Biochemical and molecular biological studies on infection (Ascochyta rabiei)-induced thaumatin-like proteins from chickpea plants (Cicer arietinum L.)

A pathogenesis-related protein induced by infection with Ascochyta rabiei was purified from intercellular washing fluid of chickpea (Cicer arietinum L.) leaves. Amino-terminal sequencing identified the protein, named PR-5a, as a thaumatin-like protein. The isoelectric point was determined with 6.5 and the molecular mass is 16 kDa. Therefore, chickpea PR-5a is the first dicot member of a TLP subgroup containing small TLPs with a molecular weight between 15 and 18 kDa. PR-5a shows no antifungal activity towards A. rabiei. Screening of a chickpea cDNA library led to the isolation of a cDNA clone (p5a-241) for this protein. A second cDNA clone (ELR112) encoding a TLP was isolated using differential hybridisation of cDNA libraries obtained from elicited and water treated cell suspension cultures of chickpea. The deduced protein (PR-5b) has a molecular mass of 22 kDa. PR-5b is postulated to be located in the vacuole due to the presence of a respective N-terminal signal peptide and a carboxy-terminal extension. Southern blot analyses showed that ELR112 and p5a-241 represent single copy genes. During fungal infection of chickpea plants expression of both genes proceeds much faster in an A. rabiei resistant cultivar than in a susceptible one.     

Resolution of the structure of the allergenic and antifungal banana fruit thaumatin-like protein at 1.7-A

The structure of a thaumatin-like protein from banana (Musa acuminata) fruit, an allergen with antifungal properties, was solved at 1.7-A-resolution, by X-ray crystallography. Though the banana protein exhibits a very similar overall fold as thaumatin it markedly differs from the sweet-tasting protein by the presence of a surface exposed electronegative cleft. Due to the presence of this electronegative cleft, the banana thaumatin-like protein (Ban-TLP) acquires a strong (local) electronegative character that eventually explains the observed antifungal activity. Our structural analysis also revealed the presence of conserved residues of exposed epitopic determinants that are presumably responsible for the allergenic properties of banana fruit towards susceptible individuals, and provided evidence that the Ban-TLP shares some structurally highly conserved IgE-binding epitopes with thaumatin-like proteins from fruits or pollen from other plants. In addition, some overlap was detected between the predicted IgE-binding epitopes of the Ban-TLP and IgE-binding epitopes previously identified in the mountain cedar Jun a 3 TLP aeroallergen. The presence of these common epitopes offers a molecular basis for the cross-reactivity between aeroallergens and fruit allergens.     

Purification and structural analysis of an abundant thaumatin-like protein from ripe banana fruit

 The pulp of ripe bananas (Musa acuminata) contains an abundant thaumatin-like protein (TLP). Characterization of the protein and molecular cloning of the corresponding gene from banana demonstrated that the native protein consists of a single polypeptide chain of 200 amino acid residues. Molecular modelling further revealed that the banana thaumatin-like protein (Ban-TLP) adopts an overall fold similar to that of thaumatin and thaumatin-like PR-5 proteins. Although the banana protein exhibits an electrostatically polarized surface, which is believed to be essential for the antifungal properties of TLPs, it is apparently devoid of antifungal activity towards pathogenic fungi. It exhibits a low but detectable in vitro endo-β-1,3-glucanase (EC 3.2.1.x) activity. As well as being present in fruits, Ban-TLP also occurs in root tips where its accumulation is enhanced by methyl jasmonate treatment of plants. Pulp of plantains (Musa acuminata) also contains a very similar TLP, which is even more abundant than its banana homologue. Our results demonstrate for the first time that fruit-specific (abundant) TLPs are not confined to dicots but occur also in fruits of monocot species. The possible role of the apparent widespread accumulation of fruit-specific TLPs is discussed. Received: 7 January 2000 / Accepted: 26 April 2000     

Biochemical, molecular and structural analysis of multiple thaumatin-like proteins from the elderberry tree (Sambucus nigra L.)

Thaumatin-like proteins (TLPs) were isolated and characterized from fruits and leaves of elderberry (Sambucus nigra) and their corresponding genes cloned. In addition, the developmental regulation and induction of the different TLPs was followed in some detail. Ripening berries accumulated a fruit-specific TLP during the final stages of maturation. This fruit-specific TLP had no antifungal activity and was devoid of beta-glucanase activity. Leaves constitutively expressed a TLP that closely resembled the fruit-specific homologue. Treatment with jasmonate methyl ester induced two additional TLPs in leaves but did not induce or enhance the expression of TLPs in immature berries. In contrast to jasmonate methyl ester, both ethephon and garlic extract induced the expression of a TLP in unripe berries that normally do not express any TLP. Sequence analysis and molecular modeling indicated that all elderberry thaumatin-like proteins share a high sequence similarity with group-5 pathogenesis-related proteins. However, the proteins encoded by the different sequences differed from each other in isoelectric point and the distribution of the charges on the surface of the molecule.     

Purification, characterization, and molecular gene cloning of an antifungal protein from Ginkgo biloba seeds

A novel basic protein with antifungal activity was isolated from the seeds of Ginkgo biloba and purified to homogeneity. The protein inhibited the growth of some fungi (Fusarium oxysporum, Trichoderma reesei, and Candida albicans) but did not exhibit antibacterial action against Escherichia coli. Furthermore, this protein showed weak inhibitory activity against the aspartic protease pepsin. To design primers for gene amplification, the NH(2)-terminal and partial internal amino acid sequences were determined using peptides obtained from a tryptic digest of the oxidized protein. The full-length cDNA of the antifungal protein was cloned and sequenced by RT-PCR and rapid amplification of cDNA ends (RACE). The cDNA contained a 402-bp open reading frame encoding a 134-aa protein with a potential signal peptide (26 residues), suggesting that this protein is synthesized as a preprotein and secreted outside the cells. The antifungal protein shows approximately 85% identity with embryo-abundant proteins from Picea abies and Picea glauca at the amino acid level; however, there is no homology between this protein and other plant antifungal proteins, such as defensin, and cyclophilin-, miraculin- and thaumatin-like proteins.     

Eryngin, a novel antifungal peptide from fruiting bodies of the edible mushroom Pleurotus eryngii

An antifungal peptide with a molecular mass of 10k Da was isolated from fruiting bodies of the mushroom Pleurotus eryngii. The peptide, designated as eryngin, inhibited mycelial growth in Fusarium oxysporum and Mycosphaerella arachidicola. It was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and S-Sepharose. Its N-terminal sequence demonstrated some similarity to the antifungal protein from the mushroom Lyophyllum shimeiji and little resemblance to thaumatin and thaumatin-like proteins.     

Induction of pathogenesis-related proteins in sugarcane leaves and cell-cultures by a glycoprotein elicitor isolated from <Emphasis Type="Italic">Colletotrichum falcatum</Emphasis>

The induction of pathogenesis-related (PR) proteins in sugarcane (Saccharum officinarum L.) leaves and suspension-cultured cells in response to treatment with a glycoprotein elicitor isolated from Colletotrichum falcatum (the red rot pathogen) was investigated. Treatment of leaves and cells with the elicitor resulted in a much marked increase in the activities of chitinase and β-1,3-glucanase in red rot resistant (BO 91) than susceptible (CoC 671) sugarcane cultivar. SDS-PAGE analysis revealed that C. falcatum elicitor induced the accumulation of several proteins in suspension-cultured cells of resistant cultivar (BO 91); among them the 35 kDa protein was predominant. Whereas, a 27 kDa protein was induced predominantly in the cells of susceptible cultivar upon treatment with the elicitor. When sugarcane leaves were treated with C. falcatum elicitor, two proteins with apparent molecular masses of 25 and 27 kDa were induced both in the resistant and susceptible cultivars. However, the induction was stronger in the resistant than the susceptible cultivar. Immunoblot analysis for chitinase indicated that a protein with an apparent molecular mass of 37 kDa cross-reacting with barley chitinase antiserum was strongly induced in the suspension cultured cells of both the cultivars. The induction of 37 kDa chitinase was more in the cells of resistant cultivar than in the susceptible cultivar. Western blot analysis revealed that a 25 kDa thaumatin-like protein (TLP) cross-reacting with bean TLP antiserum was strongly induced in leaves and cultured cells of both resistant and susceptible cultivars due to elicitor treatment.     

Isolation of fungal cell wall degrading proteins from barley (Hordeum vulgare L.) leaves infected with Rhynchosporium secalis

Proteins with antifungal activity towards Rhynchosporium secalis conidia were isolated from the intercellular washing fluid (IWF) of barley leaves. The active components were purified by high-performance liquid chromatography under conditions that maintained biological activity. Five major barley IWF proteins deleterious to the cell wall of viable R. secalis conidia were isolated and identified by a combination of N-terminal amino acid sequencing, peptide mapping, and determination of mass and isoelectric point. They were a 32-kDa beta-1,3-glucanase (Pr32), a 25-kDa chitinase (Pr25), and three 22-kDa thaumatin-like (TL) proteins (Pr22-1, Pr22-2, and Pr22-3). Pr22-1 and Pr22-2 were similar to the protein R class of TL proteins, whereas Pr22-3 was more similar to the S class. Pr22-3 was shown to digest laminarin, indicating that this TL protein has glucanase activity. In addition, Pr22-3 was more active in the spore bioassay than Pr22-2. Various combinations of the five proteins had a greater effect on R. secalis spores than did the individual proteins. The extraction of proteins with antifungal activity from the IWF of barley leaves indicates their possible role in defense against leaf pathogens. A similar bioassay may be developed for other systems to identify particular isoforms of pathogenicity-related proteins that might have a role in plant disease resistance.     

First chromatographic isolation of an antifungal thaumatin-like protein from French bean legumes and demonstration of its antifungal activity.

A protein, with a molecular weight of 20 kDa, and an N-terminal sequence analogous to those of thaumatin-like proteins (TLPs) and thaumatins, was first isolated from the legume of the French bean Phaseolus vulgaris cv Kentucky wonder using a simple procedure involving affinity and ion exchange chromatography. The protein was adsorbed on both CM-Sepharose and Affi-gel Blue Gel. It was the first leguminous TLP-like protein demonstrated to exert antifungal activity against Fusarium oxysporum, Pleurotus ostreatus, and Coprinus comatus but not against Rhizoctonia solani.     

A molecular basis for the endo-beta 1,3-glucanase activity of the thaumatin-like proteins from edible fruits

Fruit-specific thaumatin-like proteins were isolated from cherry, apple and banana, and their enzymatic and antifungal activities compared. Both the apple and cherry possess a moderate endo-beta 1,3-glucanase activity but are devoid of antifugal activity. In contrast, the banana thaumatin-like protein inhibits the in vitro hyphal growth of Verticillium albo-atrum but is virtually devoid of endo-beta 1,3-glucanase activity. Both structural and molecular modeling studies showed that all three thaumatin-like proteins possess an extended electronegatively charged cleft at their surface, which is believed to be a prerequisite for endo-beta 1,3-glucanase activity. Docking experiments showed that the positioning of linear (1,3)-beta-D-glucans in the cleft of the apple and cherry proteins allows an interaction with the glutamic acid residues that are responsible for the hydrolytic cleavage of the glucan. Due to a different positioning in the cleft of the banana thaumatin-like protein, the linear beta-glucans cannot properly interact with the catalytic glutamic acid residues and as a result the protein possesses no enzymatic activity. The possible function of the fruit-specific thaumatin-like proteins is discussed in view of the observed biological activities and structural features.     

Crystal structure of tobacco PR-5d protein at 1.8 A resolution reveals a conserved acidic cleft structure in antifungal thaumatin-like proteins

The crystal structure of tobacco PR-5d, an antifungal thaumatin-like protein isolated from cultured tobacco cells, was determined at the resolution of 1.8 A. The structure consists of 208 amino acid residues and 89 water molecules with a crystallographic R-factor of 0.169. The model has good stereochemistry, with respective root-mean-square deviations from the ideal values for bond and angle distances of 0.007 A and 1.542 degrees. Of the homologous PR-5 proteins, only those with antifungal activity had a common motif, a negatively charged surface cleft. This cleft is at the boundary between domains I and II, with a bottom part consisting of a three-stranded antiparallel beta-sheet in domain I. The acidic residues located in the hollow of the cleft form the beta-sheet region. Sequence and secondary structure analyses showed that the amino acid residues comprising the acidic cleft of PR-5d are conserved among other antifungal PR-5 proteins. This is the first report on the high-resolution crystal structure of an antifungal PR-5 protein. This structure provides insight into the function of pathogenesis-related proteins.     

Patterns of molecular evolution and predicted function in thaumatin-like proteins of Populus trichocarpa

Some pathogenesis-related proteins (PR proteins) are subject to positive selection, while others are under negative selection. Here, we report the patterns of molecular evolution in thaumatin-like protein (TLP, PR5 protein) genes of Populus trichocarpa. Signs of positive selection were found in 20 out of 55 Populus TLPs using the likelihood ratio test and ML-based Bayesian methods. Due to the connection between the acidic cleft and the antifungal activity, the secondary structure and three-dimensional structure analyses predicted antifungal activity β-1,3-glucanase activities in these TLPs. Moreover, the coincidence with variable basic sites in the acidic cleft and positively selected sites suggested that fungal diseases may have been the main environmental stress that drove rapid adaptive evolution in Populus.     

Gene note. Cloning of a cDNA encoding the thaumatin-like protein of winter rye (Secale cereale L. Musketeer) and its functional characterization

A cDNA clone, WRTLP2, encoding an open reading frame of 173 amino acids, was recovered from a cDNA library of winter rye (Secale cereale L. Musketeer). The amino acid sequence deduced from the cloned cDNA exhibits very high sequence similarity (70-95%) with those of extracellular and low molecular weight thaumatin-like proteins of other cereals. It was possible to overexpress this isolated cDNA in Escherichia coli and it was found that the encoded protein of this clone exhibited antifungal activities against several fungal strains.     

Crystal structure analysis of NP24-I: a thaumatin-like protein

The crystal structure of NP24-I, an isoform of the thaumatin-like protein (TLP) NP24 from tomato, has been reported. A prominent acidic cleft is observed between domains I and II of the three-domain structure of this antifungal protein, a feature common to other antifungal TLPs. The defensive role of the TLPs has also been attributed to their beta-1,3-glucanase activity and here too the acidic cleft is reported to play a vital role. NP24 is known to bind beta-glucans and so a linear beta-1,3-glucan molecule has been docked in the interdomain cleft of NP24-I. From the docked complex it is observed that the beta-glucan chain is so positioned in the cleft that a Glu and Asp residue on either side of it may form a catalytic pair to cause the cleavage of a glycosidic bond. NP24 has been reported to be an allergenic protein and an allergenic motif could be identified on the surface of the helical domain II of NP24-I. In addition, some allergenic motifs bearing high similarity/identity with some predicted Ig-E binding motifs of closely related allergenic TLPs like Jun a 3 (Juniperus ashei, from mountain cedar pollen) and banana-TLP have been identified on the molecular surface of NP24-I.     

Antifreeze proteins in winter rye leaves form oligomeric complexes

Antifreeze proteins (AFPs) similar to three pathogenesis-related proteins, a glucanase-like protein (GLP), a chitinase-like protein (CLP), and a thaumatin-like protein (TLP), accumulate during cold acclimation in winter rye (Secale cereale) leaves, where they are thought to modify the growth of intercellular ice during freezing. The objective of this study was to characterize the rye AFPs in their native forms, and our results show that these proteins form oligomeric complexes in vivo. Nine proteins were separated by native-polyacrylamide gel electrophoresis from apoplastic extracts of cold-acclimated winter rye leaves. Seven of these proteins exhibited multiple polypeptides when denatured and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After isolation of the individual proteins, six were shown by immunoblotting to contain various combinations of GLP, CLP, and TLP in addition to other unidentified proteins. Antisera produced against individual cold-induced winter rye GLP, CLP, and TLP all dramatically inhibited glucanase activity in apoplastic extracts from cold-acclimated winter rye leaves, and each antiserum precipitated all three proteins. These results indicate that each of the polypeptides may be exposed on the surface of the protein complexes. By forming oligomeric complexes, AFPs may form larger surfaces to interact with ice, or they may simply increase the mass of the protein bound to ice. In either case, the complexes of AFPs may inhibit ice growth and recrystallization more effectively than the individual polypeptides.     

Overexpression of thaumatin-like protein in transgenic tomato plants confers enhanced resistance to Alternaria solani

Abstract

A cDNA encoding thaumatin-like protein (TLP) from rice was cloned into the binary vector pMON410 under the control of the CaMV 35S promoter for Agrobacterium-mediated transformation of tomato. All putative transformants were tested for the integration and expression of the chimeric gene by polymerase chain reaction (PCR) for hygromycin resistance gene (hph) and enzyme-linked immunosorbent assay (ELISA) for TLP respectively. Constitutive, high-level expression of TLP was observed in transgenic plants. The transgenic lines exhibited increased resistance to Alternaria solani, the early blight pathogen compared to non-transgenic tomato plants.