Thaumatin-like Research Articles

Methyl Jasmonate Primed Defense Responses Against Penicillium expansum in Sweet Cherry Fruit

The effect of methyl jasmonate (MeJA) treatment on controlling blue mold decay caused by Penicillium expansum in sweet cherry fruit and the possible mechanisms were investigated. The results indicated that fruit treated with MeJA had significantly lower disease incidence and smaller lesion diameter than the control fruit did. The in vitro experiment showed that MeJA transiently inhibited spore germination and germ tube elongation of P. expansum. It is clear that MeJA triggers a priming mechanism in sweet cherry fruit, since only in fruit that had been pretreated with MeJA and then challenged with P. expansum was an enhanced capacity to augment defense responses observed. These augmented responses included enhanced activities of chitinase (CHI) and β-1,3-glucanase (GLU), and increased gene expression levels of catalase (CAT), calmodulin (CaM), GLU, phenylalanine ammonia-lyase (PAL), nonexpressor of pathogenesis-related genes 1 (NPR1-like), and thaumatin-like (THAU). Moreover, MeJA inhibited the increase of activities of polygalacturonase (PG) and pectinmethylesterase (PME). These results suggest that the efficacy of MeJA on controlling blue mold decay in sweet cherry fruit may be related to the transient direct inhibitory effect against the pathogens, suppressed activities of PG and PME, and the priming of defense responses.

Enzymatic hydrolysis of thermo-sensitive grape proteins by a yeast protease as revealed by a proteomic approach

Grape juice protein hydrolysis can be proposed as one of the potential alternative methods to prevent haze wine formation. Saccharomyces cerevisiae PlR1 is a wild yeast strain of grapes able to secrete an acidic proteolytic activity during growth on a synthetic medium with proline. The aim of the present work was to confirm, by SDS-PAGE and two-dimensional electrophoresis (2D-E), the hydrolysis of some grape juice proteins after incubation with a S. cerevisiae PlR1 culture supernatant, and then to identify these hydrolyzed proteins by nano-LC–MS/MS analysis. Results obtained showed that hydrolyzed proteins correspond to pathogenesis-related (PR) proteins, in particular, thaumatin-like (TL) proteins and chitinases. Some of these PR proteins are likely to be implicated in the wine haze formation. These proteins will be isolated and characterized in order to explain why they are sensitive to enzymatic hydrolysis. Otherwise, the protease will be purified, sequenced and cloned in yeast in order to improve the wine stability with regard to proteic haze.

Evaluation of pathogenesis-related protein content and protein instability of seven white grape (Vitis vinifera L.) clones from Casablanca Valley, Chile

Thaumatin-like proteins and chitinases are the main pathogenesis-related (PR) proteins found in grapes, grape juice and wine and are responsible for protein haze formation in bottled white wine during storage and transport. We have studied the effect of the content of both thaumatin-like proteins and chitinases on protein instability of Sauvignon blanc (clones 1, 107 and 242) and Chardonnay (clones 4, 5, 75 and Mendoza) grape juices from both a warm and a cold production zone in the Casablanca Valley, Chile. The PR proteins were identified and quantified by reversed-phase liquid chromatography (RP-HPLC). Protein instability was determined using a heat test and was expressed in nephelometric turbidity units (NTUs). Thaumatin-like (TL) proteins were identified as the major PR proteins present in all grape juices studied. Three TL proteins were identified and named Vitis vinifera thaumatin-like proteins 1, 2 and 3 (VVTL1, VVTL2 and VVTL3). Chitinase A (ChitA) was identified in the Sauvignon blanc and Chardonnay grape juices, and chitinase B (ChitB) was found only in Chardonnay grape juices. Significant differences in the protein content and in the type of protein were observed between grapes from different production zones and between grapes of different varieties, respectively.

Two-Step Purification of Pathogenesis-Related Proteins from Grape Juice and Crystallization of Thaumatin-like Proteins

Grape thaumatin-like (TL) proteins and chitinases play roles in plant-pathogen interactions and can cause protein haze in white wine unless removed prior to bottling. A two-step method is described that highly purified hundreds of milligrams of TL proteins and chitinases from two juices by strong cation exchange (SCX) and hydrophobic interaction chromatography (HIC). The method was fast and separated isoforms of TL proteins and chitinases from within the same juice, in most cases to >97% purity. The isolated proteins were identified by peptide nanoLC-MS/MS and crystallized using a high-throughput screening method. Crystals from three protein fractions produced high-resolution X-ray crystallography data.

Lentinula edodes tlg1 Encodes a Thaumatin-Like Protein That Is Involved in Lentinan Degradation and Fruiting Body Senescence

Lentinan is an antitumor product that is purified from fresh Lentinula edodes fruiting bodies. It is a cell wall component, comprising beta-1,3-glucan with beta-1,6-linked branches, which becomes degraded during postharvest preservation as a result of increased glucanase activity. In this study, we used N-terminal amino acid sequence to isolate tlg1, a gene encoding a thaumatin-like (TL) protein in L. edodes. The cDNA clone was approximately 1.0 kb whereas the genomic sequence was 2.1 kb, and comparison of the two indicated that tlg1 contains 12 introns. The tlg1 gene product (TLG1) was predicted to comprise 240 amino acids, with a molecular mass of 25 kD and isoelectric point value of 3.5. The putative amino acid sequence exhibits approximately 40% identity with plant TL proteins, and a fungal genome database search revealed that these TL proteins are conserved in many fungi including the basidiomycota and ascomycota. Transcription of tlg1 was not detected in vegetative mycelium or young and fresh mushrooms. However, transcription increased following harvest. Western-blot analysis demonstrated a rise in TLG1 levels following harvest and spore diffusion. TLG1 expressed in Escherichia coli and Aspergillus oryzae exhibited beta-1,3-glucanase activity and, when purified from the L. edodes fruiting body, demonstrated lentinan degrading activity. Thus, we suggest that TLG1 is involved in lentinan and cell wall degradation during senescence following harvest and spore diffusion.

Molecular characterization of a fruit-preferential thaumatin-like gene from apple (Malus domestica cv. Fuji)

Functioning of the thaumatin-like (TL) protein is known to be pathogenesis- or stress-related. Here we identified TL protein cDNA, from an apple- skin library, that was nearly identical to that of theMdTl1 gene. Transcripts of this so-named gene,MdTL1a, were highly expressed in the fruit, but rarely in other tissue types. Expression was found in both the skin and the flesh of the fruit We also examined the environmental or hormonal control ofMdTL1a expression. Exposing the fruit to light caused this gene to be induced in the skin tissues. Accumulation ofMdTL1a mRNA reached a peak between Days 1 and 5 after exposure. In the leaves,MdTL1a was induced by salicylic acid (SA), but was not significantly affected by any other stresses. Analysis of theMdTL1a genomic clone, λTL1, revealed that transcription ofMdTL1a begins 53 bp upstream of the start codon. Sequence analysis of the ca. 1.0-kbMdTL1a promoter region has enabled us to predict that it has a stress-related cis-element, such as the ABA responsive element (ABRE), as well as a light-responsive GT-1 and l-box.

Changes in Chitinase and Thaumatin-like Pathogenesis-Related Proteins of Grape Berries during the Champagne Winemaking Process

Plant defense proteins (pathogenesis-related, or PR, proteins) have been implicated in allergenic reactions in humans. Grape PR-proteins are also known to be responsible for heat-induced haze and are susceptible to precipitation in the bottle. Changes in two defense proteins, a chitinase (CHV5) and a thaumatinlike (TL), were followed during the Champagne winemaking process, using <i>Vitis vinifera</i> L. cv. Pinot noir grape berries. Determination of chitinase activity indicated that the extraction yield of chitinases is at least 22% in the must with low pressing to make Champagne and that chitinase activity regularly diminishes during the winemaking process. SDS-PAGE electrophoresis of the must revealed that CHV5 and TL are the major proteins found in grape berries cv. Pinot noir. Using specific antibodies, changes in these two proteins during the Champagne winemaking process were followed. Western blotting indicated a regular disappearance of CHV5 and TL. TL did not seem to precipitate or hydrolyze but was absent in Champagne wine. CHV5 was likely fixed on the cell wall of <i>Saccharomyces cerevisiae</i> yeast (yeast lees) after alcoholic fermentation and on the cell wall of <i>Oenococcus oeni</i> bacteria after malolactic fermentation. Fragments of low molecular weight linked to CHV5 were observed, probably indicating that CHV5 had undergone proteolysis. This observation is in good agreement with the occurrence of an acid protease activity during the Champagne winemaking process.

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.

Some thaumatin-like proteins hydrolyse polymeric beta-1,3-glucans.

Thaumatin and 12 purified thaumatin-like (TL) proteins were surveyed for their capacity to hydrolyse beta-1,3-glucans by using an in-gel glucanase assay. Six TL proteins identified by N-terminal amino acid microsequencing were found to be active on carboxymethyl(CM)-pachyman: a barley leaf stress-related permatin, two tomato fruit osmotins, a cherry fruit and two tobacco stigma proteins. TL enzymes ranged in specific activity from 0.07 to 89 nkat mg-1 with CM-pachyman as substrate. Hydrolytic activities were not restricted to TL proteins strongly binding to water-insoluble beta-1,3-glucans since the two osmotins were active without tight binding to pachyman. Some TL proteins hydrolysed crude fungal walls and one barley TL enzyme even lysed fungal spores. No activity was observed on laminarin in the in-gel hydrolase assay. Thin-layer chromatography revealed that the six enzymes acted as endo-beta-1, 3-glucanases leading to the formation of various oligoglucosides. Thus far, the TL enzymes (EC 3.2.1.x) appeared different from the well-known beta-1,3-glucanases (EC 3.2.1.39). No activity was found with thaumatin, zeamatin, tobacco leaf PR-R protein and four stress-related TL proteins from barley and pea. This is the first demonstration that diverse TL proteins are enzymatically active. The functions of some TL proteins must be reassessed because they display endo-beta-1,3-glucanase activity on polymeric beta-1, 3-glucans.

Induction of different pathogenesis‐related cDNAs in grapevine infected with powdery mildew and treated with ethephon

Uncinula necator, the causal agent of grapevine powdery mildew, was found to elevate the activity of the pathogenesis‐related (PR) proteins, chitinase and β‐1,3‐glucanase in leaves and berries of a number of susceptible grapevine (Vitis vinifera) cultivars. The increase in hydrolytic activity was directly related to the severity of powdery mildew infection on both leaves and berries but no systemic induction was observed in uninfected tissues. A number of cDNA clones encoding various PR proteins, including chitinases (PR‐2), β‐1,3‐glucanases (PR‐3) and thaumatin‐like (TL) proteins (PR‐5), were isolated and their expression in response to powdery mildew infection examined. PR genes encoding extracellular proteins were most strongly induced in infected leaves, including an acidic class III chitinase (VvChi3), a basic class I glucanase (VvGlub) and a thaumatin‐like protein (VvTl2). A basic class I chitinase (VvChi1b) was also detected, but only in severely infected leaves. A similar pattern of PR gene induction was observed in mildew‐infected preveraison berries, with the exception of VvChi1b, which was not detected. Chitinase and β‐1,3‐glucanase enzyme activities were also found to be highly induced in leaves and preveraison berries by ethephon treatment. The pattern of chitinase gene induction by ethephon, however, was markedly different for mildew infection, with no apparent induction of VvChi3 but a marked increase in the transcript level of an acidic class IV chitinase, VvChi4. These results demonstrate that the leaves and preveraison berries of grapevine respond to environmental and stress‐related stimuli by the expression of specific PR genes but that the induction of these genes during pathogen invasion does not afford complete protection of these tissues against Uncinula necator.The following abbreviations have been used: Tris: 2‐amino‐2‐(hydroxymethyl)‐1,3‐propanediol; EDTA: ethylenediaminetetra‐acetic acid; PVPP: polyvinylpolypyrrolidone. The GenBank accession numbers for VvChi1b and VvGlub are AF053341 and AF053750, respectively.

Pathogenesis-related proteins in barley leaves, induced by infection with Drechslera teres(Sacc.) Shoem. and by treatment with other biotic agents

The perthotrophic fungus Drechslera teres, the causal agent of net blotch disease in barley, induces the accumulation of pathogenesis-related (PR) proteins in barley leaves as shown by isoelectric focusing. The same protein pattern was also found in leaves treated with a toxin extract from the culture filtrate of D. teresas well as after infection with Erysiphe graminisf.sp. hordeior Puccinia hordei. Some of the proteins induced by infection with D. tereswere characterized as peroxidases, β-1,3-glucanases and chitinases by isoenzyme analysis. Immunodetection following western blots demonstrated that the induced proteins are the same as those that accumulate after inoculation of barley with E. graminis: basic PR-1a and b proteins, thaumatin-like (TL) proteins, β-1,3-glucanases and chitinases. The accumulation of PR-1 type proteins, chitinases and TL-proteins was analysed quantitatively by ELISA.

Isolation and expression of a host response gene family encoding thaumatin-like proteins in incompatible oat-stem rust fungus interactions.

Four cDNA clones (corresponding to tlp-1, -2, -3, and -4 genes) encoding thaumatin-like (TL), pathogenesis-related proteins were isolated from oat (Avena sativa) infected by an incompatible isolate Pga-1H of the oat stem rust fungus (Puccinia graminis f. sp. avenae). All four cDNA clones contained an open reading frame predicted to encode a 169-amino acid polypeptide with a signal peptide of 21 amino acids at the N-terminus, suggesting that these proteins are transported through a secretory pathway. The amino acid sequences revealed high homology among the four cDNA clones, 80 to 99% identity and 86 to 100% similarity. The tlp genes and several TL protein genes of certain cereals are clustered into a small group that is phylogenetically separate from the major group of TL protein genes of several plant species. In plants infected with the incompatible isolate Pga-1H, or an inappropriate isolate Pgt-8D of P. graminis f. sp. tritici, high levels of tlp gene transcripts accumulated at 42 to 48 h AI and thereafter when hypersensitive host cell death occurred and hyphal growth was inhibited, whereas in plants infected with a compatible isolate Pga-6A, relatively lower amounts of transcripts were detected. Overall, transcript levels were higher with tlp-1 than with the three other genes. Spray with a light mineral oil used as a spore carrier induced transient expression of tlp-1, -2, and -3 genes at 16 to 30 h AI which obscured the initial induction of the tlp genes in response to infection by the pathogens. In contrast, tlp-4 was induced very little by oil spray, so that induction was clearly observed in response to either compatible, incompatible, or inappropriate isolates at 24 to 30 h AI. Wounding leaves by either slicing or puncturing them strongly induced tlp-1 and tlp-3, moderately induced tlp-2, but had no effect on tlp-4. Taken together, the results showed that tlp genes displayed differential responses to oil spray, mechanical wounding, and pathogen infection and that the expression of tlp genes, especially tlp-1, in oat is associated with resistance reactions in response to infection by incompatible and inappropriate isolates of the stem rust fungi.

Abiotic stresses induce a thaumatin-like protein in maize; cDNA isolation and sequence analysis

The cDNA clone (CHEM4) coding for a stress-induced protein has been isolated from a mercuric chloride-treated maize ( Zea mays L.) cDNA library by differential screening. Nucleotide sequence analysis of the 694-bp-cDNA insert predicts an open reading frame of 522 nucleotides encoding a mature protein of 149 amino acids and a signal peptide of 25 amino acid residues. The sequence of CHEM4-encoded protein shows 50 to 60% sequence identity with thaumatin, bifunctional α-amylase/trypsin inhibitor from maize and thaumatin-like (TL) proteins from dicotyledonous plants. Higher sequence identities are found to wheat PWIR2 and barley Hv-1b TL-proteins. CHEM4-mRNA is not only induced by mercuric chloride treatment but also by other stresses such as high salt concentration, high temperatures, UV light, wounding and polluted rainwater. CHEM4 genes appear to be highly inducible by UV light and mercuric chloride, moderately by wounding, salt stress, polluted rainwater and heat shock and very weakly by cooling.

Identification of a basic pathogenesis-related, thaumatin-like protein of virus-infected tobacco as osmotin

A basic PR protein was isolated and characterized from Samsun NN tobacco leaves infected with tobacco mosaic virus. The protein is serologically-related to the pathogenesis-related (PR) proteins R and S, the thaumatin-like (TL) proteins so-called because of their high level of homology with thaumatin, a sweet-tasting protein from the West African shrub Thaumatococcus daniellii Benth. Its amino acid composition and its NH 2 -terminal sequence indicate that this PR protein is in fact osmotin, a protein known to accumulate in tobacco cells in response to osmotic stress. A specific serum was obtained and used in immunoblotting experiments to study the serological relationships of TL-proteins of tobacco and to compare the induction of osmotin and acidic PR proteins R and S during the hypersensitive reaction of tobacco to tobacco mosaic virus.

CDNA cloning of six mRNAs induced by TMV infection of tobacco and a characterization of their translation products

A cDNA library was constructed to 10-15 S poly(A) RNA from tobacco mosaic virus (MV)-infected Samsun NN tobacco.By differential colony hybridization of 1400 transformants,32 clones were obtained corresponding to TMV-inducible tobacco mRNAs. These clones were subdivided into six clusters on the basis of cross-hybridization of the inserts. By Northern blot hybridization it was shown that three of the corresponding mRNAs were strongly induced by spraying tobacco plants with salicylic acid, whereas one mRNA was weakly induced by this treatment. All mRNAs were systemically induced in plants in which only the lower leaves were locally infected by TMV. Hybrid-selected translation was performed, using six clones representing one cluster each, followed by immunoprecipitation using an antiserum to purified pathogenesis-related (PR) proteins. Four clones yielded precipitable translation products. One of these clones represented a cluster of PR-1 clones, another clone encoded the thaumatin-like (TL) protein of tobacco which may correspond to PR-P or -Q.