ArticleLiterature Review

The evolution, function and mechanisms of action for plant defensins

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Abstract

Plant defensins are an extensive family of small cysteine rich proteins characterised by a conserved cysteine stabilised alpha beta protein fold which resembles the structure of insect and vertebrate defensins. However, secondary structure and disulphide topology indicates two independent superfamilies of defensins with similar structures that have arisen via an extreme case of convergent evolution. Defensins from plants and insects belong to the cis-defensin superfamily whereas mammalian defensins belong to the trans-defensin superfamily. Plant defensins are produced by all species of plants and although the structure is highly conserved, the amino acid sequences are highly variable with the exception of the cysteine residues that form the stabilising disulphide bonds and a few other conserved residues. The majority of plant defensins are components of the plant innate immune system but others have evolved additional functions ranging from roles in sexual reproduction and development to metal tolerance. This review focuses on the antifungal mechanisms of plant defensins. The activity of plant defensins is not limited to plant pathogens and many of the described mechanisms have been elucidated using yeast models. These mechanisms are more complex than simple membrane permeabilisation induced by many small antimicrobial peptides. Common themes that run through the characterised mechanisms are interactions with specific lipids, production of reactive oxygen species and induction of cell wall stress. Links between sequence motifs and functions are highlighted where appropriate. The complexity of the interactions between plant defensins and fungi helps explain why this protein superfamily is ubiquitous in plant innate immunity.

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... Defensins were discovered in all multicellular organisms [15]. Amino acid sequences of plant defensins are highly variable, while the fold of the molecule stabilized by four or five (in floral defensins) disulfide bonds is similar and comprises the so-called cysteine-stabilized αβ-motif (СSαβ-motif) with an α-helix running parallel to the triple-stranded β-sheet [15]. ...
... Defensins were discovered in all multicellular organisms [15]. Amino acid sequences of plant defensins are highly variable, while the fold of the molecule stabilized by four or five (in floral defensins) disulfide bonds is similar and comprises the so-called cysteine-stabilized αβ-motif (СSαβ-motif) with an α-helix running parallel to the triple-stranded β-sheet [15]. According to Silverstein et al. [9], the cysteine motif of classical defensins is as follows: CX{4,25}CX{2,12}CX{3,4}CX{3,17}CX{4,32}CXCX{1,6}C. ...
... They inhibit the growth of fungi, bacteria, or herbivores, disturbing the membranes of the pathogens or acting as enzyme inhibitors or ion channel blockers. The role of defensins in abiotic stress tolerance and development was also demonstrated [15]. Plant defensins are synthesized as precursor proteins containing a signal peptide transferring the mature peptide to the apoplast. ...
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Cysteine-rich peptides (CRPs) play an important role in plant physiology. However, their role in resistance induced by biogenic elicitors remains poorly understood. Using whole-genome transcriptome sequencing and our CRP search algorithm, we analyzed the repertoire of CRPs in tomato Solanum lycopersicum L. in response to Fusarium oxysporum infection and elicitors from F. sambucinum. We revealed 106 putative CRP transcripts belonging to different families of antimicrobial peptides (AMPs), signaling peptides (RALFs), and peptides with non-defense functions (Major pollen allergen of Olea europaea (Ole e 1 and 6), Maternally Expressed Gene (MEG), Epidermal Patterning Factor (EPF)), as well as pathogenesis-related proteins of families 1 and 4 (PR-1 and 4). We discovered a novel type of 10-Cys-containing hevein-like AMPs named SlHev1, which was up-regulated both by infection and elicitors. Transcript profiling showed that F. oxysporum infection and F. sambucinum elicitors changed the expression levels of different overlapping sets of CRP genes, suggesting the diversification of functions in CRP families. We showed that non-specific lipid transfer proteins (nsLTPs) and snakins mostly contribute to the response of tomato plants to the infection and the elicitors. The involvement of CRPs with non-defense function in stress reactions was also demonstrated. The results obtained shed light on the mode of action of F. sambucinum elicitors and the role of CRP families in the immune response in tomato.
... These different factors decrease plant productivity due to physical damages and physiological and molecular changes they cause on the growth and development of plants (Singh et al. 2020). To counter the invasions of phytopathogens, plants have an enormous variety of secondary metabolites involved in distinctive innate defense mechanisms, pathogenesis-related proteins in plant defense mechanisms, and plant defensins (Dixon 2001;Abdelmohsen et al. 2020;Mostafa et al. 2009;Parisi et al. 2019;Yehia et al. 2020). ...
... Protein sequence analysis of different plant defensins revealed the significant amino acid variability except for the cysteine peptides and few other residues, but conservation of their three-dimensional structures was noticeable (Lacerda et al. 2014;Parisi et al. 2019). ...
... Gene expression analysis of plant defensins indicated that genes are either constitutively expressed or up-regulated in response to biotic/abiotic stresses in all plant tissues or specific plant organs (Noonan et al. 2017;Parisi et al. 2019). Recently, high-level expression of different defensin recombinant proteins has been reported. ...
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The search for effective and bioactive antimicrobial molecules to encounter the medical need for new antibiotics is an encouraging area of research. Plant defensins are small cationic, cysteine-rich peptides with a stabilized tertiary structure by disulfide-bridges and characterized by a wide range of biological functions. The heterologous expression of Egyptian maize defensin (MzDef) in Escherichia coli and subsequent purification by glutathione affinity chromatography yielded 2 mg/L of recombinant defensin peptide. The glutathione-S-transferase (GST)-tagged MzDef of approximately 30 kDa in size (26 KDa GST + ~ 4 KDa MzDef peptide) was immunodetected with anti-GST antibodies. The GST-tag was successfully cleaved from the MzDef peptide by thrombin, and the removal was validated by the Tris-Tricine gel electrophoresis. The MzDef induced strong growth inhibition of Rhizoctonia solani, Fusarium verticillioides, and Aspergillus niger by 94.23%, 93.34%, and 86.25%, respectively, whereas relatively weak growth inhibitory activity of 35.42% against Fusarium solani was recorded. Moreover, strong antibacterial activities were demonstrated against E. coli and Bacillus cereus and the moderate activities against Salmonella enterica and Staphylococcus aureus at all tested concentrations (0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 µM). Furthermore, the in vitro MTT assay exhibited promising anticancer activity against all tested cell lines (hepatocellular carcinoma, mammary gland breast cancer, and colorectal carci-noma colon cancer) with IC 50 values ranging from 14.85 to 29.85 µg/mL. These results suggest that the recombinant peptide MzDef may serve as a potential alternative antimicrobial and anticancer agent to be used in medicinal application.
... Furthermore, it has been demonstrated that apart from their key role against pathogens in planta, defensins also regulate other biological processes mainly associated with growth and development [15,16]. In several species, a body of evidence is accumulating regarding the mobilization of anti-fungal defensin mechanisms [17] and the effectiveness of defensins against several fungi, such as Phytophthora infestans [14], Verticillium dahliae, and Neurospora crassa [16]. Although evidence suggests that the defensin mode of action (MOA) is more diverse than sole disease resistance, their participation in physiological processes upon viral infections and nematode infestations has received less attention [18][19][20]. ...
... Furthermore, higher antifungal activity has been detected towards mold fungi causing growth reduction and hyper-branching in hyphal tips, which has been attributed to the generally higher positive charge affected by the above-mentioned amino acid residues [31]. Finally, it has been reported [32] that other common residues include the two glycine residues (positions 12 and 32 relative to the plant defensin NaD1), an aromatic residue (at position 10), as well as a glutamate (at position 27), all of which participate in the folding of plant defensins [17]. ...
... These mechanisms, however, are more compound than simple membrane permeabilization prompted by several small antimicrobial peptides. Common characterized mechanisms include interfacing with explicit lipids, production of Reactive Oxygen Species (ROS), and stimulation of cell wall tension [17]. Still, nematodes employ complex strategies, such as delivering elicitors to suppress or bypass the plant defense mechanisms. ...
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Defensins are small and rather ubiquitous Cysteine-rich anti-microbial peptides. These 24 proteins may act against pathogenic microorganisms either directly (by binding and disrupting 25 membranes) or indirectly (as signaling molecules that participate in the organization of the cellular 26 defense). Even though defensins are widespread across eukaryotes, still, extensive nucleotide and 27 amino acid dissimilarities hamper the elucidation of their response to stimuli and mode of function. 28 In the current study, we screened the Solanum lycopersicum genome for the identification of defensin 29 genes predicted the relating protein structures and further studied their transcriptional responses 30 to biotic (Verticillium dahliae, Meloidogyne javanica, Cucumber Mosaic Virus and Potato Virus Y 31 infections) and abiotic (cold stress) stimuli. Tomato defensin sequences were classified into two 32 groups (C8 and C12). Our data indicate that the transcription of defensin coding genes primarily 33 depends on the specific pathogen recognition patterns of V. dahliae and M. javanica. The 34 immunodetection of plant defensin 1 protein was achieved only in the root of plants inoculated with 35 V. dahliae. In contrast, the almost null effects of viral infections and cold stress, and the failure to 36 substantially induce the gene transcription suggest that these factors are probably not primarily 37 targeted by the tomato defensin network. 38
... Although progress has recently been made recently in the identification of AMPs in seeds, those corresponding to flowers have been less studied (Astafieva et al., 2012). Concentrated in epidermal and stomatal cells, defensins are produced in areas that are likely to be the initial points of contact with pathogens (Parisi et al., 2018). Additionally, it was established that in the same species there are multiple defensin genes and this redundancy, necessary to protect the plant against selections of pathogens with higher tolerance to a particular type of defence molecule, is the result of the co-evolution of the immune systems of the plant and the pathogen (Vriens et al., 2014;Schmitt et al., 2016). ...
... The main activity reported for plant defensins is antifungal, being active in micromolar concentrations (Hayes et al., 2013;Sagaram et al., 2013;Parisi et al., 2018). Besides antifungal activity, antibacterial, antiprotozoal and insecticidal action, inhibition of α-amylase, trypsin and protein synthesis as well as blockage of ion channels have been established for these molecules (Spelbrink et al., 2004;Lin et al., 2007;Vijayan et al., 2013;Nascimento et al., 2015;Parisi et al., 2018). ...
... The main activity reported for plant defensins is antifungal, being active in micromolar concentrations (Hayes et al., 2013;Sagaram et al., 2013;Parisi et al., 2018). Besides antifungal activity, antibacterial, antiprotozoal and insecticidal action, inhibition of α-amylase, trypsin and protein synthesis as well as blockage of ion channels have been established for these molecules (Spelbrink et al., 2004;Lin et al., 2007;Vijayan et al., 2013;Nascimento et al., 2015;Parisi et al., 2018). Plant defensins do not only act against plant pathogens, some of these molecules are effective against human pathogens and tumour cells as well (Vriens et al., 2015;Bleackley et al., 2016). ...
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Fusarium graminearum is the etiological agent of Fusarium head blight (FHB), a disease that produces a significant decrease in wheat crop yield and it is further aggravated by the presence of mycotoxins in the affected grains that may cause health problems to humans and animals. Plant defensins and defensin-like proteins are antimicrobial peptides (AMPs); they are small basic, cysteine-rich peptides (CRPs) ubiquitously expressed in the plant kingdom and mostly involved in host defence. They present a highly variable sequence but a conserved structure. The γ-core located in the C-terminal region of plant defensins has a conserved β-hairpin structure and is a well-known determinant of the antimicrobial activity among disulphide-containing AMPs. Another conserved motif of plant defensins is the α-core located in the N-terminal region, not conserved among the disulphide-containing AMPs, it has not been yet extensively studied. In this report, we have cloned the putative antimicrobial protein DefSm2, expressed in flowers of the wild plant Silybum marianum. The cDNA encodes a protein with two fused basic domains of an N-terminal defensin domain (DefSm2-D) and a C-terminal Arg-rich and Lys-rich domain. To further characterize the DefSm2-D domain, we built a 3D template-based model that will serve to support the design of novel antifungal peptides. We have designed four potential antifungal peptides: two from the DefSm2-D α-core region (SmAPα1-21 and SmAPα10-21) and two from the γ-core region (SmAPγ27-44 and SmAPγ29-35). We have chemically synthesized and purified the peptides and further characterized them by electrospray ionization mass spectrometry (ESI-MS) and Circular dichroism (CD) spectroscopy. SmAPα1-21, SmAPα10-21, and SmAPγ27-44 inhibited the growth of the phytopathogen F. graminearum at low micromolar concentrations. Conidia exposure to the fungicidal concentration of the peptides caused membrane permeabilization to the fluorescent probe propidium iodide (PI), suggesting that this is one of the main contributing factors in fungal cell killing. Furthermore, conidia treated for 0.5h showed cytoplasmic disorganization as observed by transmission electron microscopy (TEM). Remarkably, the peptides derived from the α-core induced morphological changes on the conidia cell wall, which is a promising target since its distinctive biochemical and structural organization is absent in plant and mammalian cells.
... Secondly, AMPs can modulate and bridge the adaptive immune response elements, e.g., human β-defensins 1, 2, and 3 (hBD-1,-2, and -3) induce the upregulation of specific cytokines, such as IL-8 and MCP-1, whereas only hBD-1,-2 induce IL-6 and IL-10 (Boniotto et al., 2006). In animals, AMPs are mostly produced by epithelial cells of skin, airways and gastrointestinal tract (Zasloff, 2002), while in plants they preferentially accumulate in the peripheral cell layer Lay and Anderson, 2005;van der Weerden et al., 2013;Parisi et al., 2019b). AMPs can be constitutively expressed e.g., hBD-1 in epithelial cells (O'Neil et al., 1999); termicin and spinigerin in termites (Lamberty et al., 2001b) and the plant defensin Psd1 in the epidermal tissues and vascular bundles of pea pods (Almeida et al., 2000(Almeida et al., , 2002. ...
... Additionally, AMPs can be expressed upon induction e.g., hBD-2,-3, and -4 in epithelial cells upon infection and inflammation (García et al., 2001;Liu et al., 2003;Sørensen et al., 2003;Gácser et al., 2014); CRAMP in mouse skin upon Candida albicans infection (López- García et al., 2005) and plant peptides e.g., AtPDF1.2, AtPDF2.3 in leaves upon microbial invasion (Penninckx et al., 1996;Broekaert et al., 1997;Manners et al., 1998;Thomma and Broekaert, 1998;Lay and Anderson, 2005;van der Weerden et al., 2013;Parisi et al., 2019b). ...
... The focus of this review will be on the structure and mode of action of membrane-interacting antifungal AMPs and on a potential link between their mode of action and membrane contact sites (MCSs), thereby focusing on AMPs of which the mode of action is already described in more detail. Additional information on AMPs can be found in Faruck et al. (2016); Parisi et al. (2019b), Buda De Cesare et al. (2020, and Mookherjee et al. (2020). Gaining insight in their mechanism of action allows to exploit this information for novel therapeutics. ...
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The incidence of invasive fungal infections is increasing worldwide, resulting in more than 1.6 million deaths every year. Due to growing antifungal drug resistance and the limited number of currently used antimycotics, there is a clear need for novel antifungal strategies. In this context, great potential is attributed to antimicrobial peptides (AMPs) that are part of the innate immune system of organisms. These peptides are known for their broad-spectrum activity that can be directed toward bacteria, fungi, viruses, and/or even cancer cells. Some AMPs act via rapid physical disruption of microbial cell membranes at high concentrations causing cell leakage and cell death. However, more complex mechanisms are also observed, such as interaction with specific lipids, production of reactive oxygen species, programmed cell death, and autophagy. This review summarizes the structure and mode of action of antifungal AMPs, thereby focusing on their interaction with fungal membranes.
... In addition to membrane permeabilisation, other downstream effects of membrane binding by defensins have been suggested, further highlighting their multifaceted mechanisms in combating microbial pathogens and tumour cells. Generally, defensins can trigger different cellular effects including, but not limited to, reactive oxygen species (ROS) and/or nitric oxide (NO) production, activation of cell wall integrity (CWI) pathway and dysregulation of ionic homeostasis, ultimately contributing to cell death (Figure 3) [45]. ...
... Defensins NaD1 and RsAPF2 have both been shown to induce the formation of ROS (and NO in the case of NaD1) in fungal cells, hence significantly damaging key cellular components and processes [45,46] (Figure 3A,B). RsAFP2 is believed to cause induction of ROS as a downstream signal from its binding to GluCer in the membrane. ...
... Various cell signalling pathways have been shown to be activated in response to defensin exposure. MsDef1 and RsAFP2 both induce increased MAPK signalling and activation of the CWI pathway in response to damage caused by defensins binding to GluCer in the membrane ( Figure 3A,D) [45]. RsAFP2 induces increased phosphorylation of Mkc1p, a downstream interaction partner of Pkc1p both involved in the CWI pathway in C. albicans ( Figure 3A) [32]. ...
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Defensins are a class of host defence peptides (HDPs) that often harbour antimicrobial and anticancer activities, making them attractive candidates as novel therapeutics. In comparison with current antimicrobial and cancer treatments, defensins uniquely target specific membrane lipids via mechanisms distinct from other HDPs. Therefore, defensins could be potentially developed as therapeutics with increased selectivity and reduced susceptibility to the resistance mechanisms of tumour cells and infectious pathogens. In this review, we highlight recent advances in defensin research with a particular focus on membrane lipid-targeting in cancer and infection settings. In doing so, we discuss strategies to harness lipid-binding defensins for anticancer and anti-infective therapies.
... Plant defensins are small cysteine-rich proteins (45-54 amino acids) found in most plant kingdom, they stabilized by disulfide bonds (Parisi et al., 2019). Plant defensins have many functions such as zinc tolerance, blocking of ion channels, antifungal and antibacterial activity (van der Weerden and Anderson 2013). ...
... Although they are very toxic to the fungal pathogens of human and plants, they are non-toxic to mammalian or plant cells. Plant defensins are low molecular weight, cationic proteins that are widespread in many parts of plants such as seeds, leaves, roots, bark, pods, tubers, fruit, and floral organs (Parisi et al., 2019). The SDS page of the purified protein from the broad bean debris ensured that It is a single band with 5.5 kDa molecular weight (Fig. 1). ...
... Plant defensins are comprised of 45-54 amino acid residues and are held together by four disulphide bonds. These disulphide bonds render the molecule highly stable to proteases and extremes of pH and temperature (Parisi et al., 2019). The amino acid analysis of the purified protein in this work showed that the most abundant amino acid in the purified protein is cysteine (Table .1). ...
Article
Fusarium oxysporum is one of the most common soil borne pathogens over the world. This work tried to introduce safe and efficient agent in controlling this pathogen infection in cucumber plants, beside that understanding the mechanism at which fabitine enables the cucumber plants to fight wilt disease. Also, this works introduce a molecular modeling for the direct interaction between fabatin and the Fusarium oxysporum. We evaluated the impact of defensin isolated from broad bean debris (fabatin) against the Fusarium wilt disease in the greenhouse, followed by the analysis of defense-related enzymes, including superoxide dismutase, polyphenol oxidase, soluble peroxidase, cell wall-bound peroxidase, catalase, lactate dehydrogenase, phenylalanine ammonia-lyase, chitinase, and glucanase. Moreover, the growth-regulating protein of the fungus (Snt2, a BAH/PHD) was docked with the fabatin. Fabatin reduced the disease severity from 18.52 to 0.0% by 100% disease reduction, 30 days after transplanting, and from 66.67 to 33.33% by 50.01% disease reduction after 60 days. Interestingly fabatin spray induced upregulation of the studied defense enzymes. The sprayed group showed significant continuous increase in defense related enzymes under infection. Furthermore, fabatin showed a high binding affinity to the Bromo-adjacent domain (BAH) of the Snt2 by - 214.42 kacl/mol and this can prevent the transcriptional regulation processes of the fungus. The present study proved that the exogenous spray fabatin is an effective way in controlling cucumber Fusarium wilt. This is achieved through the enhancement the defense related enzymes which regulate the resistance mechanism in the plants. Moreover, fabatin can directly influence the fungal growth.
... Their antimicrobial activity is mainly directed against fungi and oomycetes, with relatively few effects on bacteria [12]. Since initial isolation from barley and wheat endosperm in 1990, defensins have been subsequently isolated and identified from different tissues of various monocotyledonous and dicotyledonous plants [19]. Moreover, defensins are widely present in insects and animals for protection against invasion by bacteria, fungi, or viruses, and are an important component of the immune response system. ...
... Class I defensins enter the secretory pathway directly upon synthesis. They lack the signal sequences for post-translational modification or subcellular targeting, and accumulate in the cell wall and extracellular space [19]. With a CTPP that targets vesicles, class II defensins mostly undergo proteolysis in the vesicle to release mature short peptides [12,20]. ...
Article
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Botany-derived antimicrobial peptides (BAMPs), a class of small, cysteine-rich peptides produced in plants, are an important component of the plant immune system. Both in vivo and in vitro experiments have demonstrated their powerful antimicrobial activity. Besides in plants, BAMPs have cross-kingdom applications in human health, with toxic and/or inhibitory effects against a variety of tumor cells and viruses. With their diverse molecular structures, broad-spectrum antimicrobial activity, multiple mechanisms of action, and low cytotoxicity, BAMPs provide ideal backbones for drug design, and are potential candidates for plant protection and disease treatment. Lots of original research has elucidated the properties and antimicrobial mechanisms of BAMPs, and characterized their surface receptors and in vivo targets in pathogens. In this paper, we review and introduce five kinds of representative BAMPs belonging to the pathogenesis-related protein family, dissect their antifungal, antiviral, and anticancer mechanisms, and forecast their prospects in agriculture and global human health. Through the deeper understanding of BAMPs, we provide novel insights for their applications in broad-spectrum and durable plant disease prevention and control, and an outlook on the use of BAMPs in anticancer and antiviral drug design.
... Plant defensins are a remarkable family of proteins. They are defined by a conserved threedimensional structure consisting of three beta strands and a single alpha helix stabilized by four disulfide bonds forming a fold known as the cysteine-stabilized alpha-beta (CSαβ) motif [1]. The amino acid sequence requirements to form the CSαβ motif appear to be limited to the eight cysteines that participate in the disulfide bonds, although the spacing between these cysteines can vary, with the only other residues that are more than 80% conserved being two glycines. ...
... Plant defensins are traditionally considered to be a component of the pathogen defence system [1]. However, there are often multiple [4], even hundreds of defensin genes in a single genome [49]. ...
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Plant defensins are best known for their antifungal activity and contribution to the plant immune system. The defining feature of plant defensins is their three-dimensional structure known as the cysteine stabilized alpha-beta motif. This protein fold is remarkably tolerant to sequence variation with only the eight cysteines that contribute to the stabilizing disulfide bonds absolutely conserved across the family. Mature defensins are typically 46–50 amino acids in length and are enriched in lysine and/or arginine residues. Examination of a database of approximately 1200 defensin sequences revealed a subset of defensin sequences that were extended in length and were enriched in histidine residues leading to their classification as histidine-rich defensins (HRDs). Using these initial HRD sequences as a query, a search of the available sequence databases identified over 750 HRDs in solanaceous plants and 20 in brassicas. Histidine residues are known to contribute to metal binding functions in proteins leading to the hypothesis that HRDs would have metal binding properties. A selection of the HRD sequences were recombinantly expressed and purified and their antifungal and metal binding activity was characterized. Of the four HRDs that were successfully expressed all displayed some level of metal binding and two of four had antifungal activity. Structural characterization of the other HRDs identified a novel pattern of disulfide linkages in one of the HRDs that is predicted to also occur in HRDs with similar cysteine spacing. Metal binding by HRDs represents a specialization of the plant defensin fold outside of antifungal activity.
... As a consequence of such diversification processes, the evolutionary concept of peptide promiscuity emerged at protein level, where multiple functions could be performed by a single protein (or scaffold), depending on the biological context (Aharoni et al., 2005;Franco, 2011;Nobeli et al., 2009). Therefore, a number of distinct functions could be associated with plant defensins (Parisi et al., 2019). ...
... Depending on their precursor organization, plant defensins could be divided into two major classes (Lay and Anderson, 2005). While Class I defensins are composed of a signal peptide and a mature defensin and are directed to the secretory pathway (Parisi et al., 2019), class II defensins present an additional C-terminal prodomain, generating a precursor sequence containing about 100 amino acid residues (Balandín et al., 2005;Lay and Anderson, 2005). This prodomain is responsible for, firstly, directing the defensin to the vacuole, and secondly, inhibiting the toxicity of these defensins towards plant cells (Lay et al., 2014). ...
Article
Defensins comprise a polyphyletic group of multifunctional defense peptides. Cis-defensins, also known as cysteine stabilized αβ (CSαβ) defensins, are one of the most ancient defense peptide families. In plants, these peptides have been divided into two classes, according to their precursor organization. Class I defensins are composed of the signal peptide and the mature sequence, while class II defensins have an additional C-terminal prodomain, which is proteolytically cleaved. Class II defensins have been described in Solanaceae and Poaceae species, indicating this class could be spread among all flowering plants. Here, a search by regular expression (RegEx) was applied to the Arabidopsis thaliana proteome, a model plant with more than 300 predicted defensin genes. Two sequences were identified, A7REG2 and A7REG4, which have a typical plant defensin structure and an additional C-terminal prodomain. TraVA database indicated they are expressed in flower, ovules and seeds, and being duplicated genes, this indicates they could be a result of a subfunctionalization process. The presence of class II defensin sequences in Brassicaceae and Solanaceae and evolutionary distance between them suggest class II defensins may be present in other eudicots. Discovery of class II defensins in other plants could shed some light on flower, ovules and seed physiology, as this class is expressed in these locations.
... For these reasons, they are free to interact with other proteins or targets and this may be the explanation for the broad biological activities already described for plant defensins. In fact, for plant defensins, their inhibitory activities on protein translation (Méndez et al. 1990(Méndez et al. , 1996 and α-amylases (Bloch and Richardson 1991;Lin et al. 2007;Pelegrini et al. 2008;Dos Santos et al. 2010), tolerance to heavy metal (Mirouze et al. 2006), blockage of ion channels (Spelbrink et al. 2004), antimicrobial inhibitory spectra (Carvalho and Gomes 2011), their capacity to bind to membrane lipids (phospholipids and sphingolipids) (Baxter et al. 2015;Gonçalves et al. 2012;De Paula et al. 2011;Poon et al. 2014), dimerization (Lay et al. 2012;Song et al. 2011) and antifungal inhibitory mechanisms (Coninck et al. 2013;Parisi et al. 2019) are well described. Also, it is startling that many of these abilities are linked to the amino acid stretch that compose the γ-core which is a desirable characteristic for drug design as explained in the follow paragraph (Supplementary Table 1). ...
... It is also observed that the microorganism species is relevant to the inhibition process, because despite being inactive against yeasts, DD was active against L. amanzonensis (Souza et al. 2019). Corroborating with this suggestion are studies with fungi demonstrating that lipids, especially the negatively charged ones, that are present in the fungal membrane, such as mannosyldiinositol phosphorylceramide (M(IP) 2 C), phosphatidic acid (PA) and phosphatidylinositol 4,5 bisphosphate (PI(4,5)P 2 ), may be the targets of plant defensins and the cationic and hydrophobic amino acids of the γ-core region are important for this interaction (Parisi et al. 2019). MtDef 4 , for example, had different mechanism of action on the fungi Neurospora crassa and Fusarium graminearum, because the differential concentration of sphingolipids in those fungi, as suggested by the authors (El-Mounadi et al. 2016). ...
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Microbial resistance to available drugs is a growing health threat imposing the need for the development of new drugs. The scaffold of plant defensins, including their γ-cores, are particularly good candidates for drug design. This work aimed to improve the antifungal activity of a previous design peptide, named A36,42,44γ32–46VuDef (for short DD) against yeasts by altering its biochemical parameters. We explore the correlation of the biological activity and structure of plant defensins and compared their primary structures by superimposition with VuDef1 and DD which indicated us the favorable position and the amino acid to be changed. Three new peptides with modifications in charge, hydrophobicity (RR and WR) and chirality (D-RR) were designed and tested against pathogenic yeasts. Inhibition was determined by absorbance. Viability of mammalian cells was determined by MTT. The three designed peptides had better inhibitory activity against the yeasts with better potency and spectrum of yeast species inhibition, with low toxicity to mammalian cells. WR, the most hydrophobic and cationic, exhibited better antifungal activity and lower toxicity. Our study provides experimental evidence that targeted changes in the primary structure of peptides based on plant defensins γ-core primary structures prove to be a good tool for the synthesis of new compounds that may be useful as alternative antifungal drugs. The method described did not have the drawback of synthesis of several peptides, because alterations are guided. When compared to other methods, the design process described is efficient and viable to those with scarce resources.
... Plant defensins can have a wide variety of functions including antibacterial activity, trypsin or αamylase inhibition, and roles in plant development, but research has primarily focused on characterizing activity against economically important agricultural fungi [2,3]. Antifungal defensins generally act via disruption of the plasma membrane and while mechanisms of action (MOA) vary, they often require binding to specific cell wall lipids before inducing secondary effects such as production of reactive oxygen species, disruption of Ca 2+ signaling, or membrane lysis [4][5][6]. ...
... Plant defensins are approximately 50 residues in length and contain eight conserved cysteine residues forming four disulfide bonds that increase stability against proteases, temperature and pH [11][12][13][14]. Plant defensins belong to the cis-defensin superfamily that have a characteristic antiparallel β-sheet bound to an α-helix by two disulfide bonds (Fig. 1a) [4,15]. The significant length and complexity of defensins can hinder synthetic approaches to obtain sufficient quantities of defensins for extensive biological characterization. ...
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Background Plant defensins are a broadly distributed family of antimicrobial peptides which have been primarily studied for agriculturally relevant antifungal activity. Recent studies have probed defensins against Gram-negative bacteria revealing evidence for multiple mechanisms of action including membrane lysis and ribosomal inhibition. Herein, a truncated synthetic analog containing the γ-core motif of Amaranthus tricolor DEF2 (Atr-DEF2) reveals Gram-negative antibacterial activity and its mechanism of action is probed via proteomics, outer membrane permeability studies, and iron reduction/chelation assays. Results Atr-DEF2(G39-C54) demonstrated activity against two Gram-negative human bacterial pathogens, Escherichia coli and Klebsiella pneumoniae . Quantitative proteomics revealed changes in the E. coli proteome in response to treatment of sub-lethal concentrations of the truncated defensin, including bacterial outer membrane (OM) and iron acquisition/processing related proteins. Modification of OM charge is a common response of Gram-negative bacteria to membrane lytic antimicrobial peptides (AMPs) to reduce electrostatic interactions, and this mechanism of action was confirmed for Atr-DEF2(G39-C54) via an N-phenylnaphthalen-1-amine uptake assay. Additionally, in vitro assays confirmed the capacity of Atr-DEF2(G39-C54) to reduce Fe ³⁺ and chelate Fe ²⁺ at cell culture relevant concentrations, thus limiting the availability of essential enzymatic cofactors. Conclusions This study highlights the utility of plant defensin γ-core motif synthetic analogs for characterization of novel defensin activity. Proteomic changes in E. coli after treatment with Atr-DEF2(G39-C54) supported the hypothesis that membrane lysis is an important component of γ-core motif mediated antibacterial activity but also emphasized that other properties, such as metal sequestration, may contribute to a multifaceted mechanism of action.
... Similarly, the induction can be impaired by the M24 mutation ( Figure 6F). As major components of the plant's innate immune system [46,47], defensins are small, basic and cysteine-rich proteins with direct antimicrobial activities. P. capsici infection can induce the expression of several N. benthamiana defensin genes [48]. ...
... Our gene expression analysis reveals that PyolNLP5/7 modulates plant defense via inducing the expression of defensins and EIN3, the upstream regulator of PDF1.2. Plant defensins are toxic to pathogens but not mammalian or plant cells [46,47]. Defensins render resistance to a broad range of pathogens via their diverse antifungal, antibacterial, α-amylase and trypsin inhibitory activities [57]. ...
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As a non-pathogenic oomycete, the biocontrol agent Pythium oligandrum is able to control plant diseases through direct mycoparasite activity and boosting plant immune responses. Several P. oligandrum elicitors have been found to activate plant immunity as microbe-associated molecular patterns (MAMPs). Necrosis-and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are a group of MAMPs widely distributed in eukaryotic and prokaryotic plant pathogens. However, little is known about their distribution and functions in P. oligandrum and its sister species Pythium periplocum. Here, we identified a total of 25 NLPs from P. oligandrum (PyolNLPs) and P. periplocum (PypeNLPs). Meanwhile, we found that PyolNLPs/PypeNLPs genes cluster in two chromosomal segments, and our analysis suggests that they expand by duplication and share a common origin totally different from that of pathogenic oomycetes. Nine PyolNLPs/PypeNLPs induced necrosis in Nicotiana benthamiana by agroinfiltration. Eight partially purified PyolNLPs/PypeNLPs were tested for their potential biocontrol activity. PyolNLP5 and PyolNLP7 showed necrosis-inducing activity in N. benthamiana via direct protein infiltration. At sufficient concentrations, they both significantly reduced disease severity and suppressed the in planta growth of Phytophthora capsici in solanaceous plants including N. benthamiana (tobacco), Solanum lycopersicum (tomato) and Capsicum annuum (pepper). Our assays suggest that the Phytophthora suppression effect of PyolNLP5 and PyolNLP7 is irrelevant to reactive oxygen species (ROS) accumulation. Instead, they induce the expression of antimicrobial plant defensin genes, and the induction depends on their conserved nlp24-like peptide pattern. This work demonstrates the biocontrol role of two P. oligandrum NLPs for solanaceous plants, which uncovers a novel approach of utilizing NLPs to develop bioactive formulae for oomycete pathogen control with no ROS-caused injury to plants.
... Plant defensins can be divided into two classes according to their precursors (Lay & Anderson, 2005). Class I defensins composed of signal peptides and mature defensins and secreted into the extracellular space (Parisi et al., 2019). Class II defensins contained a C-terminal propeptide, and is mainly expressed constitutively in flowers and fruits of Solanaceae plants (Balandín et al., 2003). ...
... Under potassium deficiency, it was noted that the expression of most BnaPDF DEGs was significantly induced in the shoots but was inhibited in the roots (Fig. 10). Plant defensins are used as potassium channel blockers (Parisi et al., 2019;Vriens et al., 2016). For example, the sequence of AtPDF2.3 contains a toxin characteristic sequence (K-C5-R-G) that can block potassium channels (Vriens et al., 2016). ...
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Plant defensins (PDFs), short peptides with strong antibacterial activity, play important roles in plant growth, development, and stress resistance. However, there are few systematic analyses on PDFs in Brassica napus. Here, bioinformatics methods were used to identify genome-wide PDFs in Brassica napus, and systematically analyze physicochemical properties, expansion pattern, phylogeny, and expression profiling of BnaPDFs under diverse nutrient stresses. A total of 37 full-length PDF homologs, divided into two subgroups (PDF1s and PDF2s), were identified in the rapeseed genome. A total of two distinct clades were identified in the BnaPDF phylogeny. Clade specific conserved motifs were identified within each clade respectively. Most BnaPDFs were proved to undergo powerful purified selection. The PDF members had enriched cis-elements related to growth and development, hormone response, environmental stress response in their promoter regions. The expression patterns of BnaPDFs were analyzed in different tissues. BnaPDF1.2bs was mainly expressed in the roots, whereas BnaPDF2.2s and BnaPDF2.3s were both expressed in stamen, pericarp, silique, and stem. However, the other BnaPDF members showed low expression levels in various tissues. Differential expression of BnaPDFs under nitrate limitation, ammonium excess, phosphorus starvation, potassium deficiency, cadmium toxicity, and salt stress indicated that they might participate in different nutrient stress resistance. The genome-wide identification and characterization of BnaPDFs will enrich understanding of their molecular characteristics and provide elite gene resources for genetic improvement of rapeseed resistance to nutrient stresses.
... Finally, a conceptual plant defensin (PDF) pathway is presented for Arabidopsis thaliana. PDFs are cysteine-rich, structurally conserved antimicrobial peptides, responsive to biotic stress, including bacteria [18], fungi [19] and insects [20,21]. Besides the fact that PDF has previously been studied in many plants [21], most of the information about its regulation is scattered in the literature. ...
... PDFs are cysteine-rich, structurally conserved antimicrobial peptides, responsive to biotic stress, including bacteria [18], fungi [19] and insects [20,21]. Besides the fact that PDF has previously been studied in many plants [21], most of the information about its regulation is scattered in the literature. Here we show the potential of LAITOR4HPC to gather comprehensive information on biological cooccurrences, allowing a conceptual and dynamic view of pathways. ...
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Background: The amount of published full-text articles has increased dramatically. Text mining tools configure an essential approach to building biological networks, updating databases and providing annotation for new pathways. PESCADOR is an online web server based on LAITOR and NLProt text mining tools, which retrieves protein-protein co-occurrences in a tabular-based format, adding a network schema. Here we present an HPC-oriented version of PESCADOR's native text mining tool, renamed to LAITOR4HPC, aiming to access an unlimited abstract amount in a short time to enrich available networks, build new ones and possibly highlight whether fields of research have been exhaustively studied. Results: By taking advantage of parallel computing HPC infrastructure, the full collection of MEDLINE abstracts available until June 2017 was analyzed in a shorter period (6 days) when compared to the original online implementation (with an estimated 2 years to run the same data). Additionally, three case studies were presented to illustrate LAITOR4HPC usage possibilities. The first case study targeted soybean and was used to retrieve an overview of published co-occurrences in a single organism, retrieving 15,788 proteins in 7894 co-occurrences. In the second case study, a target gene family was searched in many organisms, by analyzing 15 species under biotic stress. Most co-occurrences regarded Arabidopsis thaliana and Zea mays. The third case study concerned the construction and enrichment of an available pathway. Choosing A. thaliana for further analysis, the defensin pathway was enriched, showing additional signaling and regulation molecules, and how they respond to each other in the modulation of this complex plant defense response. Conclusions: LAITOR4HPC can be used for an efficient text mining based construction of biological networks derived from big data sources, such as MEDLINE abstracts. Time consumption and data input limitations will depend on the available resources at the HPC facility. LAITOR4HPC enables enough flexibility for different approaches and data amounts targeted to an organism, a subject, or a specific pathway. Additionally, it can deliver comprehensive results where interactions are classified into four types, according to their reliability.
... Our research adds to the existing evidence on the potential of chicken β Gal-3 as a genetic target for developing diseaseresistant crops. More research in this direction could open many doors for developing safe ways of combating human and animal pathogens (Cools et al., 2017;Islam et al., 2017;Parisi et al., 2018). Although, we have tested the transgenic plant materials on mice models for toxicity, allergies, and possible horizontal gene transfer to the environment or to other species. ...
Article
Gallinacin-3 (Gal-3) is a newly discovered epithelial beta-defensin that acts as cationic antimicrobial peptides, and plays an important role in chicken innate immunity. However, the gallinacin-3 precursor contained a lengthy C-terminal region, which often hindered its expression. After codon optimization of Gal-3 and construction of an expression vector, the transgenic plants of Medicago sativa were obtained. Transgenic plants were validated and expression of proteins was detected. The antimicrobial activity of chicken β Gal-3 was analyzed and effects of chicken β Gal-3 on the body weight and intestinal microflora of mice were described. Our results demonstrated that the codon optimized chicken Gal-3 was stably expressed in transgenic Medicago sativa using the pCAMBIA3301 expression vector under the control of protein phosphatase (Ppha) promoter. Five transgenic plants with the highest expression of chicken β Gal-3 were selected, and were evaluated for the in vitro antimicrobial activity against Escherichia coli, Staphylococcus aureus and Salmonella typhi. Our findings confirmed that the Minimum Inhibitory Concentration (MIC) of the three bacterial strains were 32, 16 and 128 μg/mL, respectively. In addition, the effect of chicken Gal-3 on the body weight of mice fed with transgenic plants showed no significant deviation compared with that of the control group. Similarly, no loss of intestinal microflora was evident in the experimental group compared with the control group. Together, our findings demonstrate an alternative method for the stable expression of chicken Gal-3 with significant antibacterial effects and potential probiotics uses. In addition, this study may also be useful in the development of resistant M. sativa plants against pathogenic bacteria in future studies.
... In case of antifungal activity, plant defensins are known to target a variety of intracellular targets, such as interaction with nucleic acids and inhibition of ion channels, among others (reviewed by Parisi et al. 88 ). Antibacterial activity of plant defensins has been less studied but it was recently reported that 2 Medicago truncatula defensins had different mode of action against Pseudomonas species. ...
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Plant defensins are small, basic peptides that have a characteristic three-dimensional folding pattern which is stabilized by four disulfide bridges. We show here that Arabidopsis contains in addition to the proper plant defensins a group of 9 plant defensin-like (PdfL) genes. They are all expressed at low levels while GUS fusions of the promoters showed expression in most tissues with only minor differences. We produced two of the encoded peptides in E. coli and tested the antimicrobial activity in vitro. Both were highly active against fungi but had lower activity against bacteria. At higher concentrations hyperbranching and swollen tips, which are indicative of antimicrobial activity, were induced in Fusarium graminearum by both peptides. Overexpression lines for most PdfL genes were produced using the 35S CaMV promoter to study their possible in planta function. With the exception of PdfL4.1 these lines had enhanced resistance against F. oxysporum. All PDFL peptides were also transiently expressed in Nicotiana benthamiana leaves with agroinfiltration using the pPZP3425 vector. In case of PDFL1.4 this resulted in complete death of the infiltrated tissues after 7 days. All other PDFLs resulted only in various degrees of small necrotic lesions. In conclusion, our results show that at least some of the PdfL genes could function in plant resistance.
... Later, the term "γ-thionin" was replaced by "defensin" based on the higher number of primary and tertiary structures of these proteins and also on their antifungal activities more related to insect and mammalian defensins than to plant thionins. 53 Plant defensins belong to a diverse protein superfamily called cis-defensin 54 and exhibit cationic charge, consisting of 45 to 54 aa with 2 to 4 disulfide bonds. 53,55 Plant defensins share similar tertiary structures and typically exhibit a triple-stranded antiparallel β sheet, enveloped by an α-helix and confined by intramolecular disulfide bonds 1 (Figure 2A). ...
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ABSTRACT: Even before the perception or interaction with pathogens, plants rely on constitutively guardian molecules, often specific to tissue or stage, with further expression after contact with the pathogen. These guardians include small molecules as antimicrobial peptides (AMPs), generally cysteine-rich, functioning to prevent pathogen establishment. Some of these AMPs are shared among eukaryotes (eg, defensins and cyclotides), others are plant specific (eg, snakins), while some are specific to certain plant families (such as heveins). When compared with other organisms, plants tend to present a higher amount of AMP isoforms due to gene duplications or polyploidy, an occurrence possibly also associated with the sessile habit of plants, which prevents them from evading biotic and environmental stresses. Therefore, plants arise as a rich resource for new AMPs. As these molecules are difficult to retrieve from databases using simple sequence alignments, a description of their characteristics and in silico (bioinformatics) approaches used to retrieve them is provided, considering resources and databases available. The possibilities and applications based on tools versus database approaches are considerable and have been so far underestimated. KEywORdS: Defensin, lipid transfer protein, hevein, cyclotide, snakin, knotin, macadamia β-barrelins, impatiens-like, puroindoline, thaumatin
... Plant defensins form a large family of $45-54 amino acid residue cationic proteins Kovaleva et al., 2020;Parisi et al., 2019;Thomma et al., 2002). The signature feature of plant defensins is the presence of conserved cysteine residues. ...
Article
Plant defensins demonstrate high structural stability at extreme temperatures and pH values and, in general, are non-toxic to mammalian cells. These properties make them attractive candidates for use in biotechnology and biomedicine. Knowing the structure-function relationship is desirable to guide the design of plant defensin-based applications. Thus far, the broad range of biological activities was described only for one defensin from gymnosperms, the defensin PsDef1 from Scots pine. Here, we report that closely related defensin from the same taxonomy group, PsDef2, differing from PsDef1 by six amino acids, also possesses antimicrobial, antibacterial, and insect α-amylase inhibitory activities. We also report the solution structure and dynamics properties of PsDef2 assessed using a combination of experimental nuclear magnetic resonance (NMR) techniques. Lastly, we perform a comparative analysis of PsDef2 and PsDef1 gaining a molecular-level insight into their structure-dynamics-function relationship.
... Consequently, CRPs are under investigation as molecular scaffolds for engineering pharmaceutically useful peptides. CRPs have been studied from numerous plant families [39,40], and the squash family (Cucurbitaceae) is known as a rich source of these stabilized peptides [5,41]. In this study, we isolated and characterized novel CRPs from C. colocynthis a member of the Cucurbitaceae family. ...
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The plant Citrullus colocynthis, a member of the squash (Cucurbitaceae) family, has a long history in traditional medicine. Based on the ancient knowledge about the healing properties of herbal preparations, plant-derived small molecules, e.g., salicylic acid, or quinine, have been integral to modern drug discovery. Additionally, many plant families, such as Cucurbitaceae, are known as a rich source for cysteine-rich peptides, which are gaining importance as valuable pharmaceuticals. In this study, we characterized the C. colocynthis peptidome using chemical modification of cysteine residues, and mass shift analysis via matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. We identified the presence of at least 23 cysteine-rich peptides in this plant, and eight novel peptides, named citcol-1 to -8, with a molecular weight between ~3650 and 4160 Da, were purified using reversed-phase high performance liquid chromatography (HPLC), and their amino acid sequences were determined by de novo assignment of b- and y-ion series of proteolytic peptide fragments. In silico analysis of citcol peptides revealed a high sequence similarity to trypsin inhibitor peptides from Cucumis sativus, Momordica cochinchinensis, Momordica macrophylla and Momordica sphaeroidea. Using genome/transcriptome mining it was possible to identify precursor sequences of this peptide family in related Cucurbitaceae species that cluster into trypsin inhibitor and antimicrobial peptides. Based on our analysis, the presence or absence of a crucial Arg/Lys residue at the putative P1 position may be used to classify these common cysteine-rich peptides by functional properties. Despite sequence homology and the common classification into the inhibitor cysteine knot family, these peptides appear to have diverse and additional bioactivities yet to be revealed.
... Marker genes for SA, JA, and ET did not display altered transcript levels, whereas PDF1.1 and PDF1.2 transcript levels were low in sr1-4D (Figs. 5 and 6). Plant defensins have been proposed to contribute to the resistance against fungal infection through a variety of molecular functions including lipid binding, inhibition of protein synthesis and regulation of ion channel activity (Parisi et al. 2019). Recently, PDF1.1 was proposed to act as an apoplastic iron chelator, resulting in resistance to the necrotroph Pectobacterium carotovorum subsp. ...
Article
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Plants require interaction between signaling pathways to differentiate and integrate stress responses and deploy appropriate defenses. The hormones ethylene, salicylic acid (SA), and jasmonic acid (JA) are important regulators of plant defenses. Numerous interactions between these signaling pathways are the cornerstone of robust plant immunity. Additionally, during the early response to pathogens, reactive oxygen species (ROS) act as signaling molecules. Here, we examined the extent of signal interaction in the early stages of Botrytis cinerea infection. To enable a comparison between B. cinerea infection with ROS signaling, we subjected plants to ozone treatment, which stimulates an apoplastic ROS burst. We used a collection of single, double, and triple signaling mutants defective in hormone signaling and biosynthesis and subjected them to B. cinerea infection and ozone treatment at different timepoints. We examined lesion size, cell death, and gene expression (both quantitatively and spatially). The two treatments shared many similarities, especially in JA-insensitive mutants, which were sensitive to both treatments. Unexpectedly, a B. cinerea–susceptible JA-insensitive mutant (coi1), became tolerant when both SA biosynthesis and signaling was impaired (coi1 npr1 sid2), demonstrating that JA responses may be under the control of SA. Extensive marker gene analysis indicated JA as the main regulator of both B. cinerea and ozone defenses. In addition, we identified the transcription factor SR1 as a crucial regulator of PLANT DEFENSIN expression and cell-death regulation, which contributes to resistance to B. cinerea. Overall, our work further defines the context of ROS in plant defense signaling. [Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .
... AMPs have a wide spectrum of activities (antibacterial, antifungal, insecticidal, and antiviral), and some AMPs also inhibit hydrolases and protein biosynthesis [12]. Due to their chemical properties, plant AMPs also demonstrate antiproliferative action [13,14]. The above properties of AMPs can be used for the development of new drugs or ...
Article
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Plants are good sources of biologically active compounds with antimicrobial activity, including polypeptides. Antimi-crobial peptides (AMPs) represent one of the main barriers of plant innate immunity to environmental stress factors and are attracting much research interest. There are some extraction methods for isolation of AMPs from plant organs based on the type of extractant and initial fractionation stages. But most methods are directed to obtain some specific structural types of AMPs and do not allow to understand the molecular diversity of AMP inside a whole plant. In this mini-review, we suggest an optimized scheme of AMP isolation from plants followed by obtaining a set of peptides belonging to various structural families. This approach can be performed for large-scale screening of plants to identify some novel or homologous AMPs for fundamental and applied studies.
... In the recent publication of Aumer et al. [133], the use of a proteomic approach has allowed evidencing the alteration of spliceosome, ribosome protein processing in endoplasmic reticulum, endocytosis, MAPK signaling pathway and oxidative phosphorylation in B. cinerea exposed to an analogue of the insect defensin heliomicin. In addition to being comprehensively reviewed by Parisi et al. [134] and Struyfs et al. [135], the antifungal activity of different defensins can result from different mechanisms. While some defensins require crossing the fungal cell wall and plasma membrane to induce cell death, others can exert their toxic effects from the extracellular side of the fungal cells. ...
Article
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Crops are threatened by numerous fungal diseases that can adversely affect the availability and quality of agricultural commodities. In addition, some of these fungal phytopathogens have the capacity to produce mycotoxins that pose a serious health threat to humans and livestock. To facilitate the transition towards sustainable environmentally friendly agriculture, there is an urgent need to develop innovative methods allowing a reduced use of synthetic fungicides while guaranteeing optimal yields and the safety of the harvests. Several defensins have been reported to display antifungal and even—despite being under-studied—antimycotoxin activities and could be promising natural molecules for the development of control strategies. This review analyses pioneering and recent work addressing the bioactivity of defensins towards fungal phytopathogens; the details of approximately 100 active defensins and defensin-like peptides occurring in plants, mammals, fungi and invertebrates are listed. Moreover, the multi-faceted mechanism of action employed by defensins, the opportunity to optimize large-scale production procedures such as their solubility, stability and toxicity to plants and mammals are discussed. Overall, the knowledge gathered within the present review strongly supports the bright future held by defensin-based plant protection solutions while pointing out the obstacles that still need to be overcome to translate defensin-based in vitro research findings into commercial products.
... Plant defensins constitute a large and evolutionarily diverse family of antimicrobial peptides (AMPs), being important components of the plant's innate immune system (Lay and Anderson 2005;Parisi et al. 2018). The first plant defensins were identified in nettle seeds (Urtica dioica L.), wheat (Triticum aestivum L.), and barley (Hordeum vulgare L.) (Broekaert et al. 1989;Colilla et al. 1990;Mendez et al. 1990). ...
Article
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Plant defensins and lipid transfer proteins (LTPs) constitute a large and evolutionarily diverse family of antimicrobial peptides. Defensins and LTPs are two pathogenesis-related proteins (PR proteins) whose characterization may help to uncover aspects about the sugarcane response to pathogens attack. LTPs have also been investigated for their participation in the response to different types of stress. Despite the important roles of defensins and LTPs in biotic and abiotic stresses, scarce knowledge is found about these proteins in sugarcane. By using bioinformatics approaches, we characterized defensins and LTPs in the sugarcane wild species and modern cultivar genomes. The identification of defensins and LTPs showed that all five defensins groups and eight of the nine LTPs have their respective genes loci, although some was only identified in the cultivar genome. Phylogenetic analysis showed that defensins appear to be more conserved among groups of plants than LTPs. Some defensins and LTPs showed opposite expression during pathogenic and benefic bacterial interactions. Interestingly, the expression of defensins and LTPs in shoots and roots was completely different in plants submitted to benefic bacteria or water depletion. Finally, the modeling and comparison of isoforms of LTPs and defensins in wild species and cultivars revealed a high conservation of tertiary structures, with variation of amino acids in different regions of proteins, which could impact their antimicrobial activity. Our data contributed to the characterization of defensins and LTPs in sugarcane and provided new elements for understanding the involvement of these proteins in sugarcane response to different types of stress.
... Plant defensins (formerly known as γ-thionins) possesses different structural configuration (β1-α-β2-β3) from mammalian defensins (α-β1-β2-β3). They are small cationic peptides consisting of 45-54 amino acids that comprise four to five intramolecular disulfide bonds [4][5][6]. These defensins exhibit diverse biological functions that include antifungal [7][8][9][10][11][12] and antibacterial [13,14] activities, and α-amylase and trypsin inhibitory properties [15,16]. ...
Article
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Plant defensins possess diverse biological functions that include antifungal and antibacterial activities and α-amylase and trypsin inhibitory properties. Two mutations, G9R and V39R, were confirmed to increase the antifungal activity of Raphanus sativus antifungal protein 2 (RsAFP2). Accelerated Molecular Dynamics (aMD) were carried out to examine the confor-mational changes present in these RsAFP2 mutants, and its two closest homologs compared to the wild-type protein. Specifically, the root mean square fluctuation values for the eight cysteine amino acids involved in the four disulfide bonds were low in the V39R mutant compared to the wild-type. Additionally, analysis of the free energy change revealed that G9R and V39R mutations exert a neutral and stabilizing effect on RsAFP2 conformation, and this is supported by the observed lower total energy of mutants compared to the wild-type, suggesting that enhanced stability of the mutants. However, MD simulations to a longer time scale would aid in capturing more conformational state of the wild-type and mutants defensin protein. Furthermore, the aMD simulations on fungal mimic membranes with RsAFP2 and its mutants and homologs showed that the mutant proteins caused higher deformation and water diffusion than the native RsAFP2, especially the V39R mutant. The mutant variants seem to interact by specifically targeting the POPC and POPI lipids amongst others. This work highlights the stabilizing effect of mutations at the 9 th and 39 th positions of RsAFP2 and their increased membrane deformation activity.
... They also have a conserved cysteine that stabilizes protein structure by interacting with negatively charged microbial membranes. PR-12 proteins are subdivided into 2 major groups: cis-defensin (found in plants and insects) and trans-defensin (mammals), which have a similar structure [65]. Defensins are antimicrobial peptides that have different mechanisms of action against pathogens such as viruses, fungi and bacteria. ...
Article
Many unfavorable stress conditions, such as wounding, drought, extreme temperatures, salinity and pathogen attacks control growth, development and plant yield. To survive under such environments, plants have developed many strategies. They are able to induce the expression of a large number of genes that encodes effectors, receptors, as well as signaling proteins and protective molecules. Among all, we find pathogenesis-related proteins (PRs) which were found to be activated in response to a large number of biotic and abiotic threats. Those proteins have a wide range of functions; acting as chitinases, peroxidases, anti-microbial agents, hydrolases, protease inhibitors, and other activities. Activation of PR proteins has been demonstrated in different plant families as a response to different stresses. In this review, we have summarized the structural, biological and functional characteristics of the different PRs families in plants, their regulation, as well as their roles in plant defense against abiotic and biotic stresses.
... Fungal diseases, in addition to cause the destruction of several agricultural crops, have become a serious threat to human health [1,2]. Fungal diseases are estimated to affect more than one billion people worldwide, of whom 150 million suffer from serious infections [3,4]. ...
Article
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Antimicrobial peptides (AMPs) are molecules present in several life forms, possess broad-spectrum of inhibitory activity against pathogenic microorganisms, and are a promising alternative to combat the multidrug resistant pathogens. The aim of this work was to identify and characterize AMPs from Capsicum chinense fruits and to evaluate their inhibitory activities against yeasts of the genus Candida and α-amylases. Initially, after protein extraction from fruits, the extract was submitted to anion exchange chromatography resulting two fractions. Fraction D1 was further fractionated by molecular exclusion chromatography, and three fractions were obtained. These fractions showed low molecular mass peptides, and in fraction F3, only two protein bands of approximately 6.5 kDa were observed. Through mass spectrometry, we identified that the lowest molecular mass protein band of fraction F3 showed similarity with AMPs from plant defensin family. We named this peptide CcDef3 (Capsicum chinense defensin 3). The antifungal activity of these fractions was analyzed against yeasts of the genus Candida. At 200 μg/mL, fraction F1 inhibited the growth of C. tropicalis by 26%, fraction F2 inhibited 35% of the growth of C. buinensis, and fraction F3 inhibited all tested yeasts, exhibiting greater inhibition activity on the growth of the yeast C. albicans (86%) followed by C. buinensis (69%) and C. tropicalis (21%). Fractions F1 and F2 promoted membrane permeabilization of all tested yeasts and increased the endogenous induction of reactive oxygen species (ROS) in C. buinensis and C. tropicalis, respectively. We also observed that fraction F3 at a concentration of 50 µg/mL inhibited the α-amylase activities of Tenebrio molitor larvae by 96% and human salivary by 100%. Thus, our results show that fraction F3, which contains CcDef3, is a very promising protein fraction because it has antifungal potential and is able to inhibit the activity of different α-amylase enzymes.
... Defensins are composed of three antiparallel βsheets and one α-helix stabilized by four or five disulfide bonds (Almeida et al., 2000;Lacerda et al., 2014;Vriens et al., 2014). The first and crucial step in plant defensin activity is an interaction with membrane targets such as sphingolipids and phospholipids (Cools et al., 2017;Parisi et al., 2019). Fungal glucosylceramide-enriched membranes are critical targets for some plant defensins. ...
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Approximately four million people contract fungal infections every year in Brazil, primarily caused by Aspergillus spp . The ability of these fungi to form biofilms in tissues and medical devices complicates treatment and contributes to high rates of morbidity and mortality in immunocompromised patients. Ps d2 is a pea defensin of 5.4 kDa that possesses good antifungal activity against planktonic cells of representative pathogenic fungi. Its function depends on interactions with membrane and cell wall lipid components such as glucosylceramide and ergosterol. In the present study, we characterized Aspergillus nidulans biofilm formation and determined the effect of Ps d2 on A. nidulans biofilms. After 4 hours, A. nidulans conidia adhered to polystyrene surfaces and formed a robust extracellular matrix-producing biofilm at 24 h, increasing thickness until 48 h Ps d2 inhibited A. nidulans biofilm formation in a dose-dependent manner. Most notably, at 10 μM Ps d2 inhibited 50% of biofilm viability and biomass and 40% of extracellular matrix production. Ps d2 significantly decreased the colonized surface area by the biofilm and changed its level of organization, causing a shortening of length and diameter of hyphae and inhibition of conidiophore formation. This activity against A. nidulans biofilm suggests a potential use of Ps d2 as a prototype to design new antifungal agents to prevent biofilm formation by A. nidulans and related species.
... S. cerevisiae is also used in functional studies as a model organism [38]. In contrast, so-called plant γ-thionins, which have been classified into the defensin family, have anti-yeast activity [39]. This study is the first report of thionin activity against clinical yeast isolates. ...
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High-cationic biologically active peptides of the thionins family were isolated from black cumin (Nigella sativa L.) seeds. According to their physicochemical characteristics, they were classified as representatives of the class I thionin subfamily. Novel peptides were called “Nigellothionins”, socalled because of their source plant. Thionins are described as components of plant innate immunity to environmental stress factors. Nine nigellothionins were identified in the plant in different amounts. Complete amino acid sequences were determined for three of them, and a high degree of similarity was detected. Three nigellothionins were examined for antifungal properties against collection strains. The dominant peptide, NsW2, was also examined for activity against clinical isolates of fungi. Cytotoxic activity was determined for NsW2. Nigellothionins activity against all collection strains and clinical isolates varied from absence to a value comparable to amphotericin B, which can be explained by the presence of amino acid substitutions in their sequences. Cytotoxic activity in vitro for NsW2 was detected at sub-micromolar concentrations. This has allowed us to propose an alteration of the molecular mechanism of action at different concentrations. The results obtained suggest that nigellothionins are natural compounds that can be used as antimycotic and anti-proliferative agents.
... Most plant defensins have been found to have antifungal activity, and some of them also possess biological activity against bacteria (Chen et al. 2005). In addition, other biological functions, such as inhibiting protein synthesis (Liu et al. 2006) and enzyme activity (Pelegrini et al. 2008), blocking ion channels (Almeida et al. 2002), and their role in heavy metal tolerance and plant development, have also been reported (Parisi et al. 2018). ...
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Defensins, as part of the PR protein family, not only have antimicrobial activity in vitro, but also play an important role in plant disease resistance. To explore the molecular function of defensin in spruce, a defensin gene PaDef was isolated and characterized from Picea asperata. The full-length cDNA sequence of the PaDef was 264 bp and contained an ORF of 252 bp, encoding a total of 83 amino acids. The deduced PaDef protein belongs to the gamma-thionin family and contains the Knot1 domain and eight conserved cysteine residues. Blast and phylogenetic analysis showed that the PaDef protein shared high homology with other plant defensin proteins. The tertiary structure of PaDef was obtained by homologous modelling using the structure of Pinus sylvestris defensin as a template and exhibited a βαββ structure. SDS-PAGE analysis showed that the molecular mass of recombinant PaDef expressed in Escherichia coli was 23.8 kDa. Plate assay showed that purified PaDef exhibited strong antifungal activity inhibiting the growth of Pestalotiopsis neolitseae, but not Colletotrichum gloeosporioides and Botrytis cinerea. Microscopic observation revealed that purified PaDef had a negative effect on the hyphal morphology of all three phytopathogens. qRT-PCR analysis indicated that the PaDef gene had the highest expression level in needles of P. asperata compared with roots, phloem, twigs, and could be significantly upregulated by the pathogenic fungus Lophodermium piceae, which causes needle cast disease. These results provide a molecular basis for determining the molecular function of defensin PaDef in P. asperata and its potential as an antifungal agent.
... Induction of DEFL0770 genes in response to A. brassicicola and downregulation of expression in syncytia indicated an involvement of the genes in plant defense. It is known that many plant defensins and DEFL peptides have antimicrobial activity in vitro [45,46] Examples of DEFL peptides from plants include, for instance, peptides from Phaseolus vulgaris [47,48] Trigonella foenum-graecum seeds [49] and from Gymnocladus chinensis seeds [50]. We have recently shown that two plant defensin-related peptides, which we called plant defensin-like, have antimicrobial activity against bacteria and especially fungi (Omidvar and Bohlmann, unpublished results). ...
Article
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Arabidopsis contains 317 genes for defensin-like (DEFL) peptides. DEFLs have been grouped into different families based mainly on cysteine motifs. The DEFL0770 group contains seven genes, of which four are strongly expressed in roots. We found that the expression of these genes is downregulated in syncytia induced by the beet cyst nematode Heterodera schachtii as revealed by RNAseq analysis. We have studied one gene of this group, At3g59930, in detail. A promoter::GUS line revealed that the gene is only expressed in roots but not in other plant organs. Infection of the GUS line with larvae of H. schachtii showed a strong downregulation of GUS expression in infection sites as early as 1 dpi, confirming the RNAseq data. The At3g59930 peptide had only weak antimicrobial activity against Botrytis cinerea. Overexpression lines had no enhanced resistance against this fungus but were more resistant to H. schachtii infection. Our data indicate that At3g59930 is involved in resistance to nematodes which is probably not due to direct nematicidal activity.
... Defensins are considered as a part of the antimicrobial protein family and are rich in cysteine. Furthermore, defensins offer assistance to cells in combating bacterial (Menendez and Finlay, 2007), viral (Wilson et al., 2013) and fungal infections (Parisi et al., 2019;Sathoff and Samac, 2019) by destroying the structural integrity of bacterial cell membranes (Bun Ng et al., 2013;De Coninck et al., 2013;de Oliveira Dias and Franco, 2015). Precisely, defensins bind to the microbial cell membrane forming a pore-like channel in the membrane which cause ions and nutrients to leak through biphasic permeabilization (Jarczak et al., 2013). ...
Article
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Nearly all living species comprise of host defense peptides called defensins, that are crucial for innate immunity. These peptides work by activating the immune system which kills the microbes directly or indirectly, thus providing protection to the host. Thus far, numerous preclinical and clinical trials for peptide-based drugs are currently being evaluated. Although, experimental methods can help to precisely identify the defensin peptide family and subfamily, these approaches are often time-consuming and cost-ineffective. On the other hand, machine learning (ML) methods are able to effectively employ protein sequence information without the knowledge of a protein's three-dimensional structure, thus highlighting their predictive ability for the large-scale identification. To date, several ML methods have been developed for the in silico identification of the defensin peptide family and subfa-mily. Therefore, summarizing the advantages and disadvantages of the existing methods is urgently needed in order to provide useful suggestions for the development and improvement of new computational models for the identification of the defensin peptide family and subfamily. With this goal in mind, we first provide a comprehensive survey on a collection of six state-of-the-art computational approaches for predicting the defensin peptide family and subfamily. Herein, we cover different important aspects, including the dataset quality, feature encoding methods, feature selection schemes, ML algorithms, cross-validation methods and web server availability/usabi-lity. Moreover, we provide our thoughts on the limitations of existing methods and future perspectives for improving the prediction performance and model interpretability. The insights and suggestions gained from this review are anticipated to serve as a valuable guidance for researchers for the development of more robust and useful predictors.
... PR are usually hydrolytic enzymes such as chitinases, glucanases or peroxidases, etc., that attack the cell wall of the pathogens. Defensins are small proteins that show antimicrobial activity by disrupting the pathogens' membrane but their role is still controversial, since they have been involved in abiotic stress tolerance and plant developmental processes as well [11]. Moreover, when Trichoderma spp. ...
Article
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Here, we analyzed the effects on Capsicum annuum plants of Trichoderma atroviride P. Karst strains altered in the expression of SWOLLENIN (SWO1), a protein with amorphogenic activity on plant cell wall components. Strains of T. atroviride that overexpressed the Taswo1 gene were constructed as well as deletion mutants. A novel, cheap and accurate method for assessing root colonization was developed. Colonization assays showed that the Taswo1 overexpressing strains invaded the host root better than the WT, resulting in a stronger plant growth-promoting effect. The expression of plant defense marker genes for both the systemic acquired resistance and induced systemic resistance pathways was enhanced in plants inoculated with Taswo1 overexpressing strains, while inoculation with deletion mutant strains resulted in a similar level of expression to that observed upon inoculation with the wild-type strain. Response to pathogen infection was also enhanced in the plants inoculated with the Taswo1 overexpressing strains, and surprisingly, an intermediate level of protection was achieved with the mutant strains. Tolerance to abiotic stresses was also higher in plants inoculated with the Taswo1 overexpressing strains but was similar in plants inoculated with the wild-type or the mutant strains. Compatible osmolyte production in drought conditions was studied. This study may contribute to improving Trichoderma biocontrol and biofertilization abilities.
... The membrane-targeting mechanisms include either the embedding of peptides into the cell membrane and the formation of pores that result in cytoplasmic leakage and cell death (the pole model), or the arrangement of peptides parallel to the cell membrane surface and its destruction in a detergent-like manner (the carpet model) [13]. By contrast, the non-membrane-targeting peptides can enter host cells and induce cell death by inhibiting key metabolic processes such as protein translation, nucleic acid biosynthesis, protease activity, and cell division [13,32,33]. The mechanism of translocation of the latter class of AMPs across the membrane remains unclear. ...
Article
Fungicide use is one of the core elements of intensive agriculture because it is necessary to fight pathogens that would otherwise cause large production losses. Oomycete and fungal pathogens are kept under control using several active compounds, some of which are predicted to be banned in the near future owing to serious concerns about their impact on the environment, non-targeted organisms, and human health. To avoid detrimental repercussions for food security, it is essential to develop new biomolecules that control existing and emerging pathogens but are innocuous to human health and the environment. This review presents and discusses the use of novel low-risk biological compounds based on small RNAs and short peptides that are attractive alternatives to current contentious fungicides.
... Plant defensins possess many biological activities; among them, antifungal properties are the most studied (Cools et al. 2017;Parisi et al. 2019). All the recombinant defensins from conifers produced to date showed antifungal activity. ...
Article
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Main conclusion The recombinantPsDef5.1 defensin inhibits the growth of phytopathogenic fungi, Gram-positive and Gram-negative bacteria, and human pathogen Candida albicans. Expression of seed-derived Scots pine defensins is tissue-specific and developmentally regulated. Abstract Plant defensins are ubiquitous antimicrobial peptides that possess a broad spectrum of activities and multi-functionality. The genes for these antimicrobial proteins form a multigenic family in the plant genome and are expressed in every organ. Most of the known defensins have been isolated from seeds of various monocot and dicot species, but seed-derived defensins have not yet been characterized in gymnosperms. This study presents the isolation of two new 249 bp cDNA sequences from Scots pine seeds with 97.9% nucleotide homology named PsDef5.1 and PsDef5.2. Their deduced amino acid sequences have typical plant defensin features, including an endoplasmic reticulum signal sequence of 31 amino acids (aa), followed by a characteristic defensin domain of 51 aa. To elucidate the functional activity of new defensins, we expressed the mature form of PsDef5.1 in a prokaryotic system. The purified recombinant peptide exhibited activity against the phytopathogenic fungi and Gram-negative and Gram-positive bacteria with the IC50 of 5–18 µM. Moreover, it inhibited the growth of the human pathogen Candida albicans with the IC50 of 6.0 µM. Expression analysis showed that transcripts of PsDef5.1–2 genes were present in immature and mature pine seeds and different parts of seedlings at the early stage of germination. In addition, unlike the PsDef5.2, the PsDef5.1 gene was expressed in the reproductive organs. Our findings indicate that novel defensins are promising candidates for transgenic application and the development of new antimicrobial drugs.
... At the early stage, the mode of action was thought to only have electrostatic interactions between cationic peptides and negatively charged cell surface proteins. However, studies on anionic peptides revealed that they were also able to interact with the cell membrane by adopting α-helixes and β-sheets [153]. The nonspecific region targeting ability largely depends on the peptide's vibrant confirmation, with transitional stages taking place earlier or throughout the binding. ...
Article
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Even in a natural ecosystem, plants are continuously threatened by various microbial diseases. To save themselves from these diverse infections, plants build a robust, multilayered immune system through their natural chemical compounds. Among the several crucial bioactive compounds possessed by plantsʹ immune systems, antimicrobial peptides (AMPs) rank in the first tier. These AMPs are environmentally friendly, anti-pathogenic, and do not bring harm to humans. Antimi-crobial peptides can be isolated in several ways, but recombinant protein production has become increasingly popular in recent years, with the Escherichia coli expression system being the most widely used. However, the efficacy of this expression system is compromised due to the difficulty of removing endotoxin from its system. Therefore, this review suggests a high-throughput cDNA library-based plant-derived AMP isolation technique using the Bacillus subtilis expression system. This method can be performed for large-scale screening of plant sources to classify unique or ho-mologous AMPs for the agronomic and applied field of plant studies. Furthermore, this review also focuses on the efficacy of plant AMPs, which are dependent on their numerous modes of action and exceptional structural stability to function against a wide range of invaders. To conclude, the findings from this study will be useful in investigating how novel AMPs are distributed among plants and provide detailed guidelines for an effective screening strategy of AMPs.
... Antimicrobial peptides (AMPs) seem to be distributed ubiquitously in multicellular organisms. Plants have also been shown to contain a variety of different AMPs, including thionins [4,5] and plant defensins [6,7]. It is thought that the molecular targets of the majority of the usually basic AMPs are acidic phospholipids in biomembranes [8]. ...
Article
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Thionins are antimicrobial peptides found only in plants. They are first produced as preproproteins and then processed to yield the usually 5 kDa, basic thionin peptide with three or four disulfide bridges. So far, thionins had only been found in some plant families of angiosperms. The One Thousand Plant Transcriptomes Initiative (1KP project) has sequenced the transcriptomes of more than 1000 plant species. We have used these data to search for new thionin sequences which gave 225 hits. After removing doublets these resulted in 133 new thionins. No sequences were found in algae and mosses. The phylogenetically earliest hits were from Selaginella species and from conifers. Many hits were from angiosperm plant families which were previously not known to contain thionins. A large gene family for thionins was found in Papaver. We isolated a genomic clone from Papaver somniferum which confirmed the general genomic structure with two small introns within the acidic domain. We also expressed the thionin encoded by the genomic clone and found that it had antimicrobial activity in vitro, especially against fungi. Previously, we had grouped thionins into four classes. The new data reported here led us to revise this classification. We now recognize only class 1 thionins with eight cysteine residues and class 2 thionins with six cysteine residues. The different variants that we found (and also previously known variants) can all be traced back to one of these two classes. Some of the variants had an uneven number of cysteine residues and it is not clear at the moment what that means for their threedimensional structure.
... S. cerevisiae is also used in functional studies as a model organism [38]. In contrast, so-called plant γ-thionins, which have been classified into the defensin family, have anti-yeast activity [39]. This study is the first report of thionin activity against clinical yeast isolates. ...
Conference Paper
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Antimicrobial peptides in the thionin family are known to have inhibitory activity against a wide range of microorganisms and tumor cells. Here, we represent the results of a study on thionins that were isolated from black cumin (Nigella sativa L.). N. sativa is a common species that is used as a medicinal plant. Ten novel thionins (NsW 1–10) were isolated from N. sativa seeds using acetic acid extraction followed by liquid chromatographic fractionating. The molecules obtained were assigned to the thionin family according to their primary structure. Four of the molecules were tested in the functional study. Activity against three clinical Aspergillus species was detected for three of the tested peptides. Activity against Candida albicans and Gram-positive bacteria was also shown. C. albicans cell death occurred by disruption of cell membrane integrity. This mode of action on the yeast cells was confirmed by studying the effect of peptides on the tumor cells in vitro. Upon incubation of the peptides with four different cancer cell lines (AsPC-1, Colo357, RD, Jukart), a dose-dependent cytotoxic effect was found. The recombinant peptide NsW2 was obtained using heterologous expression in a prokaryotic model of the Escherichia coli Origami strain. The target peptide was obtained in association using a chimerical construction with the original E. coli protein thioredoxin, which provides normal peptide molecule folding. Insecticidal action of NsW 1-2 toward a mealworm (Tenebrio molitor) and a flesh fly (Sarcophaga carnaria) via direct injection to the hemolymph was detected. There was no cytotoxic effect toward the Sf9 insect cell line. Thus, there is likely another molecular mechanism of insectotoxicity that may be associated with neuromuscular transmission blocking or have specific activity on ion channels. This work was supported by the Russian Science Foundation (grant № 18-74-10073)
... NCR peptides are classified as defensin-like proteins. A representative defensin structure is composed of a double-or triple-stranded β-sheet and typically, an α-helix 30,31 . Normally, the loop between β-strands is responsible for the function of the defensin 30 . ...
Article
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A model legume, Medicago truncatula , has over 600 nodule-specific cysteine-rich (NCR) peptides required for symbiosis with rhizobia. Among them, NCR169, an essential factor for establishing symbiosis, has four cysteine residues that are indispensable for its function. However, knowledge of NCR169 structure and mechanism of action is still lacking. In this study, we solved two NMR structures of NCR169 caused by different disulfide linkage patterns. We show that both structures have a consensus C-terminal β-sheet attached to an extended N-terminal region with dissimilar features; one moves widely, whereas the other is relatively stapled. We further revealed that the disulfide bonds of NCR169 contribute to its structural stability and solubility. Regarding the function, one of the NCR169 oxidized forms could bind to negatively charged bacterial phospholipids. Furthermore, the positively charged lysine-rich region of NCR169 may be responsible for its antimicrobial activity against Escherichia coli and Sinorhizobium meliloti . This active region was disordered even in the phospholipid bound state, suggesting that the disordered conformation of this region is key to its function. Morphological observations suggested the mechanism of action of NCR169 on bacteria. The present study on NCR169 provides new insights into the structure and function of NCR peptides.
... From a structural point of view, defensins show a N-terminal loop and an α-helix region followed by an antiparallel β-sheet [30,31]. Several insect defensins showed antibacterial activity against both Gram-positive and Gram-negative bacteria [32,33], including Defensin B from Anomala cuprea [34], the defensin peptide isolated from Pyrrhocoris apterus [35], Coprisin from Copris tripartitus [36], Defensin 1 isolated from Acalolepta luxuriosa [37] and the Defensin from Bombus pascuorum [38]. Insect defensins may kill bacteria through a membranolytic mechanism leading to pore formation on the bacterial membranes or could interact with phospholipids inducing microheterogeneity in the membrane [39,40]. ...
Article
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Antibiotics are commonly used to treat pathogenic bacteria, but their prolonged use contributes to the development and spread of drug-resistant microorganisms raising the challenge to find new alternative drugs. Antimicrobial peptides (AMPs) are small/medium molecules ranging 10–100 residues synthesized by all living organisms and playing important roles in the defense systems. These features, together with the inability of microorganisms to develop resistance against the majority of AMPs, suggest that these molecules might represent effective alternatives to classical antibiotics. Because of their high biodiversity, with over one million described species, and their ability to live in hostile environments, insects represent the largest source of these molecules. However, production of insect AMPs in native forms is challenging. In this work we investigate a defensin-like antimicrobial peptide identified in the Hermetia illucens insect through a combination of transcriptomics and bioinformatics approaches. The C-15867 AMP was produced by recombinant DNA technology as a glutathione S-transferase (GST) fusion peptide and purified by affinity chromatography. The free peptide was then obtained by thrombin proteolysis and structurally characterized by mass spectrometry and circular dichroism analyses. The antibacterial activity of the C-15867 peptide was evaluated in vivo by determination of the minimum inhibitory concentration (MIC). Finally, crystal violet assays and SEM analyses suggested disruption of the cell membrane architecture and pore formation with leaking of cytosolic material.
Chapter
Bacterial resistance to conventional antibiotics is a growing issue, and the development of novel antimicrobial compounds has not been able to keep pace with the emergence of multi drug resistant pathogens. Antimicrobial peptides are source of inspiration for the development of next generation antimicrobial drugs; however, there are key limitations to the use of natural antimicrobial peptides, owing in large part to common pharmacological features that preclude their use outside of topical applications. The development of novel antimicrobial peptides and peptidomimetics via strategic chemical alterations is a means of improving on the functionality of natural antimicrobial peptides while tuning their pharmacological properties and in some instances, imparting novel mechanisms of action and functions outside of bactericidal activity as well.
Article
Insect defensins are effector components of the innate defense system. Defensins, which are widely distributed among insects, are a type of small cysteine-rich plant antimicrobial peptides with broad-spectrum antimicrobial activity. Here, the cDNAs of the black soldier fly, Hermetia illucens (L.), encoding six defensins, designated herein as Hidefensin1-1, 2, 3, 4, 5, 6. Moreover, Hidefensin1-1, 2, and 5 were identified for the first time by genome-targeted analysis. These Hidefensins were found to mainly adopt α-helix and β-sheet conformation homology as modeled by PRABI, Swiss-Model and ProFunc server. Six conserved cysteine residues that contribute to three disulfide bonds formed the spacing pattern “C-X12-C-X3-C-X9-C-X5-C-X-C”, which play a vital role in the molecular stability of Hidefensins. Phylogenetic analysis revealed that the homology of five Hidefensins (except Hidefensin4) was about 59%–92% compared with other insect defensins, indicating that they are novel antimicrobial peptides genes in black soldier fly. Furthermore, the Hidefensin1-1 was expressed in the Escherichia coli strain BL21(DE3) as a fusion protein with thioredoxin. Results showed that the purified TRX-Hidefensin1-1 exerted strong inhibitory effects against the Gram-positive bacteria Staphylococcus aureus and the Gram-negative bacteria Escherichia coli. The inhibitory efficacy of TRX-Hidefensin1-1 against Gram-positive bacteria was better than that against Gram-negative bacteria. These results indicated that Hidefensin1-1 has potent antimicrobial activities against test pathogens.
Article
Plant defensins (γ-thionins) are antimicrobial peptides (AMPs) constituting the host defense system. They are known to interact with the cell membranes to exhibit antifungal and antibacterial activity. Though defensin genes are inducible under biotic and abiotic stress, their involvement in insect herbivore defense is not well studied. In this work we studied the phylogeny, structural diversity, biochemical and functional properties of defensins from Capsicum annuum L with particular emphasis on their interaction with insect and fungal pathogens. Phylogenetic analysis of defensin peptides from Solanaceae, Pinaceae, Fabaceae, Asteraceae, Brassicaceae and similar peptides from invertebrates divided them into ten groups and Capsicum peptides were represented in most of them. Three thionin (CanThio-1, −2, −3) and two defensin (CanDef-20, −21) genes isolated from the flowers of Capsicum annuum were cloned and recombinant proteins were expressed. The mature peptide amino acid sequences of CanThio and CanDef were 54–75 aa with 21.82 to 73.81% similarity. They contained the conserved features of defensin peptides like the gamma core region and the presence of eight cysteine residues. Predicted structural alignment indicated that CanThio and CanDef had structural diversity and structurally unaligned loop regions. The recombinant CanThio and CanDef inhibited 5% - 65% Aspergillus oryzae amylase activity at 20 μM and ~ 20% of Helicoverpa armigera gut amylase activity at 50 μM peptide concentrations. CanThio-2 displayed bovine trypsin inhibition (~14%) while all the CanThio and CanDef inhibited ~20% of H. armigera gut proteinase activity at 20 μM. H. armigera larvae fed on an artificial diet incorporated with recombinant CanThio-2 and CanDef-20 (125 μg/ml) exhibited a 15% reduction in larval mass and 13% reduction in pupal mass. The CanThio and CanDef peptides also exhibited antifungal properties against pathogenic fungus Fusarium oxysporum at 5 μg/ml, where CanThio-2 and CanDef-20 showed the strongest activity, which may be attributed to the positively charged amino acids in the γ-core region. We conclude that despite having relatively similar conserved three-dimensional structures CanThio and CanDef display functional variability, which can be utilized for enhancing insect and fungal resistance in plants.
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Kentucky bluegrass (Poa pratensis L . ) is an excellent cool-season turfgrass utilized widely in Northern China. However, turf quality of Kentucky bluegrass declines significantly due to drought. Ethephon seeds-soaking treatment has been proved to effectively improve the drought tolerance of Kentucky bluegrass seedlings. In order to investigate the effect of ethephon leaf-spraying method on drought tolerance of Kentucky bluegrass and understand the underlying mechanism, Kentucky bluegrass plants sprayed with and without ethephon are subjected to either drought or well watered treatments. The relative water content and malondialdehyde conent were measured. Meanwhile, samples were sequenced through Illumina. Results showed that ethephon could improve the drought tolerance of Kentucky bluegrass by elevating relative water content and decreasing malondialdehyde content under drought. Transcriptome analysis showed that 58.43% transcripts (254,331 out of 435,250) were detected as unigenes. A total of 9.69% (24,643 out of 254,331) unigenes were identified as differentially expressed genes in one or more of the pairwise comparisons. Differentially expressed genes due to drought stress with or without ethephon pre-treatment showed that ethephon application affected genes associated with plant hormone, signal transduction pathway and plant defense, protein degradation and stabilization, transportation and osmosis, antioxidant system and the glyoxalase pathway, cell wall and cuticular wax, fatty acid unsaturation and photosynthesis. This study provides a theoretical basis for revealing the mechanism for how ethephon regulates drought response and improves drought tolerance of Kentucky bluegrass.
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Chronic wound infections have caused an increasing number of deaths and economic burden, which necessitates wound treatment options. Hitherto, the development of functional wound dressings has achieved reasonable progress. Antibacterial agents, growth factors, and miRNAs are incorporated in different wound dressings to treat various types of wounds. As an effective antimicrobial agent and emerging wound healing therapeutic, antimicrobial peptides (AMPs) have attracted significant attention. The present study focuses on the application of AMPs in wound healing and discusses the types, properties and formulation strategies of AMPs used for wound healing. In addition, the clinical trial and the current status of studies on “antimicrobial peptides and wound healing” are elaborated through bibliometrics. Also, the challenges and opportunities for further development and utilization of AMP formulations in wound healing are discussed. Nowadays, antimicrobial peptides (AMPs) are expected to be potential candidate therapeutics for infections and wound healing due to promising activities. Though AMP formulation strategies have achieved progresses in promoting wound healing therapy, several drawbacks and challenges still need to be overcome before these could have clinical and commercial application.
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Antimicrobial peptides (AMPs) are a class of short, usually positively charged polypeptides that exist in humans, animals, and plants. Considering the increasing number of drug-resistant pathogens, the antimicrobial activity of AMPs has attracted much attention. AMPs with broad-spectrum antimicrobial activity against many gram-positive bacteria, gram-negative bacteria, and fungi are an important defensive barrier against pathogens for many organisms. With continuing research, many other physiological functions of plant AMPs have been found in addition to their antimicrobial roles, such as regulating plant growth and development and treating many diseases with high efficacy. The potential applicability of plant AMPs in agricultural production, as food additives and disease treatments, has garnered much interest. This review focuses on the types of plant AMPs, their mechanisms of action, the parameters affecting the antimicrobial activities of AMPs, and their potential applications in agricultural production, the food industry, breeding industry, and medical field.
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In recent years, it has been recognized that epigenetic alterations play an important role in the development and maintenance of cancer, including leukemias. Furthermore, it is known that these alterations are involved in the emergence of resistance to conventional chemotherapeutics. Consequently, molecules with an anticancer activity whose activity is ruled by epigenetic modifications are attractive to search for new therapies against cancer. The plant antimicrobial peptides have been widely evaluated as molecules with anticancer activity; however, the analysis of the epigenetic regulation induced by these molecules associated with this activity is scarce and still is an unexplored field. In this work, we show that the PaDef defensin, a plant antimicrobial peptide from Mexican avocado fruit ( Persea americana var. drymifolia) is cytotoxic for Jurkat cell line from acute lymphoid leukemia cells, through an apoptotic process. PaDef inhibited cell viability in a concentration-dependent manner, with an IC 50 = 47.3 μM. Treatment of Jurkat cells with PaDef (IC 50 ) induced cell death by apoptosis dependent on caspases 8 and 9; besides, it was related to an increase in the production of reactive oxygen species and the loss of mitochondrial membrane potential. Interestingly, the inhibition of caspase activation by inhibitors of caspases 8 and 9 does not revert the reduction in viability, suggesting that other mechanisms, in addition to caspase activity, could be participating in the PaDef cytotoxic effect. Also, the modifications in the histone 3 tails induced by PaDef in Jurkat cells were evaluated, specifically acetylation and methylation. PaDef increased global histone 3 acetylation and lysine 9 specific marks (2-fold and up to 4-fold, respectively). These effects correlated with the reduction of the Histone Deacetylase activity (HDAC, ∼50%). Based on methylation marks, PaDef treatment increased lysine 9 di- and tri-methylation tags (2-fold in both cases). The epigenetic modulation induced by PaDef on Jurkat cells could be related to the chromatin compaction-decompaction promoting gene expression or repression; however, further studies are necessary to correlate these marks with the transcription of specific genes. Therefore, the study of new molecules that may have anticancer activity through epigenetic modulation is interesting.
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Clinical fungal infections always cause a negative impact on human health. Moreover, during the interaction of pathogenic fungi with the environment and host, many biologically active substances are produced. Here, we report a new toxin-like defensin of purlisin derived from a clinical pathogenic isolate of Purpureocillium lilacinum. The analysis of its genomic and mRNA sequences revealed an open reading frame of 444 bp without introns. The deduced precursor peptide was composed of 147 amino acids, and the mature peptide were identified at protein level by LC-ESI-Q-TOF-MS/MS. After posttranslational processing, the precursor peptide of purlisin was split into two independent peptides. The two mature defensins, purlisin-NT and purlisin-CT, are consisting of 36 and 38 amino acid residues, which can form three and four intramolecular disulfide bonds, respectively. The results of circular dichroism and homology modeling revealed that they adopted a representative cysteine-stabilized α-helical and β-sheet motif. The purlisin-NT showed a dose-dependent selective inhibition of immune-related hKv1.3 target channel with IC50 value of 0.2 ± 0.04 μM but no obvious antibacterial activity, while the purlisin-CT displayed antimicrobial activities against gram-positive bacteria as well as clinical isolates of MRSA and low affinities for potassium channels. Our findings suggest that purlisin-NT with immunosuppressive effects and purlisin-CT possessing antibacterial activities are adapted to the survival and pathogenicity of clinical P lilacinumis. Moreover, they can also be used as templates for the design of novel antibacterial peptide and immunosuppressive agents.
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Plant pathogens emerging as threat to human and animal health has been a matter of concern within the scientific community. Fusarium oxysporum, predominantly a phytopathogen, can infect both plants and animals. As a plant pathogen, F. oxysporum is one of the most economically damaging pathogen. In humans, F. oxysporum can infect immunocompromised individuals and is increasingly being considered as a problematic pathogen. Mycotoxins produced by F. oxysporum supress the innate immune pathways in both plants and animals. Hence, F. oxysporum is the perfect example for studying similarities and differences between defence strategies adopted by plants and animals. In this review we will discuss the innate immune response of plant and animal hosts for protecting against F. oxysporum infection. Such studies will be helpful for identifying genes, protein and metabolites with antifungal properties suitable for protecting humans.
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Chemical crop protection is widely used to control plant diseases. However, the adverse effects of pesticide use on human health and environment, resistance development and the impact of regulatory requirements on the crop protection market urges the agrochemical industry to explore innovative and alternative approaches. In that context, we demonstrate here the potential of camelid single domain antibodies (VHHs) generated against fungal glucosylceramides (fGlcCer), important pathogenicity factors. To this end, llamas were immunized with purified fGlcCer and a mixture of mycelium and spores of the fungus Botrytis cinerea, one of the most important plant pathogenic fungi. The llama immune repertoire was subsequently cloned in a phage display vector to generate a library with a diversity of at least 10⁸ different clones. This library was incubated with fGlcCer to identify phages that bind to fGlcCer, and VHHs that specifically bound fGlcCer but not mammalian or plant-derived GlcCer were selected. They were shown to inhibit the growth of B. cinerea in vitro, with VHH 41D01 having the highest antifungal activity. Moreover, VHH 41D01 could reduce disease symptoms induced by B. cinerea when sprayed on tomato leaves. Based on all these data, anti-fGlcCer VHHs show the potential to be used as an alternative approach to combat fungal plant diseases. © 2017 De Coninck, Verheesen, Vos, Van Daele, De Bolle, Vieira, Peferoen, Cammue and Thevissen.
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Fungi cause more than a billion skin infections, more than 100 million mucosal infections, 10 million serious allergies and more than a million deaths each year. Global mortality owing to fungal infections is greater than for malaria and breast cancer and is equivalent to that owing to tuberculosis (TB) and HIV. These statistics evidence fungal infections as a major threat to human health and a major burden to healthcare budgets worldwide. Those patients who are at greatest risk of life-threatening fungal infections include those who have weakened immunity or have suffered trauma or other predisposing infections such as HIV. To address these global threats to human health, more research is urgently needed to understand the immunopathology of fungal disease and human disease susceptibility in order to augment the advances being made in fungal diagnostics and drug development. Here, we highlight some recent advances in basic research in medical mycology and fungal immunology that are beginning to inform clinical decisions and options for personalized medicine, vaccine development and adjunct immunotherapies. This article is part of the themed issue ‘Tackling emerging fungal threats to animal health, food security and ecosystem resilience’.
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