Defensin Gene Research Articles

Effects of Dietary Lactic Acid Supplementation on the Activity of Digestive and Antioxidant Enzymes, Gene Expressions, and Bacterial Communities in the Intestine of Common Carp, Cyprinus carpio

The present study investigated the effects of dietary lactic acid (LA) supplementation on the growth performance, intestinal digestive/antioxidant enzymes’ activities, gene expression, and bacterial communities in common carp, Cyprinus carpio. Four diets were formulated to contain 0 g/kg LA (control), at 2.5 g/kg LA (2.5LAC), 5 g/kg LA (5LAC), and 10 g/kg LA (10LAC) and offered to the fish over a period of 56 days. The results showed that dietary 5 g/kg LA supplementation improved growth performance and feed efficiency in the fish. All LA treatments exhibited significant elevations in the intestinal trypsin and chymotrypsin activities, whereas the intestinal lipase, amylase, and alkaline phosphatase activities exhibited significant elevations in the 5LAC and 10LAC treatments. All LA treatments exhibited significant elevations in the intestinal heat shock protein 70, tumor necrosis factor-alpha, interleukin-1 beta, and defensin gene expressions, and the highest expression was observed in the 5LAC treatment. Additionally, dietary LA treatment significantly increased the lysozyme expression and Lactobacillus sp. population in the intestine of the fish, and the highest values were observed in the 5LAC and 10LAC treatments. Aeromonas sp. and Vibrio sp. populations decreased in the LA treatments, and the lowest Aeromonas sp. population was observed in the 10LAC treatment. The intestinal mucin2 and mucin5 expressions, and the hepatic reduced glutathione content, significantly increased, whereas hepatic glutathione peroxidase, glutathione reductase, and malondialdehyde significantly decreased in the 5LAC and 10LAC treatments. In conclusion, dietary 5 g/kg LA is recommended for common carp feeding to improve growth rate, antioxidant capacity, and intestinal health.

Effects of the pyrE deletion mutant from Bacillus thuringiensis on gut microbiota and immune response of Spodoptera exigua.

The gut microbiota is essential for the growth and development of insects, and the intestinal immune system plays a critical role in regulating the homeostasis of intestinal microorganisms and their interactions with pathogenic bacteria. Infection with Bacillus thuringiensis (Bt) can disrupt the gut microbiota of insects, but the regulatory factors governing the interaction between Bt and gut bacteria are not well understood. Uracil secreted by exogenous pathogenic bacteria can activate DUOX-mediated reactive oxygen species (ROS) production, which helps maintain intestinal microbial homeostasis and immune balance. To elucidate the regulatory genes involved in the interaction between Bt and gut microbiota, we investigate the effects of uracil derived from Bt on gut microbiota, and host immunity using a uracil deficient Bt strain (Bt GS57△pyrE) obtained by homologous recombination. We analyze the biological characteristics of the uracil deficient strain and found that the deletion of uracil in Bt GS57 strain changed the diversity of gut bacteria in Spodoptera exigua, as investigated using Illumina HiSeq sequencing. Furthermore, qRT-PCR analysis showed that compared with Bt GS57 (control), the expression of the SeDuox gene and the level of ROS were significantly decreased after feeding with Bt GS57△pyrE. Adding uracil to Bt GS57△pyrE restored the expression level of DUOX and ROS to a higher level. Additionally, we observed that PGRP-SA, attacin, defensin and ceropin genes were significant different in the midgut of S. exigua infected by Bt GS57 and Bt GS57△pyrE, with a trend of increasing first and then decreasing. These results suggest that uracil regulates and activates the DUOX-ROS system, affects the expression of antimicrobial peptide genes, and disturb intestinal microbial homeostasis. We preliminarily speculate that uracil is a key factor in the interaction between Bt and gut microbiota, and these findings provide a theoretical basis for clarifying the interaction between Bt, host, and intestinal microorganisms, as well as for gaining new insights into the insecticidal mechanism of B. thuringiensis in insects.

Endometrial gene expression of DEFB4B, LYZ, and SLPI in biopsy samples of mares resistant or susceptible to endometritis

Endometritis is one of the main causes of subfertility in mares and therefore one of the most common reasons for economic losses in the horse breeding industry. Multiparous older mares are more susceptible to the development of persistent endometritis after mating, artificial insemination, or foaling since aging is associated with an altered systemic immune response and anatomical defects that interfere with physiological uterine clearance (Katila et.al. Animals. 2022; 12:779). In this context, their inability to recover from endometritis can lead to degenerative changes in the endometrium (e.g., glandular degenerative changes and associated peri-glandular and/or diffuse stromal fibrosis) (Canisso et.al. International Journal of Molecular Sciences 2020; 21:1432). This study aimed to evaluate possible variations in endometrial gene expression of the genes Defensin Beta 4B (DEFB4B), Lysozyme (LYZ), and Secretory Leukocyte Peptidase Inhibitor (SLPI) between resistant and susceptible mares. These three genes have already been identified as involved in the equine innate immune response and in the chronic uterine inflammatory response (Marth et.al. Reproduction, Fertility, Development 2018; 30:533-545). Endometrial biopsy samples were collected from 19 mares. Mares were divided into two groups concerning their age and reproductive history: the "resistant group" (RES) (N=4) consisted of maiden mares 3.00±0.00 years old with endometrial biopsy grade I according to Kenney and Doig classification (1986); the "susceptible group" (SUS) (N=15)was composed of multiparous mares 17.33±3.28 years old, with endometrial biopsy of grade IIA, IIB or III. Total RNA was extracted from each sample and the corresponding cDNAs were prepared by retro-transcription using dedicated commercial kits. The samples were then amplified by qRT-PCR procedure as described by Marth and colleagues (Marth et.al. Reproduction, Fertility, Development 2018; 30:533-545). The housekeeping gene GADPH was amplified in each sample as internal control and for data normalization. To minimize possible experimental errors, for each sample three technical replicates were performed and the quality of the extracted RNA was evaluated by spectrophotometry and gel electrophoresis. The results showed a statistically significant difference in the expression of the genes LYZ (p=0.0003) and DEFB4B (p=0.0273) which were upregulated in SUS versus RES mares. This suggests that lower gene expression found in young mares are attributable to a more balanced local immunomodulation, with consequent reduction of the inflammatory pathways and slower progression towards the deposition of fibrotic tissue compared to older mares. No differences in the expression of the SLPI gene were found between the two groups (p=0.8854). In conclusion, despite these promising results, larger studies are needed before LYZ and DEFB4B genes could be used as biomarkers to identify the mares susceptible to endometritis.

RNAi-mediated gene silencing of Phlebotomus papatasi defensins favors Leishmania major infection.

Introduction: Production of different antimicrobial peptides (AMPs) is one of the insect's prominent defense strategies, regulated mainly by Toll and immune deficiency (IMD) humoral pathways. Here we focused mainly on two AMPs of Phlebotomus papatasi, vector of Leishmania major parasites, their association with the relish transcription factor and the effective participation on Leishmania infection. Methods and results: We further characterized the role of previously described gut-specific P. papatasi defensin (PpDef1) and identified the second defensin (PpDef2) expressed in various sand fly tissues. Using the RNAi-mediated gene silencing, we report that the silencing of PpDef1 gene or simultaneous silencing of both defensin genes (PpDef1 and PpDef2) resulted in increased parasite levels in the sand fly (detectable by PCR) and higher sand fly mortality. In addition, we knocked down relish, the sole transcription factor of the IMD pathway, to evaluate the association of the IMD pathway with AMPs expression in P. papatasi. We demonstrated that the relish gene knockdown reduced the expression of PpDef2 and attacin, another AMP abundantly expressed in the sand fly body. Conclusions: Altogether, our experiments show the importance of defensins in the sand fly response toward L. major and the role of the IMD pathway in regulating AMPs in P. papatasi.

Expression of Mn-sod, PAL1, aos1 and HPL genes in soybean plants overexpressing the NmDef02 defensin.

Plant defensins are a potential tool in crop improvement programs through biotechnology. Their antifungal action makes them attractive molecules for the production of transgenic plants. Information is currently lacking on what happens to the expression of defense genes in transgenic plants that overexpress a defensin. Here we show the relative expression of four defense-related genes: Mn-sod, PAL1, aos1 and HPL evaluated in two transgenic soybean events (Def1 and Def17) constitutively expressing the NmDef02 defensin gene from Nicotiana megalosiphon. The expression of these defense genes showed a differential profile in the transgenic events, with the increased expression of the aos1 gene and the repression of the Mn-sod gene in both events, when compared to the non-transgenic control. Furthermore, the expression of the PAL1 gene only increased in the Def17 event. The results indicate that although there were some changes in the expression of defense genes in transgenic plants overexpressing the defensin NmDef02; the morphoagronomic parameters evaluated were similar to the non-transgenic control. Understanding the molecular changes that occur in these transgenic plants could be of interest in the short, medium and long term.

Changes in the expression of antimicrobial peptide genes in poultry under the influence of glyphosate and probiotic

Relevance. There is increasing information that glyphosates can have a range of adverse effects on animal, bird and human health, raising serious concerns about global feed and animal and poultry product safety. Methods. The experiments were carried out in the vivarium of OOO “BIOTROF+” on broilers of the Ross 308 cross. The birds were divided into 3 groups: 1st (control), which received a diet without additives, 2nd (experimental), which received a diet with the addition of glyphosate, 3rd (experimental), which received a diet with the addition of glyphosate and a strain of the microorganism Bacillus sp. GL-8. Analysis of the gene expression of the caecum of the intestines of broilers was carried out using quantitative PCR with reverse transcription. To analyze mRNA expression, specific primers for antimicrobial peptide genes were selected. Amplification reactions were performed using “SsoAdvanced™ Universal SYBR® Green Supermix” (“Bio-Rad”). Results showed that the residual amounts of glyphosates which are present at sterns of broilers influence an expression of genes of antimicrobic peptides AvBD1, AvBD2, AvBD4, AvBD6, CATH-2, NK-lysin, strengthening it. For example, in experimental group 2, there was an increase in gene expression of defensins AvBD1, AvBD2, AvBD4, AvBD6 by 21.9, 29.9, 35.1 and 33.5 times, respectively, compared to control group 1 (Р ≤ 0,001). A decrease (31 to 41%) in LEAP-2 gene expression when feed is contaminated with glyphosates may likely lead to a decrease in resistance to bacterial pathogens such as Salmonella enterica typhimurium, Streptococcus spp. and increased severity by the symptom of coccidiosis in poulty. The probiotic had a «leveling» effect on the expression of AvBD1, AvBD2, AvBD4 and AvBD6 defensin genes. This is likely due to the enhancement in the probiotic-modified gut microbiota of glyphosate metabolic opportunities, which may have acted as a physical barrier.

Integration of Transcriptome and Metabolome Analyses Reveals the Mechanistic Basis for Cadmium Accumulation in Maize.

Cadmium (Cd) pollution in soil has become a major environmental issue worldwide. However, the underlying molecular mechanism of low grain-Cd accumulation (GCA) in maize is still largely unknown. Herein, we report the mechanistic basis for low GCA in maize by a multiomics approach. The low GCA genotype L63 showed normal vacuolar formation and a lower capacity of xylem loading of Cd than the high-accumulator L42 under Cd stress. Transcriptomic sequencing identified 84 low-GCA-associated genes which are mainly involved in the S-adenosylmethionine (SAM) cycle, metal transport, and vacuolar sequestration. A metabolome analysis revealed that L63 plants had a more active SAM cycle and a greater capacity for terpenoid synthesis and phenylalanine metabolism than L42. Combining the analysis of transcriptome and metabolome characterized several genes as key genes involved in the determination of Cd accumulation. Our study identifies a mechanistic basis for low Cd accumulation in maize grains and provides candidate genes for genetic improvement of crops.

Genetic Characterization of Some Saudi Arabia's Accessions from Commiphora gileadensis Using Physio-Biochemical Parameters, Molecular Markers, DNA Barcoding Analysis and Relative Gene Expression.

Commiphora gileadensis L. is a medicinal plant, known as balsam, with pharmaceutical potential for its phytochemical activities and chemical constituents. Genetic diversity is a genetic tool used in medicinal plant evolution and conservation. Three accessions from C. gileadensis were collected from three localities in Saudi Arabia (Jeddah, Jizan and Riyadh). Genetic characterization was carried out using physio-biochemical parameters, molecular markers (inter-simple sequence repeat (ISSR) and start codon targeted (SCoT)), DNA barcoding (18 S rRNA and ITS rDNA regions), relative gene expressions (phenylalanine ammonia-lyase 1 (PAL1), defensin (PR-12)) and pathogenesis-related protein (AFPRT). The results of this study showed that C. gileadensis accession C3, collected from Riyadh, had the highest content from the physio-biochemical parameters perspective, with values of 92.54 mg/g and 77.13 mg/g for total phenolic content (TPC) and total flavonoid content (TFC), respectively. Furthermore, the highest content of antioxidant enzyme activity was present in accession C3 with values of 16.87, 60.87, 35.76 and 27.98 U mg-1 for superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) (mol/min/mg FW) and ascorbate peroxidase (APX) (U mg-1 protein), respectively. The highest total number of bands and number of unique bands were 138 and 59, respectively, for the SCoT marker. The SCoT marker was the most efficient for the genetic diversity of C. gileadensis by producing the highest polymorphism (75.63%). DNA barcoding using 18 S and ITS showed the nearby Commiphora genus and clustered C. gileadensis accessions from Jeddah and Jizan in one clade and the C. gileadensis accession from Ryiadh in a separate cluster. Moreover, relative gene expression of the PAL1, defensin (PR-12) and AFPRT (PR1) genes was upregulated in the C. gileadensis accession from Ryiadh. In conclusion, ecological and environmental conditions in each locality affect the genomic expression and genetic diversity, which can help the evolution of important medicinal plants and improve breeding and conservation systems.

Expression of radish defensin (RsAFP2) gene in chickpea (Cicer arietinum L.) confers resistance to Fusarium wilt disease.

Chickpea (Cicer arietinum L.), a major nutritional source cultivated worldwide, is vulnerable to several abiotic and biotic stresses, including different types of soil-borne pathogens like Fusarium oxysporum f. sp. ciceri, which causes root rot disease and severely affects productivity. In this study, putative transgenic plants were obtained with the Radish defensin (Rs-AFP2) gene through Agrobacterium tumefaciens mediated transformation using the embryo axis explants. Transgenes were confirmed in 18 putative transgenic plants with PCR-specific primers for nptII and Rs-AFP2 genes. Twelve transgenic plants were established successfully under greenhouse conditions. The T0 plants were allowed for self-pollination to obtain T1 seeds. The T1 plants, selected for Fusarium wilt assay using Fusarium oxysporum f. sp. Cicero, showed different resistance levels, from moderate to high levels in comparison to control plants (wild-type) which exhibited severe wilt symptoms. Our results suggest the application of Radish defensins (RsAFP1/RsAFP2 genes) for improving pathogen resistance in chickpea.

Agrobacterium-mediated transformation and in vitro evaluation of dehydration tolerance in transgenic tobacco harboring Ca-AFP gene

We have known that plant defensins have the main roles in biotic stress, especially antifungal activities. In addition, their functions on abiotic stress tolerance have been demonstrated on previous studies, but that information was still limited. Ca-AFP, a plant defensin gene derived from chickpea, was transformed into tobacco leaf disks by Agrobacterium method. After 16 weeks on selection media containing 15 mg/L hygromycin, some regeneration shoot lines were obtained. Four of them showed the positive bands on hpt gene and Ca-AFP gene by PCR screening. These lines were applied on in vitro evaluation of dehydration tolerance using rotary liquid culture, and the water-deficit stress was caused by 2% (w/v) PEG 6000. These four transgenic lines had better growth of adventitious roots (number of adventitious roots, total adventitious root length, and average of root length) and higher rate of biomass increase than the non-transgenic line. The result illustrated that the expression of Ca-AFP gene could mitigate the negative effect of water-deficit stress on transgenic tobacco lines under the in vitro stress condition. These transgenic tobacco lines are potential for water-deficit stress trials on greenhouse in the future.

Assessment of antibacterial genes of emerging queen Apis mellifera L. induced by the bacterium Paenibacillus larvae larvae

This study evaluated the expression of antimicrobial abaecin and defensin genes in virgin honey-bee queens (Apis mellifera) following induction of 4th larval instar with the bacterium Paenibacillus larvae larvae (1.07 × 102 CFU/queen); to investigate whether the presence of bacteria affects the immune response as well as gene transcript levels for the immune proteins. The total body proteins of bacteria-treated queens showed a highly significant increase, and the appearance of new proteins patterns and/or disappearance of others. The gene expression profile of treated queens showed up-regulation equalled four-fold of the defensin gene, whereas no progression occurred in the abacien gene. This finding likewise greatly affects the diseases challenging this pollinator.

Transcriptome Sequencing and Comparison of Venom Glands Revealed Intraspecific Differentiation and Expression Characteristics of Toxin and Defensin Genes in Mesobuthus martensii Populations.

Mesobuthus martensii, a famous and important Traditional Chinese Medicine has a long medical history and unique functions. It is the first scorpion species whose whole genome was sequenced worldwide. In addition, it is the most widespread and infamous poisonous animal in northern China with complex habitats. It possesses several kinds of toxins that can regulate different ion channels and serve as crucial natural drug resources. Extensive and in-depth studies have been performed on the structures and functions of toxins of M. martensii. In this research, we compared the morphology of M. martensii populations from different localities and calculated the COI genetic distance to determine intraspecific variations. Transcriptome sequencing by RNA-sequencing of the venom glands of M. martensii from ten localities and M. eupeus from one locality was analyzed. The results revealed intraspecific variation in the expression of sodium channel toxin genes, potassium channel toxin genes, calcium channel toxin genes, chloride channel toxin genes, and defensin genes that could be related to the habitats in which these populations are distributed, except the genetic relationships. However, it is not the same in different toxin families. M. martensii and M. eupeus exhibit sexual dimorphism under the expression of toxin genes, which also vary in different toxin families. The following order was recorded in the difference of expression of sodium channel toxin genes: interspecific difference; differences among different populations of the same species; differences between sexes in the same population, whereas the order in the difference of expression of potassium channel toxin genes was interspecific difference; differences between both sexes of same populations; differences among the same sex in different populations of the same species. In addition, there existed fewer expressed genes of calcium channel toxins, chloride channel toxins, and defensins (no more than four members in each family), and their expression differences were not distinct. Interestingly, the expression of two calcium channel toxin genes showed a preference for males and certain populations. We found a difference in the expression of sodium channel toxin genes, potassium channel toxin genes, and chloride channel toxin genes between M. martensii and M. eupeus. In most cases, the expression of one member of the toxin gene clusters distributed in series on the genome were close in different populations and genders, and the members of most clusters expressed in same population and gender tended to be the different. Twenty-one toxin genes were found with the MS/MS identification evidence of M. martensii venom. Since scorpions were not subjected to electrical stimulation or other special treatments before conducting the transcriptome extraction experiment, the results suggested the presence of intraspecific variation and sexual dimorphism of toxin components which revealed the expression characteristics of toxin and defensin genes in M. martensii. We believe this study will promote further in-depth research and use of scorpions and their toxin resources, which in turn will be helpful in standardizing the identification and medical applications of Quanxie in traditional Chinese medicine.

Dual-function DEFENSIN 8 mediates phloem cadmium unloading and accumulation in rice grains.

Grain cadmium (Cd) is translocated from source to sink tissues exclusively via phloem, though the phloem Cd unloading transporter has not been identified yet. Here, we isolated and functionally characterized a defensin-like gene DEFENSIN 8 (DEF8) highly expressed in rice (Oryza sativa) grains and induced by Cd exposure in seedling roots. Histochemical analysis and subcellular localization detected DEF8 expression preferentially in pericycle cells and phloem of seedling roots, as well as in phloem of grain vasculatures. Further analysis demonstrated that DEF8 is secreted into extracellular spaces possibly by vesicle trafficking. DEF8 bound to Cd in vitro, and Cd efflux from protoplasts as well as loading into xylem vessels decreased in the def8 mutant seedlings compared with the wild type. At maturity, significantly less Cd accumulation was observed in the mutant grains. These results suggest that DEF8 is a dual function protein that facilitates Cd loading into xylem and unloading from phloem, thus mediating Cd translocation from roots to shoots and further allocation to grains, representing a phloem Cd unloading regulator. Moreover, essential mineral nutrient accumulation as well as important agronomic traits were not affected in the def8 mutants, suggesting DEF8 is an ideal target for breeding low grain Cd rice.

Isolation and characterization of peroxidase P7-like gene and Rab-GDI like gene from potential medicinal plants: A step toward understanding cell defense signaling.

Defensin genes form part of a plant’s defense system and are activated when exposed to biotic or abiotic stress. They play a vital role in controlling many signaling pathways involved in various plant defense mechanisms. This research aimed to isolate and characterize novel defensin genes from selected medicinally important plants to explore their signaling mechanisms and defense associated roles for breeding. The DNA of Albizia lebbeck and Moringa oleifera was subjected to PCR amplification using gene-specific primers of defensin genes. Two novel defensin genes were isolated in each species, with sequence lengths of 300 bp in A. lebbeck and 150 bp in M. oleifera. In-silico analysis undertaken to retrieve and align their orthologous sequences revealed 100% similarity of the A. lebbeck gene with the Musa acuminate peroxidase P7-like gene and 85% similarity of the M. oleifera gene with the Manihot esculenta GDP dissociation inhibitor gene. The reliability, stability and physiochemical properties of homology models of these sequences was confirmed through online computational studies. This preliminary study confirmed the presence of novel genes with peroxidase P7 and Rab GDP dissociation inhibitor gene-like activity in A. lebbeck and M. oleifera, respectively, and their potential defense role in plants. Thus, the defensin genes of both species could be used in the synthesis of transgenic self-defensive plants with increased disease resistance and as potential candidates for improved crop production and thraputic formulation in the future.

Cloning and Functional Characterization of a Novel β-GRP Gene From Melanotus cribricollis

In this study, a novel β-1,3-glucan recognition protein gene (β-GRP) was identified from Melanotus cribricollis, and its potential role in the immune response was investigated. The full length of β-GRP cDNA (Accession Number: MT941530) was 1644 bp, encoding a protein composed of 428 amino acids. The theoretical molecular weight and the isoelectric point were 51.53 kDa and 6.17, respectively. The amino acid sequence of β-GRP from M. cribricollis was closely related to that of. β-GRP-like from Photinus pyralis, and was predicted to contain conserved GH16 domain without glucanase active site. The results of real-time quantitative PCR showed that fungal infection of Metarhizium pingshaense may significantly upregulated the expression level of β-GRP gene. The RNAi suppression of β-GRP gene expression significantly increased the corrected cumulative mortality. Meanwhile, antimicrobial peptide genes defensin and lysozyme were significantly downregulated after interference of β-GRP. Taken together, these results suggest that β-GRP of M. cribricollis probably participates in the host immune system by mediating the expression of antimicrobial peptides. This study provides comprehensive insights into the innate immune system of insect larvae.

Silicon supplementation induces expression of a histidine-rich defensin gene family in Nicotiana tabacum

Silicon (Si) is known to protect plants from a variety of environmental and biological stressors. The mode of action for these Si responses is unclear. In this study, a family of 14 histidine-rich defensin genes (HRDs) in Nicotiana tabacum, previously described as Defensin 19-like were identified and induced following Si treatment. These genes formed the Nicotiana tabacum histidine-rich defensin 1 (NtHRD1) family. All but one of the NtHRD1 genes were induced in roots with Si treatment, with different members showing differential regulation in upper and lower roots. More than 60% of the NtHRD1 genes showed induced expression with Si treatment in leaves from the upper portion of the plant, while only two genes showed induced expression in lower leaves. Histidine-rich defensins are a newly described group of plant cis-defensins that may play roles in both antimicrobial activity and metal binding. Induction of these genes in N. tabacum following Si treatment could explain some of the beneficial properties of this nutrient.

Keratinocyte-derived defensins activate neutrophil-specific receptors Mrgpra2a/b to prevent skin dysbiosis and bacterial infection

Healthy skin maintains a diverse microbiome and a potent immune system to fight off infections. Here, we discovered that the epithelial-cell-derived antimicrobial peptides defensins activated orphan G-protein-coupled receptors (GPCRs) Mrgpra2a/b on neutrophils. This signaling axis was required for effective neutrophil-mediated skin immunity and microbiome homeostasis. We generated mutant mouse lines lacking the entire Defensin (Def) gene cluster in keratinocytes or Mrgpra2a/b. Def and Mrgpra2 mutant animals both exhibited skin dysbiosis, with reduced microbial diversity and expansion of Staphylococcus species. Defensins and Mrgpra2 were critical for combating S.aureus infections and the formation of neutrophil abscesses, a hallmark of antibacterial immunity. Activation of Mrgpra2 by defensin triggered neutrophil release of IL-1β and CXCL2 which are vital for proper amplification and propagation of the antibacterial immune response. This study demonstrated the importance of epithelial-neutrophil signaling via the defensin-Mrgpra2 axis in maintaining healthy skin ecology and promoting antibacterial host defense.

In silico analysis of the C-terminal domain of big defensin from the Pacific oyster

Defensins are antimicrobial peptides consisting of intramolecular disulphide bonds in a complex folded arrangement of two or three antiparallel β-sheets with or without an α-helical structure. They are produced by a vast range of organisms being constitutively expressed or induced in various tissues against different stimuli like infection, injury or other inflammatory factors. Two classes of invertebrate defensin exist, namely CS-αβ and big defensin, the latter being predominantly present in molluscs. Intriguingly, an invertebrate big defensin gene has been hypothesized as the most probable ancestor of vertebrate β-defensins. Here, conserved residues were identified for both big defensin and β-defensin. In silico mutation on conserved amino acid positions of the β-defensin-like domain of big defensin from Crassostrea gigas was carried out to understand the effects of mutation on the structure and function of the protein. R64A and E71A have been identified as deleterious as well as destabilizing for the protein. Changes in amino acid network and aggregation propensity were also observed upon mutating these two charged residues. 100 ns molecular dynamics simulations of wild-type, R64A and E71A structures revealed significant conformational changes in the case of mutants. Furthermore, molecular docking highlighted the significance of R64 in ligand interaction. In conclusion, these results provide the first in-depth understanding of the structural and functional importance imparted by two conserved charged residues in the C-terminal region of big defensin. It also enhances the existing knowledge about this antimicrobial peptide for application in therapeutics and other aspects of protein engineering. Communicated by Ramaswamy H. Sarma

Panax notoginseng WRKY Transcription Factor 9 Is a Positive Regulator in Responding to Root Rot Pathogen Fusarium solani.

Panax notoginseng (Burk) F.H. Chen is a rare and valuable Chinese herb, but root rot mainly caused by Fusarium solani severely affects the yield and quality of P. notoginseng herbal materials. In this study, we isolated 30 P. notoginseng WRKY transcription factors (TFs), which were divided into three groups (I, II, and III) on the basis of a phylogenetic analysis. The expression levels of 10 WRKY genes, including PnWRKY9, in P. notoginseng roots increased in response to a methyl jasmonate (MeJA) treatment and the following F. solani infection. Additionally, PnWRKY9 was functionally characterized. The PnWRKY9 protein was localized to the nucleus. The overexpression of PnWRKY9 in tobacco (Nicotiana tabacum) considerably increased the resistance to F. solani, whereas an RNAi-mediated decrease in the PnWRKY9 expression level in P. notoginseng leaves increased the susceptibility to F. solani. The RNA sequencing and hormone content analyses of PnWRKY9-overexpression tobacco revealed that PnWRKY9 and the jasmonic acid (JA) signaling pathway synergistically enhance disease resistance. The PnWRKY9 recombinant protein was observed to bind specifically to the W-box sequence in the promoter of a JA-responsive and F. solani resistance-related defensin gene (PnDEFL1). A yeast one-hybrid assay indicated that PnWRKY9 can activate the transcription of PnDEFL1. Furthermore, a co-expression assay in tobacco using β-glucuronidase (GUS) as a reporter further verified that PnWRKY9 positively regulates PnDEFL1 expression. Overall, in this study, we identified P. notoginseng WRKY TFs and demonstrated that PnWRKY9 positively affects plant defenses against the root rot pathogen. The data presented herein provide researchers with fundamental information regarding the regulatory mechanism mediating the coordinated activities of WRKY TFs and the JA signaling pathway in P. notoginseng responses to the root rot pathogen.

Isolation and In Silico Characterization of Defensin Genes from Some Indigenous Plant Species of Pakistan

Aims: Damage of crop plants due to pathogenic attacks, postharvest crops spoilage and lethal effects of chemical pesticides has enforced scientists to find some potential natural alternative. Plants produce different types of antimicrobial peptides including defensins in response to biotic and abiotic stresses. Defensins are small cysteine rich, cationic peptide with 40-45 amino acid residues with a variety of biological activities. Hence defensins have pharmaceutical and agricultural significance especially wide range of antifungal activities.
 Methodology: Nine plant species viz Brassica napus, Brassica nigra, Conyza bonariensis, Alhagi marorum, Sonchus arvensis, Brassica compestris, Eruca sativa, Cirsium arvensis and Brassica juncea were selected for the study. Four set of primers were applied on extracted genomic DNAs and four amplified genes were isolated from different plants species by PCR. The amplified genes were cloned via pTZ57R/T in E.coli. The cloned fragments were sequenced and characterized by different bioinformatics tool such as coding length and peptide sequence, site of cell aggregation, peptide physico-chemical properties, antimicrobial properties and gene expression pattern.
 Result: The amplified products from B. napus, B. nigra, B. compestris, E. sativa and B. juncea were about 330 bp in length and showed upto 85% nucleotide homology to the reported defensins. Deduced amino acid sequence from Bn-Def, Es-Def Bj-Def and Bc- Def showed the conserved defensin domain. These sequences were also characterized for different characteristic like charged amino acids, pI value, shelflife and stability. Sonchus arvensis didn’t give any specific product by prescribed set of primers. However the products from Alhagi marorum was about 770 bp and B. napus was of 270 bp but both give homology below 30%.
 Conclusion: This study showed that in silico characterization of deduced antimicrobial peptides from different plant species has depicted these as an effective alternative to communal therapeutics. However, further work is needed to verify defensins specific activity at protein level.