Polygalacturonase-inhibiting Protein Research Articles

Generation of transgenic sugar beet (Beta vulgarism L.) overexpressing the polygalacturonase inhibiting protein 1 of Phaseolus vulgaris (PvPGIP1) through Agrobacterium-mediated transformation

Numerous diseases caused by fungal pathogens influence the annual production of sugar beet. In order to obtain a plant resistant to fungi, genetic transformation has been applied to the sugar beet. To invade a plant tissue, phytopathogenic fungi produce several cell wall-degrading enzymes (CWDEs); polygalacturonases (PGs) are pathogenicity factors produced at the earlier stages of a fungal infection that depolymerize the homogalacturonan. One of the strategies used by plants to limit the degradation of the cell wall polysaccharides by fungal CWDEs is the production of proteinaceous inhibitors. Against fungal, microbial, and insect PGs, plants produce cell wall-associated polygalacturonase-inhibiting proteins (PGIPs). The overexpression of PGIPs improves the resistance to fungal and bacterial necrotrophs in different plants. In this research, the gene encoding the PGIP1 fused downstream of the leader sequence for secretion in the extracellular environment was isolated from Phaseolus vulgaris and cloned into the expression vector pBI121 for the Agrobacterium-mediated transformation of sugar beet. Modified transformation protocol and selection strategies were developed. In comparison with the preexisting methods, the transformation efficiency was increased and different cultivars were transformed, highlighting the general effectiveness of the method applied. The presence of the transgene and the activity of PvPGIP1 were confirmed by PCR and agarose diffusion assay analyses, respectively, and the present and copy number of the transgene in the T0 plants' genome were demonstrated by Southern blot.

Recent Progress in Technologies for Enzymatic Peeling of Fruit

Enzymatic peeling of fruit and vegetables is considered superior to conventional peeling methods in terms of minimizing product damage and indeed is expected to improve them. This technology was first proposed for the peeling of citrus fruit and numerous studies on its practical use have been conducted. However, its application to non-citrus fruit and vegetables remains limited. A novel process, including heat treatment prior to enzymatic treatment, is proposed to peel persimmon fruit. Heat treatment induces fine and even cracks on the fruit surface of certain persimmon cultivars, which act as channels via which enzyme solution can enter the peel tissue. Another function of the heat treatment is to inactivate polygalacturonase-inhibiting protein (PGIP), which is inherent in persimmon fruit. Inactivation of PGIP improves the efficiency of enzymatic peeling with exogenous polygalacturonase. Chemical pre-treatments with a food-grade emulsifier solution and a weak basic solution also enhance the permeability of enzyme solution into the peel tissue. These physical and chemical treatments are useful for applying enzymatic peeling to various persimmon cultivars. In future, enzymatic peeling technology is expected to be applied to various agricultural crops as the technology is further refined.

Identification of a wheat polygalacturonase-inhibiting protein involved in Fusarium head blight resistance

The plant cell wall is the first barrier to obstruct attack by pathogens. The polygalacturonase-inhibiting protein (PGIP) can specifically recognize and inhibit polygalacturonase (PG), a hydrolase that is secreted by fungi to invade plants, thus protecting plants from fungal infection by reducing the hydrolytic activity of PGs. In this study, we cloned a novel PGIP gene, Tapgip3, that is located on the 7D chromosome and encoded a 791 amino acid protein in the Fusarium head blight (FHB)-resistant wheat cultivar Ning7840. The real-time PCR analysis showed that this gene was upregulated sharply in spikes at 48 h after infection by either Fusarium graminearum or the trichothecene deoxynivalenol (DON). Subcellular localization analysis revealed that TaPGIP3 was secreted on the plant cell wall. When the transcripts of Tapgip3 were knocked down by barley stripe mosaic virus—virus-induced gene silencing (BSMV-VIGS), the growth of F. graminearum hyphae was promoted with larger lesions on wheat leaves. This suggests that Tapgip3 plays an important role in the response to F. graminearum infection. Although salicylic acid (SA), methyl jasmonate (MeJA) and abscisic acid (ABA) have been reported to be involved in wheat FHB resistance, we demonstrate in our study that the two copies of Tapgip3 are negatively regulated by these three hormones and positively regulated by indoleacetic acid (IAA).

The pearl millet mitogen-activated protein kinase PgMPK4 is involved in responses to downy mildew infection and in jasmonic- and salicylic acid-mediated defense.

Plant mitogen-activated protein kinases (MPKs) transduce signals required for the induction of immunity triggered by host recognition of pathogen-associated molecular patterns. We isolated a full-length cDNA of a group B MPK (PgMPK4) from pearl millet. Autophosphorylation assay of recombinant PgMPK4 produced in Escherichia coli confirmed it as a kinase. Differential accumulation of PgMPK4 mRNA and kinase activity was observed between pearl millet cultivars 852B and IP18292 in response to inoculation with the downy mildew oomycete pathogen Sclerospora graminicola. This increased accumulation of PgMPK4 mRNA, kinase activity as well as nuclear-localization of PgMPK protein(s) was only detected in the S. graminicola resistant cultivar IP18292 with a ~tenfold peak at 9h post inoculation. In the susceptible cultivar 852B, PgMPK4 mRNA and immuno-detectable nuclear PgMPK could be induced by application of the chemical elicitor β-amino butyric acid, the non-pathogenic bacteria Pseudomonas fluorescens, or by the phytohormones jasmonic acid (JA) or salicylic acid (SA). Furthermore, kinase inhibitor treatments indicated that PgMPK4is involved in the JA- and SA-mediated expression of three defense genes, lipoxygenase, catalase 3 and polygalacturonase-inhibitor protein. These findings indicate that PgMPK/s contribute to pearl millet defense against the downy mildew pathogen by activating the expression of defense proteins.

Functional analysis of OsPGIP1 in rice sheath blight resistance.

As one of the most devastating diseases of rice, sheath blight causes severe rice yield loss. However, little progress has been made in rice breeding for sheath blight resistance. It has been reported that polygalacturonase inhibiting proteins can inhibit the degradation of the plant cell wall by polygalacturonases from pathogens. Here, we prokaryotically expressed and purified OsPGIP1 protein, which was verified by Western blot analysis. Activity assay confirmed the inhibitory activity of OsPGIP1 against the PGase from Rhizoctonia solani. In addition, the location of OsPGIP1 was determined by subcellular localization. Subsequently, we overexpressed OsPGIP1 in Zhonghua 11 (Oryza sativa L. ssp. japonica), and applied PCR and Southern blot analysis to identify the positive T0 transgenic plants with single-copy insertions. Germination assay of the seeds from T1 transgenic plants was carried out to select homozygous OsPGIP1 transgenic lines, and the expression levels of OsPGIP1 in these lines were analyzed by quantitative real-time PCR. Field testing of R. solani inoculation showed that the sheath blight resistance of the transgenic rice was significantly improved. Furthermore, the levels of sheath blight resistance were in accordance with the expression levels of OsPGIP1 in the transgenic lines. Our results reveal the functions of OsPGIP1 and its resistance mechanism to rice sheath blight, which will facilitate rice breeding for sheath blight resistance.

GmPGIP3 enhanced resistance to both take-all and common root rot diseases in transgenic wheat.

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.

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.

T-RESVERATROL ACCUMULATION AND POLYGALACTURONASE INHIBITION DURING INFECTION OF GRAPE BERRIES BY BOTRYTIS CINEREA

SUMMARY The resistance of immature grape berries to Botrytis cinerea Pers. ex Fr. results from a combination of factors, among which the accumulation of the stilbene phytoalexin t-resveratrol. Stilbene accumulation is thought to be induced by oligogalacturonides (OGs) which are released from the plant cell wall during tissue maceration catalysed by polygalacturonases (PGs) produced by necrotrophic fungal pathogens. Inhibitors of PG activity, the polygalacturonase-inhibiting proteins (PGIPs), have the capability to slow the hydrolytic activity of PGs and favour the accumulation of active OGs. In the present work we have observed that artificial inoculation of grape inflorescences and immature berries at bloom and post bloom, leads to severe infection of the rachis, while lesions on young berries did not result in the development of the disease, but in the development of necrotic spots and rings. PG was detectable in berry tissues, but its secretion was significantly lower than in infected leaves. Tissues of immature berries reacted to B. cinerea inoculation by a marked accumulation of t-resveratrol. PG produced during spore germination of B. cinerea elicited t-resveratrol accumulation. Apoplastic fluids (AFs) from immature berries and leaves were found to inhibit total PG activity produced by B. cinerea, indicating the involvement of a proteinaceous or non-proteinaceous PG inhibitor, or both, as intercellular defence mechanisms. Taken together, our results suggest that the inhibition of PG produced by B. cinerea mediated by a putative PG inhibitor during the early stages of infection of immature grape berries plays a central role in promoting accumulation of t-resveratrol and, consequently, the restriction of pathogen spread.

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.

Phylogenetic analysis of pectin-related gene families in Physcomitrella patensand nine other plant species yields evolutionary insights into cell walls

BackgroundPectins are acidic sugar-containing polysaccharides that are universally conserved components of the primary cell walls of plants and modulate both tip and diffuse cell growth. However, many of their specific functions and the evolution of the genes responsible for producing and modifying them are incompletely understood. The moss Physcomitrella patens is emerging as a powerful model system for the study of plant cell walls. To identify deeply conserved pectin-related genes in Physcomitrella, we generated phylogenetic trees for 16 pectin-related gene families using sequences from ten plant genomes and analyzed the evolutionary relationships within these families.ResultsContrary to our initial hypothesis that a single ancestral gene was present for each pectin-related gene family in the common ancestor of land plants, five of the 16 gene families, including homogalacturonan galacturonosyltransferases, polygalacturonases, pectin methylesterases, homogalacturonan methyltransferases, and pectate lyase-like proteins, show evidence of multiple members in the early land plant that gave rise to the mosses and vascular plants. Seven of the gene families, the UDP-rhamnose synthases, UDP-glucuronic acid epimerases, homogalacturonan galacturonosyltransferase-like proteins, β-1,4-galactan β-1,4-galactosyltransferases, rhamnogalacturonan II xylosyltransferases, and pectin acetylesterases appear to have had a single member in the common ancestor of land plants. We detected no Physcomitrella members in the xylogalacturonan xylosyltransferase, rhamnogalacturonan I arabinosyltransferase, pectin methylesterase inhibitor, or polygalacturonase inhibitor protein families.ConclusionsSeveral gene families related to the production and modification of pectins in plants appear to have multiple members that are conserved as far back as the common ancestor of mosses and vascular plants. The presence of multiple members of these families even before the divergence of other important cell wall-related genes, such as cellulose synthases, suggests a more complex role than previously suspected for pectins in the evolution of land plants. The presence of relatively small pectin-related gene families in Physcomitrella as compared to Arabidopsis makes it an attractive target for analysis of the functions of pectins in cell walls. In contrast, the absence of genes in Physcomitrella for some families suggests that certain pectin modifications, such as homogalacturonan xylosylation, arose later during land plant evolution.

Expression of Defense Genes in Strawberry Fruits Treated with Different Resistance Inducers

The expression of 18 defense genes in strawberry fruit treated with elicitors: chitosan, BTH, and COA, at 0.5, 6, 24, and 48 h post-treatment was analyzed. The genes were up-regulated differentially, according to the elicitor. Chitosan and COA treatments promoted the expression of key phenylpropanoid pathway genes, for synthesis of lignin and flavonoids; only those associated with flavonoid metabolism were up-regulated by BTH. The calcium-dependent protein kinase, endo-β 1,4-glucanase, ascorbate peroxidase, and glutathione-S-transferase genes were up-regulated by BTH. The K+ channel, polygalacturonase, polygalacturonase-inhibiting protein, and β-1,3-glucanase, increased in response to all tested elicitors. The enzyme activities of phenylalanine ammonia lyase, β-1,3-glucanase, Chitinase, and guaiacol peroxidase supported the gene expression results. Similarity of gene expression was >72% between chitosan and COA treatments, while BTH showed lower similarity (38%) with the other elicitors. This study suggests the relationship between the composition of the elicitors and a specific pattern of induced defense genes.

English

In plants, polygalacturonase inhibitor proteins (PGIPs) are very important to inactivate polygalacturonases secreted by pathogens. Vitis thunbergii Sieb. et Zucc. polygalacturonase inhibitor proteins (VtPGIP) was first isolated from the wild grape Vitis thunbergii Sieb. et Zucc., which exhibits high resistance to disease. VtPGIP is sublocated in the plant cell wall, and this location is consistent with the function of PGIPs in the first line of host defense. The promoter of VtPGIP contains salicylic acid (SA), abscisic acid (ABA), and fungus infection response elements. Results from real-time quantitative reverse transcriptase (RT)-PCR analysis showed that VtPGIP expression was induced by SA, ABA, and fungi. The results indicated that VtPGIP may have important functions in defense-related responses of V. thunbergii against pathogenic fungi.

Characterization of a novel anther-specific gene encoding a leucine-rich repeat protein in petunia.

In Petunia x hybrida 'Fantasy Red', a leucine-rich repeat (LRR) gene referred to as PhLRR, was identified in a flower bud cDNA library. The open reading frame sequence of PhLRR was 1251 bp, encoding a putative 46.2-kDa protein of 416 amino acids. The PhLRR protein showed high similarity to members of polygalacturonase inhibitor proteins (PGIPs), contained 11 conserved LRR domains, and was an extracellular localization protein. Phylogenetic analysis showed that PhLRR belonged to the same PGIPs subfamily as SHY, indicating that PhLRR may be involved in the development of pollen-like SHY. Expression analysis revealed that PhLRR was abundantly expressed during early stages of flower bud and anther development, while it was not detected in any other examined organs, such as sepals, petals, pistils, roots, stems, leaves, or open flowers. Furthermore, many cis-acting elements (such as AGAAA and GTGA) related to anther-specific gene expression were identified in the PhLRR gene promoter region, indicating that the promoter is also anther-specific. These results suggested that PhLRR is a novel anther-specific gene that may be essential for the early development of anthers.

Isolation of differentially expressed genes related to salt tolerance in peanut

Salinity is one of the most serious environmental factors influencing crop growth and development, especially during germination period. In peanut, screening for salinity tolerance has been reported by several investigators, but there are few reports regarding isolation and identification of salt responsive genes from salt tolerant genotypes. In this study, we compared gene expression profile of a large-seeded saline tolerant peanut line 10L19 imbibed in 1.0% sodium chloride solution and seeds of the same genotype treated with distilled water, using the annealing control primer (ACP)-based GeneFishing technology. Total RNAs were extracted from seeds sampled at 1, 6 and 24 h after imbibition initiation. With 40 ACPs, a total of 20 differentially expressed transcripts were isolated. Thirteen of them were found to have high homology to genes with known functions in GenBank nr database. BLAST search revealed that those genes were related to gene expression regulation, signalling transduction, development and stress tolerance, among which S-38 was of direct relevance to hyperosmotic salinity response. S-14, S-15, S-17, S-28, S-31, S-32 and S-38, encoding ethylene-responsive element binding factor 4, DNA-directed RNA polymerase subunit, oleosin 5, polygalacturonase-inhibiting protein, vacuolarprocessing enzyme precursor, ribosomal protein s7 and nac domain protein, respectively, were selected and their relative expression levels determined by qRT-PCR using β-actin as internal control. The results confirmed that all the seven genes were expressed differentially in stressed and control peanut seeds. The highest relative expression for S-17 and S-38 was both at 6 h, while that for the rest genes was at 24 h.

Detection of the genetic variation of polygalacturonase-inhibiting protein gene 2 in autotetraploid alfalfa (Medicago sativa) using an improved SSCP technique.

In this study, 2 approaches were adopted to obtain good single-strand conformation polymorphism (SSCP) data for autotetraploid alfalfa; primers were added to PCR products, and fluorescent-labeled primers were utilized. PCR-SSCP conditions for a 331-bp fragment in the coding region of polygalacturonase-inhibiting protein gene 2 in alfalfa (MsPGIP2) were optimized, and the results showed that the best SSCP gel pattern could be obtained when the loading mixture was made by mixing 1 μL PCR products, 0.2 to 0.8 μL unlabeled primers (50 μM) and 4 to 16 μL loading buffer. Furthermore, the use of the fluorescent-labeled primers resulted in 2 separated electrophoresis images from 2 complementary single DNA strands, thus making the determination of alleles and idiotypes a relatively easy task. In addition, the results of sequencing prove that the determination of alleles and idiotypes were accurate based on SSCP analysis. Finally, a total of 9 alleles with 18 SNP sites were identified for MsPGIP2 in the alfalfa variety 'Algonquin'. In conclusion, MsPGIP2 possessed great genetic variation, and the addition of primers to the PCR products in combination with the fluorescent labeling of primers could significantly improve the sensitivity and resolution of SSCP analysis. This technique could be used for genetic diversity detection and marker-assisted breeding of useful genes in autopolyploid species such as alfalfa.

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.

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.

Biochemical changes and defence responses during the development of peach gummosis caused by Lasiodiplodia theobromae

Peach gummosis is a disease caused by Lasiodiplodia theobromae and a major problem in peach production. The disease is characterized by gum exudation from fungal infection sites on tree trunks, branches, and fruits. In this study, we found that high humidity and mechanical wounds promote gummosis development, whereas different light intensities and temperature do not affect gummosis after infecting detached current year shoots of the susceptible peach cultivar ‘Spring snow’ with L. theobromae in a laboratory. The chlorophyll a and b contents of the infected shoots were significantly lower than those of the uninfected control shoots, but those of H2O2 and malondialdehyde were increased in the infected tissues. Anthocyanin content and phenylalanine ammonia lyase (PAL) activity were significantly higher in the infected shoots at 1 day after inoculation, which is consistent with significant increases in the transcript levels of genes encoding enzymes involved anthocyanin biosynthesis, such as PAL, chalcone synthase, and flavanone 3-hydroxylase. The transcript levels were induced for defence-related genes, namely, chitinase, beta-1,3-glucanase, polygalacturonase-inhibiting protein, and pathogenesis-related protein 4, but down-regulated for pathogenesis-related protein 10. Cell walls were seriously degraded in response to L. theobromae infection, as observed under transmission electron microscopy. Accordingly, the transcript levels of genes that encode cell wall–degrading enzymes, such as beta-galactosidase, pectin methylesterase 1, and endo-polygalacturonase, were significantly increased in infected peach shoots.

Claviceps purpurea expressing polygalacturonases escaping PGIP inhibition fully infects PvPGIP2 wheat transgenic plants but its infection is delayed in wheat transgenic plants with increased level of pectin methyl esterification

Claviceps purpurea is a biotrophic fungal pathogen of grasses causing the ergot disease. The infection process of C. purpurea on rye flowers is accompanied by pectin degradation and polygalacturonase (PG) activity represents a pathogenicity factor. Wheat is also infected by C. purpurea and we tested whether the presence of polygalacturonase inhibiting protein (PGIP) can affect pathogen infection and ergot disease development. Wheat transgenic plants expressing the bean PvPGIP2 did not show a clear reduction of disease symptoms when infected with C. purpurea. To ascertain the possible cause underlying this lack of improved resistance of PvPGIP2 plants, we expressed both polygalacturonases present in the C. purpurea genome, cppg1 and cppg2 in Pichia pastoris. In vitro assays using the heterologous expressed PGs and PvPGIP2 showed that neither PG is inhibited by this inhibitor. To further investigate the role of PG in the C. purpurea/wheat system, we demonstrated that the activity of both PGs of C. purpurea is reduced on highly methyl esterified pectin. Finally, we showed that this reduction in PG activity is relevant in planta, by inoculating with C. purpurea transgenic wheat plants overexpressing a pectin methyl esterase inhibitor (PMEI) and showing a high degree of pectin methyl esterification. We observed reduced disease symptoms in the transgenic line compared with null controls. Together, these results highlight the importance of pectin degradation for ergot disease development in wheat and sustain the notion that inhibition of pectin degradation may represent a possible route to control of ergot in cereals.

Expression and regulation of a pear polygalacturonase inhibitor protein gene (PpPGIP1) during fruit development, under salicylic acid treatment, and in diseased fruit

To elucidate the roles of genes during fruit development, a fruit-predominant cDNA (designated PpPGIP1) encoding a polygalacturonase inhibitor protein (PGIP) was isolated from a pear (Pyrus pyrifolia Nakai. cv. Whangkeumbae) fruit cDNA library. The PpPGIP1 gene shares high sequence homology at the nucleotide level in the coding region and at the amino acid level with other plant PGIP genes. Subsequently, the corresponding PpPGIP1 gene was isolated from pear genomic DNA. Real-time quantitative PCR analyses revealed that PpPGIP1 transcripts were functionally accumulated during the developing fruit of pear. The PpPGIP1 expression signals were detected at relatively low level in early stage of pear fruit development (30–140 days after full bloom). With the fruit ripening, softening and senescence, the expression of PpPGIP1 gene increased to a relative high level in 150–180 days after full bloom fruit. In addition, PpPGIP1 gene was dramatically up-regulated in 180 days after full bloom diseased fruit and by 0.2 mM salicylic acid (SA) treatment for 12 h in 150 days after full bloom fruit. These results suggested that PpPGIP1 might play an important role in response to disease resistance and SA during fruit development of pear.