Pgip Gene Research Articles

Genome-wide identification and characterization of PGIP gene family in Vigna radiata L. Wilczek and its expression in wild non-progenitor, Vigna umbellata L. Thunb. modulate bruchid resistance

Genome-wide identification and characterization of PGIP gene family in Vigna radiata L. Wilczek and its expression in wild non-progenitor, Vigna umbellata L. Thunb. modulate bruchid resistance

Transcriptome Analysis of Air Space-Type Variegation Formation in Trifolium pratense.

Air space-type variegation is the most diverse among the species of known variegated leaf plants and is caused by conspicuous intercellular spaces between the epidermal and palisade cells and among the palisade cells at non-green areas. Trifolium pratense, a species in Fabaceae with V-shaped air space-type variegation, was selected to explore the application potential of variegated leaf plants and accumulate basic data on the molecular regulatory mechanism and evolutionary history of leaf variegation. We performed comparative transcriptome analysis on young and adult leaflets of variegated and green plants and identified 43 candidate genes related to air space-type variegation formation. Most of the genes were related to cell-wall structure modification (CESA, CSL, EXP, FLA, PG, PGIP, PLL, PME, RGP, SKS, and XTH family genes), followed by photosynthesis (LHCB subfamily, RBCS, GOX, and AGT family genes), redox (2OG and GSH family genes), and nitrogen metabolism (NodGS family genes). Other genes were related to photooxidation, protein interaction, and protease degradation systems. The downregulated expression of light-responsive LHCB subfamily genes and the upregulated expression of the genes involved in cell-wall structure modification were important conditions for air space-type variegation formation in T. pratense. The upregulated expression of the ubiquitin-protein ligase enzyme (E3)-related genes in the protease degradation systems were conducive to air space-type variegation formation. Because these family genes are necessary for plant growth and development, the mechanism of the leaf variegation formation in T. pratense might be a widely existing regulation in air space-type variegation in nature.

Comparative expression profile of some putative resistance genes of chickpea genotypes in response to ascomycete fungus, Ascochyta rabiei (Pass.) Labr.

Chickpea (Cicer arietinum L.) is one of the most important legumes infected with many pathogens including fungal pathogens. One of the most important devastating fungal pathogens in chickpea is Ascochyta rabiei (Pass.) Labr. which causes up to 100% reduction in yield. In the present work, the expression patterns of AFP-ca, CaD2, LRR and PGIP genes were studied in response to Ascochyta rabiei in two susceptible and resistant chickpea genotypes. The experimental system was conducted in greenhouse, for both inoculated and mock-inoculated plants (control). RNA was isolated from Icc 12004 (resistant) and FLIP 82-150c (susceptible) genotypes in 0, 6, 12, 24, 48, 72 and 96 h after inoculation. The expression of the genes was measured in susceptible and resistant plants via semiquantitative RT-PCR. Results showed higher expression of all three genes in resistant genotype compared with susceptible one. Results showed that the candidate genes from antimicrobial families (CaD2 and AFP-ca) were up-regulated in resistant genotypes at early hours after inoculation (6–24 hpi), and also for PGIP from galacturonase-inhibiting protein families, the maximum expression was observed at early hours of inoculation to 48 hpi. In general, we concluded that all genes studied in this investigation contribute to plant–pathogen interaction and all of them can increase the resistance responses to Ascochyta blight disease.

Study of defense-related gene expression in grapevine infested by Colomerus vitis (Acari: Eriophyidae)

Real-time quantitative polymerase chain reaction was used to study the expression of some marker genes involved in the interaction between grape (Vitis vinifera L.) and the erineum mite Colomerus vitis Pagenstecher (Acari: Eriophyidae). Potted vines of cultivars Atabaki (resistant to C. vitis), Ghalati (susceptible to C. vitis) and Muscat Gordo (moderately resistant to C. vitis) were infested at the six-leaf stage. The expression of protease inhibitor (PIN), beta-1,3-glucanase (GLU), polygalacturonase inhibitor (PGIP), Vitis vinifera proline-rich protein 1 (PRP1), stilbene synthase (STS), and lipoxygenase (LOX) genes was assessed on young leaves collected 96, 120 and 144h after mite infestation (hami). As a control, non-infested leaves collected 24h before mite infestations were used. Differences were detected in expression of the selected genes during the C. vitis-grapevine interaction. The resistant cultivar Atabaki increased the expression of LOX, STS, GLU, PGIP and PRP1 genes during the first 120 hami. On the contrary, in the susceptible Ghalati, all selected genes showed an expression level similar or lower than non-infested leaves. Muscat Gordo increased the expression of all selected genes in comparison with non-infested leaves, but it was lower than in Atabaki. Significant transcript accumulation of PIN gene was detected for Muscat Gordo whereas it was slightly up-regulated in Ghalati and Atabaki. LOX, STS, PIN, GLU, PGIP and PRP1 genes were clearly expressed in response to C. vitis infestation. We therefore infer that expression of PGIP, PIN and PRP1 genes could represent a defense strategy against C. vitis infestations in grapevine leaves.

Independent Preharvest Applications of Methyl Jasmonate and Chitosan Elicit Differential Upregulation of Defense-Related Genes with Reduced Incidence of Gray Mold Decay during Postharvest Storage of Fragaria chiloensis Fruit.

The Chilean strawberry (Fragaria chiloensis) fruit has interesting organoleptic properties, but its postharvest life is affected by gray mold decay caused by Botrytis cinerea. The effect of preharvest applications of methyl jasmonate (MeJA) or chitosan on the molecular defense-related responses and protection against gray mold decay were investigated in Chilean strawberry fruit during postharvest storage. Specifically, we inoculated harvested fruit with B. cinerea spores and studied the expression of genes encoding for the pathogenesis-related (PR) proteins β-1,3-glucanases (FcBG2-1, FcBG2-2 and FcBG2-3) and chitinases (FcCHI2-2 and FcCHI3-1), and for polygalacturonase inhibiting proteins (FcPGIP1 and FcPGIP2) at 0, 2, 24, 48, and 72 h post inoculation (hpi). Remarkably, MeJA- and chitosan-treated fruit exhibited a lower incidence of B. cinerea infection than the control-treated at 48 and 72 hpi. At the molecular level, both are efficient elicitors for priming in F. chiloensis fruit since we observed an upregulation of the FcBG2-1, FcBG2-3, FcPGIP1, and FcPGIP2 at 0 hpi. Moreover, a chitosan-mediated upregulation of FcPGIPs at early times post inoculation (2–24 hpi) and MeJA upregulated FcBGs (24–72 hpi) and FcPGIP1 at later times could contribute to reduce B. cinerea incidence by differential upregulation of defense genes. We concluded that preharvest applications of MeJA or chitosan had a long-lasting effect on the reduction of B. cinerea incidence during postharvest as well as an enhancer effect on the induction of PR and PGIP gene expression.

Sunflower polygalacturonase-inhibiting proteins (HaPGIP) are genetically conserved in cultivated sunflower (Helianthus annuus L.) but diverse in wild species

Cultivation of sunflower is often associated with substantial pathogen-related yield losses. Natural defense mechanisms of plants might be improved by advantageous combination of genes encoding proteins that suppress important pathogenicity factors. Endopolygalacturonases (endoPGs) as cell-wall-degrading enzymes contribute to the virulence of fungi, bacterial pathogens, and oomycetes. However, plants have evolved families of polygalacturonase-inhibiting proteins (PGIPs) that specifically recognize and inactivate these polygalacturonases (PGs). Here, we describe the identification of four sunflower pgip genes. The genes were cloned, genetically mapped, in silico characterized, and subjected to expression analyses in response to pathogen infection. Three Helianthus annuus pgip genes (Hapgip2–4) are located on linkage group (LG) 10 and phylogenetically closely related to other dicot pgips. In contrast, Hapgip1 was mapped on LG1 and formed a separate Asteraceae branch, suggesting that HaPGIPs may represent structurally and functionally distinct proteins. All HaPGIPs displayed characteristic sequence structures such as the tandem repetition of leucine-rich domains. WRKY transcription factor-binding sites identified through in silico promoter analyses differed between Hapgips in number and architecture. A comprehensive diversity analysis indicated that HaPGIP1–HaPGIP4 are rather conserved in current breeding material but genetically diverse in wild species. Based on homologous positions determined as amino acids with known influence on PvPGIP–PG interaction, non-synonymous substitutions in HaPGIPs were identified in H. maximiliani, H. ciliaris, H. paradoxus, H. tuberosis, and H. petiolaris. Furthermore, relative real-time quantitative PCR analyses revealed enhanced Hapgip1–Hapgip4 expression upon Botrytis cinerea inoculation and an increased expression level of Hapgip3 in response to the oomycete Plasmopara halstedii.

An update on polygalacturonase-inhibiting protein (PGIP), a leucine-rich repeat protein that protects crop plants against pathogens

Polygalacturonase inhibiting proteins (PGIPs) are cell wall proteins that inhibit the pectin-depolymerizing activity of polygalacturonases secreted by microbial pathogens and insects. These ubiquitous inhibitors have a leucine-rich repeat structure that is strongly conserved in monocot and dicot plants. Previous reviews have summarized the importance of PGIP in plant defense and the structural basis of PG-PGIP interaction; here we update the current knowledge about PGIPs with the recent findings on the composition and evolution of pgip gene families, with a special emphasis on legume and cereal crops. We also update the information about the inhibition properties of single pgip gene products against microbial PGs and the results, including field tests, showing the capacity of PGIP to protect crop plants against fungal, oomycetes and bacterial pathogens.

The pgip family in soybean and three other legume species: evidence for a birth-and-death model of evolution.

BackgroundPolygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) plant cell wall glycoproteins involved in plant immunity. They are typically encoded by gene families with a small number of gene copies whose evolutionary origin has been poorly investigated. Here we report the complete characterization of the full complement of the pgip family in soybean (Glycine max [L.] Merr.) and the characterization of the genomic region surrounding the pgip family in four legume species.ResultsBAC clone and genome sequence analyses showed that the soybean genome contains two pgip loci. Each locus is composed of three clustered genes that are induced following infection with the fungal pathogen Sclerotinia sclerotiorum (Lib.) de Bary, and remnant sequences of pgip genes. The analyzed homeologous soybean genomic regions (about 126 Kb) that include the pgip loci are strongly conserved and this conservation extends also to the genomes of the legume species Phaseolus vulgaris L., Medicago truncatula Gaertn. and Cicer arietinum L., each containing a single pgip locus. Maximum likelihood-based gene trees suggest that the genes within the pgip clusters have independently undergone tandem duplication in each species.ConclusionsThe paleopolyploid soybean genome contains two pgip loci comprised in large and highly conserved duplicated regions, which are also conserved in bean, M. truncatula and C. arietinum. The genomic features of these legume pgip families suggest that the forces driving the evolution of pgip genes follow the birth-and-death model, similar to that proposed for the evolution of resistance (R) genes of NBS-LRR-type.

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

Polygalacturonases (PGs) are hydrolytic enzymes employed by several phytopathogens to weaken the plant cell wall by degrading homopolygalacturonan, a major constituent of pectin. Plants fight back by employing polygalacturonase-inhibitor proteins (PGIPs). The present study compared the inhibition potential of pearl millet PGIP (Pennisetum glaucum; PglPGIP1) with the known inhibition of Phaseolus vulgaris PGIP (PvPGIP2) against two PGs, the PG-II isoform from Aspergillus niger (AnPGII) and the PG-III isoform from Fusarium moniliforme (FmPGIII). The key rationale was to elucidate the relationship between the extent of sequence similarity of the PGIPs and the corresponding PG inhibition potential. First, a pearl millet pgip gene (Pglpgip1) was isolated and phylogenetically placed among monocot PGIPs alongside foxtail millet (Setaria italica). Upstream sequence analysis of Pglpgip1 identified important cis-elements responsive to light, plant stress hormones, and anoxic stress. PglPGIP1, heterologously produced in Escherichia coli, partially inhibited AnPGII non-competitively with a pH optimum between 4.0 and 4.5, and showed no inhibition against FmPGIII. Docking analysis showed that the concave surface of PglPGIP1 interacted strongly with the N-terminal region of AnPGII away from the active site, whereas it weakly interacted with the C-terminus of FmPGIII. Interestingly, PglPGIP1 and PvPGIP2 employed similar motif regions with few identical amino acids for interaction with AnPGII at non-substrate-binding sites; however, they engaged different regions of AnPGII. Computational mutagenesis predicted D126 (PglPGIP1)-K39 (AnPGII) to be the most significant binding contact in the PglPGIP1-AnPGII complex. Such protein-protein interaction studies are crucial in the future generation of designer host proteins for improved resistance against ever-evolving pathogen virulence factors.

Functional characterisation of wheat Pgip genes reveals their involvement in the local response to wounding

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 TaPGIP1 nor TaPGIP2 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.

Effect of antifungal genes expressed in transgenic pea (Pisum sativum L.) on root colonization with Glomus intraradices

Pathogenic fungi have always been a major problem in agriculture. One of the effective methods for controlling pathogen fungi to date is the introduction of resistance genes into the genome of crops. It is interesting to find out whether the induced resistance in crops will have a negative effect on non-target organisms such as root colonization with the AM fungi. The objective of the present research was to study the influence of producing antifungal molecules by four transgenic pea (Pisum sativum L.) lines expressing PGIP gene from raspberry, VST-stilbene synthase from vine, a hybrid of PGIP/VST and bacterial Chitinase gene (Chit30) from Streptomyces olivaceoviridis respectively on the colonization potential of Glomus intraradices. Four different experiments were done in greenhouse and climate chamber, colonization was observed in all replications. The following parameters were used for evaluation: frequency of mycorrhization, the intensity of mycorrhization, the average presence of arbuscules within the colonized areas and the presence of arbuscules in the whole root system which showed insignificant difference between transgenic and non-transgenic plants. The root/shoot ratio exhibited different values according to the experiment condition. Compared with negative non-transgenic control all transgenic lines showed the ability to establish symbiosis and the different growth parameters had insignificant effect due to mycorrhization. The results of the present study proved that the introduced pathogen resistance genes did not affect the mycorrhization allocations in pea.

Prokaryotic Expression of Rice Ospgip1 Gene and Bioinformatic Analysis of Encoded Product

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.

PCR identification of rpgip1 transgene in Pisum sativum L.

Recent efforts to increase Ascochyta blight resistance of pea have focused on the introduction of foreign genes by genetic engineering. The rpgip1 gene from Rubus idaeus was introduced by Agrobacterium-mediated transformation into Pisum sativum, cv. Baroness with the aim to increase pea resistance to fungal diseases. Notwithstanding this success, practical applications have to be preceded by the development of analytical methods for screening. Singleplex and multiplex PCR assays were employed to test primer efficiency in identifying the rpgip1 transgene in 11 pea genotypes. Five from ten primer combinations were effective in identifying transgene or insert sequences. PCR amplification using five other primer pairs revealed unspecific amplicons. According to in silico analyses, they arose from retrotransposons and pea genes including homologues of rpgip1. Two sets of primers were prepared with the aim of simultaneous amplification of different rpgip1 fragments. Fingerprints were sums of bands observed from individual pairs so the utility of multiplex assays was demonstrated. An additional advantage of multiplex PCR was clear differentiation between the transgene and endogenous pgip genes present in the donor species, R. idaeus. Sequencing of two PCR products confirms that no substantial rearrangements at the rpgip1 transgene arose during development of transgenic plants. However, a deletion occurred at 59 bp in the PGIP+VST line and a substitution at 392 bp in the PGIP line. The frequency of point mutations was not high (1.1 x 10-3) and comparable with the frequency expected for host genes based on the neutral theory of molecular evolution.

Native and sulfated oligoglucuronans as elicitors of defence-related responses inducing protection against Botrytis cinerea of Vitis vinifera

During the past two decades, β-(1,4)-d-polyglucuronic acids (glucuronans) have been intensively described in literature for their physico-chemical and biological properties in different models. In this study, a bacterial glucuronan partially acetylated was degraded by a fungal glucuronan lyase to obtain unsaturated oligogluguronans, finally tested for their potential as elicitors of natural defences in Vitis vinifera. The objective was to correlate biological activities with specific structural features using oligoglucuronans varying in degree of polymerization, acetylation and sulfation. The results demonstrated that different defence reactions elicited by these anionic oligomers were dependent on degree of polymerization and nature of substituents (acetyl or sulfate groups). Acetylated oligoglucuronans with degrees of polymerization of 10 and 14 induced transient production of H2O2 and expression of some defence-related genes (PAL, STS, Chit4c and PGIP). The infection of grapevine leaves by Botrytis cinerea was reduced (23%) after treatment with acetylated oligoglucuronans having degree of polymerization of 14. Sulfated oligoglucuronans were not effective for production of H2O2 but induced strong expression of other defence-related PIN, PGIP genes. This work opens the way to new and valuable strategies to protect V. vinifera and other plants against pathogens.

PCR identification of rpgip1 transgene in Pisum sativum L.

Recent efforts to increase Ascochyta blight resistance of pea have focused on the introduction of foreign genes by genetic engineering. The rpgip1 gene from Rubus idaeus was introduced by Agrobacterium-mediated transformation into Pisum sativum, cv. Baroness with the aim to increase pea resistance to fungal diseases. Notwithstanding this success, practical applications have to be preceded by the development of analytical methods for screening. Singleplex and multiplex PCR assays were employed to test primer efficiency in identifying the rpgip1 transgene in 11 pea genotypes. Five from ten primer combinations were effective in identifying transgene or insert sequences. PCR amplification using five other primer pairs revealed unspecific amplicons. According to in silico analyses, they arose from retrotransposons and pea genes including homologues of rpgip1. Two sets of primers were prepared with the aim of simultaneous amplification of different rpgip1 fragments. Fingerprints were sums of bands observed from individual pairs so the utility of multiplex assays was demonstrated. An additional advantage of multiplex PCR was clear differentiation between the transgene and endogenous pgip genes present in the donor species, R. idaeus. Sequencing of two PCR products confirms that no substantial rearrangements at the rpgip1 transgene arose during development of transgenic plants. However, a deletion occurred at 59 bp in the PGIP+VST line and a substitution at 392 bp in the PGIP line. The frequency of point mutations was not high (1.1 x 10 -3 ) and comparable with the frequency expected for host genes based on the neutral theory of molecular evolution.

English

shown to inhibit the activity of fungal endopolygalacturonase (endo-PGs) and modulate their activity and has the potential to be developed as a disease or resistance biomarker for the oil palm. The identification and isolation of this gene in oil palm allowed for the study of its differential expression during the fungal infection. The oil palm PGIP gene (EgPGIP) has between 60-100% similarities with the database sequence of PGIP from other monocotyledons. Interestingly, we found that the expression of EgPGIP gene measured using Real-Time PCR showed that the expression level of EgPGIP in infected oil palm was temporally down regulated. The results suggest that, down regulation of the EgPGIP is related to the establishment of infection by G. boninense.

Brassica napus possesses an expanded set of polygalacturonase inhibitor protein genes that are differentially regulated in response to Sclerotinia sclerotiorum infection, wounding and defense hormone treatment

Most plants encode a limited set of polygalacturonase inhibitor (PGIP) genes that may be involved in aspects of plant development, but more importantly in the inactivation of polygalacturonases (PG) secreted by pathogens. Previously, we characterized two Brassica napus PGIP genes, BnPgip1 and BnPgip2, which were differentially expressed in response to pathogen infection and wounding. Here we report that the B. napus genome encodes a set of at least 16 PGIP genes that are similar to BnPgip1 or BnPgip2. This is the largest Pgip gene family reported to date. Comparison of the BnPGIPs revealed several sites within the xxLxLxx region of leucine rich repeats that form beta-sheets along the interacting face of the PGIP that are hypervariable and represent good candidates for generating PGIP diversity. Characterization of the regulatory regions and RT-PCR studies with gene-specific primers revealed that individual genes were differentially responsive to pathogen infection, mechanical wounding and signaling molecules. Many of the BnPgip genes responded to infection by the necrotic pathogen, Sclerotinia sclerotiorum; however, these genes were also induced either by jasmonic acid, wounding and salicylic acid or some combination thereof. The large number of PGIPs and the differential manner in which they are regulated likely ensures that B. napus can respond to attack from a broad spectrum of pathogens and pests.

A LTR copia retrotransposon and Mutator transposons interrupt Pgip genes in cultivated and wild wheats

Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) proteins involved in plant defence. Wheat pgip genes have been isolated from the B (Tapgip1) and D (Tapgip2) genomes, and now we report the identification of pgip genes from the A genomes of wild and cultivated wheats. By Southern blots and sequence analysis of BAC clones we demonstrated that wheat contains a single copy pgip gene per genome and the one from the A genome, pgip3, is inactivated by the insertion of a long terminal repeat copia retrotranspon within the fourth LRR. We demonstrated also that this retrotransposon insertion is present in Triticum urartu and all the polyploidy wheats assayed, but is absent in T. monococcum (Tmpgip3), suggesting that this insertion took place after the divergence between T. monococcum and T. urartu, but before the formation of the polyploid wheats. We identified also two independent insertion events of new Class II transposable elements, Vacuna, belonging to the Mutator superfamily, that interrupted the Tdipgip1 gene of T. turgidum ssp. dicoccoides. The occurrence of these transposons within the coding region of Tdipgip1 facilitated the mapping of the Pgip locus in the pericentric region of the short arm of chromosome group 7. We speculate that the inactivation of pgip genes are tolerated because of redundancy of PGIP activities in the wheat genome.

Characterization of expressed Pgip genes in rice and wheat reveals similar extent of sequence variation to dicot PGIPs and identifies an active PGIP lacking an entire LRR repeat

Polygalacturonase-inhibiting proteins (PGIPs) are leucine-rich repeat (LRR) proteins involved in plant defence. A number of PGIPs have been characterized from dicot species, whereas only a few data are available from monocots. Database searches and genome-specific cloning strategies allowed the identification of four rice (Oryza sativa L.) and two wheat (Triticum aestivum L.) Pgip genes. The rice Pgip genes (Ospgip1, Ospgip2, Ospgip3 and Ospgip4) are distributed over a 30 kbp region of the short arm of chromosome 5, whereas the wheat Pgip genes, Tapgip1 and Tapgip2, are localized on the short arm of chromosome 7B and 7D, respectively. Deduced amino acid sequences show the typical LRR modular organization and a conserved distribution of the eight cysteines at the N- and C-terminal regions. Sequence comparison suggests that monocot and dicot PGIPs form two separate clusters sharing about 40% identity and shows that this value is close to the extent of variability observed within each cluster. Gene-specific RT-PCR and biochemical analyses demonstrate that both Ospgips and Tapgips are expressed in the whole plant or in a tissue-specific manner, and that OsPGIP1, lacking an entire LRR repeat, is an active inhibitor of fungal polygalacturonases. This last finding can contribute to define the molecular features of PG-PGIP interactions and highlights that the genetic events that can generate variability at the Pgip locus are not only limited to substitutions or small insertions/deletions, as so far reported, but can also involve variation in the number of LRRs.

Cloning of polygalacturonase inhibitor protein genes from Solanum brevidens Fill.

New data were obtained for the Solanum brevidens Fill. nucleotide sequences coding for polygalacturonase inhibitor proteins (PGIPs), which are involved in plant defense against phytopathogenic fungi. Highly degenerate primers directed to the conserved regions of the known PGIP genes of tomato, kiwi, apple, carrot, and grape were used to clone four pgip genes and one pseudogene from the genome of S. brevidens, a species that is closely related to cultivated potato, forms no tubers, is highly resistant to phytopathogens, and is often employed in potato breeding. The sequenced part of the coding region of the new genes is 924 bp and codes for a protein of 308 amino acid residues (without the leader peptide). The genes were designated as pgipSbr1(1), pgipSbr1 (2). pgipSbr2, pgipSbr3, and pgipSbr4. The amino acid sequences of the S. brevidens PGIPs have 90.9-99.4% identity to each other and 94% identity to PGIP of Lycopersicon esculentum Mill., another member of the family Solanaceae. The amino acid residues differing between S. brevidens PGIPs were assumed to determine the selectivity of interactions with particular polyglucuronases of phytopathogenic fungi.