Whole-genome sequencing and identification of antimicrobial peptide coding genes in parsley (Petroselinum crispum), an important culinary and medicinal Apiaceae species.

Parsley is a commonly cultivated Apiaceae species of culinary and medicinal importance. Parsley has several recognized health benefits and the species has been utilized in traditional medicine since ancient times. Although parsley is among the most commonly cultivated members of Apiaceae, no systematic genomic research has been conducted on parsley. In the present work, parsley genome was sequenced using the long-read HiFi (high fidelity) sequencing technology and a draft contig assembly of 1.57 Gb that represents 80.9% of the estimated genome size was produced. The assembly was highly repeat-rich with a repetitive DNA content of 81%. The assembly was phased into a primary and alternate assembly in order to minimize redundant contigs. Scaffolds were constructed with the primary assembly contigs, which were used for the identification of AMP (antimicrobial peptide) genes. Characteristic AMP domains and 3D structures were used to detect and verify antimicrobial peptides. As a result, 23 genes (PcAMP1-23) representing defensin, snakin, thionin, lipid transfer protein and vicilin-like AMP classes were identified. Bioinformatic analyses for the characterization of peptide physicochemical properties indicated that parsley AMPs are extracellular peptides, therefore, plausibly exert their antimicrobial effects through the most commonly described AMP action mechanism of membrane attack. AMPs are attracting increasing attention since they display their fast antimicrobial effects in small doses on both plant and animal pathogens with a significantly reduced risk of resistance development. Therefore, identification and characterization of AMPs is important for their incorporation into plant disease management protocols as well as medicinal research for the treatment of multi-drug resistant infections.

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.

The Antimicrobial Peptide γ-Thionin from Habanero Chile (Capsicum chinense) Induces Caspase-Independent Apoptosis on Human K562 Chronic Myeloid Leukemia Cells and Regulates Epigenetic Marks.

Cancer is a relevant health problem worldwide. In 2020, leukemias represented the 13th most commonly reported cancer cases worldwide but the 10th most likely to cause deaths. There has been a progressive increase in the efficacy of treatments for leukemias; however, these still generate important side effects, so it is imperative to search for new alternatives. Defensins are a group of antimicrobial peptides with activity against cancer cells. However, the cytotoxic mechanism of these peptides has been described mainly for animal defensins. This study shows that defensin γ-thionin (Capsicum chinense) is cytotoxic to the K562 leukemia cells with an IC50 = 290 µg/mL (50.26 µM) but not for human peripheral blood mononuclear cells. Results showed that γ-thionin did not affect the membrane potential; however, the peptide modified the mitochondrial membrane potential (ΔΨm) and the intracellular calcium release. In addition, γ-thionin induced apoptosis in K562 cells, but the activation of caspases 8 and 9 was not detected. Moreover, the activation of calpains was detected at one hour of treatment, suggesting that γ-thionin activates the caspase-independent apoptosis. Furthermore, the γ-thionin induced epigenetic modifications on histone 3 in K562 cells, increased global acetylation (~2-fold), and specific acetylation marks at lysine 9 (H3K9Ac) (~1.5-fold). In addition, γ-thionin increased the lysine 9 methylation (H3K9me) and dimethylation marks (H3K9me2) (~2-fold), as well as the trimethylation mark (H3K9me3) (~2-fold). To our knowledge, this is the first report of a defensin that triggers caspase-independent apoptosis in cancer cells via calpains and regulating chromatin remodelation, a novel property for a plant defensin.

Production of transgenic Allium cepa by nanoparticles to resist Aspergillus niger infection.

BACKGROUND: Transgenic plants are becoming a more powerful tool in modern biotechnology. Genetic engineering was used in biotech-derived products to create genetically modified (GM) plants resistant to diseases. The onion (Allium cepa, L.) is a common, important perennial vegetable crop grown in Egypt for food and economic value. Onions are susceptible to a variety of fungal infections and diseases. Aspergillus niger is a common onion phytopathogen that causes diseases such as black mould (or black rot), which is a major issue, particularly when exporting onions. A. niger grows between the bulb's outer (dead, flaky) skin and the first fleshy scales, which become water-soaked. Thionin genes produce thionin proteins, which have antimicrobial properties against a variety of phytopathogens, including A. niger. Chitosan nanoparticles act as a carrier for the thionin gene, which allows A. cepa to resist infection by A. niger. METHODS AND RESULTS: Thionin gene (Thio-60) was transformed into A. cepa to be resistance to fungal infection. The gene was loaded on chitosan nanoparticles to be transformed into plants. Transgenic A. cepa had a 27% weight inhibition compared to non-transgenic one, which had a 69% inhibition. The expressed thionin protein has a 52% inhibitory effect on A. niger spore germination. All these findings supported thionin protein's antifungal activity as an antimicrobial peptide. Furthermore, the data presented here demonstrated the efficacy of chitosan nanoparticles in gene transformation. CONCLUSION: The present study describes the benefits of producing transgenic onion resistance to black rot diseases via expression of thionin proteins.

Spatial structure and oligomerization of viscotoxin A3 in detergent micelles: Implication for mechanisms of ion channel formation and membrane lysis.

Thionins are the family of small (∼5 kDa) cationic cysteine-rich peptides involved in the immune response in plants. Viscotoxin A3 (VtA3) is the thionin from mistletoe (Viscum album) demonstrating antimicrobial and cytotoxic activity against cancer cells in vitro. VtA3 (charge +6) interacts with the membranes containing anionic lipids and forms cation-selective ion channels. Here we studied the VtA3 structure in membrane-mimicking media by NMR spectroscopy. Spatial structure of VtA3, consisting of a helical hairpin and a short ß-sheet, was stable and did not undergo significant changes during micelle binding. VtA3 molecule bound with high affinity to the surface of zwitterionic dodecylphosphocholine (DPC) micelle by hydrophobic patch in the helical hairpin. Oligomerization of VtA3 was observed in the anionic micelles of sodium dodecylsulphate (SDS). No direct contacts between the peptide molecules were observed and the possible interfaces of detergent-assisted oligomerization were revealed. The data obtained suggest that the VtA3 membrane activity, depending on the concentration, obeys the 'toroidal' pore model or the 'carpet' mechanism. The model of the membrane disrupting complex, which explains the ion channel formation in the partially anionic membranes, was proposed.

Comparative physiological and transcriptomic analyses illuminate common mechanisms by which silicon alleviates cadmium and arsenic toxicity in rice seedlings.

The inessential heavy metal/loids cadmium (Cd) and arsenic (As), which often co-occur in polluted paddy soils, are toxic to rice. Silicon (Si) treatment is known to reduce Cd and As toxicity in rice plants. To better understand the shared mechanisms by which Si alleviates Cd and As stress, rice seedlings were hydroponically exposed to Cd or As, then treated with Si. The addition of Si significantly ameliorated the inhibitory effects of Cd and As on rice seedling growth. Si supplementation decreased Cd and As translocation from roots to shoots, and significantly reduced Cd- and As-induced reactive oxygen species generation in rice seedlings. Transcriptomics analyses were conducted to elucidate molecular mechanisms underlying the Si-mediated response to Cd or As stress in rice. The expression patterns of the differentially expressed genes in Cd- or As-stressed rice roots with and without Si application were compared. The transcriptomes of the Cd- and As-stressed rice roots were similarly and profoundly reshaped by Si application, suggesting that Si may play a fundamental, active role in plant defense against heavy metal/loid stresses by modulating whole genome expression. We also identified two novel genes, Os01g0524500 and Os06g0514800, encoding a myeloblastosis (MYB) transcription factor and a thionin, respectively, which may be candidate targets for Si to alleviate Cd and As stress in rice, as well as for the generation of Cd- and/or As-resistant plants. This study provides valuable resources for further clarification of the shared molecular mechanisms underlying the Si-mediated alleviation of Cd and As toxicity in rice.

In vitro and in silico studies on membrane interactions of diverse Capsicum annuum flower γ-thionin peptides.

Thionins are small, cysteine-rich peptides that play an important role in plant defense, primarily through their interactions with membranes. Eight novel γ-thionin peptides (CanThio1-8) were isolated from the flower of Capsicum annuum. Sequence analysis revealed that the peptides cluster into three groups. A representative peptide from each group (CanThio1, 2, and 3) was used for experimental characterization. Interestingly, peptides were found to possess some cytotoxic activity against normal human embryonic kidney cell line but higher cytotoxicity against cancer cell line MCF-7. CanThio3 peptide was chosen as a representative peptide to study the molecular mechanism of action on membranes. Microsecond timescale atomistic simulations of CanThio3 were performed in the presence of a POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) lipid bilayer. Simulations revealed that CanThio3 interacts with the bilayer and causes lipid thinning in the vicinity. Nonpolar amino acids specific to the α-core region of CanThio3 along with nonpolar residues in the γ-core region are seen to interact with the lipid tails. The differences in the amino acid sequence of CanThio peptides in these regions explain the variability in cytotoxic activities. In summary, our results demonstrate the membrane-mediated activity of a novel series of γ-thionin peptides from C. annuum.

Enhanced Resistance to Fusarium graminearum in Transgenic Arabidopsis Plants Expressing a Modified Plant Thionin.

The fungal pathogen Fusarium graminearum causes Fusarium head blight (FHB) on wheat, barley, and other grains. FHB results in yield reductions and contaminates grain with trichothecene mycotoxins, which threaten food safety and food security. Innovative mechanisms for controlling FHB are urgently needed. We have previously shown that transgenic tobacco and citrus plants expressing a modified thionin (Mthionin) exhibited enhanced resistance toward several bacterial pathogens. The aim of this study was to investigate whether overexpression of Mthionin could be similarly efficacious against F. graminearum, and whether transgenic expression of Mthionin impacts the plant microbiome. Transgenic Arabidopsis plants expressing Mthionin were generated and confirmed. When challenged with F. graminearum, Mthionin-expressing plants showed less disease and fungal biomass in both leaves and inflorescences compared with control plants. When infiltrated into leaves, macroconidia of F. graminearum germinated at lower rates and produced less hyphal growth in Arabidopsis leaves expressing Mthionin. Moreover, marker genes related to defense signaling pathways were expressed at significantly higher levels after F. graminearum infection in Mthionin transgenic Arabidopsis plants. However, Mthionin expression did not appreciably alter the overall microbiome associated with transgenic plants grown under controlled conditions; across leaves and roots of Mthionin-expressing and control transgenic plants, only a few bacterial and fungal taxa differed, and differences between Mthionin transformants were of similar magnitude compared with control plants. In sum, our data indicate that Mthionin is a promising candidate to produce transgenic crops for reducing FHB severity and ultimately mycotoxin contamination.

Label-free electrochemical aptasensor for detection of alpha-fetoprotein based on AFP-aptamer and thionin/reduced graphene oxide/gold nanoparticles.

Sensitive and accurate detection of tumor markers is critical to early diagnosis, point-of-care and portable medical supervision. Alpha fetoprotein (AFP) is an important clinical tumor marker for hepatocellular carcinoma (HCC), and the concentration of AFP in human serum is related to the stage of HCC. In this paper, a label-free electrochemical aptasensor for AFP detection was fabricated using AFP-aptamer as the recognition molecule and thionin/reduced graphene oxide/gold nanoparticles (TH/RGO/Au NPs) as the sensor platform. With high electrocatalytic property and large specific surface area, RGO and Au NPs were employed on the screen-printed carbon electrode to load TH molecules. The TH not only acted as a bridging molecule to effectively capture and immobilize AFP-aptamer, but as the electron transfer mediator to provide the electrochemical signal. The AFP detection was based on the monitoring of the electrochemical current response change of TH by the differential pulse voltammetry. Under optimal conditions, the electrochemical responses were proportional to the AFP concentration in the range of 0.1-100.0 µg/mL. The limit of detection was 0.050 µg/mL at a signal-to-noise ratio of 3. The proposed method may provide a promising application of aptamer with the properties of facile procedure, low cost, high selectivity in clinic.

Antifungal activity of synthetic cowpea defensin Cp-thionin II and its application in dough.

Plant defensins are small, cysteine-rich antimicrobial peptides of the immune system found in several organs during plant development. A synthetic peptide, KT43C, a linear analogue of the native Cp-thionin II found in cowpea seeds, was evaluated for its antifungal potential. It was found that KT43C displayed antifungal activity against Fusarium culmorum, Penicillium expansum and Aspergillus niger. Like native plant defensins, KT43C showed thermostability up to 100 °C and cation sensitivity. The synthetic peptide decreased the fungal growth without inducing morphogenic changes in the fungal hyphae. Non-inhibitory concentrations of the peptide induced permeabilization of the fungal membrane. In addition, high concentrations of KT43C induced the production of reactive oxygen species in the granulated cytoplasm. To investigate potential applications, the peptide was used as an additive in the preparation of dough which did not contain yeast. This peptide delayed the development of fungal growth in the dough by 2 days. Furthermore, KT43C did not induce red blood cell lysis up to a concentration of 200 µg.ml-1. These results highlight the potential for the use of synthetic antimicrobial defensins for shelf-life extension of food products.

The Defensins Consist of Two Independent, Convergent Protein Superfamilies.

The defensin and defensin-like proteins are an extensive group of small, cationic, disulfide-rich proteins found in animals, plants, and fungi and mostly perform roles in host defense. The term defensin was originally used for small mammalian proteins found in neutrophils and was subsequently applied to insect proteins and plant γ-thionins based on their perceived sequence and structural similarity. Defensins are often described as ancient innate immunity molecules and classified as a single superfamily and both sequence alignments and phylogenies have been constructed. Here, we present evidence that the defensins have not all evolved from a single ancestor. Instead, they consist of two analogous superfamilies, and extensive convergent evolution is the source of their similarities. Evidence of common origin necessarily gets weaker for distantly related genes, as is the case for defensins, which are both divergent and small. We show that similarities that have been used as evidence for common origin are all expected by chance in short, constrained, disulfide-rich proteins. Differences in tertiary structure, secondary structure order, and disulfide bond connectivity indicate convergence as the likely source of the similarity. We refer to the two evolutionarily independent groups as the cis-defensins and trans-defensins based on the orientation of the most conserved pair of disulfides.

Quantification of large scale DNA organization for predicting prostate cancer recurrence.

This study investigates whether Genomic Organization at Large Scales (which we propose to call GOALS) as quantified via nuclear phenotype characteristics and cell sociology features (describing cell organization within tissue) collected from prostate tissue microarrays (TMAs) can separate biochemical failure from biochemical nonevidence of disease (BNED) after radical prostatectomy (RP). Of the 78 prostate cancer tissue cores collected from patients treated with RP, 16 who developed biochemical relapse (failure group) and 16 who were BNED patients (nonfailure group) were included in the analyses (36 cores from 32 patients). A section from this TMA was stained stoichiometrically for DNA using the Feulgen-Thionin methodology, and scanned with a Pannoramic MIDI scanner. Approximately 110 nuclear phenotypic features, predominately quantifying large scale DNA organization (GOALS), were extracted from each segmented nuclei. In addition, the centers of these segmented nuclei defined a Voronoi tessellation and subsequent architectural analysis. Prostate TMA core classification as biochemical failure or BNED after RP using GOALS features was conducted (a) based on cell type and cell position within the epithelium (all cells, all epithelial cells, epithelial >2 cell layers away from basement membrane) from all cores, and (b) based on epithelial cells more than two cell layers from the basement membrane using a Classifier trained on Gleason 6, 8, 9 (16 cores) only and applied to a Test set consisting of the Gleason 7 cores (20 cores). Successful core classification as biochemical failure or BNED after RP by a linear classifier was 75% using all cells, 83% using all epithelial cells, and 86% using epithelial >2 layers. Overall success of predicted classification by the linear Classifier of (b) was 87.5% using the Training Set and 80% using the Test Set. Overall success of predicted progression using Gleason score alone was 75% for Gleason >7 as failures and 69% for Gleason >6 as failures. © 2017 International Society for Advancement of Cytometry.

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.

Self-processing of a barley subtilase expressed in E. coli.

The barley protease BAJ93208 belongs to the subtilase family of serine proteases. We have expressed BAJ93208 in the cytoplasm of the Escherichiacoli strain SHuffle C3030 using a rhamnose-inducible promoter. The expression construct included a (His)6-tag at the N-terminus and a strep-tag at the C-terminus. Western blot analysis revealed that the protein was processed at the N- and C-terminus. To exclude that this processing was due to contaminating E. coli proteases, a mutated BAJ93208 protease was constructed. This inactive mutant was not processed, demonstrating that the processing was an autocatalytic process. To define the exact cleavage sites mass spectrometry was used which detected four differently processed versions of the protease. At the N-terminus, the self-processing removed the internal inhibitor and an additional 19 amino acids. At the C-terminus there was a cleavage site after Ala(765) which also removed the strep-tag. This explained the inability to detect the purified (His)6-BAJ93208-strep protease with an anti-strep-tag antibody. Finally, an additional alanine was removed either at the N-terminus (Ala(119)) or at the C-terminus (Ala(764)).

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.

Enhanced Synthesis of Foreign Nuclear Protein Stimulates Viral Reproduction via the Induction of γ-Thionin Expression.

Plants are a promising platform for recombinant protein production. Here we propose a novel approach to increase the level of viral vector-mediated recombinant protein synthesis. This approach is based on the hypothesis that antiviral protection is weakened during the antibacterial cellular response. We suggested that introduced to the cell foreign nuclear localized proteins, including effectors such as bacterial nucleomodulins, can interfere with the import of cellular nuclear proteins and launch antibacterial defense reactions, creating favorable conditions for cytoplasmic virus reproduction. Here, we performed synthesis of an artificial nuclear protein-red fluorescent protein (mRFP) fused with a nuclear localization sequence (NLS)-in plant cells as a mimetic of a bacterial effector. Superproduction of mRFP:NLS induced Nicotiana benthamiana γ-thionin (NbγThio) mRNA accumulation. Both NLS-containing protein synthesis and increased NbγThio expression stimulated reproduction of the viral vector based on the genome of crucifer-infecting tobacco mosaic virus (crTMV) in N. benthamiana leaves. We isolated the NbγThio gene promoter (PrγThio) and showed that PrγThio activity sharply increased in response to massive synthesis of GFP fused with NLS. We conclude that NLS-induced PrγThio activation and increased accumulation of Nbγthio mRNA led to the stimulation of GFP expression from crTMV: GFP vector in the transient expression system.

Production of transgenic Paulownia tomentosa (Thunb.) steud. using chitosan nanoparticles to express antimicrobial genes resistant to bacterial infection.

Paulownia tomentosa (Thunb.) Steud. is a very important hard woody plant, an extremely fast-growing tree and produce timber. Therefore, there is a demand to produce transgenic Paulownia plant resistant to bacterial infection. Microbial infection (especially bacterial one) is serious sever and cause a loss in plant productivity as they bear upon the character and amount of plant product. Two phytopathogenic bacteria were chosen to consider their effect on Paulownia tomentosa. These two bacterial species were Erwinia carotovora and Pseudomonas aeruginosa. Two thionin genes (AT1G12660 and AT1G12663) were selected. They produce antimicrobial peptides to resist this bacterial infection. Chitosan nanoparticle is a novel technology in genetic transformation into plant tissues. Chitosan nanoparticles were used in a ratio of 1:1 with the plasmid DNA carrying thionin genes independently. Characterization for chitosan nanoparticles was applied to determine the conditions of genetic transformation. The new transgenic P. tomentosa lines produced are partially resistant to these two bacterial infections compared to non-transgenic lines. The inhibitory percentage in the transgenic lines ranged from 8 to 21% wherein the non-transgenic the inhibitory percentage of P. tomentosa leaves ranged from 53-24%. Likewise, it is noticed that is Paulownia tomentosa less infectious than Erwinia carotovora. In conclusion, I recommend using chitosan nanoparticle is an excellent way for gene transformation into plant tissues. Also, manipulate the idea of using thionin as antimicrobial genes to resist bacterial infection for different plant species.

The Versatile Roles of Sulfur-Containing Biomolecules in Plant Defense-A Road to Disease Resistance.

Sulfur (S) is an essential plant macronutrient and the pivotal role of sulfur compounds in plant disease resistance has become obvious in recent decades. This review attempts to recapitulate results on the various functions of sulfur-containing defense compounds (SDCs) in plant defense responses to pathogens. These compounds include sulfur containing amino acids such as cysteine and methionine, the tripeptide glutathione, thionins and defensins, glucosinolates and phytoalexins and, last but not least, reactive sulfur species and hydrogen sulfide. SDCs play versatile roles both in pathogen perception and initiating signal transduction pathways that are interconnected with various defense processes regulated by plant hormones (salicylic acid, jasmonic acid and ethylene) and reactive oxygen species (ROS). Importantly, ROS-mediated reversible oxidation of cysteine residues on plant proteins have profound effects on protein functions like signal transduction of plant defense responses during pathogen infections. Indeed, the multifaceted plant defense responses initiated by SDCs should provide novel tools for plant breeding to endow crops with efficient defense responses to invading pathogens.

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.

Thin-layer MoS2 and thionin composite-based electrochemical sensing platform for rapid and sensitive detection of zearalenone in human biofluids.

In this study, a facile electrochemical sensing platform based on thin-layer MoS2 and thionin (MoS2-Thi) composites was constructed for the sensitive and rapid detection of zearalenone (ZEA) in human biofluids. MoS2-Thi composites with a thin-layer MoS2 structure and large specific surface area were synthesized via a simple ultrasound exfoliation method. To further enhance the performance of the platform but without increasing the complexity of the fabrication process, ZEA antibodies were modified with Pt nanoparticles (Pt NPs) using an environmentally friendly method, where they had a much wider linear range and four times higher sensitivity compared with the original ZEA monoclonal antibody (ZEA-MAb). Under the optimal conditions, the electrochemical responses of the MoS2-Thi composite-based immunosensor were linear (R2 = 0.9915) when the ZEA concentrations ranged from 0.01 to 50 ng mL-1. The proposed immunosensor could detect ZEA at concentrations as low as 0.005 ng mL-1 with excellent selectivity. The immunosensor exhibited acceptable stability with high reproducibility and accuracy, and it was applied successfully for determining ZEA in human plasma and urine samples. Our findings indicated that the proposed MoS2-Thi composite-based electrochemical sensing platform provides a new approach for the rapid and sensitive bioanalysis of ZEA, and it may also be extended to other mycotoxins for multiplex detection.