna aconitifolia [Jacq.] Maréchal) accession TN67 (resistant) and cultivated moth-bean accession ICPMO056 (susceptible) revealed that resistance to the azuki bean weevil (Callosobruchus chinensis L.) in TN67 was regulated by a single gene located in the major quantitative trait locus— qVacBrc2.1. In this study, qVacBrc2.1 was finely mapped and candidate genes in this locus were identified using F2OA and another large F 2 population (F2NB) derived from the cross mentioned previously. In contrast to the previous study, segregation analysis in the F2NB population revealed that resistance against this pest was controlled by two genes. Furthermore, the addition of novel markers to qVacBrc2.1 and reanalysis of the QTL in the F2OA population demonstrated that qVacBrc2.1 constituted two linked QTLs— qVacBrc2.1-A and qVacBrc2.1-B. The presence of qVacBrc2.1-B was verified using the population F2NB. Comparative genomics using three Vigna spp. strongly suggested the presence of two tandemly duplicated genes, VacPGIP1 and VacPGIP2 , which encoded polygalacturonase inhibitors (polygalacturonase-inhibiting proteins) as the candidates for conferring resistance, but only VacPGIP1 could be successfully cloned and sequenced. The alignment of VacPGIP1 coding sequences of TN67 and ICPMO056 revealed eight single nucleotide polymorphisms, three of which altered the amino-acid sequence of the predicted domains of polygalacturonase inhibitors in ICPMO056. Overall, these findings indicate that VacPGIP1 and VacPGIP2 regulated C. chinensis resistance in TN67. • Fine mapping revealed closely linked genes, VacBrc2.1-A and VacBrc2.1-B , controls C. chinensis resistance in moth bean TN67. • Comparative genome analysis revealed two polygalacturonase-inhibiting protein gene as candidate gene for the resistance. • VacPGIP1 sequence alignment revealed nucleotide polymorphism causing amino acid changes. [ABSTRACT FROM AUTHOR]

Take-all (caused by the fungal pathogen Gaeumannomyces graminis var. tritici, Ggt) and common root rot (caused by Bipolaris sorokiniana) are devastating root diseases of wheat ( Triticum aestivum L.). Development of resistant wheat cultivars has been a challenge since no resistant wheat accession is available. GmPGIP3, one member of polygalacturonase-inhibiting protein (PGIP) family in soybean ( Glycine max), exhibited inhibition activity against fungal endopolygalacturonases (PGs) in vitro. In this study, the GmPGIP3 transgenic wheat plants were generated and used to assess the effectiveness of GmPGIP3 in protecting wheat from the infection of Ggt and B. sorokiniana. Four independent transgenic lines were identified by genomic PCR, Southern blot, and reverse transcription PCR (RT-PCR). The introduced GmPGIP3 was integrated into the genomes of these transgenic lines and could be expressed. The expressing GmPGIP3 protein in these transgenic wheat lines could inhibit the PGs produced by Ggt and B. sorokiniana. The disease response assessments postinoculation showed that the GmPGIP3-expressing transgenic wheat lines displayed significantly enhanced resistance to both take-all and common root rot diseases caused by the infection of Ggt and B. sorokiniana. These data suggested that GmPGIP3 is an attractive gene resource in improving resistance to both take-all and common root rot diseases in wheat. [ABSTRACT FROM AUTHOR]

Polygalacturonase-inhibiting proteins ( PGIPs) are cell wall leucine-rich repeat ( LRR) proteins involved in plant defence. The hexaploid wheat ( Triticum aestivum, genome AABBDD) genome contains one Pgip gene per genome. Tapgip1 (B genome) and Tapgip2 (D genome) are expressed in all tissues, whereas Tapgip3 (A genome) is inactive because of a long terminal repeat, Copia retrotransposon insertion within the coding region. To verify whether Tapgip1 and Tapgip2 encode active PGIPs and are involved in the wheat defence response, we expressed them transiently and analysed their expression under stress conditions. Neither Ta PGIP1 nor Ta PGIP2 showed inhibition activity in vitro against fungal polygalacturonases. Moreover, a wheat genotype ( T. turgidum ssp. dicoccoides) lacking active homologues of Tapgip1 or Tapgip2 possesses PGIP activity. At transcript level, Tapgip1 and Tapgip2 were both up-regulated after fungal infection and strongly induced following wounding. This latter result has been confirmed in transgenic wheat plants expressing the β-glucuronidase ( GUS) gene under control of the 5′-flanking region of Tdpgip1, a homologue of Tapgip1 with an identical sequence. Strong and transient GUS staining was mainly restricted to the damaged tissues and was not observed in adjacent tissues. Taken together, these results suggest that Tapgips and their homologues are involved in the wheat defence response by acting at the site of the lesion caused by pathogen infection. [ABSTRACT FROM AUTHOR]

Abstract: Blueberry is a widely grown and easily perishable fruit crop. An efficient post-harvest handling is critical, and for that purpose gene technology methods have been part of ongoing programmes to improve crops with high food values such as blueberry. Here we report the isolation, cloning, characterization and differential expression levels of two cDNAs encoding Polygalacturonase-Inhibitor Protein (PGIP) and Cinnamoyl-Coa Reductase (CCR) from blueberry fruits in relation to various storage conditions. The open reading frame of PGIP and CCR encodes a polypeptide of 329 and 347 amino acids, respectively. To assess changes in the expression of blueberry PGIP and CCR after harvest, a storage trial was initiated. The northern blots hybridization showed a clear differential expression level of PGIP and CCR between freshly harvested and stored fruits as well as between fruits stored under various storage conditions. Although the prospects of exploiting such a strategy for crop improvement are limited, the results provide further insight into the control of the quality over the storage period at the molecular level. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Abstract: Using the reference sequences of pgip genes in GenBank, a fragment of 930 bp covering the open reading frame (ORF) of rice Ospgip1 (Oryza sativa polygalacturonase-inhibiting protein 1) was amplified. The prokaryotic expression product of the gene inhibited the growth of Rhizoctonia solani, the causal agent of rice sheath blight, and reduced its polygalacturonase activity. Bioinformatic analysis showed that OsPGIP1 is a hydrophobic protein with a molecular weight of 32.8 kDa and an isoelectric point (pI) of 7.26. The protein is mainly located in the cell wall of rice, and its signal peptide cleavage site is located between the 17th and 18th amino acids. There are four cysteines in both the N- and C-termini of the deduced protein, which can form three disulfide bonds (between the 56th and 63rd, the 278th and 298th, and the 300th and 308th amino acids). The protein has a typical leucine-rich repeat (LRR) domain, and its secondary structure comprises α-helices, β-sheets and irregular coils. Compared with polygalacturonase-inhibiting proteins (PGIPs) from other plants, the 7th LRR is absent in OsPGIP1. The nine LRRs could form a cleft that might associate with proteins from pathogenic fungi, such as polygalacturonase. [Copyright &y& Elsevier]

Abstract: Polygalacturonases (PGs) of wild-type and non-virulent phenotype conversion mutant (PC) strains of Ralstonia solanacearum were compared by investigating their activities and their inhibition by polygalacturonase-inhibiting proteins (PGIPs) from tomato stems. In cultures of wild-type strain ToUdk2, slimy (s), retarded slimy (rs) and non-slimy (ns) colonies appeared. The conversion of the ‘s’ into the ‘rs’ colony form coincided with the beginning of PG production. PG activity of the PC strain increased about 5 h earlier (at 6 hpi), and was up to 35 times higher in media supplemented with two different tomato stem extracts or polygalacturonic acid, compared to the wild-type at 6 hpi, and generally 4–8 times higher across test media and time. By hydrophobic interaction chromatography (HIC), fluorophor-assisted carbohydrate–polyacrylamid-gel electrophoresis (FACE–PAGE) and mass spectrometry analyses, endo-PG PehA, exo-PGs PehB and PehC were identified. PGs of the PC mutant consisted mainly of endo-PG. The increased PG production after supplementing the medium with tomato cell wall extract was reflected by a higher activity of exo-PGs for both strains. Total PGs (endo-PG and exo-PGs) activities were inhibited by PGIPs of tomato stem extracts. PGIP activity was concentration dependent, constitutively present, and not related to resistance nor susceptibility of tomato recombinant inbred lines to R. solanacearum. The proteinaceous character of the inhibiting component was inferred from ammonium sulphate precipitation. For the first time a plant PGIP activity against a bacterial pathogen is reported. Observations support that endo- and exo-PG synthesis is governed by a sensitive regulatory network, which, in interaction with PGIP and cell wall degradation products, leads to generation or avoidance of elicitor-active oligomers, and, thus, may contribute to the development of the compatible or incompatible interaction. [Copyright &y& Elsevier]

Seed dormancy is essential for most plants to control the timing of germination. In Arabidopsis thaliana, PED3 is a single-copy gene encoding an ATP-binding cassette transporter that is required for peroxisomal fatty acid β-oxidation. PED3 is involved in the import of several biologically important molecules into the peroxisome, including very-long-chain fatty acids associated with the breakdown of seed-reserve lipids, and precursors of auxin and jasmonic acid. The germination of ped3 mutants is significantly impaired, suggesting that PED3 regulates dormancy and germination. A transcriptome analysis revealed that many genes containing the core motif of the ABA responsive element (ABRE) in their promoter regions, and the ABA insensitive 5 ( ABI5) transcription factor that binds to ABRE, are abnormally up-regulated in imbibed ped3 seeds. Expression of polygalacturonase inhibiting proteins (PGIPs) is also up-regulated specifically in ped3 after imbibition. By contrast, the ped3 abi5 double mutant does not show any of these expression patterns. The results indicate that the abi5 mutation normalizes PGIP expression and rescues the impaired germination phenotype of the ped3 mutant. PGIPs are known to act as inhibitors of polygalacturonases that degrade pectin. The amount of PGIP1 transcript regulates the timing of radicle protrusion. The impaired germination of ped3 could also be rescued by removal of pectin from the seed coat using exogenous polygalacturonase or acidic conditions. Overall, our results suggest that PED3, a peroxisomal ABC transporter, promotes seed germination by suppressing PGIPs under the control of ABI5. [ABSTRACT FROM AUTHOR]

Polygalacturonase-inhibiting proteins (PGIPs) are plant cell wall proteins that specifically inhibit the activity of endopolygalacturonases (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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Background: Grape allergy is considered rare; grape lipid transfer protein (LTP; Vit v 1), an endochitinase and a thaumatin-like protein (TLP) have been reported as grape allergens. A considerable number of patients have referred to our department for severe reactions to grapes, and several IgE binding proteins were detected. Objectives: The aim of this study was to identify and characterise the allergens involved in severe allergic reactions to grapes and describe the population in which they occur. Methods: Patients with reported severe allergic reactions to grapes (n = 37) are described. Grape allergens were purified/fractionated by a combination of chromatographic techniques, identified by proteomic analysis and biochemically characterised. Immunoreactivity was assessed by blot (inhibitions) and RAST (inhibitions), and skin prick tests were performed with the isolated allergens. Results: All subjects were polyallergic, sensitised and reactive to several additional foods and pollen. All patients were sensitised to grape LTP. A 28-kDa expansin, a 37.5-kDa polygalacturonase-inhibiting protein, a 39-kDa β-1,3-glucanase and a 60-kDa protein were identified as minor grape allergens. Endochitinase and TLP did not play a role. Inhibition experiments revealed the possible cross-reactive role of LTP for clinical sensitivities to other LTP-containing plant foods, but also the involvement of cross-reactive carbohydrate determinants of minor allergens in IgE cross-reactivity. Conclusions: LTP is the major grape allergen, while additional minor allergens may contribute to clinical reactivity. Severe grape allergy presents in atopic patients who frequently react to other LTP-containing, plant-derived foods. The ‘LTP syndrome’ is the appropriate term to describe this condition. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Summary: The rough lemon (Citrus jambhiri) gene encoding polygalacturonase-inhibiting protein (RlemPGIPA) was overexpressed in the pathogenic fungus Alternaria citri. The overexpression mutant AcOPI6 retained the ability to utilize pectin as a sole carbon source, and the overexpression of polygalacturonase-inhibiting protein did not have any effect on the growth of AcOPI6 in potato dextrose and pectin medium. The pathogenicity of AcOPI6 to cause a black rot symptom in citrus fruits was also unchanged. Polygalacturonase-inhibiting protein was secreted together with endopolygalacturonase into culture filtrates of AcOPI6, and oligogalacturonides were digested from polygalacturonic acid by both proteins in the culture filtrates. The reaction mixture containing oligogalacturonides possessed activity for induction of defense-related gene, RlemLOX, in rough lemon leaves. [Copyright &y& Elsevier]

Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) proteins that inhibit fungal endopolygalacturonases (PGs). They are encoded by multigene families whose members show functional redundancy and subfunctionalization for recognition of fungal PGs. In order to expand the information on the structure and functional features of legume PGIP, we have isolated and characterized four members of the soybean Pgip gene family and determined the properties of the encoded protein products. Sequence analysis showed that these genes form two clusters: one cluster of about 5 kbp containing Gmpgip1 and Gmpgip2, and the other containing Gmpgip3 and Gmpgip4 within a 60 kb fragment of a separate BAC clone. Sequence diversification of the four members resides mainly in the xxLxLxx region that includes residues forming the β-sheet B1. When compared with other legume Pgip genes, Gmpgip3 groups with the bean genes Pvpgip1 and Pvpgip2, suggesting that these genes are closer to the ancestral gene. At the protein level, only GmPGIP3 shows the capability to inhibit fungal PGs. The spectrum of inhibition of GmPGIP3 against eight different fungal PGs mirrors that of the PGIP purified from soybean tissues and is similar to that of the bean PvPGIP2, one of the most efficient inhibitors so far characterized. We also report that the four Gmpgip genes are differentially regulated after wounding or during infection with the fungal pathogen Sclerotinia sclerotiorum. Following fungal infection Gmpgip3 is up regulated promptly, while Gmpgip2 is delayed. [ABSTRACT FROM AUTHOR]

Abstract: The genome of the necrotrophic fungus Botrytis cinerea encodes at least six endopolygalacturonases; among them BcPG1 is constitutively expressed and is required for full virulence. To counteract fungal polygalacturonase, plants have evolved families of polygalacturonases-inhibiting proteins (PGIPs) with specific recognition capabilities against PGs from different fungal species. However, it is not clear whether PGIPs can specifically recognize single PG isozymes important for Botrytis virulence. To identify PGIPs that are able to efficiently inhibit BcPG1, we expressed it in the heterologous host Pichia pastoris, purified and tested its activity in the presence of PGIPs of Phaseolus vulgaris and Arabidopsis thaliana. We found that PvPGIP2 of P. vulgaris is a very efficient inhibitor of BcPG1 and that, when overexpressed in transgenic tobacco and Arabidopsis plants, it restricts B. cinerea colonization. We conclude that the ability of PvPGIP2 to limit fungal colonization in transgenic plants correlates with its inhibitory effect on BcPG1, and discuss the potential of PGIPs and the encoding genes as biotechnological tools to control the phytopathogenic fungus B. cinerea. [Copyright &y& Elsevier]

Abstract: Polygalacturonase-inhibiting proteins (PGIPs) are plant proteins believed to play a role in the defence against pathogenic fungi. Purified apple PGIP1 inhibited polygalacturonases (PGs) secreted by Verticillium dahliae grown on potato root cell walls and pectin. We therefore hypothesised that apple PGIP1 could be used to confer resistance against Verticillium-wilt, a major disease of potato caused by the fungus V. dahliae. Transgenic lines containing the apple pgip1 gene under control of the enhanced CaMV 35S (e35S) promoter were generated. Stable integration of the apple pgip1 transgene into the potato genome was shown by the polymerase chain reaction (PCR) and Southern blot. Cross hybridisation with potato pgip(s) was not observed. High-level expression of the apple PGIP1 in several independent transgenic potato events was verified by silver staining of SDS-PAGE separated proteins and Western blot. All but one of the PGIP1 extracts prepared from the transgenic potato lines were successful in inhibiting V. dahliae PGs. Active PGIP1 was expressed in the leaves as well as the roots of the transgenic plants. The apoplastic localisation of PGIP activity in the pgip-transgenic potato plants was demonstrated by a vacuum infiltration–extraction experiment. A glasshouse trial indicated that six transgenic lines (A10, B10, B13, A3, A14 and B16) had significantly reduced disease symptoms compared to the untransformed control and other lines when grown in the inoculated soil, but five of them also showed significantly slower senescence symptoms when grown in the control soil. It is proposed that an extended juvenile phase in the transgenic lines resulted in the apparent increased disease resistance, and this could not be attributed to inhibition of V. dahliae PGs, despite high-level expression of the apple PGIP1. [Copyright &y& Elsevier]

Polygalacturonase-inhibiting proteins (PGIPs) have been demonstrated to play a role in host defence in several plants. The PGIP now cloned from strawberry (Fragaria x ananassa) showed a high degree of homology to other fruit PGIPs. The gene expression of strawberry PGIP was monitored in healthy leaves, flowers and fruit at different maturity stages. PGIP transcript levels were also analysed following fruit inoculation with the fungal pathogen Botrytis cinerea in strawberry cultivars displaying variation in susceptibility. Healthy mature berries showed the highest constitutive PGIP gene expression levels compared with leaves, flowers and immature fruit, indicating that the gene is developmentally regulated. Among the cultivars studied ('Elsanta', 'Korona', 'Polka', 'Senga sengana', 'Tenira'), 'Polka' had the highest constitutive expression level of PGIP. After inoculation with B. cinerea, all five cultivars displayed a significant induction of PGIP gene expression, but the differences between them were not statistically significant. The high induction of the PGIP gene after inoculation with B. cinerea indicates that PGIP has a role in defence of strawberry. [ABSTRACT FROM AUTHOR]

Polygalacturonases (PGs) are produced by fungal pathogens during early plant infection and are believed to be important pathogenicity factors. Polygalacturonase-inhibiting proteins (PGIPs) are plant defense proteins which reduce the hydrolytic activity of endoPGs and favor the accumulation of long-chain oligogalacturonides (OGs) which are elicitors of a variety of defense responses. PGIPs belong to the superfamily of leucine reach repeat (LRR) proteins which also include the products of several plant resistance genes. A number of evidence demonstrates that PGIPs efficiently inhibit fungal invasion. [Copyright &y& Elsevier]

Venturia nashicola is the causal agent of scab, a fungal disease affecting Asian pears. The Japanese pear cv. ‘Kousui’ is highly susceptible to the race 1 of this fungus whereas the cv. ‘Kinchaku’ and the non-host European pear cv. ‘Flemish Beauty’ are resistant. The aim of this work is to investigate the role of polygalacturonase-inhibiting proteins (PGIPs) of pear during the interactions with V. nashicola leading to susceptibility or resistance. PGIP protein was detected from immature fruit of Kousui and Kinchaku. It showed a molecular mass of 42 kDa that shifted to 35 kDa after chemical deglycosylation. The gene pgip was amplified by PCR using genomic DNA and/or cDNA from young leaves of Kousui, Kinchaku, and European pear cvs. Flemish Beauty, ‘Bartlett’, and an Asian wild pear strain ‘Mamenashi 12’, then sequenced after sub-cloning. Some conserved variations were identified in the sequence indicating that gene family also exists in pgip of Japanese pear and confirmed by Southern blot analysis. The expression of PGIP was studied in scab-inoculated leaves of the susceptible Kousui and the resistant Kinchaku and Flemish Beauty. pgip Gene and its encoding protein were highly and rapidly activated in these resistant plants. In addition, PGIP extracts derived from Kinchaku and Flemish Beauty partially inhibited the activity of polygalacturonase (PG) from V. nashicola suggesting a possible role of PGIP in limiting fungal growth frequently observed in these resistant cultivars. [Copyright &y& Elsevier]

A method for determination of the interaction between pectins and proteins was developed using cross-linked polygalacturonic acid (CLPG) as the pectic substrate and polygalacturonase-inhibiting proteins (PGIPs). Defined water-insoluble pectins were prepared by chemical substitutions with acetyl or methoxyl groups on CLPG. In the presence of 0.1 M NaCl, PGIPs fully bound to CLPG but not to cross-linked alginic acid (CLAL), which had a similar pKa to CLPG, suggesting that the inhibitor was not simply bound to the substrate by nonspecific electrostatic interaction. Optimum binding of PGIPs to CLPG occurred at pH 2.4 to 4.7. The binding ability of the inhibitor to CLPGs with degree of methylation (DM) of 66% or degree of acetylation (DAc) of 133% was not significantly changed. In contrast, the DM of 82% or 95% decreased the binding. These results indicated that the carboxylic groups of galacturonic acid residues were involved in the recognition of the substrate by PGIPs. [Copyright &y& Elsevier]

The effects of the polygalacturonase-inhibiting protein (PGIP) on the rate of oligouronide formation were studied in a model system containing polygalacturonic acid and polygalacturonase (PG) from the culture medium of phytopathogenic fungi. PGIP preparations were prepared from stored potato tubers and sprouts and also from apple fruits. The PGIP effects on oligouronide synthesis depended markedly on the physiological state of the source plant. Apple cultivars differing in their earliness differed in PGIP effects as well. The PGIP from potato tubers, which were in deep dormancy, suppressed oligouronide formation. The inhibitory PGIP action was decreased after dormancy release and tuber sprouting, which resulted in the oligouronide accumulation. The effects of PGIP from apple fruits on the oligouronide synthesis in the system containing PG from various phytopathogenic fungi were not correlated with tissue damage induced by these fungi. The PGIP effects on oligouronide formation are evident; however, their role in plant-cell processes related to the pectin compound conversions and plant resistance to diseases remains to be elucidated. [ABSTRACT FROM AUTHOR]

A cDNA encoding a polygalacturonase-inhibiting protein gene ( SaiPGIPA) was identified from the citrus cultivar Sainumphung ( Citrus sp.), one of the most popular cultivars in northern Thailand. SaiPGIPA was expressed in Escherichia coli cells, and the functional properties of citrus PGIP were analyzed. The PGIP fusion protein inhibited by a maximum of about 60% of the endopolygalacturonase activity, and a mixture of the PGIP and fungal endopoly-galacturonase released oligogalacturonides from polygalacturonic acid. The mixture containing the oligogalactur-onides, endopolygalacturonase and PGIP induced expression of the PGIP gene and a chalcone synthase gene in citrus leaves. The mixture also induced resistance in cucumber leaves against Colletotrichum lagenarium. [ABSTRACT FROM AUTHOR]

A cDNA encoding the polygalacturonase-inhibiting protein gene ( RlemPGIPl) was identified from rough lemon ( Citrus jambhiri Lush.). The deduced amino acid sequence is 46-75% identical to other plant PGIPs. The transcripts of the PGIP gene were weakly but constitutively expressed in leaves and increased in leaves within 30 min after wounding or inoculation with nonpathogenic Alternaria alternata or A. citri. However, expression of the PGIP and chalcone synthase genes did not increase after inoculation with an endopolygalacturonase (endoPG)-disrupted mutant of A. citri. These results indicate that endoPG may act as an elicitor for induction of these genes in citrus, most likely caused by PGIP interactions. [ABSTRACT FROM AUTHOR]

Plant polygalacturonase-inhibiting proteins (PGIPs) inhibit endo-polygalacturonases (endo-PGs) released by invasive fungi. Two potential PGlP cDNAs have been cloned from raspberry (Rubus idaeus). PGIP1 corresponds to a full-length PGIP cDNA with a high degree of identity with previously isolated genes and maintains all the characteristic features of PGIP peptides. A genomic fragment of PGIP1 identifies a single 243 bp intron which is efficiently spliced out of the PGIP1 premRNA transcript. PGIP2 contains a frame shift mutation which would lead to the loss of 105 amino acids off the C-terminus. Contrary to PGIP activity levels in raspberry (Johnston et al., 1993), expression analysis detected PGIP1 at equal levels throughout fruit development from closed flower, through to ripe fruit. [ABSTRACT FROM AUTHOR]

Polygalacturonase-inhibiting proteins (PGIPs), leucine-rich repeat (LRR) proteins evolutionarily related to several plant resistance genes, bind to and regulate the action of fungal endopolygalacturonases. In Phaseolus vulgaris L., PGIPs are encoded by a gene family comprising at least five members. As a start for a systematic analysis of the regulation of the pgip family, we have analysed the ability of the promoter of the bean gene pgip-1 to direct expression of β-glucuronidase (GUS) in transfected tobacco protoplasts, microbombarded bean and tobacco leaves, and transgenic tobacco plants. In protoplasts, the pgip-1 gene region from nucleotide (nt) −2004 to nt +27 directed a level of expression that was as high as that directed by the cauliflower mosaic virus (CaMV) 35S promoter and could not be further induced by elicitor treatment; alteration of the region immediately following the TATAA sequence at nt −29 abolished expression. Upon stable integration into tobacco plants of the pgip-1 promoter-GUS construct, as well as of a −394 deletion, expression was detected for both constructs mainly in the stigma and, to a lesser extent, in the anthers and in the conductive vascular tissue. The promoter responded to wounding but not to oligogalacturonides, fungal glucan, salicylic acid, cryptogein, or pathogen infection. This expression pattern does not mirror that of the whole pgip gene family. [ABSTRACT FROM AUTHOR]

Polygalacturonase-inhibiting protein ( PGIP) is a cell wall protein which inhibits fungal endopolygalacturonases. A small gene family encodes PGIP in the genome of common bean, as indicated by Southernblot experiments performed at high-stringency conditions. Southern-blot analysis of DNA extracted from different cultivars of Phaseolus vulgaris and from Phaseolus coccineus showed length polymorphism of the hybridizing restriction fragments. The cytological localization of the PGIP genes was determined in polytene chromosomes of the P. vulgaris embryo suspensor cells. In-situ hybridization experiments using the cloned PGIP gene revealed labelling over a single region of the pericentromeric heterochromatin of chromosome pair X, next to the euchromatin, suggesting that PGIP gene family may be clustered in one chromosomal region. [ABSTRACT FROM AUTHOR]