Thaumatin-like Protein Gene Research Articles

Genome-wide identification, characterization and expression analysis of the TLP gene family in melon (Cucumis melo L.)

Thaumatin-like proteins (TLPs), which belong to pathogenesis-related (PR) protein family 5 (PR5), are involved in plant host defense and various developmental processes. The functions of the TLP family have been extensively discussed in multiple organisms, whereas the detailed information of this family in melon has not been reported yet. In this study, we identified 28 TLP genes in the melon genome and a N-terminal signal peptide was found highly conserved within each member of this family. Phylogeny analysis indicated that TLPs from melon and other plant species were clustered into ten groups. Twelve segmental and seven tandem duplication gene pairs that underwent purifying selection were identified. TLP genes expressed differentially in different tissues/organs, and were significantly induced after Podosphaera xanthii infection. TLPs in breeding line MR-1 tend to express early after pathogen infection compared with cultivar Top Mark. Our study provides a comprehensive understanding of the melon TLP family and demonstrates their potential roles in disease resistance, therefore provides more reference for further research.

PR-proteins as markers of winter wheat (<i>Triticum aestivum L.</i>) resistance to leaf pathogens

Using real-time PCR analysis, the constitutive expression of PR-protein genes encoding thaumatin-like protein (TLP), peroxidase III (TaPero), chitinase (Chitin), glucanase (Glucan), protease inhibitor (PrInh), oxalate oxidase (OxOxid) and lipid transfer protein (Ltp) was studied in collection varieties of winter wheat. It has been shown that plants of varieties with increased resistance to a complex of leaf pathogens have higher constitutive expression levels of Chitin and PrInh genes, and, to a greater extent, of TLP, TaPero and Glucan genes, compared with non-resistant varieties. It is proposed to use constitutive levels of expression of TLP, TaPero and Glucan genes for the selection of winter wheat varietal samples with increased resistance to the complex of leaf diseases.

Antifungal properties of a thaumatin-like protein from watermelon

Fusarium wilt is a causal disease that threatens watermelon production. In this work, we report the identification and antifungal activity of a thaumatin-like protein (ClTLP27) that was identified from a Fusarium oxysporum (F. oxysporum)-responsive proteomic analysis during watermelon and F. oxysporum interaction. A two-dimensional gel electrophoresis approach was used to compare changes in the leaf proteome profiles of rootstock-grafted watermelon upon F. oxysporum inoculation, and the abundance of a thaumatin-like protein was found to be differentially accumulated. This thaumatin-like protein gene was further cloned from watermelon and named ClTLP27 (accession no. MF445020). ClTLP27 contains 807 nucleotides and encodes a putative polypeptide of 268 amino acids with a calculated molecular mass of 28.93 kDa and a theoretical pI of 7.65. Sequence alignment showed that ClTLP27 contains the conserved motif with 16 cysteines. Phylogenetic analysis indicated that ClTLP27 belongs to the thaumatin-like protein cluster, and is closely related to the Cucumis TLP gene with a sequence identity of 90%. Real-time PCR analysis revealed that ClTLP27 was expressed in all tissues examined, with the highest levels of expression occurring in the roots. Expression profiles of ClTLP27 following F. oxysporum inoculation revealed that the transcript level of ClTLP27 varied in susceptible and resistant seedlings. ClTLP27 was further cloned into the PET28a(+) vector to obtain a bacterially expressed recombinant His-ClTLP27 protein. Antifungal activity analysis showed that the His-ClTLP27 protein significantly inhibited the mycelial growth of F. oxysporum f.sp. niveum race 1, Fusarium solani f.sp. cucurbitae race 1, F. oxysporum f.sp. melonis, Fusarium verticillioides and Didymella bryoniae. This work implies that ClTLP27 could be used as botanical fungicide or as a potential gene in the engineering of disease-resistant watermelon.

Characterization of a Pinus sylvestris thaumatin-like protein gene and determination of antimicrobial activity of the in vitro expressed protein

Thaumatin-like proteins (TLPs) are pathogenesis-related proteins, which are involved in plant defense responses to pathogen infection. Expression of the Pinus sylvestris L. TLP gene is up-regulated by methyl jasmonate treatment and inoculation with Heterobasidion annosum. A full-length Pinus taeda TLP gene sequence was used to design PCR primers for amplification of the full-length TLP gene from P. sylvestris. A putative 705-bp open reading frame of TLP gene was cloned into Escherichia coli cells, and then subcloned into the overexpression vector pET100 using BL21 Star expression bacteria. Optimization of the expression of recombinant TLP was achieved by decreasing both expression temperature and IPTG concentration. The purified 24.6-kDa TLP shows antimicrobial activity against 12 fungal species.

Functional analysis of a pathogenesis-related thaumatin-like protein gene TaLr35PR5 from wheat induced by leaf rust fungus

BackgroundPlants have evolved multifaceted defence mechanisms to resist pathogen infection. Production of the pathogenesis-related (PR) proteins in response to pathogen attack has been implicated in plant disease resistance specialized in systemic-acquired resistance (SAR). Our earlier studies have reported that a full length TaLr35PR5 gene, encoding a protein exhibiting amino acid and structural similarity to a sweet protein thaumatin, was isolated from wheat near-isogenic line TcLr35. The present study aims to understand the function of TaLr35PR5 gene in Lr35-mediated adult resistance to Puccinia triticina.ResultsWe determined that the TaLr35PR5 protein contained a functional secretion peptide by utilizing the yeast signal sequence trap system. Using a heterologous expression assay on onion epidermal cells we found that TaLr35PR5 protein was secreted into the apoplast of onion cell. Expression of TaLr35PR5 was significantly reduced in BSMV-induced gene silenced wheat plants, and pathology test on these silenced plants revealed that Lr35-mediated resistance phenotype was obviously altered, indicating that Lr35-mediated resistance was compromised.ConclusionsAll these findings strongly suggest that TaLr35PR5 is involved in Lr35-mediated adult wheat defense in response to leaf rust attack.

Transgenic Tea Over-expressing Solanum tuberosum Endo-1,3-beta-d-glucanase Gene Conferred Resistance Against Blister Blight Disease

Tea (Camellia sinensis [L.] O. Kuntze) plant, one of the most important plantation crops in the world, is infected by a fungus called Exobasidium vexans leading to dreaded blister blight disease. The disease may result in crop losses up to 35% which directly affect the tea industry. Solanum tuberosum endo-1,3-beta-d-glucanase was cloned into tea genome via Agrobacterium-mediated transformation. The transformation event initially gave 32 kanamycin-resistant plantlets, out of which PCR analysis confirmed only 10 plantlets about the integration of transgene in the plant genome. Real-time PCR study detected transgene expression in six transgenic plantlets. Upregulation of endogenous C. sinensis pathogenesis-related (PR) genes like PR3 (chitinase I) gene and PR5 (thaumatin-like protein) gene also occurred in transgenic plantlets. Detached leaf infection assay showed resistance to E. vexans in greenhouse-acclimated transgenic plantlets. An inhibitory activity against E. vexans was noticed on the detached leaves of transgenic plantlets compared to control. Transgenic plantlets showed resistance to inoculated fungal pathogen by the formation of hypersensitivity reaction area unlike the formation of fungal lesion on control plantlet. Thus, it can be inferred that constitutive expression of the potato endo-1,3-beta-d-glucanase gene can be a strategy to produce blister blight-resistant tea.

Overexpression of rice thaumatin-like protein (Ostlp) gene in transgenic cassava results in enhanced tolerance to Colletotrichum gloeosporioides f. sp. manihotis.

Cassava (Manihot esculenta Crantz) is the most important staple food for more than 300 million people in Africa, and anthracnose disease caused by Colletotrichum gloeosporioides f. sp. manihotis is the most destructive fungal disease affecting cassava production in sub-Saharan Africa. The main objective of this study was to improve anthracnose resistance in cassava through genetic engineering. Transgenic cassava plants harbouring rice thaumatin-like protein (Ostlp) gene, driven by the constitutive CaMV35S promoter, were generated using Agrobacterium-mediated transformation of friable embryogenic calli (FEC) of cultivar TMS 60444. Molecular analysis confirmed the presence, integration, copy number of the transgene all the independent transgenic events. Semi-quantitative RT-PCR confirmed high expression levels of Ostlp in six transgenic lines tested. The antifungal activity of the transgene against Colletotrichum gloeosporioides pathogen was evaluated using the leaves and stem cuttings bioassay. The results demonstrated significantly delayed disease development and reduced size of necrotic lesions in leaves and stem cuttings of all transgenic lines compared to the leaves and stem cuttingss of non-transgenic control plants. Therefore, constitutive overexpression of rice thaumatin-like protein in transgenic cassava confers enhanced tolerance to the fungal pathogen C. gloeosporioides f. sp. manihotis. These results can therefore serve as an initial step towards genetic engineering of farmer-preffered cassava cultivars for resistance to anthracnose disease.

Assessment of gene copy number variation of Scots pine thaumatin-like protein gene using real-time PCR based methods

The importance and impact of gene copy number variations (CNVs) as a source of polymorphism in the human and other genomes is being increasingly recognized. Less information is available about CNVs in forest tree species, mainly due to the relative lack of genomic resources. In this study, several methods—quantitative polymerase chain reaction, comparative high-resolution melting curve analysis (C-HRM), and digital polymerase chain reaction (dPCR)—were used to investigate CNV of the Scots pine thaumatin-like protein gene (PsTLP). The obtained results were supported by transcriptome analysis of a single Pinus sylvestris individual and publically available pine genome sequences. Although estimations of gene copy number (CN) varied, depending on the region of the PsTLP gene investigated and the endogenous control utilized, our results revealed the existence of copy number variations of the PsTLP gene between Scots pine individuals. Of 23 individuals analyzed, two had an increased calculated relative CN regardless of the analyzed gene region and endogenous control used, while several samples had increased copy numbers of regions of the PsTLP gene. C-HRM results were highly correlated with qPCR data (R 2 TLP3′ = 0.88; R 2 TLPc = 0.92), but interpretation of gene CN from C-HRM results proved to be difficult. The results from selected samples analyzed by digital PCR also were highly correlated with qPCR results (R 2 = 0.90).

Hop stunt viroid: Effect on Host (Humulus lupulus) Transcriptome and Its Interactions With Hop Powdery Mildew (Podospheara macularis).

Viroids are the smallest known plant pathogens that exploit host systems for their replication and cause diseases in many hosts. In this study, the host response of hop plants to Hop stunt viroid (HSVd) infection was studied through transcriptome analysis. RNA sequence analysis of hop leaves infected with HSVd revealed dynamic changes in hop gene expression. Defense-related genes and genes involved in lipid and terpenoid metabolism are the major categories that showed differential expression due to HSVd infection. Additionally, the effect of HSVd on development of hop powdery mildew (Podospheara macularis) (HPM) was studied. Transcriptome analysis followed by quantitative reverse transcription-polymerase chain reaction analysis showed that transcript levels of pathogenesis-related (PR) genes such as PR protein 1, chitinase, and thaumatin-like protein genes are induced in leaves infected with HPM alone. The response in these genes to HPM is significantly down-regulated in leaves with HSVd-HPM mixed infection. These results confirm that HSVd alters host metabolism, physiology, and plant defense responses. Nevertheless, in detached leaf assays, HPM consistently expanded faster on HSVd-negative leaves relative to HSVd-positive leaves. Although HSVd infection suppresses elements associated with the host immunity response, infection by HSVd is antagonistic to HPM infection of hops.

The effect of potassium phosphite on PR genes expression and the phenylpropanoid pathway in cucumber (Cucumis sativus) plants inoculated with Pseudoperonospora cubensis

In the present study, the impact of potassium phosphite (KPhi) was investigated at molecular and biochemical levels in response to Pseudoperonospora cubensis infection in cucumber plants. Real-time PCR was employed to explore the differential expression of defense genes against P. cubensis. The highest expression for thaumatin-like protein (TLP), Ribosome-inactivating protein (RIP) and Defensin genes was observed in pre-inoculation plants at 96, 72 and 48h, respectively. These findings revealed the involvement of these genes in the defense response of cucumber leaves after KPhi treatment and pathogen inoculation. At the biochemical level, more induction in the contents of some end-products of phenylpropanoid pathway, such as phytoalexin, phenolic component, flavonoid and anthocyanin as well as phenylalanine ammonia-lyase (PAL) enzymatic activity was detected in pre-inoculation plants compared to post-inoculation plants at all-time points. Data suggest that KPhi primes rapid and robust response in plants against infection via activation of defense responses. The negative effects of P. cubensis on cucumber plants could be considerably mitigated by KPhi application before infection.

Analysis of the grape (Vitis vinifera L.) thaumatin-like protein (TLP) gene family and demonstration that TLP29 contributes to disease resistance

Thaumatin-like protein (TLP) is present as a large family in plants, and individual members play different roles in various responses to biotic and abiotic stresses. Here we studied the role of 33 putative grape (Vitis vinifera L.) TLP genes (VvTLP) in grape disease resistance. Heat maps analysis compared the expression profiles of 33 genes in disease resistant and susceptible grape species infected with anthracnose (Elsinoe ampelina), powdery mildew (Erysiphe necator) or Botrytis cinerea. Among these 33 genes, the expression level of TLP29 increased following the three pathogens inoculations, and its homolog from the disease resistant Chinese wild grape V. quinquangularis cv. ‘Shang-24’, was focused for functional studies. Over-expression of TLP29 from grape ‘Shang-24’ (VqTLP29) in Arabidopsis thaliana enhanced its resistance to powdery mildew and the bacterium Pseudomonas syringae pv. tomato DC3000, but decreased resistance to B. cinerea. Moreover, the stomatal closure immunity response to pathogen associated molecular patterns was strengthened in the transgenic lines. A comparison of the expression profiles of various resistance-related genes after infection with different pathogens indicated that VqTLP29 may be involved in the salicylic acid and jasmonic acid/ethylene signaling pathways.

Identification of candidate genes for fiber length quantitative trait loci through RNA-Seq and linkage and physical mapping in cotton

BackgroundCotton (Gossypium spp.) fibers are single-celled elongated trichomes, the molecular aspects of genetic variation in fiber length (FL) among genotypes are currently unknown. In this study, two backcross inbred lines (BILs), i.e., NMGA-062 (“Long”) and NMGA-105 (“Short”) with 32.1 vs. 27.2 mm in FL, respectively, were chosen to perform RNA-Seq on developing fibers at 10 days post anthesis (DPA). The two BILs differed in 4 quantitative trait loci (QTL) for FL and were developed from backcrosses between G. hirsutum as the recurrent parent and G. barbadense.ResultsIn total, 51.7 and 54.3 million reads were obtained and assembled to 49,508 and 49,448 transcripts in the two genotypes, respectively. Of 1551 differentially expressed genes (DEGs) between the two BILs, 678 were up-regulated and 873 down-regulated in “Long”; and 703 SNPs were identified in 339 DEGs. Further physical mapping showed that 8 DEGs were co-localized with the 4 FL QTL identified in the BIL population containing the two BILs. Four SNP markers in 3 DEGs that showed significant correlations with FL were developed. Among the three candidate genes encoding for proline-rich protein, D-cysteine desulfhydrase, and thaumatin-like protein, a SNP of thaumatin-like protein gene showed consistent correlations with FL across all testing environments.ConclusionsThis study represents one of the first investigations of positional candidate gene approach of QTL in cotton in integrating transcriptome and SNP identification based on RNA-Seq with linkage and physical mapping of QTL and genes, which will facilitate eventual cloning and identification of genes responsible for FL QTL. The candidate genes may serve as the foundation for further in-depth studies of the molecular mechanism of natural variation in fiber elongation.

Cloning and expression of three thaumatin-like protein genes from Polyporus umbellatus

Genes encoding thaumatin-like protein (TLPs) are frequently found in fungal genomes. However, information on TLP genes in Polyporus umbellatus is still limited. In this study, three TLP genes were cloned from P. umbellatus. The full-length coding sequence of PuTLP1, PuTLP2 and PuTLP3 were 768, 759 and 561bp long, respectively, encoding for 256, 253 and 187 amino acids. Phylogenetic trees showed that P. umbellatus PuTLP1, PuTLP2 and PuTLP3 were clustered with sequences from Gloeophyllum trabeum, Trametes versicolor and Stereum hirsutum, respectively. The expression patterns of the three TLP genes were higher in P. umbellatus with Armillaria mellea infection than in the sclerotia without A. mellea. Furthermore, over-expression of three PuTLPs were carried out in Escherichia coli BL21 (DE3) strain, and high quality proteins were obtained using Ni-NTA resin that can be used for preparation of specific antibodies. These results suggest that PuTLP1, PuTLP2 and PuTLP3 in P. umbellatus may be involved in the defense response to A. mellea infections.

Molecular Characterization, Structural Modeling, and Evaluation of Antimicrobial Activity of Basrai Thaumatin-Like Protein against Fungal Infection.

A thaumatin-like protein gene from Basrai banana was cloned and expressed in Escherichia coli. Amplified gene product was cloned into pTZ57R/T vector and subcloned into expression vector pET22b(+) and resulting pET22b-basrai TLP construct was introduced into E. coli BL21. Maximum protein expression was obtained at 0.7 mM IPTG concentration after 6 hours at 37°C. Western blot analysis showed the presence of approximately 20 kDa protein in induced cells. Basrai antifungal TLP was tried as pharmacological agent against fungal disease. Independently Basrai antifungal protein and amphotericin B exhibited their antifungal activity against A. fumigatus; however combined effect of both agents maximized activity against the pathogen. Docking studies were performed to evaluate the antimicrobial potential of TLP against A. fumigatus by probing binding pattern of antifungal protein with plasma membrane ergosterol of targeted fungal strain. Ice crystallization primarily damages frozen food items; however addition of antifreeze proteins limits the growth of ice crystal in frozen foods. The potential of Basrai TLP protein, as an antifreezing agent, in controlling the ice crystal formation in frozen yogurt was also studied. The scope of this study ranges from cost effective production of pharmaceutics to antifreezing and food preserving agent as well as other real life applications.

Selection and Cloning of Thaumatin-like Protein ( TLP ) Gene from Winter Brassica rapa and Its Expression under Low Temperature Stress

Selection and Cloning of Thaumatin-like Protein ( <i>TLP</i> ) Gene from Winter <i>Brassica rapa</i> and Its Expression under Low Temperature Stress

Expansion and evolution of thaumatin-like protein (TLP) gene family in six plants

Thaumatin-like protein (TLP) is one of pathogenesis-related protein family, which plays an important role in plant defense system. While its origin and expansion is unknown in plants. Here, we investigated the evolution of this gene family by performing a genome-wide identification and comparison in Arabidopsis, Oryza, Populus, Zea, Physcomitrella and Chlamydomonas. We found a birth process of this gene family in evolution. Tandem and segmental duplication played dominant roles for their expansion. In addition, TLPs were under purifying selection, and most members were responding to some biotic or abiotic stresses. Functional network analysis exhibited some resistance genes working together with TLPs. Our findings shed some light on this family gene evolution in plants, which might provide a base for further functional investigation of this family.

In silico and experimental evaluation of DNA-based detection methods for the ability to discriminate almond from other Prunus spp.

Ten published DNA-based analytical methods aiming at detecting material of almond (Prunus dulcis) were in silico evaluated for potential cross-reactivity with other stone fruits (Prunus spp.), including peach, apricot, plum, cherry, sour cherry and Sargent cherry. For most assays, the analysis of nucleotide databases suggested none or insufficient discrimination of at least some stone fruits. On the other hand, the assay targeting non-specific lipid transfer protein (Röder et al., 2011, Anal Chim Acta 685:74–83) was sufficiently discriminative, judging from nucleotide alignments. Empirical evaluation was performed for three of the published methods, one modification of a commercial kit (SureFood allergen almond) and one attempted novel method targeting thaumatin-like protein gene. Samples of leaves and kernels were used in the experiments. The empirical results were favourable for the method from Röder et al. (2011) and a modification of SureFood allergen almond kit, both showing cross-reactivity <10−3 compared to the model almond.

Chestnut, European (Castanea sativa).

Development of a system for direct transfer of antifungal candidate genes into European chestnut (Castanea sativa) would provide an alternative approach to conventional breeding for production of chestnut trees that are tolerant to ink disease caused by Phytophthora spp. Overexpression of genes encoding PR proteins (such as thaumatin-like proteins), which display antifungal activity, may represent an important advance in control of the disease. We have used a chestnut thaumatin-like protein gene (CsTL1) isolated from European chestnut cotyledons and have achieved overexpression of the gene in chestnut somatic embryogenic lines used as target material. We have also acclimatized the transgenic plants and grown them on in the greenhouse. Here, we describe the various steps of the process, from the induction of somatic embryogenesis to the production of transgenic plants.

Characterization of a pathogen induced thaumatin-like protein gene AdTLP from Arachis diogoi, a wild peanut.

Peanut (Arachis hypogaea L) is one of the widely cultivated and leading oilseed crops of the world and its yields are greatly affected by various biotic and abiotic stresses. Arachis diogoi, a wild relative of peanut, is an important source of genes for resistance against various stresses that affect peanut. In our previous study a thaumatin-like protein gene was found to be upregulated in a differential expression reverse transcription PCR (DDRT-PCR) study using the conidial spray of the late leaf spot pathogen, Phaeoisariopsis personata. In the present study, the corresponding full length cDNA was cloned using RACE-PCR and has been designated as AdTLP. It carried an open reading frame of 726 bp potentially capable of encoding a polypeptide of 241 amino acids with 16 conserved cysteine residues. The semi-quantitative RT-PCR analysis showed that the transcript level of AdTLP increased upon treatment with the late leaf spot pathogen of peanut, P. personata and various hormone treatments indicating its involvement in both, biotic and abiotic stresses. The antifungal activity of the purified recombinant protein was checked against different fungal pathogens, which showed enhanced anti-fungal activity compared to many other reported TLP proteins. The recombinant AdTLP-GFP fusion protein was found to be predominantly localized to extracellular spaces. Transgenic tobacco plants ectopically expressing AdTLP showed enhanced resistance to fungal pathogen, Rhizoctonia solani. The seedling assays showed enhanced tolerance of AdTLP transgenic plants against salt and oxidative stress. The transcript analysis of various defense related genes highlighted constitutively higher level expression of PR1a, PI-I and PI-II genes in transgenic plants. These results suggest that the AdTLP is a good candidate gene for enhancing stress resistance in crop plants.

Cloning and characterization of a thaumatin-like protein gene PeTLP in Populus deltoides × P. euramericana cv. ‘Nanlin895’

Thaumatin-like protein (TLP) genes constitute a large, highly complex gene family involved in host defense against abiotic stresses, as well as other physiological processes. However, their functions in the abiotic stress-induced defense response in poplar remain largely unknown. In this study, one full-length cDNA from a hybrid poplar (Populusdeltoides × P. euramericana cv. ‘Nanlin895’) that encodes a TLP was isolated by RACE and designated PeTLP. The deduced amino acid sequence of PeTLP encodes a protein of a predicted molecular mass 24.5 kDa and a pI of 8.19. Transcript of PeTLP accumulated in stems, but the gene showed low transcriptional levels in roots and leaves. PeTLP was upregulated in leaves after abiotic treatments, including 200 mM NaCl, 10 % PEG6000, 100 μM gibberellin, 1 mM salicylic acid and 100 μM abscisic acid. Subcellular localization analysis showed that GFP-tagged PeTLP predominantly localizes as discrete points in the cytoplasm and the periphery of the plasma membrane. Recombinant PeTLP obtained using Escherichiacoli expression produced antifungal activities to Rhizopus sp., Aspergillusniger and Penicilliumcitrinum. These results suggest that PeTLP is an abiotic stresses-induced protein and plays roles in physiological processes of poplar development, and is involved in the defense of poplar against abiotic stresses. Manipulation of PeTLP in poplar or other trees should improve their performance under various abiotic stress conditions.