Fungal Endopolygalacturonases Research Articles

Structural Insights into the Mechanisms Underlying the Kinetic Stability of GH28 Endo-Polygalacturonase

Thermostability is a key property of industrial enzymes. Endo-polygalacturonases of the glycoside hydrolase family 28 have many practical applications, but only few of their structures have been determined, and the reasons for their stability remain unclear. We identified and characterized the Talaromyces leycettanus JCM12802 endo-polygalacturonase TlPGA, which differs from other GH28 family members because of its high catalytic activity, with an optimum temperature of 70 °C. Distinctive features were revealed by comparison of thermophilic TlPGA and all known structures of fungal endo-polygalacturonases, including a relatively large exposed polar accessible surface area in thermophilic TlPGA. By mutating potentially important residues in thermophilic TlPGA, we identified Thr284 as a critical residue. Mutant T284A was comparable to thermophilic TlPGA in melting temperature but exhibited a significantly lower half-life and half-inactivation temperature, implicating residue Thr284 in the kinetic stability of thermophilic TlPGA. Structure analysis of thermophilic TlPGA and mutant T284A revealed that a carbon-oxygen hydrogen bond between the hydroxyl group of Thr284 and the Cα atom of Gln255, and the stable conformation adopted by Gln255, contribute to its kinetic stability. Our results clarify the mechanism underlying the kinetic stability of GH28 endo-polygalacturonases and may guide the engineering of thermostable enzymes for industrial applications.

Probing the role of cation-π interaction in the thermotolerance and catalytic performance of endo-polygalacturonases.

Understanding the dynamics of the key pectinase, polygalacturonase, and improving its thermotolerance and catalytic efficiency are of importance for the cost-competitive bioconversion of pectic materials. By combining structure analysis and molecular dynamics (MD) simulations, eight mutagenesis sites having the potential to form cation-π interactions were identified in the widely used fungal endo-polygalacturonase PG63. In comparison to the wild-type, three single mutants H58Y, T71Y and T304Y showed improved thermostability (the apparent Tms increased by 0.6−3.9 °C) and catalytic efficiency (by up to 32-fold). Chromatogram analysis of the hydrolysis products indicated that a larger amount of shorter sugars were released from the polygalacturonic acid by these three mutants than by the wild-type. MD analysis of the enzyme-substrate complexes illustrated that the mutants with introduced cation-π interaction have modified conformations of catalytic crevice, which provide an enviable environment for the catalytic process. Moreover, the lower plasticity of T3 loop 2 at the edge of the subsite tunnel appears to recruit the reducing ends of oligogalacturonide into the active site tunnel and initiates new hydrolysis reactions. This study demonstrates the importance of cation-π interaction in protein conformation and provides a realistic strategy to enhance the thermotolerance and catalytic performance of endo-polygalacturonases.

A Novel Acid-Stable Endo-Polygalacturonase from Penicillium oxalicum CZ1028: Purification, Characterization, and Application in the Beverage Industry.

Acidic endo-polygalacturonases are the major part of pectinase preparations and extensively applied in the clarification of fruits juice, vegetables extracts, and wines. However, most of the reported fungal endo-polygalacturonases are active and stable under narrow pH range and low temperatures. In this study, an acidic endo-polygalacturonase (EPG4) was purified and characterized from a mutant strain of Penicillium oxalicum. The N-terminal amino acid sequence of EPG4 (ATTCTFSGSNGAASASKSQT) was different from those of reported endopolygalacturonases. EPG4 displayed optimal pH and temperature at 5.0 and 60-70°C towards polygalacturonic acid (PGA), respectively, and was notably stable at pH 2.2-7.0. When tested against pectins, EPG4 showed enzyme activity over a broad acidic pH range (>15.0% activity at pH 2.2-6.0 towards citrus pectin; and >26.6% activity at pH 2.2-7.0 towards apple pectin). The Km and Vmax values were determined as 1.27 mg/ml and 5,504.6 U/mg, respectively. The enzyme hydrolyzed PGA in endo-manner, releasing oligo-galacturonates from PGA, as determined by TLC. Addition of EPG4 (3.6 U/ml) significantly reduced the viscosity (by 42.4%) and increased the light transmittance (by 29.5%) of the papaya pulp, and increased the recovery (by 24.4%) of the papaya extraction. All of these properties make the enzyme a potential application in the beverage industry.

Two thermophilic fungal pectinases from Neosartorya fischeri P1: Gene cloning, expression, and biochemical characterization

In addition to cellulose and hemicellulose, pectin is an important renewable resource for biorefinery. Endo- and exo-polygalacturonases are two essential enzymes required for complete deconstruction of pectin into simple and fermentable sugars. In biorefinery, thermophilic pectinases are more appealing than their mesophilic counterparts. Two pectinase genes encoding a putative exo-polygalacturonase (NfPG4) and a putative endopolygalacturonase (NfPG5), respectively, were thus cloned from a thermophilic filamentous fungus Neosartorya fischeri P1 and successfully expressed in Pichia pastoris. NfPG4 and NfPG5 share 79% and 58% identity, respectively, with their closest characterized homologs from Aspergillus tubingensis and Achaetomium sp. Xz-8. They are both acidic and thermophilic, with maximum activities at pH 3.5 and 65°C for NfPG4 and pH 4.5 and 70°C for NfPG5, respectively. In comparison with other characterized thermophilic polygalacturonases, NfPG5 is the most thermophilic fungal endopolygalacturonase. Both enzymes have excellent stability at 55°C and over a broad pH range (pH 3.0–8.0 for NfPG4 and pH 2.0–11.0 for NfPG5), and are strongly resistant to many metal ions and chemical reagents. NfPG5 has a typical endo-mode of action on polygalacturonic acid, releasing galacturonic acid, di-galacturonic acid, and tri-galacturonic acid. In contrast, NfPG4 only releases galacturonic acid as the end hydrolysis product, typified by exo-polygalacturonases. NfPG4 has a specific activity of 700.9U/mg, higher than most characterized exo-polygalacturonases. NfPG5 is also a robust polygalacturonase with a specific activity of 3630.4U/mg. These superior properties make the two polygalacturonases interesting candidates for industrial applications with a preference for thermophilic pectinases.

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.

Phenyl derivative of pyranocoumarin precludes Fusarium oxysporum f.sp. Lycopersici infection in Lycopersicon esculentum via induction of enzymes of the phenylpropanoid pathway.

Binding of phenyl derivative of pyranocoumarin (PDP) modulated activity of fungal endopolygalacturonase in silico. Induced fit docking study of PDP with endopolygalacturonase (1HG8) showed a bifurcated hydrogen bond interaction with the protein at Lys 244 with a docking score of -3.6 and glide energy of -37.30 kcal/mol. Docking with endopolygalacturonase II (1CZF) resulted hydrogen bond formation with Lys 258 with a docking score of -2.3 and glide energy of -30.42 kcal/mol. It was hypothesized that this modulation favors accumulation of cell wall fragments (oligogalacturonides) which act as elicitors of plant defense responses. In order to prove the same, in vivo studies were carried out using a formulation developed from PDP (PDP 5EC) on greenhouse grown Lycopersicon esculentum L. The formulation was effective at different concentrations in reduction of seed infection, improvement of vigor and control of Fusarium oxysporum f.sp. lycopersici infection in L. esculentum. At a concentration of 2 %, PDP 5EC significant reduction in seed infection (95.83 %), improvement in seed vigor (64.31 %) and control of F. oxysporum f.sp. lycopersici infection (96.15 %) were observed. Further application of PDP 5EC to L. esculentum challenged with F. oxysporum f.sp. lycopersici significantly increased the activity of enzymes of the phenylpropanoid pathway, namely, peroxidase (PO), polyphenol oxidase (PPO), phenylalanine ammonia lyase (PAL), and enhanced the total phenolic content when compared to the control.

Wound healing response and xylem differentiation in tobacco plants over-expressing a fungal endopolygalacturonase is mediated by copper amine oxidase activity

In this work, we have investigated the involvement of copper amine oxidase (CuAO; EC 1.4.3.21) in wound healing and xylem differentiation of Nicotiana tabacum plants over-expressing a fungal endopolygalacturonase (PG plants), which show constitutively activated defence responses. In petioles and stems of PG plants, we found higher CuAO activity and lower polyamine (PA) levels, particularly putrescine (Put), with respect to wild-type (WT) plants. Upon wounding, a more intense autofluorescence of cell wall phenolics was observed in correspondence of wound surface, extending to epidermis and cortical parenchima only in PG plants. This response was mostly dependent on CuAO activity, as suggested by the reversion of autofluorescence upon supply of 2-bromoethylamine (2-BrEt), a CuAO specific inhibitor. Moreover, in unwounded plants, histochemical analysis revealed a tissue-specific expression of the enzyme in the vascular cambium and neighboring derivative cells of both petioles and stems of PG plants, whereas the corresponding WT tissues appeared unstained or faintly stained. A higher histochemical CuAO activity was also observed in xylem cells of PG plants as compared to WT xylem tissues suggesting a peculiar role of CuAO activity in xylem differentiation in PG plants. Indeed, roots of PG plants exhibited early xylem differentiation, a phenotype consistent with both the higher CuAO and the lower Put levels observed and supported by the 2-BrEt-mediated reversion of early root xylem differentiation and H2O2 accumulation. These results strongly support the relevance of PA-catabolism derived H2O2 in defence responses, such as those signaled by a compromised status of cell wall pectin integrity.

Fungal Endopolygalacturonases Are Recognized as Microbe-Associated Molecular Patterns by the Arabidopsis Receptor-Like Protein RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1

Plants perceive microbial invaders using pattern recognition receptors that recognize microbe-associated molecular patterns. In this study, we identified RESPONSIVENESS TO BOTRYTIS POLYGALACTURONASES1 (RBPG1), an Arabidopsis (Arabidopsis thaliana) leucine-rich repeat receptor-like protein, AtRLP42, that recognizes fungal endopolygalacturonases (PGs) and acts as a novel microbe-associated molecular pattern receptor. RBPG1 recognizes several PGs from the plant pathogen Botrytis cinerea as well as one from the saprotroph Aspergillus niger. Infiltration of B. cinerea PGs into Arabidopsis accession Columbia induced a necrotic response, whereas accession Brno (Br-0) showed no symptoms. A map-based cloning strategy, combined with comparative and functional genomics, led to the identification of the Columbia RBPG1 gene and showed that this gene is essential for the responsiveness of Arabidopsis to the PGs. Transformation of RBPG1 into accession Br-0 resulted in a gain of PG responsiveness. Transgenic Br-0 plants expressing RBPG1 were equally susceptible as the recipient Br-0 to the necrotroph B. cinerea and to the biotroph Hyaloperonospora arabidopsidis. Pretreating leaves of the transgenic plants with a PG resulted in increased resistance to H. arabidopsidis. Coimmunoprecipitation experiments demonstrated that RBPG1 and PG form a complex in Nicotiana benthamiana, which also involves the Arabidopsis leucine-rich repeat receptor-like protein SOBIR1 (for SUPPRESSOR OF BIR1). sobir1 mutant plants did not induce necrosis in response to PGs and were compromised in PG-induced resistance to H. arabidopsidis.

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

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

A Novel Polygalacturonase-Inhibiting Protein (PGIP) from Lathyrus sativus L. Seeds

Polygalacturonase-inhibiting proteins (PGIPs) are extracellular plant proteins bound to the plant cell wall containing leucine-rich repeats (LRR). They play an important role in plant defence being able to inhibit fungal endopolygalacturonases (EPGs), the first enzymes secreted by phytopathogenic fungi during plant infection. In the present work, a novel PGIP (LsPGIP) has been isolated from Lathyrus sativus seeds. LsPGIP exhibited an inhibitory activity towards EPGs from Aspergillus niger and Rhizopus spp. A pI value of 8.3 and a molecular mass of 40 kDa were determined for the purified inhibitor. Furthermore, N-terminal sequence up to residue 20 revealed that LsPGIP exhibit a high percentage of identity with PGIP from Actinidia deliciosa. A secondary structure similar to those of other polygalacturonase inhibitors was also inferred form circular dichroism data.

The Ectopic Expression of a Pectin Methyl Esterase Inhibitor Increases Pectin Methyl Esterification and Limits Fungal Diseases in Wheat

Cell wall pectin methyl esterification can influence plant resistance because highly methyl-esterified pectin can be less susceptible to the hydrolysis by pectic enzymes such as fungal endopolygalacturonases (PG). Pectin is secreted into the cell wall in a highly methyl-esterified form and, here, is de-methyl esterified by pectin methyl esterase (PME). The activity of PME is controlled by specific protein inhibitors called PMEI; consequently, an increased inhibition of PME by PMEI might modify the pectin methyl esterification. In order to test the possibility of improving wheat resistance by modifying the methyl esterification of pectin cell wall, we have produced durum wheat transgenic lines expressing the PMEI from Actinidia chinensis (AcPMEI). The expression of AcPMEI endows wheat with a reduced endogenous PME activity, and transgenic lines expressing a high level of the inhibitor showed a significant increase in the degree of methyl esterification. These lines showed a significant reduction of disease symptoms caused by the fungal pathogens Bipolaris sorokiniana or Fusarium graminearum. This increased resistance was related to the impaired ability of these fungal pathogens to grow on methyl-esterified pectin and to a reduced activity of the fungal PG to hydrolyze methyl-esterified pectin. In addition to their importance for wheat improvement, these results highlight the primary role of pectin despite its low content in the wheat cell wall.

ARTIFICIAL EVOLUTION CORRECTS A REPULSIVE AMINO ACID IN POLYGALACTURONASE INHIBITING PROTEINS (PGIPs)

Polygalacturonase-inhibiting proteins (PGIPs), extracellular proteins that specifically inhibit fungal endopolygalacturonases (PGs), play a critical role in plant protection by favouring the accumulation of oligogalacturonides (OGs), which are elicitors of plant defence responses. The genes encoding PGIP2 of P. vulgaris and the variant PGIP2.Q224K were subjected to error prone PCR (epPCR) to generate mutated inhibitors with novel and improved recognition capabilities. Using a Pichia pastoris expression library and a high-throughput screening method, two mutated PvPGIP2.Q224Kderived inhibitors active against the PG produced by the phytopathogenic fungus F. phyllophilum (FpPG) were isolated. Both variants were better inhibitors than PGIP2.Q224K and were characterized by the replacement of the lysine in position 224, supporting the view that the absence of this positively charged amino acid at position 224 is a primary requirement for gaining the inhibition capability against FpPG.

Protein trafficking to the cell wall occurs through mechanisms distinguishable from default sorting in tobacco

The secretory pathway in plants involves sustained traffic to the cell wall, as matrix components, polysaccharides and proteins reach the cell wall through the endomembrane system. We studied the secretion pattern of cell-wall proteins in tobacco protoplasts and leaf epidermal cells using fluorescent forms of a pectin methylesterase inhibitor protein (PMEI1) and a polygalacturonase inhibitor protein (PGIP2). The two most representative protein fusions, secGFP-PMEI1 and PGIP2-GFP, reached the cell wall by passing through ER and Golgi stacks but using distinct mechanisms. secGFP-PMEI1 was linked to a glycosylphosphatidylinositol (GPI) anchor and stably accumulated in the cell wall, regulating the activity of the endogenous pectin methylesterases (PMEs) that are constitutively present in this compartment. A mannosamine-induced non-GPI-anchored form of PMEI1 as well as a form (PMEI1-GFP) that was unable to bind membranes failed to reach the cell wall, and accumulated in the Golgi stacks. In contrast, PGIP2-GFP moved as a soluble cargo protein along the secretory pathway, but was not stably retained in the cell wall, due to internalization to an endosomal compartment and eventually the vacuole. Stable localization of PGIP2 in the wall was observed only in the presence of a specific fungal endopolygalacturonase ligand in the cell wall. Both secGFP-PMEI1 and PGIP2-GFP sorting were distinguishable from that of a secreted GFP, suggesting that rigorous and more complex controls than the simple mechanism of bulk flow are the basis of cell-wall growth and differentiation.

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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.

Characterization of the dry bean polygalacturonase-inhibiting protein (PGIP) gene family during Sclerotinia sclerotiorum (Sclerotiniaceae) infection

Polygalacturonase-inhibiting proteins are leucine-rich repeat proteins that inhibit fungal endopolygalacturonases. The interaction of polygalacturonase-inhibiting protein with endopolygalacturonases limits the destructive potential of endopolygalacturonases and may trigger plant defense responses induced by oligogalacturonides. We examined the expression of fungal pg and plant Pvpgip genes in bean (Phaseolus vulgaris) stems infected with Sclerotinia sclerotiorum to determine whether any of them are associated with the infection process. Transcriptional analysis was carried out by means of semi-quantitative reverse transcription PCR or real-time PCR. The sspg1 gene was highly expressed during infection; sspg3 was regulated during the later phases of infection; sspg5 was more uniformly expressed during infection, whereas sspg6 was only weakly expressed. During the course of infection, Pvpgip1 transcripts were not detected at early stages, but they appeared 72 h post-inoculation. High levels of Pvpgip2 expression were observed during the initial phase of infection; the transcript peaked by 48 h post-inoculation and declined by 72 h post-inoculation. Pvpgip3 expression increased strongly at 96 h post-inoculation. Pvpgip4 was constantly present from 24 h post-inoculation until the end of the experiment. However, we detected higher levels of the Pvpgip4 transcript in the necrotic lesion area than in plants that had been mechanically wounded. Remarkably, only Pvpgip4 appeared to be moderately induced by mechanical wounding. These results provide evidence that endopolygalacturonases contribute to the infection process during host colonization by promoting the release of plant cell oligogalacturonides, which are powerful signaling molecules and may also activate plant defenses, such as polygalacturonase-inhibiting proteins.

The Expression of a Bean PGIP in Transgenic Wheat Confers Increased Resistance to the Fungal Pathogen Bipolaris sorokiniana

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

The characterization of the soybean polygalacturonase-inhibiting proteins (Pgip) gene family reveals that a single member is responsible for the activity detected in soybean tissues

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 beta-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.

Cloning and analysis of pppg1, an inducible endopolygalacturonase gene from the oomycete plant pathogen Phytophthora parasitica

Phytophthora parasitica is an oomycete plant pathogen that causes severe disease in a wide variety of crops. Here, we report the isolation of a gene, named pppg1, which encodes an extracellular endopolygalacturonase in P. parasitica. Both cDNA and a genomic clone were isolated and sequenced. The pppg1 gene showed standard characteristics with respect to core promoter and intron sequences of Phytophthora. The predicted protein of pppg1 has a calculated molecular mass of 39.7 kDa and a p I value of 5.2, and contains a putative signal peptide of 20 amino acid residues on the N-terminus. The deduced amino acid sequence is highly conserved with those of other Phytophthora and fungal endopolygalacturonases. Analysis by reverse transcription followed by real-time quantitative polymerase chain reaction showed that transcription of pppg1 was repressed by glucose, but induced by pectin in the culture. Moreover, pppg1 is highly expressed during interaction of P. parasitica with the host plant, suggesting its involvement in the process of host infection. Heterologous expression of pppg1 in Pichia pastoris produced proteins with molecular mass ranging from 75 to 200 kDa, very likely due to differential glycosylation by the yeast. Deglycosylation of the recombinant protein resulted in a complete loss of the endopolygalacturonase activity.

Evaluation of tolerance to Pierce's disease and Botrytis in transgenic plants of Vitis vinifera L. expressing the pear PGIP gene

SUMMARY Polygalacturonase-inhibiting proteins (PGIPs) are plant cell-wall proteins that specifically inhibit fungal endo-polygalacturonases (PGs) that contribute to the aggressive decomposition of susceptible plant tissues. The inhibition of fungal PGs by PGIPs suggests that PGIPs have a role in plant tolerance to fungal infections and this has been observed in transgenic plants expressing PGIPs. Xylella fastidiosa, the causal agent of Pierce's disease (PD) in grapevines, has genes that encode cell-wall-degrading enzymes, including a putative PG. Therefore, we hypothesized that PGIP expression could confer tolerance against this bacterium as well as against the fungal pathogen Botrytis cinerea. To test this hypothesis, Vitis vinifera cvs. 'Thompson Seedless' and 'Chardonnay' were transformed to express pear fruit PGIP-encoding gene (pPGIP) under the control of the CaMV 35S promoter. Substantial pear PGIP (pPGIP) activity was found in crude extracts from leaves and in xylem exudate of transgenic lines obtained from independent transformation events, but not in untransformed controls. pPGIP activity was detected in xylem exudate of untransformed scions grafted on to transgenic rootstocks expressing pPGIP. Leaves of transgenic plants infected with B. cinerea had reduced rates of lesion expansion. The development of PD was delayed in some transgenic lines with increased pPGIP activity. PD-tolerant transgenic lines had reduced leaf scorching, lower Xylella titres and better re-growth after pruning than the untransformed controls.

Role of extracytoplasmic leucine rich repeat proteins in plant defence mechanisms

Plant-pathogen interactions involve highly complex series of reactions in disease development. Plants are endowed with both, resistance and defence genes. The activation of defence genes after contact with avirulence gene products of pathogens depends on signals transduced by leucine-rich repeats (LRRs) contained in resistance genes. Additionally, LRRs play roles for various actions following ligand recognition. Polygalacturonase inhibiting proteins (PGIPs), the only plant LRR protein with known ligands, are pectinase inhibitors, bound by ionic interactions to the extracellular matrix (ECM) of plant cells. They have a high affinity for fungal endopolygalacturonases (endoPGs). PGIP genes are organised in families encoding proteins with similar physical characteristics but different specificities. They are induced by infection and stress related signals. The molecular basis of PG-PGIP interaction serves as a model to understand the evolution of plant LRR proteins in recognising non-self-molecules. Extensins form a different class of structural proteins with repetitive sequences. They are also regulated by wounding and pathogen infection. Linkage of extensins with LRR motifs is highly significant in defending host tissues against pathogen invasion. Overexpression of PGIPs or expression of several PGIPs in a plant tissue, and perhaps manipulation of extensin expression could be possible strategies for disease management.