The Bursaphelenchus xylophilus Effector BxNMP1 Targets PtTLP-L2 to Mediate PtGLU Promoting Parasitism and Virulence in Pinus thunbergii.

Pinus is an important economic tree species, but pine wilt disease (PWD) seriously threatens the survival of pine trees. PWD caused by Bursaphelenchus xylophilus is a major quarantine disease worldwide that causes significant economic losses. However, more information about its molecular pathogenesis is needed, resulting in a lack of effective prevention and treatment measures. In recent years, effectors have become a hot topic in exploring the molecular pathogenic mechanism of pathogens. Here, we identified a specific effector, BxNMP1, from B. xylophilus. In situ hybridization experiments revealed that BxNMP1 was specifically expressed in dorsal gland cells and intestinal cells, and RT-qPCR experiments revealed that BxNMP1 was upregulated in the early stage of infection. The sequence of BxNMP1 was different in the avirulent strain, and when BxNMP1-silenced B. xylophilus was inoculated into P. thunbergii seedlings, the disease severity significantly decreased. We demonstrated that BxNMP1 interacted with the thaumatin-like protein PtTLP-L2 in P. thunbergii. Additionally, we found that the ß-1,3-glucanase PtGLU interacted with PtTLP-L2. Therefore, we hypothesized that BxNMP1 might indirectly interact with PtGLU through PtTLP-L2 as an intermediate mediator. Both targets can respond to infection, and PtTLP-L2 can enhance the resistance of pine trees. Moreover, we detected increased salicylic acid contents in P. thunbergii seedlings inoculated with B. xylophilus when BxNMP1 was silenced or when the PtTLP-L2 recombinant protein was added. In summary, we identified a key virulence effector of PWNs, BxNMP1. It positively regulates the pathogenicity of B. xylophilus and interacts directly with PtTLP-L2 and indirectly with PtGLU. It also inhibits the expression of two targets and the host salicylic acid pathway. This study provides theoretical guidance and a practical basis for controlling PWD and breeding for disease resistance.

Ectopic expression of a thaumatin-like protein impairs the timely deposition and dissolution of callose during microsporogenesis, leading to microspore death and male sterility in Arabidopsis.

KEY MESSAGE: Two pollen-preferential thaumatin-like proteins show both common and distinctive expression profiles. Precocious expression of one of them drastically disturbs timely deposition and dissolution of callose during microsporogenesis, leading to microspore death. Thaumatin-like proteins (TLPs), members of the pathogenesis-related protein family 5 (PR-5), are involved in plant defenses against biotic and abiotic stresses through antifungal activity and enhanced tolerance. Accordingly, studies on TLPs have focused on their responses to various pathogens and stresses and on engineering agronomically valuable crops that can be cultivated in suboptimal environments. On the other hand, the role of TLP members in plant development and their genetic regulation remains largely unexplored. Recently, we reported that the generative cell internalization after pollen mitosis I, an essential pollen patterning step for the nonmotile sperm cell delivery through a pollen tube, depends on STICKY GENERATIVE CELL which suppresses callose deposition in the nascent generative cell and interacts with a germline cell preferential GCTLP1 in Arabidopsis. Here, we additionally identified GCTLP2 which is similarly expressed in the germline cells. We generated various transgenic lines and examined their expressions and phenotypes to elucidate GCTLP functions during pollen development. Expression profiles suggest two GCTLP proteins may have common but also distinctive roles during pollen development. Importantly, ectopic expression analyses show that precocious expression of GCTLP2 severely disturbs the timely deposition and degradation of callose during microsporogenesis which is essential to produce viable microspores. Therefore, our study broadens the knowledge of TLP function and callose regulation for successful pollen development in Arabidopsis.

Characterization and expression analysis of a thaumatin-like protein PpTLP1 from ground cherry Physalis pubescens.

Ground cherry, Physalis pubescens, is mainly cultivated as a fruit worldwide and popularly used as a food supplement and traditional Chinese medicine. Plants are challenged by external environmental stress and can initiate resistance to the stress through the regulation of pathogenesis-related (PR) proteins. Among PR proteins, PR-5, a thaumatin-like protein (TLP), was identified in many plants and found to be able to enhance stress resistance. However, PR-5 in ground cherry is not characterized and its expression is yet to be understood. In this study, a PR-5 protein PpTLP1 in P. pubescens was firstly identified. Analysis of the amino acid sequences revealed that PpTLP1 was highly similar to PR-NP24 identified in tomato with a difference in only one amino acid. Expression analysis indicated that the PpTLP1 gene was highly expressed in leaf while the PpTLP1 protein was tissue-specifically accumulated in cherry exocarp. Furthermore, the down-regulation of PpTLP1 in ground cherry was induced by NaCl treatment while the up-regulation was promoted by the infection of Sclerotinia sclerotiorum and Botrytis cinerea. This study will provide a new plant resource containing a TLP in Physalis genus and a novel insight for the improvement of postharvest management of ground cherry and other Solanaceae plants.

Identification and Characterization of Neuroprotective Properties of Thaumatin-like Protein 1a from Annurca Apple Flesh Polyphenol Extract.

BACKGROUND: Alzheimer's disease (AD) and Parkinson's disease (PD) are multifactorial neurodegenerative disorders that are mostly treated with drugs inhibiting key enzymes of cholinergic and aminergic neurotransmission, such as acetyl and butyryl cholinesterase (AChE, BuChE) or monoamine oxidases (MAO)-A/B, and of Aß1-40 aggregation. Diet plant components with multitarget functions are promising compounds in the prevention of AD and PD. Our aim was to identify neuroprotective compounds from Annurca apple polyphenol extract (AFPE). METHODS: AFPE was fractionated by gel filtration, and the eluted peaks were subjected to chemical analyses (i.e., RP-HPLC and mass spectrometry), determination of inhibitory enzyme activity and cell effects by MTT, and morphology assays. RESULTS: In AFPE, we identified thaumatin-like protein 1a, belonging to the pathogenesis-related protein (PR) family. This protein showed the best inhibitory activity on AChE, MAO-A (IC50 = 5.53 µM and 1.71 µM, respectively), and Aß1-40 fibril aggregation (IC50 = 9.16 µM), compared to AFPE and other polyphenol-containing fractions. Among the latter, Peak 4 reverted Aß fibril formation (IC50 = 104.87 µM). Moreover, thaumatin-like protein 1a protected AGS and MKN-28 cells from serum-deprivation-induced stress conditions. CONCLUSIONS: We showed that AFPE exerted neuroprotective functions not only through its polyphenols but also through thaumatin-like protein 1a, which acted like a multitarget molecule.

PRMT5 regulates the polysaccharide content by controlling the splicing of thaumatin-like protein in Ganoderma lucidum.

IMPORTANCE: PRMT5 contributes to secondary metabolite biosynthesis in Ganoderma lucidum. However, the mechanism through which PRMT5 regulates the biosynthesis of secondary metabolites remains unclear. In the current study, PRMT5 silencing led to a significant decrease in the biosynthesis of polysaccharides from G. lucidum through the action of the alternative splicing of TLP. A shorter TLP2 isoform can directly bind to PGI and regulated polysaccharide biosynthesis. These results suggest that PRMT5 enhances PGI activity by regulating TLP binding to PGI. The results of the current study reveal a novel target gene for PRMT5-mediated alternative splicing and provide a reference for the identification of PRMT5 regulatory target genes.

Tomato-Thaumatin-like Protein Genes Solyc08g080660 and Solyc08g080670 Confer Resistance to Five Soil-Borne Diseases by Enhancing ß-1,3-Glucanase Activity.

Although thaumatin-like proteins (TLPs) are involved in resistance to a variety of fungal diseases, whether the TLP5 and TLP6 genes in tomato plants (Solanum lycopersicum) confer resistance to the pathogenesis of soil-borne diseases has not been demonstrated. In this study, five soil-borne diseases (fungal pathogens: Fusarium solani, Fusarium oxysporum, and Verticillium dahliae; bacterial pathogens: Clavibacter michiganense subsp. michiganense and Ralstonia solanacearum) were used to infect susceptible "No. 5" and disease-resistant "S-55" tomato cultivars. We found that SlTLP5 and SlTLP6 transcript levels were higher in susceptible cultivars treated with the three fungal pathogens than in those treated with the two bacterial pathogens and that transcript levels varied depending on the pathogen. Moreover, the SlTLP5 and SlTLP6 transcript levels were much higher in disease-resistant cultivars than in disease-susceptible cultivars, and the SlTLP5 and SlTLP6 transcript levels were higher in cultivars treated with the same fungal pathogen than in those treated with bacterial pathogens. SlTLP6 transcript levels were higher than SlTLP5. SlTLP5 and SlTLP6 overexpression and gene-edited transgenic mutants were generated in both susceptible and resistant cultivars. Overexpression and knockout increased and decreased resistance to the five diseases, respectively. Transgenic plants overexpressing SlTLP5 and SlTLP6 inhibited the activities of peroxidase (POD), superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT) after inoculation with fungal pathogens, and the activities of POD, SOD, and APX were similar to those of fungi after infection with bacterial pathogens. The activities of CAT were increased, and the activity of ß-1,3-glucanase was increased in both the fungal and bacterial treatments. Overexpressed plants were more resistant than the control plants. After SlTLP5 and SlTLP6 knockout plants were inoculated, POD, SOD, and APX had no significant changes, but CAT activity increased and decreased significantly after the fungal and bacterial treatments, contrary to overexpression. The activity of ß-1,3-glucanase decreased in the treatment of the five pathogens, and the knocked-out plants were more susceptible to disease than the control. In summary, this study contributes to the further understanding of TLP disease resistance mechanisms in tomato plants.

Time-specific ultrasonic treatment of litchi thaumatin-like protein inhibits inflammatory response in RAW264.7 macrophages via NF-κB and MAPK transduction pathways.

The pro-inflammation activity of litchi thaumatin-like protein (LcTLP) led to be responsible for the occurrence of adverse reactions after excessive consumption of litchi. This study aimed to characterize the changes in the structure and inflammatory activity of LcTLP induced by ultrasound treatment. Significant molecular structure of LcTLP changes occured at 15 min ultrasound treatment, and then tended to recover with subsequent treatment. Secondary structure (α-helices decreased from 17.3% to 6.3%), tertiary structure (the maximum endogenous fluorescence intensity decreased), and microstructure (mean hydrodynamic diameter reduced from 4 µm to 50 nm) of the LcTLP treated for 15 min (LT15) were significantly affected, which led to the inflammatory epitope of LcTLP (domain II and V-cleft) unfolded. In vitro, LT15 had a significant anti-inflammatory response, which inhibited NO production and had the best effect at 50 ng/mL in RAW264.7 macrophages (73.24%). Moreover, proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) secretion and mRNA expression levels were also significantly lower compared with untreated LcTLP (p < 0.05). Western blot further confirmed that the expressions of IκB-α, p65, p38, ERK and JNK reduced markedly (p < 0.05), which indicated LT15 inhibited the inflammatory response through NF-κB and MAPK transduction pathways. Overall, it can be hypothesized that LT15 exposed to low frequency ultrasonic fields have a direct effect on the protein surface structure and thus on the entry of LT15 into cells, making 15-minute ultrasound treatment potentially useful in reducing the pro-inflammatory properties of litchi or related liquid products.

Genome-Wide Analyses of Thaumatin-like Protein Family Genes Reveal the Involvement in the Response to Low-Temperature Stress in Ammopiptanthus nanus.

Thaumatin-like proteins (TLPs), a family of proteins with high sequence similarity to thaumatin, are shown to be involved in plant defense, and are thus classified into the pathogenesis related protein family 5. Ammopiptanthus nanus is a rare evergreen broad-leaved shrub distributed in the temperate zone of Central Asia, which has a high tolerance to low-temperature stress. To characterize A. nanus TLPs and understand their roles in low-temperature response in A. nanus, a comprehensive analysis of the structure, evolution, and expression of TLP family proteins was performed. A total of 31 TLP genes were detected in the A. nanus genome, and they were divided into four groups based on their phylogenetic positions. The majority of the AnTLPs contained the conserved cysteine residues and were predicted to have the typical three-dimensional structure of plant TLPs. The primary modes of gene duplication of the AnTLP family genes were segmental duplication. The promoter regions of most AnTLP genes contain multiple cis-acting elements related to environmental stress response. Gene expression analysis based on transcriptome data and fluorescence quantitative PCR analysis revealed that several AnTLP genes were involved in cold-stress response. We further showed that a cold-induced AnTLP gene, AnTLP13, was localized in apoplast, and heterologous expression of the AnTLP13 in Escherichia coli and yeast cells and tobacco leaves enhanced low-temperature stress tolerance when compared with the control cells or seedlings. Our study provided important data for understanding the roles of TLPs in plant response to abiotic stress.

Recombinant Thaumatin-Like Protein (rTLP) and Chitinase (rCHI) from Vitis vinifera as Models for Wine Haze Formation.

Cross-linking net aggregates of thermolabile thaumatin-like proteins (TLPs) and chitinases (CHIs) are the primary source of haze in white wines. Although bentonite fining is still routinely used in winemaking, alternative methods to selectively remove haze proteins without affecting wine organoleptic properties are needed. The availability of pure TLPs and CHIs would facilitate the research for the identification of such technological advances. Therefore, we proposed the usage of recombinant TLP (rTLP) and CHI (rCHI), expressed by Komagataella phaffii, as haze-protein models, since they showed similar characteristics (aggregation potential, melting point, functionality, glycosylation levels and bentonite adsorption) to the native-haze proteins from Vitis vinifera. Hence, rTLP and rCHI can be applied to study haze formation mechanisms on a molecular level and to explore alternative fining methods by screening proteolytic enzymes and ideal adsorptive resins.

Litchi thaumatin-like protein induced the liver inflammation and altered the gut microbiota community structure in mice.

The pro-inflammation activity of litchi thaumatin-like protein (LcTLP) was speculated to be the cause of the adverse reaction after excessive consumption of litchi. This study evaluated the pro-inflammation activity of LcTLP in vitro and in vivo. Results showed that LcTLP digested in vitro exhibited the pro-inflammation activity in RAW264.7 cells, as evidenced by an increase in pro-inflammation cytokines tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6). After LcTLP supplementation in mice, pathological examination revealed the edematous cytoplasm and inflammatory cell accumulation in liver tissue. The levels of TNF-α, IL-1ß, IL-6 and interleukin-10 in liver also increased by 60.43%, 74.86%, 120.18% and 129.91% in high-dose group, respectively, due to the upregulation of p-p65 and p-p38 levels and further activation of NF-κB and MAPK signaling pathways. Additionally, LcTLP altered the composition of gut microbiota, such as the reduction of Bacteroidota, norank_f__Muribaculaceae and norank_f__Lachnospiraceae abundances which were negatively correlated with inflammation cytokines levels.

Genome-wide analysis of the Thaumatin-like gene family in Qingke (Hordeum vulgare L. var. nudum) uncovers candidates involved in plant defense against biotic and abiotic stresses.

Thaumatin-like proteins (TLPs) participate in the defense responses of plants as well as their growth and development processes, including seed germination. Yet the functioning of TLP family genes, in addition to key details of their encoded protein products, has not been thoroughly investigated for Qingke (Hordeum vulgare L. var. nudum). Here, a total of 36 TLP genes were identified in the genome of Qingke via HMM profiling. Of them, 25 TLPs contained a signal peptide at the N-terminus, with most proteins predicted to localize in the cytoplasm or outer membrane. Sequence alignment and motif analysis revealed that the five REDDD residues required for ß-1,3-glucanase activity were conserved in 21 of the 36 Qingke TLPs. Phylogenetically, the TLPs in plants are clustered in 10 major groups. Our analysis of gene structure did not detect an intron in 15 Qingke TLPs whereas the other 21 did contain 1-7 introns. A diverse set of cis-acting motifs were found in the promoters of the 36 TLPs, including elements related to light, hormone, and stress responses, growth and development, circadian control, and binding sites of transcription factors, thus suggesting a multifaceted role of TLPs in Qingke. Expression analyses revealed the potential involvement of TLPs in plant defense against biotic and abiotic stresses. Taken together, the findings of this study deepen our understanding of the TLP family genes in Qingke, a staple food item in Tibet, which could strengthen future investigations of protein function in barley and its improved genetic engineering.

Genome-wide comprehensive characterization and expression analysis of TLP gene family revealed its responses to hormonal and abiotic stresses in watermelon (Citrullus lanatus).

Thaumatin-like protein (TLP) is the well-known sweetest protein which plays a crucial role in diverse developmental processes and different stress conditions in plants, fungi and animals. The TLP gene family is extensively studied in different plant species including crop plants. Watermelon (Citrullus lanatus) is an important cucurbit crop cultivated worldwide; however, the comprehensive information about the TLP gene family is not available in watermelon. In the present study, we identified the 29 TLP genes as gene family members in watermelon using various computational methods to understand its role in different developmental processes and stress conditions. ClaTLP gene family members were not uniformly distributed on 22 chromosomes. Phylogenetic analysis revealed that the ClaTLP gene family members were grouped into 10 sub-groups. Further, gene duplication analysis showed thirteen gene duplication events which included one tandem and twelve segmental duplications. Amino acid sequence alignment has shown that ClaTLP proteins shared 16 conserved cysteine residues in their THN domain. Furthermore, cis-acting regulatory elements analysis also displayed that ClaTLP gene family members contain diverse phytohormone, various defense, and stress-responsive elements in their promoter region. The expression profile of the ClaTLP gene family revealed the differential expression of gene family members in different tissues and abiotic stresses conditions. Moreover, the expression profile of ClaTLP genes was further validated by semi-quantitative reverse transcriptase PCR. Taken together, these results indicate that ClaTLP genes might play an important role in developmental processes and diverse stress conditions. Therefore, the outcome of this study brings forth the valuable information for further interpret the precise role of ClaTLP gene family members in watermelon.

Chemical and Sensory Profiles of Sauvignon Blanc Wine Following Protein Stabilization Using a Combined Ultrafiltration/Heat/Protease Treatment.

Ultrafiltration (UF) was evaluated as a process by which proteins can be selectively removed from white wine as an alternative approach to protein stabilization than traditional bentonite fining. Unfined Sauvignon Blanc wine (50 L) was fractionated by UF and the retentate stabilized either by heat and/or protease treatment or bentonite fining before being recombined with the permeate. The heat stability of recombined wine was significantly improved when retentate was heated following protease (Aspergillopepsin) addition and subsequently stabilized by bentonite treatment. The combined UF/heat/protease treatment removed 59% of protein and reduced the quantity of bentonite needed to achieve protein stability by 72%, relative to bentonite treatment alone. This innovative approach to protein stabilization had no significant impact on wine quality or sensory characteristics, affording industry greater confidence in adopting this technology as a novel approach to achieving protein stability.

Expression of the Thaumatin-Like Protein-1 Gene (Bx-tlp-1) from Pine Wood Nematode Bursaphelenchus xylophilus Affects Terpene Metabolism in Pine Trees.

Pine wilt disease is a major forest disease worldwide, including in China, where it has severely damaged pine forest ecosystems, and the pathogen is pine wood nematode (Bursaphelenchus xylophilus). The thaumatin-like protein-1 gene (Bx-tlp-1) is a key gene associated with B. xylophilus pathogenicity, which is also responsive to α-pinene. In this study, an examination of Pinus massoniana seedlings infected by B. xylophilus revealed that monoterpene (sesquiterpene) levels peaked on days 15 and 27 (days 18 and 27). Meanwhile, P. massoniana Pm-tlp expression levels were high on days 3, 12, and 27, which were consistent with the expression of key enzymes genes in the terpene biosynthesis pathway. The functional similarity of B. xylophilus Bx-TLP-1 and P. massoniana Pm-TLP suggests Bx-TLP-1 and Pm-TLP may have similar roles in P. massoniana. There was also no secondary accumulation of terpenes in P. massoniana seedlings during B. xylophilus treated with dsRNA targeting Bx-tlp-1 (dsTLP1) infections, reflecting the decreased pathogenicity of B. xylophilus and the delayed disease progression in pine trees. And the results of micro-CT showed that the degree of cavitation for the trees inoculated with Bx-TLP-1 (0.3811 mm3) was greater than that for the trees inoculated with dsTLP1 PWNs (0.1204 mm3) on day 15 after inoculation. Results from this study indicated that B. xylophilus Bx-tlp-1 gene may induce the upregulated expression of related genes encoding enzymes in the terpene synthesis pathway of P. massoniana, resulting in the accumulation of terpenes, which also provided an insight to investigate the B. xylophilus pathogenicity in the future.

Structural and inflammatory characteristics of Maillard reaction products from litchi thaumatin-like protein and fructose.

The structural characteristics and inflammatory activity of Maillard reaction products (MRPs) from fructose (Fru) and litchi thaumatin-like protein (LcTLP) with a pro-inflammatory activity were investigated. The structural changes of LcTLP-Fru MRPs were divided into two stages during the Maillard reaction. In 0-6 h, the unfolding and degradation of the LcTLP were dominant, resulting in a looser structure; the increase of ß-sheets was 13.02%; the decrease of α-helices was 9.21%; and both the molecular weight and gyration radius Rg decreased. After 6 h, the enhanced glycosylation caused the molecular weight to increase, while Rg remained low, implying that the molecular structure became more compact. In addition, LcTLP-Fru MRPs reduced the inflammation response by significantly reducing the gene and protein expressions of tumor necrosis factor-α, interleukin-1ß, and interleukin-6 compared with the LcTLP group in RAW264.7 macrophages. The findings provided a theoretical foundation for addressing the inflammatory response caused by litchi products consumption.

Haze Formation and the Challenges for Peptidases in Wine Protein Fining.

To meet consumer expectations, white wines must be clear and stable against haze formation. Temperature variations during transport and storage may induce protein aggregation, mainly caused by thaumatin like-proteins (TLPs) and chitinases (CHIs), which thus need to be fined before bottling of the wine. Currently, bentonite clay is employed to inhibit or minimize haze formation in wines. Alternatively, peptidases have emerged as an option for the removal of these thermolabile proteins, although their efficacy under winemaking conditions has not yet been fully demonstrated. The simultaneous understanding of the chemistry behind the cleavage of haze proteins and the haze formation may orchestrate alternative methods of technological and economic importance in winemaking. Therefore, we provide an overview of wine fining by peptidases, and new perspectives are developed to reopen discussions on the aforementioned challenges.

Membrane localized thaumatin-like protein from tea (CsTLP) enhanced seed yield and the plant survival under drought stress in Arabidopsis thaliana.

Thaumatin-like proteins (TLPs) are pathogenesis-related (PR5) proteins, which are induced in response to various biotic and abiotic stresses. The present work was carried out to clone TLP of Camellia sinensis (CsTLP) and to evaluate the response of transgenic lines of Arabidopsis constitutively expressing CsTLP under drought conditions. Data showed that transgenic lines exhibited lower relative electrolyte leakage and higher water retention capacity as compared to the wild-type (WT) plants under drought stress. In addition, results with confocal microscopy showed CsTLP + GFP fusion protein to be localized in the cell membrane which moved to the intercellular spaces under prolonged drought stress. Expression of CsTLP enhanced seed yield and the plant survival in transgenic lines as compared to the WT plants under drought stress. Results suggested the importance of CsTLP in improving drought tolerance in Arabidopsis.