Development of transgenic Paulownia trees expressing antimicrobial thionin genes for enhanced resistance to fungal infections using chitosan nanoparticles.

There is an increasing focus on genetically altering Paulownia trees to enhance their resistance against fungal infections, given their rapid growth and quality wood production. The aim of this research was to establish a technique for incorporating two antimicrobial thionin genes, namely thionin-60 (thio-60) and thionin-63 (thio-63), into Paulownia tomentosa and Paulownia hybrid 9501 through the utilization of chitosan nanoparticles. The outcomes revealed the successful gene transfer into Paulownia trees utilizing chitosan nanoparticles. The effectiveness of thionin proteins against plant pathogens Fusarium and Aspergillus was examined, with a specific focus on Fusarium equiseti due to limited available data. In non-transgenic Paulownia species, the leaf weight inhibition percentage varied from 25 to 36 %, whereas in transgenic species, it ranged from 22 to 7 %. In general, Paulownia species expressing thio-60 displayed increased resistance to F. equiseti, while those expressing thio-63 exhibited heightened resistance to A. niger infection. The thionin proteins displayed a strong affinity for the phospholipid bilayer of the fungal cell membrane, demonstrating their capability to disrupt its structure. The transgenic plants created through this technique showed increased resistance to fungal infections. Thionin-60 demonstrated superior antifungal properties in comparison to thio-63, being more effective at disturbing the fungal cell membrane. These findings indicate that thio-60 holds potential as a novel antifungal agent and presents a promising approach for enhancing the antimicrobial traits of genetically modified Paulownia trees.

Plant Defensin-Dissimilar Thionin OsThi9 Alleviates Cadmium Toxicity in Rice Plants and Reduces Cadmium Accumulation in Rice Grains.

Thionins are important antibacterial peptides in plants. However, the roles of plant thionins, especially the defensin-dissimilar thionins, in alleviating heavy-metal toxicity and accumulation remain unclear. Here, cadmium (Cd)-related functions and mechanisms of the defensin-dissimilar rice thionin OsThi9 were investigated. OsThi9 was significantly upregulated in response to Cd exposure. OsThi9 was localized to the cell wall and was shown to bind Cd; these characters help to increase Cd tolerance. In Cd-exposed rice plants, OsThi9 overexpression significantly increased cell wall Cd binding, decreasing upward Cd translocation and subsequent Cd accumulation in shoots and straw, while OsThi9 knockout had inverse effects. Importantly, in rice plants grown in Cd-contaminated soils, OsThi9 overexpression significantly reduced Cd accumulation in brown rice (decrease of ≥ 51.8%) without negatively impairing the crop yield and essential elements. Thus, OsThi9 plays an important role in the alleviation of Cd toxicity and accumulation and has significant potential for developing low-Cd rice.

The plant defensins PaDef and γ-thionin inhibit the endothelial cell response to VEGF.

Angiogenesis is involved in wound repair and tissue maintenance but is associated with diverse diseases. Pro-angiogenic factors such as vascular endothelial growth factor (VEGF) regulate this process. Therefore, searching for treatments to inhibit or promote angiogenesis is attractive. Reports from our group showed that plant antimicrobial peptides (PAPs) PaDef from avocado and γ-thionin from habanero pepper are cytotoxic on cancer cells. However, their functions as angiogenic regulators are unknown. In this work, we evaluate the effect of PaDef and γ-thionin on the angiogenic processes of two different endothelial cell lines: bovine endothelial cells (BUVEC) and the human endothelial cell line EA.hy926. The results showed that VEGF (10 ng/mL) stimulated the BUVEC (40 ± 7 %) and EA.hy926 cell proliferation (30 ± 9 %); however, peptides (5-500 ng/mL) reverted this effect. Besides, VEGF increased the migration of BUVEC (20 ± 8 %) and EA.hy926 cells (50 ± 6 %), but both PAPs (5 ng/mL) inhibited the VEGF stimulus (100 %). Furthermore, DMOG 50 µM (an inhibitor of HIF-hydroxylase) was used in BUVEC and EA.hy926 cells to determine the effect of hypoxia on VEGF and peptide activities. The DMOG reverted the inhibitory action of both peptides (100 %), indicating that peptides act through a HIF-independent pathway. Also, the PAPs do not affect the tube formation but decrease it in EA.hy926 cells stimulated with VEGF (100 %). Additionally, docking assays showed a possible interaction between PAPs and the VEGF receptor. These results suggest that plant defensins PaDef and γ-thionin are potential angiogenic modulators of the VEGF activity on endothelial cells.

Pharmacokinetic profile of sarcin and thionin from Aspergillus giganteus and in vitro validation against human fungal pathogen.

Fungal infections are more predominant in agricultural and clinical fields. Aspergillosis caused by Aspergillus fumigatus leads to respiratory failure in patients along with various illnesses. Due to the limitation of antifungal therapy and antifungal drugs, there is an emergence to develop efficient antifungal compounds (AFCs) from natural sources to cure and prevent fungal infections. The present study deals with the investigation of the mechanism of the active compounds from Aspergillus giganteus against aspergillosis. Primarily, the bioavailability and toxicological properties of antifungal proteins such as, sarcin, thionin, chitinase and their derivatives have proved the efficiency of pharmacokinetic properties of selected compounds. Molecular interactions of selected compounds from A. giganteus with the virulence proteins of A. fumigatus (UDP-N-acetylglucosamine pyrophosphorylase, N-myristoyl transferase and Chitinase) have exhibited a good glide score and druggable nature of the AFCs. The antagonistic potential of AFCs on the pathogen was confirmed by SEM analysis where the shrunken and damaged spores of AFCs treated pathogen were observed. The integrity of A. fumigatus cell membrane and nuclear membrane treated with AFCs were analyzed by determining the release of cellular materials. The effective concentration of AFCs was found to be 250 µg/ml (P<0.0001). The GC-MS profiling has revealed the volatile bioactive metabolites present in A. giganteus. Further, interaction studies might provide more information on the synergism activity with the non-volatile metabolites which leads to the development of novel drugs for the treatment of aspergillosis.

Neurogenesis and neuronal differentiation in the postnatal frontal cortex in Down syndrome.

Although Down syndrome (DS), the most common developmental genetic cause of intellectual disability, displays proliferation and migration deficits in the prenatal frontal cortex (FC), a knowledge gap exists on the effects of trisomy 21 upon postnatal cortical development. Here, we examined cortical neurogenesis and differentiation in the FC supragranular (SG, II/III) and infragranular (IG, V/VI) layers applying antibodies to doublecortin (DCX), non-phosphorylated heavy-molecular neurofilament protein (NHF, SMI-32), calbindin D-28K (Calb), calretinin (Calr), and parvalbumin (Parv), as well as ß-amyloid (APP/Aß and Aß1-42) and phospho-tau (CP13 and PHF-1) in autopsy tissue from age-matched DS and neurotypical (NTD) subjects ranging from 28-weeks (wk)-gestation to 3 years of age. Thionin, which stains Nissl substance, revealed disorganized cortical cellular lamination including a delayed appearance of pyramidal cells until 44 wk of age in DS compared to 28 wk in NTD. SG and IG DCX-immunoreactive (-ir) cells were only visualized in the youngest cases until 83 wk in NTD and 57 wk DS. Strong SMI-32 immunoreactivity was observed in layers III and V pyramidal cells in the oldest NTD and DS cases with few appearing as early as 28 wk of age in layer V in NTD. Small Calb-ir interneurons were seen in younger NTD and DS cases compared to Calb-ir pyramidal cells in older subjects. Overall, a greater number of Calb-ir cells were detected in NTD, however, the number of Calr-ir cells were comparable between groups. Diffuse APP/Aß immunoreactivity was found at all ages in both groups. Few young cases from both groups presented non-neuronal granular CP13 immunoreactivity in layer I. Stronger correlations between brain weight, age, thionin, DCX, and SMI-32 counts were found in NTD. These findings suggest that trisomy 21 affects postnatal FC lamination, neuronal migration/neurogenesis and differentiation of projection neurons and interneurons that likely contribute to cognitive impairment in DS.

Arabidopsis thionin-like genes are involved in resistance against the beet-cyst nematode (Heterodera schachtii).

Plants express various antimicrobial peptides including thionins to protect themselves against pathogens. It was recently found that, in addition to four thionin genes, Arabidopsis contains 67 thionin-like (ThiL) genes including six pseudogenes. It is known that thionins have antimicrobial activity and are part of the plant defense system, however, nothing is known about ThiL genes. In this study, we present a bioinformatic analysis of the (ThiL) gene family in Arabidopsis. We identified 15 different motifs which positioned the ThiL peptides in four groups. A comparison of amino acid sequences showed that the ThiL peptides are actually more similar to the acidic domain of thionin proproteins than to the thionin domain. We selected 10 ThiL genes to study the expression and possible function in the Arabidopsis plant. RT-PCR and promoter:GUS fusions showed that most genes were expressed at a very low level but in several organs and at different developmental stages. Some genes were also expressed in syncytia induced by the beet cyst nematode Heterodera schachti in roots while others were downregulated in syncytia. Some overexpression lines supported lower number of nematodes that developed on the roots after inoculation. Two of the genes resulted in a strong hypersensitive response when infiltrated into leaves of Nicotiana benthamiana. These results indicate that ThiL genes might be involved in the response to biotic stress. ThiL genes have been expanded in the Brassicales and specifically the Brassicaceae. The most extreme example is the CRP2460 subfamily that contains 28 very closely related genes from Arabidopsis which are mostly the result of tandem duplications.

Isolation and Characterization of a Thionin Proprotein-processing Enzyme from Barley.

Thionins are plant-specific antimicrobial peptides that have been isolated from the endosperm and leaves of cereals, from the leaves of mistletoes, and from several other plant species. They are generally basic peptides with three or four disulfide bridges and a molecular mass of ~5 kDa. Thionins are produced as preproproteins consisting of a signal peptide, the thionin domain, and an acidic domain. Previously, only mature thionin peptides have been isolated from plants, and in addition to removal of the signal peptide, at least one cleavage processing step between the thionin and the acidic domain is necessary to release the mature thionin. In this work, we identified a thionin proprotein-processing enzyme (TPPE) from barley. Purification of the enzyme was guided by an assay that used a quenched fluorogenic peptide comprising the amino acid sequence between the thionin and the acidic domain of barley leaf-specific thionin. The barley TPPE was identified as a serine protease (BAJ93208) and expressed in Escherichia coli as a strep tag-labeled protein. The barley BTH6 thionin proprotein was produced in E. coli using the vector pETtrx1a and used as a substrate. We isolated and sequenced the BTH6 thionin from barley to confirm the N and C terminus of the peptide in planta. Using an in vitro enzymatic assay, the recombinant TPPE was able to process the quenched fluorogenic peptide and to cleave the acidic domain at least at six sites releasing the mature thionin from the proprotein. Moreover, it was found that the intrinsic three-dimensional structure of the BTH6 thionin domain prevents cleavage of the mature BTH6 thionin by the TPPE.

Jasmonate and ethylene dependent defence gene expression and suppression of fungal virulence factors: two essential mechanisms of Fusarium head blight resistance in wheat?

BACKGROUND: Fusarium head blight (FHB) caused by Fusarium species like F. graminearum is a devastating disease of wheat (Triticum aestivum) worldwide. Mycotoxins such as deoxynivalenol produced by the fungus affect plant and animal health, and cause significant reductions of grain yield and quality. Resistant varieties are the only effective way to control this disease, but the molecular events leading to FHB resistance are still poorly understood. Transcriptional profiling was conducted for the winter wheat cultivars Dream (moderately resistant) and Lynx (susceptible). The gene expressions at 32 and 72 h after inoculation with Fusarium were used to trace possible defence mechanisms and associated genes. A comparative qPCR was carried out for selected genes to analyse the respective expression patterns in the resistant cultivars Dream and Sumai 3 (Chinese spring wheat). RESULTS: Among 2,169 differentially expressed genes, two putative main defence mechanisms were found in the FHB-resistant Dream cultivar. Both are defined base on their specific mode of resistance. A non-specific mechanism was based on several defence genes probably induced by jasmonate and ethylene signalling, including lipid-transfer protein, thionin, defensin and GDSL-like lipase genes. Additionally, defence-related genes encoding jasmonate-regulated proteins were up-regulated in response to FHB. Another mechanism based on the targeted suppression of essential Fusarium virulence factors comprising proteases and mycotoxins was found to be an essential, induced defence of general relevance in wheat. Moreover, similar inductions upon fungal infection were frequently observed among FHB-responsive genes of both mechanisms in the cultivars Dream and Sumai 3. CONCLUSIONS: Especially ABC transporter, UDP-glucosyltransferase, protease and protease inhibitor genes associated with the defence mechanism against fungal virulence factors are apparently active in different resistant genetic backgrounds, according to reports on other wheat cultivars and barley. This was further supported in our qPCR experiments on seven genes originating from this mechanism which revealed similar activities in the resistant cultivars Dream and Sumai 3. Finally, the combination of early-stage and steady-state induction was associated with resistance, while transcript induction generally occurred later and temporarily in the susceptible cultivars. The respective mechanisms are attractive for advanced studies aiming at new resistance and toxin management strategies.

Dopamine cell loss in the periaqueductal gray in multiple system atrophy and Lewy body dementia.

BACKGROUND: Experimental studies indicate that dopaminergic neurons in the ventral periaqueductal gray matter (PAG) are involved in maintenance of wakefulness. Excessive daytime sleepiness (EDS) is a common manifestation of multiple system atrophy (MSA) and dementia with Lewy bodies (DLB) but involvement of these neurons has not yet been explored. METHODS: We sought to determine whether there is loss of dopaminergic neurons in the ventral PAG in MSA and DLB. We studied the midbrain obtained at autopsy from 12 patients (9 male, 3 female, age 61 +/- 3) with neuropathologically confirmed MSA, 12 patients (11 male, 1 female, age 79 +/- 4) with diagnosis of DLB and limbic or neocortical Lewy body disease, and 12 controls (7 male, 5 female, ages 67 +/- 4). Fifty-micron sections were immunostained for tyrosine hydroxylase (TH) or alpha-synuclein and costained with thionin. Cell counts were performed every 400 mum throughout the ventral PAG using stereologic techniques. RESULTS: Compared to the total estimated cell numbers in controls (21,488 +/- 8,324 cells), there was marked loss of TH neurons in the ventral PAG in both MSA (11,727 +/- 5,984; p < 0.01) and DLB (5,163 +/- 1,926; p < 0.001) cases. Cell loss was more marked in DLB than in MSA. There were characteristic alpha-synuclein inclusions in the ventral PAG in both MSA and DLB. CONCLUSIONS: There is loss of putative wake-active ventral periaqueductal gray matter dopaminergic neurons in both multiple system atrophy and dementia with Lewy bodies, which may contribute to excessive daytime sleepiness in these conditions.

Interactions of viscotoxins with vesicles of genuine plant membranes.

Extracts of Viscum album L. produced by a specific homogenization procedure contain viscotoxins (VT) and liposome-like membrane vesicles, formed from cellular membranes. Interactions between these membrane structures and viscotoxins are characterized in this work. Binding properties of viscotoxins with mistletoe extracts or isolated membrane vesicles were analyzed by gel permeation chromatography (GPC) and centrifugation, followed by HPLC/UV for viscotoxin detection. The experiments show that a part of the viscotoxins is bound to membrane vesicles, and that this binding to the membrane structures is reversible. In the case of the vesicles studied from an extract of 100 mg plant material per mL (0.30 mM phospholipids, 244 microg/mL VT), 64 microg/mL VTs are bound to the membranes. The binding properties of the viscotoxin isoforms are different. VTA3 clearly binds more intensively to membrane structures than VTA2 or VTA1. Possible interaction of viscotoxins with DNA, which is also discussed as a mechanism of viscotoxin action, could be shown to be negligible in the framework of these experiments.