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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... Among the pathogenesis-related (PR) proteins, antimicrobial peptides (AMPs) are essential weapons in the fight against plant microbial pathogens [1]. Defensins are cationic AMPs with a surprising conservation of structure and function across the animal and plant kingdoms [2,3]. The tertiary structure of plant defensins is characterized by the presence of a single alpha-helix and three antiparallel beta-sheets, which are stabilized by four disulfide bridges formed by eight conservative cysteine residues [4,5]. ...
... Defensins have been reported to be highly active against bacteria [7,8], fungi [2,[9][10][11][12], insects [13,14], and viruses [15]. The leakage caused by the interaction of defensins with the negatively charged membranes of pathogens is considered to be the main reason for their antibacterial and antifungal properties [3], but some authors have also suggested an active role played by the reactive oxygen species (ROS) produced inside the pathogen [2,5,12]. ...
... Moreover, besides their important role in innate host resistance, such a large number of genes supports the idea of different roles played by these AMPs. In fact, several plant peptides with novel and non-defense-related functions, including development and reproduction, derived from gene duplication and neo-functionalization events of AMPs, supporting this hypothesis [3,17,48,[54][55][56][57]. ...
... (2) Defensins and related small peptides/proteins Plant defensins and defensin-like proteins are a large group of small cysteine-rich proteins generally abundant in seeds and all plant organs. Class I defensins are synthesised in the cytoplasm with an N-terminal signal sequence and a mature defensin domain (Parisi et al., 2019) of about 4-6 kDa. Together with other small proteins, e.g. ...
... Together with other small proteins, e.g. lipid transfer proteins, thionins, glycine-rich peptides, cyclotides, systemin and protease inhibitors, they are constitutively expressed and partially allocated into the apoplastic space ( Fig. 1) with a broad spectrum of protective activities against fungi, bacteria, viruses and insects (Dang & Van Damme, 2015;Parisi et al., 2019;Farvardin et al., 2020;Mammari et al., 2021). Several possible protective mechanisms have been proposed for plant defensins, e.g. ...
... Production of some DNA fragments, peptides and systemin are triggered by wounding and herbivory attack, and the release of these small molecules into the apoplastic space is considered to constitute endogenous secondary danger signals (Erb & Reymond, 2019;Tanaka & Heil, 2021). Defensins, together with other peptides/proteins, also constitute a novel class of messengers involved in cell-to-cell communication and long-range signalling (Parisi et al., 2019;Takahashi & Shinozaki, 2019;Farvardin et al., 2020). Huang et al. (2008) found that the small cysteine-rich protein defensin SPD 1 (GenBank accession no. ...
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In their environment, plants are exposed to a multitude of abiotic and biotic stresses that differ in intensity, duration and severity. As sessile organisms, they cannot escape these stresses, but instead have developed strategies to overcome them or to compensate for the consequences of stress exposure. Defence can take place at different levels and the mechanisms involved are thought to differ in efficiency across these levels. To minimise metabolic constraints and to reduce the costs of stress defence, plants prioritise first‐line defence strategies in the apoplastic space, involving ascorbate, defensins and small peptides, as well as secondary metabolites, before cellular processes are affected. In addition, a large number of different symplastic mechanisms also provide efficient stress defence, including chemical antioxidants, antioxidative enzymes, secondary metabolites, defensins and other peptides as well as proteins. At both the symplastic and the apoplastic level of stress defence and compensation, a number of specialised transporters are thought to be involved in exchange across membranes that still have not been identified, and information on the regeneration of different defence compounds remains ambiguous. In addition, strategies to overcome and compensate for stress exposure operate not only at the cellular, but also at the organ and whole‐plant levels, including stomatal regulation, and hypersensitive and systemic responses to prevent or reduce the spread of stress impacts within the plant. Defence can also take place at the ecosystem level by root exudation of signalling molecules and the emission of volatile organic compounds, either directly or indirectly into the rhizosphere and/or the aboveground atmosphere. The mechanisms by which plants control the production of these compounds and that mediate perception of stressful conditions are still not fully understood. Here we summarise plant defence strategies from the cellular to ecosystem level, discuss their advantages and disadvantages for plant growth and development, elucidate the current state of research on the transport and regeneration capacity of defence metabolites, and outline insufficiently explored questions for further investigation.
... AFPs are small (45-57 amino acids) and cationic defensin-like proteins that are produced and secreted to the culture medium by filamentous ascomycetes and exhibit anti-fungal activity (Hegedüs and Marx 2013). In general, plant defensins and AFPs show inhibitory activity against both plant and human pathogens, mainly of fungal nature, but occasionally bacterial and in some cases against virus (Garrigues et al. 2018;Hajji et al. 2010;Huber et al. 2018;Sathoff and Samac 2019), and show no toxicity to plants or animal cells (Hegedüs and Marx 2013;Parisi et al. 2019b; van der Weerden and Anderson 2013). ...
... Similarly, in the radish Raphanus sativus, two defensins RsAFP1 and RsAFP2 have long been described and well characterized (Aerts et al. 2009(Aerts et al. , 2007Tavares et al. 2008;Thevissen et al. 2012;Vriens et al. 2016). The diversity and function of plant defensins have been extensively reviewed elsewhere (Kovaleva et al. 2020;Parisi et al. 2019b). ...
... Plant defensins share an eight cysteine-stabilized CSαβ motif. This motif is formed by a triple-stranded β-sheet linked to an α-helix by three disulfide bonds in the center of the structure and a fourth one formed between the first and the last cysteines, which bind the N-and C-terminal regions and render the protein pseudo cyclic (Almeida et al. 2002;Kovaleva et al. 2020;Parisi et al. 2019b) (Fig. 1C). The structural conservation of these proteins is reflected in the spacing and positions of the eight cysteines found in the amino acid sequence of plant defensins. ...
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Fungal infections represent a significant health risk worldwide. Opportunistic infections caused by yeasts, particularly by Candida spp. and their virulent emerging isolates, have become a major threat to humans, with an increase in fatal cases of infections attributed to the lack of effective anti-yeast therapies and the emergence of fungal resistance to the currently applied drugs. In this regard, the need for novel anti-fungal agents with modes of action different from those currently available is undeniable. Anti-microbial peptides (AMPs) are promising candidates for the development of novel anti-fungal biomolecules to be applied in clinic. A class of AMPs that is of particular interest is the small cysteine-rich proteins (CRPs). Among CRPs, plant defensins and anti-fungal proteins (AFPs) of fungal origin constitute two of the largest and most promising groups of CRPs showing anti-fungal properties, including activity against multi-resistant pathogenic yeasts. In this review, we update and compare the sequence, structure, and properties of plant defensins and AFPs with anti-yeast activity, along with their in vitro and in vivo potency. We focus on the current knowledge about their mechanism of action that may lead the way to new anti-fungals, as well as on the developments for their effective biotechnological production. Key points • Plant defensins and fungal AFPs are alternative anti-yeast agents • Their multi-faceted mode of action makes occurrence of resistance rather improbable • Safe and cost-effective biofactories remain crucial for clinical application
... This sequence variability accounts for the wide range of functions and mechanisms of action exhibited by different plant defensins. They include antimicrobial activity against Gram-negative and Gram-positive bacteria, parasites, viruses and fungi, protein synthesis inhibition through interactions with nucleic acids, trypsin and α-amylase inhibition which interfere with insect digestion, roles in heavy metal tolerance, plant development and sexual reproduction and the inhibition of ion channels in mammalian and plant cells [6][7][8]. Plant defensins are most commonly described as antifungal molecules, and even here, the sequence hypervariability contributes to at least six different mechanisms of action [8]. Defensins have seven loops, defined as the sequences between the four conserved disulphide bonds, which have a major role in the biological activity of defensins [6,8]. ...
... They include antimicrobial activity against Gram-negative and Gram-positive bacteria, parasites, viruses and fungi, protein synthesis inhibition through interactions with nucleic acids, trypsin and α-amylase inhibition which interfere with insect digestion, roles in heavy metal tolerance, plant development and sexual reproduction and the inhibition of ion channels in mammalian and plant cells [6][7][8]. Plant defensins are most commonly described as antifungal molecules, and even here, the sequence hypervariability contributes to at least six different mechanisms of action [8]. Defensins have seven loops, defined as the sequences between the four conserved disulphide bonds, which have a major role in the biological activity of defensins [6,8]. ...
... Plant defensins are most commonly described as antifungal molecules, and even here, the sequence hypervariability contributes to at least six different mechanisms of action [8]. Defensins have seven loops, defined as the sequences between the four conserved disulphide bonds, which have a major role in the biological activity of defensins [6,8]. Loop 5 is the flexible region between β-strands 2 and 3 that is essential for the specificity and function of plant defensins. ...
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Plant defensins are a large family of small cationic proteins with diverse functions and mechanisms of action, most of which assert antifungal activity against a broad spectrum of fungi. The partial mechanism of action has been resolved for a small number of members of plant defensins, and studies have revealed that many act by more than one mechanism. The plant defensin Ppdef1 has a unique sequence and long loop 5 with fungicidal activity against a range of human fungal pathogens, but little is known about its mechanism of action. We screened the S. cerevisiae non-essential gene deletion library and identified the involvement of the mitochondria in the mechanism of action of Ppdef1. Further analysis revealed that the hyperpolarisation of the mitochondrial membrane potential (MMP) activates ROS production, vacuolar fusion and cell death and is an important step in the mechanism of action of Ppdef1, and it is likely that a similar mechanism acts in Trichophyton rubrum.
... Defensins are small, cationic proteins rich in cysteine, present across various forms of life such as vertebrate and invertebrate animals, plants, and fungi. In humans, they are mainly located in leukocytes and epithelial cells, serving a crucial role in both innate and adaptive immune responses to combat microbial infections [1][2][3][4][5]. Defensins, despite not having fully understood molecular mechanisms, are key players in innate immune defense against infectious pathogens like viruses and bacteria [6][7][8].These short peptides, typically 29-34 amino acids in length, possess an amphipathic nature. ...
... Defensins, despite not having fully understood molecular mechanisms, are key players in innate immune defense against infectious pathogens like viruses and bacteria [6][7][8].These short peptides, typically 29-34 amino acids in length, possess an amphipathic nature. They can insert into membranes and trigger pore formation, leading to cell death through lysis [1][2][3][4][5][6][7][8]. ...
... α defensins are primarily produced by neutrophils (found in primary granules) and Paneth cells. They are initially produced and secreted in an inactive propeptide form, becoming active through proteolytic cleavage by trypsin [1][2][3][4][5]. Beta defensins are produced by epithelial cells outside the digestive tract, including those in the respiratory tract, integument, urogenital tract, and tongue [1][2][3][4][5]. ...
Article
In a comprehensive Molecular Docking investigation using Autodock Vina with the Pyrx program, six Human Defensins (Alpha Defensin-1, Alpha Defensin-3, Alpha Defensin-4, Beta-Defensin-1, Beta-Defensin-2, Beta Defensin-3) were studied alongside various natural compounds. Notably, all investigated compounds exhibited similar binding energy scores with Human Alpha Defensin-1 and Alpha Defensin-3, around-6.0 kcal/mol. However, with Human Defensin-4, Astringin, and Rhapontin, greater energetic affinities were observed, approximately-7.5 kcal/mol, compared to Polydatin and Eleutheroside-B. In the case of Beta-defensins, the natural compounds demonstrated higher binding capacities compared to Alpha defensins. Specifically, Polydatin exhibited a noteworthy binding affinity with Human Beta-Defensin-2, with a Vina Score of approximately-9.2 kcal/mol. Astringins and Rhapontis also showed significant binding energies with Human Beta-Defensin-2, with Vina scores of-8.8 kcal/mol and-8.6 kcal/mol, respectively. While these computational findings are preliminary and rely on a Blind Docking approach, they suggest that Polydatin and its derivatives may have a higher binding affinity for Beta-Defensins, particularly Beta-Defensin-2, providing a theoretical basis for further biological and molecular studies.
... Plants are facing a variety of environmental factors during their ontogenesis. Antimicrobial peptides (AMPs) represent an ancient molecular instrument that provides defense from biotic and abiotic stresses [1,2]. AMPs are known to participate in plant innate immunity. ...
... They participate in the molecular defense against phytopatogenic microorganisms and pests. Additionally, they are involved in physiological processes such as fertilization and fruit ripening [1,3,4]. Several peptides are shown to be a part of the defense against abiotic stress factors (cold, drought, and heavy metals) [1,3,5]. ...
... Additionally, they are involved in physiological processes such as fertilization and fruit ripening [1,3,4]. Several peptides are shown to be a part of the defense against abiotic stress factors (cold, drought, and heavy metals) [1,3,5]. ...
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Black cumin (Nigella sativa L.) is known to possess a wide variety of antimicrobial peptides belonging to different structural families. Three novel antimicrobial peptides have been isolated from black cumin seeds. Two of them were attributed as members of the non-specific lipid transfer proteins family, and one as a defensin. We have made an attempt of using the proteomic approach for novel antimicrobial peptides search in N. sativa seeds as well. The use of a well-established approach that includes extraction and fractionation stages remains relevant even in the case of novel peptides search because of the lacking N. sativa genome data. Novel peptides demonstrate a spectrum of antimicrobial activity against plant pathogenic organisms that may cause economically important crop diseases. These results obtained allow considering these molecules as candidates to be applied in "next-generation" biopesticides development for agricultural use.
... Considering Cys-rich antimicrobial proteins, defensins and thionins have been studied well. Defensins are cationic small proteins (< 10 kDa) produced by essentially all eukaryotes; they contain 6, 8, or 10 cysteine residues and share a similar three-dimensional structure, despite considerable sequence variation [47][48][49]. Two mushroom defensins, copsin isolated from the basidiomycete Coprinopsis cinerea [7,8,10] and plectasin from the ascomycete Pseudoplectania nigrella [11], show antibacterial activities toward Gram-positive bacteria: neither of them has sequence similarity to Cc-PRI3. ...
... Thionins are small (~5 kDa) proteins produced by plants; they contain 6 or 8 cysteine residues and are highly homologous in their primary structures and their three-dimensional structures [50]. The antimicrobial activities of defensins and thionins have been largely attributed to their ability to permeabilize and disrupt membranes, which initially occurs through association with bacterial or fungal cell membranes via electrostatic interactions between cationic charges on the proteins and anionic charges of microbial phospholipids [47][48][49][50][51]. In the antifungal activity of recombinant Cc-PRI3(37-95), the cationic residues (Lys-50, Arg-58, Lys-71, Arg-72, Arg-79, Lys-94) that are conserved among PRI3 proteins are likely involved in targeting to the fungal plasma membrane. ...
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Mushrooms are the fruiting bodies of fungi and are important reproductive structures that produce and disseminate spores. The Pri3 gene was originally reported to be specifically expressed in the primordia (a precursor to the mature fruiting body) of the edible mushroom Cyclocybe aegerita. Here, we cloned a Pri3‐related cDNA from Cyclocybe cylindracea, another species in the same genus, and showed that the gene is specifically expressed at the pileus surface of the immature fruiting body but not in the primordia. Immunohistochemistry showed that the translated protein is secreted into a polysaccharide layer of the pileus surface. The recombinant C‐terminal Cys‐rich domain of the protein showed antifungal activity against three filamentous fungi and inhibited hyphal growth and conidiogenesis. These results suggest that the PRI3‐related protein of C. cylindracea, named cylindracin, plays an important role in the defense against pathogens.
... They accumulate in cell walls and the extracellular space. In contrast, class II defensins, guided by a C-terminal pro-peptide, are directed to the vacuole, where they undergo proteolytic processing to release the mature defensin, subsequently being stored (Parisi et al. 2019). Distinct variations in the peptide surface loops and minor conformational changes in the tertiary structure confer diverse roles to different defensins. ...
... Distinct variations in the peptide surface loops and minor conformational changes in the tertiary structure confer diverse roles to different defensins. These roles encompass enzyme-inhibitory activities, heavy metal tolerance, inhibition of protein synthesis through interactions with nucleic acids, antibacterial effects, and modulation of plant development (van der Weerden and Anderson 2013; Parisi et al. 2019). ...
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Expression of antimicrobial peptides (AMPs) in economically significant plant species, particularly in popular crops, serves a dual purpose. Firstly, their antimicrobial properties can be harnessed for safeguarding plants in agriculture and, secondly, for the development of novel antimicrobial agents for medical applications. Moreover, the process of heterologous expression in plant-based systems, known as plant molecular farming, offers the advantage of yielding AMPs with proper eukaryotic folding and necessary posttranslational modifications, resulting in the production of biologically active substances. The emergence of second-generation biotechnology, enabling the isolation of genes governing desired traits and their precise modification or transfer into targeted varieties (such as intragenesis, cisgenesis, and genome editing), presents a valuable approach for enhancing plant defense responses to phytopathogens by boosting or altering the production of AMPs and defense-oriented phytochemicals. However, several other factors demand careful consideration. These include product yield, efficient extraction, evaluation of functionality and stability, appropriate storage procedures, and overall production costs. This chapter provides a comprehensive overview of AMPs generated in transgenic plants to confer resistance against phytopathogens. It also explores advancements in crop improvement through genome editing, specifically aiming to foster the production of AMPs for plant protection. Furthermore, this chapter highlights key technologies with the potential to significantly enhance the production and productivity of AMPs in agricultural settings.
... Defensins are a major family of plant AMPs harbouring broad-spectrum antifungal activity at low micromolar or submicromolar concentrations Parisi et al., 2019). More than 1200 plant defensin sequences are currently available in the Defensins Sequence Space database (Shafee & Anderson, 2018). ...
... Antifungal defensins use a multistep process to kill fungal pathogens. During the past two decades, the MoA of several plant defensins that bind to plasma membrane-resident phospholipids, induce plasma membrane permeabilization and gain entry into fungal cells have been studied Parisi et al., 2019). ...
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Due to rapidly emerging resistance to single‐site fungicides in fungal pathogens of plants, there is a burgeoning need for safe and multisite fungicides. Plant antifungal peptides with multisite modes of action (MoA) have potential as bioinspired fungicides. Medicago truncatula defensin MtDef4 was previously reported to exhibit potent antifungal activity against fungal pathogens. Its MoA involves plasma membrane disruption and binding to intracellular targets. However, specific biochemical processes inhibited by this defensin and causing cell death have not been determined. Here, we show that MtDef4 exhibited potent antifungal activity against Botrytis cinerea . It induced severe plasma membrane and organelle irregularities in the germlings of this pathogen. It bound to fungal ribosomes and inhibited protein translation in vitro. A MtDef4 variant lacking antifungal activity exhibited greatly reduced protein translation inhibitory activity. A cation‐tolerant MtDef4 variant was generated that bound to β‐glucan of the fungal cell wall with higher affinity than MtDef4. It also conferred a greater reduction in the grey mould disease symptoms than MtDef4 when applied exogenously on Nicotiana benthamiana plants, tomato fruits and rose petals. Our findings revealed inhibition of protein synthesis as a likely target of MtDef4 and the potential of its cation‐tolerant variant as a peptide‐based fungicide.
... Several mechanisms have been described for the cytosolic translocation of AFPs and other proteins across fungal cell walls and membranes. These include direct penetration, vacuolar localization and expansion, limited disruption of the plasma membrane, formation of transition pores, and endocytosis [26,27]. To ascertain whether AtTCP21 translocation into the cytosol was facilitated by membrane damage, we used a membraneimpermeable probe, SYTOX Green, which emits green fluorescence upon binding to nucleic acids and can only infiltrate cells when the membrane integrity is compromised or when antifungal agents create pores. ...
... Several mechanisms have been described for the cytosolic translocation of AFPs and other proteins across fungal cell walls and membranes. These include direct penetration, vacuolar localization and expansion, limited disruption of the plasma membrane, formation of transition pores, and endocytosis [26,27]. To ascertain whether AtTCP21 translocation into the cytosol was facilitated by membrane damage, we used a membrane-impermeable probe, SYTOX Green, which emits green fluorescence upon binding to nucleic acids and can only infiltrate cells when the membrane integrity is compromised or when antifungal agents create pores. ...
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The realm of antimicrobial proteins in plants is extensive but remains relatively uncharted. Understanding the mechanisms underlying the action of plant antifungal proteins (AFPs) holds promise for antifungal strategies. This study aimed to bridge this knowledge gap by comprehensively screening Arabidopsis thaliana species to identify novel AFPs. Using MALDI-TOF analysis, we identified a member of the TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1 (TCP) family of transcription factors as a novel AFP, A. thaliana TCP21 (AtTCP21; accession number NP_196450). Bacterially purified recombinant AtTCP21 inhibited the growth of various pathogenic fungal cells. AtTCP21 was more potent than melittin, a well-known AFP, in combating Colletotrichum gloeosporioides. Growth inhibition assays against various fungal pathogens and yeasts confirmed the pH-dependent antimicrobial activity of AtTCP21. Without inducing any membrane alterations, AtTCP21 penetrates the fungal cell wall and membrane, where it instigates a repressive milieu for fungal cell growth by generating intracellular reactive oxygen species and mitochondrial superoxides; resulting in morphological changes and apoptosis. Our findings demonstrate the redox-regulating effects of AtTCP21 and point to its potential as an antimicrobial agent.
... Defensin is a generic term encompassing PLANT DEFENSINs (PDFs) and PLANT DEFENSIN-LIKE (DEFLs) peptides, which are both integral parts of the plant immune system (Lay and Anderson, 2005;Stotz et al., 2013;Lacerda et al., 2014). Defensins are involved in a wide range of biological activities and physiological processes (Carvalho andGomes, 2009, 2011;Parisi et al., 2019); as such, they are downstream elements of these biotic and abiotic stress signalling cascades. PDFs and DEFLs are members of the large ANTIMICROBIAL PEPTIDES (AMPs) family (Van der Weerden and Anderson, 2013; Shafee et al., 2016aShafee et al., , 2017. ...
... The antifungal activity of defensins has been widely studied (Lay and Anderson, 2005;De Coninck et al., 2013;Lacerda et al., 2014;Parisi et al., 2019;Sher Khan et al., 2019). Defensins have been described to bind preferentially to lipid II (Wilmes et al., 2011), to the fungal acidic sphingolipids mannosyldiinositol phosphorylceramide [M(IP)2C; Thevissen et al., 2003], and the neutral sphingolipids glucosylceramide (GlcCer; Thevissen et al., 2004), to phosphatidic acid (PA; Sagaram et al., 2013;Kvansakul et al., 2016;Payne et al., 2016), and to phosphatidylinositol 4,5 bis-phosphate [PI(4,5)P2; Poon et al., 2014;Baxter et al., 2015]. ...
Article
Ectopic expression of defensins in plants correlates with their increased capacity to withstand abiotic and biotic stresses. This applies to Arabidopsis thaliana, where some of the seven members of the Plant Defensin 1 family (AtPDF1) are recognised to improve plant responses to necrotrophic pathogens and increase seedling tolerance to excess zinc (Zn). However, few studies have explored the effects of decreased endogenous defensin expression on these stress responses. Here, we carried out an extensive physiological and biochemical comparative characterisation of i) novel amiRNA lines silenced for the five most similar AtPDF1s, and ii) a double null mutant for the two most distant AtPDF1s. Silencing of five AtPDF1 genes was specifically associated with increased aboveground dry mass production in mature plants under Zn excess conditions, and with increased plant tolerance to different pathogens – one fungus, one oomycete and one bacterium, while the double mutant behaved similarly to the WT. These unexpected results challenge the current paradigm describing the role of PDFs in the plant response to stresses. Additional roles of plant endogenous defensins are discussed, which open new perspectives for their functions.
... The mechanism of action for defensins in Group 6 remains largely unknown [13], while. group 7 contains defensins such as NaD1 from Nicotiana alata, which function through the production of reactive oxygen species (ROS). ...
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Plant defensins are small antimicrobial proteins (AMP) that participate in the immune defense of plants through their antibacterial, antiviral and antifungal activities. PgD1 is a defensin from Picea glauca (Canadian Pine) and has antifungal activity against plant pathogens. This activity positions it as an alternative biotechnological agent to pesticides commonly used against these plant fungi diseases. The present study aimed to recombinantly produce PgD1 in Escherichia coli to characterize its in vitro antifungal potential against different phytopathogens. To achieve this, the coding gene was amplified and cloned into pET30a( +). Recombinant plasmid was subsequently introduced into E. coli for the soluble expression of defensin PgD1. To evaluate the antifungal activity of the expressed protein, the growth inhibition test was used in solid and liquid media for approximately 7 days against significant plant pathogens, that cause significant crop damage including: Botrytis cinerea, Colletotrichum gloeosporioides, Colletotrichum musae, Colletotrichum graminicola and Fusarium oxysporum. Additionally, stability assessments included temperature variation experiments and inhibition tests using dithiothreitol (DTT). The results showed that there was significant inhibition of the fungal species tested when in the presence of PgD1. Furthermore, defensin proved to be resistant to temperature variations and demonstrated that part of its stability is due to its primary structure rich in cysteine residues through the denaturation test with dithiothreitol (DTT) where the antifungal activity of PgD1 defensin was inhibited. These data indicate that recombinant PgD1 could be utilized as a plant protection technology in agriculture.
... However, they are best known for their antimicrobial activity against bacteria (Gram-positive and Gram-negative), fungi, viruses and parasites [148]. But their activity is not limited to a response to plant pathogens, and their mechanism of action ranges from the interaction with specific lipids to the generation of ROS to induction of programmed cell death [149]. ...
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Dynamic climate changes pose a significant challenge for plants to cope with numerous abiotic and biotic stressors of increasing intensity. Plants have evolved a variety of biochemical and molecular defense mechanisms involved in overcoming stressful conditions. Under environmental stress, plants generate elevated amounts of reactive oxygen species (ROS) and, subsequently, modulate the activity of the antioxidative enzymes. In addition, an increase in the biosynthesis of important plant compounds such as anthocyanins, lignin, isoflavonoids, as well as a wide range of low molecular weight stress-related proteins (e.g., dehydrins, cyclotides, heat shock proteins and pathogenesis-related proteins), was evidenced. The induced expression of these proteins improves the survival rate of plants under unfavorable environmental stimuli and enhances their adaptation to sequentially interacting stressors. Importantly, the plant defense proteins may also have potential for use in medical applications and agriculture (e.g., biopesticides). Therefore, it is important to gain a more thorough understanding of the complex biological functions of the plant defense proteins. It will help to devise new cultivation strategies, including the development of genotypes characterized by better adaptations to adverse environmental conditions. The review presents the latest research findings on selected plant defense proteins.
... This omission arises from their limited characterization thus far, with their enzymatic activity and mode of action yet to be elucidated (Ali et al., 2018(Ali et al., ). et al., 2015Parisi et al., 2019). PR6, PR12, PR13, and PR14, due to their low molecular weight and antimicrobial activity, are classified as antimicrobial peptides (Sels et al., 2008). ...
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Plants respond to pathogen exposure by activating the expression of a group of defense-related proteins known as Pathogenesis-Related (PR) proteins, initially discovered in the 1970s. These PR proteins are categorized into 17 distinct families, denoted as PR1-PR17. Predominantly secreted, most of these proteins execute their defensive roles within the apoplastic space. Several PR proteins possess well-defined enzymatic functions, such as β-glucanase (PR2), chitinases (PR3, 4, 8, 11), proteinase (PR7), or RNase (PR10). Enhanced resistance against pathogens is observed upon PR protein overexpression, while their downregulation renders plants more susceptible to pathogen infections. Many of these proteins exhibit antimicrobial activity in vitro, and due to their compact size, some are classified as antimicrobial peptides. Recent research has unveiled that phytopathogens, including nematodes, fungi, and phytophthora, employ analogous proteins to bolster their virulence and suppress plant immunity. This raises a fundamental question: how can these conserved proteins act as antimicrobial agents when produced by the host plant but simultaneously suppress plant immunity when generated by the pathogen? In this hypothesis, we investigate PR proteins produced by pathogens, which we term “PR-like proteins,” and explore potential mechanisms by which this class of virulence factors operate. Preliminary data suggests that these proteins may form complexes with the host’s own PR proteins, thereby interfering with their defense-related functions. This analysis sheds light on the intriguing interplay between plant and pathogen-derived PR-like proteins, providing fresh insights into the intricate mechanisms governing plant-pathogen interactions.
... It is worth mentioning that the process of disulphide reduction can transform the bactericidal activity of HBD1, which is initially modest, into a highly effective antimicrobial peptide that can effectively combat opportunistic pathogenic fungi and Gram-positive commensal bacteria [140]. There are also highly favourable evaluations of the antifungal efficacy of defensins, as documented by Ordonez et al. [118] and Parisi et al. [121]. ...
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Antimicrobial peptides (AMPs), also known as host defense peptides, are petite molecules with inherent microbicidal properties that are synthesized by the host’s innate immune response. These peptides serve as an initial barrier against pathogenic microorganisms, effectively eliminating them. Human defensin (HD) AMPs represent a prominent group of peptides involved in the innate immune response of humans. These peptides are primarily produced by neutrophils and epithelial cells, serving as a crucial defense mechanism against invading pathogens. The extensive research conducted has focused on the broad spectrum of antimicrobial activities and multifaceted immunomodulatory functions exhibited by human defensin AMPs. During the process of co-evolution between hosts and bacterial pathogens, bacteria have developed the ability to recognize and develop an adaptive response to AMPs to counterattack their bactericidal activity by different antibiotic-resistant mechanisms. However, numerous non-pathogenic commensal bacteria elicit the upregulation of defensins as a means to surmount the resistance mechanisms implemented by pathogens. The precise mechanism underlying the induction of HD by commensal organisms remains to be fully understood. This review summarizes the most recent research on the expression of human defensin by pathogens and discusses the various defense mechanisms used by pathogens to counter host AMP production. We also mention recent developments in the commensal induction of defensin AMPs. A better knowledge of the pathogens’ defensin AMP resistance mechanisms and commensals’ induction of AMP expression may shed light on the creation of fresh antibacterial tactics to get rid of bacterial infection.
... Genes from different sources have different functions, with some being induced by pathogenic bacteria while others are expressed only in specific plant tissues or organs (Ferreira et al. 2007;Li et al. 2021). At micromolar concentrations, plant defensins have antibacterial, antifungal, and insecticidal activities, can inhibit α-amylase, trypsin, and protein synthesis, and can block ion channels (Tam et al. 2015;Parisi et al. 2019;Sathoff and Samac, 2019). ...
... Disulfide linkages between the eight cysteine residues (C1-C8/C2-C5/C3-C6/C4-C7) offer great stability to chemical proteolysis and high temperatures and a conserved tertiary structure. 11 They have a broad spectrum of activity and are capable of targeting various pathogens, including fungi (both yeast and filamentous), protozoa, enveloped viruses, Gram-positive bacteria, and Gram-negative bacteria. [12][13][14] The pathogen-killing mechanisms of defensins are highly dependent on the interaction between the AMP structure and the pathogen. ...
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In the present study, we identified and characterized two defensin‐like peptides in an antifungal fraction obtained from Capsicum chinense pepper fruits and inhibited the growth of Colletotrichum scovillei, which causes anthracnose. AMPs were extracted from the pericarp of C. chinense peppers and subjected to ion exchange, molecular exclusion, and reversed‐phase in a high‐performance liquid chromatography system. We investigated the endogenous increase in reactive oxygen species (ROS), the loss of mitochondrial functioning, and the ultrastructure of hyphae. The peptides obtained from the G3 fraction through molecular exclusion chromatography were subsequently fractionated using reverse‐phase chromatography, resulting in the isolation of fractions F1, F2, F3, F4, and F5. The F1‐Fraction suppressed C. scovillei growth by 90, 70.4, and 44% at 100, 50, and 25 μg mL⁻¹, respectively. At 24 h, the IC50 and minimum inhibitory concentration were 21.5 μg mL⁻¹ and 200 μg mL⁻¹, respectively. We found an increase in ROS, which may have resulted in an oxidative burst, loss of mitochondrial functioning, and cytoplasm retraction, as well as an increase in autophagic vacuoles. MS/MS analysis of the F1‐Fraction indicated the presence of two defensin‐like proteins, and we were able to identify the expression of three defensin sequences in our C. chinense fruit extract. The F1‐Fraction was also found to inhibit the activity of insect α‐amylases. In summary, the F1‐Fraction of C. chinense exhibits antifungal activity against a major pepper pathogen that causes anthracnose. These defensin‐like compounds are promising prospects for further research into antifungal and insecticide biotechnology applications. © 2024 Society of Chemical Industry.
... The peak SNP is near (2 Mbp) two adjacent defensin-like proteins, Zm00001eb247810 (Def4, Defensin-4) and Zm00001eb247820 (Def1, Defensin-1), as well as near (∼5 Mbp) an LRR family protein (Zm00001eb247140). Plant defensins are components of the plant innate immune system and have a wide range of functions such as inhibiting the growth of a broad range of fungi, bacteria, and insects (Mulla et al. 2021;Parisi et al. 2019). Although most R-genes contain a NBS and an LRR region (Williamson and Kumar 2006), proteins without the NBS have been involved in nematode resistance. ...
Article
This study provides the first report of a quantitative trait locus (QTL) in maize (Zea mays) for resistance to the southern root-knot nematode (SRKN) (Meloidogyne incognita). The SRKN can feed on the roots of maize in the U.S. Southern Coastal Plain region and can cause yield losses of 30% or greater in heavily infested fields. Increases in SRKN density in the soil may reduce the yield for subsequently planted susceptible crops. The use of maize hybrids with resistance to SRKN could prevent an increase in SRKN density, yet no genetic regions have been identified that confer host resistance. In this study, a B73 (susceptible) x Ky21 (resistant) S5 recombinant inbred line (RIL) population was phenotyped for total number of eggs (TE) and root weight. This population has been previously genotyped using single nucleotide polymorphisms (SNPs). By utilizing the SNP data with the phenotype data, a single QTL was identified on chromosome 5 that explained 15% of the phenotypic variation (PV) for the number of eggs and 11% of the PV for the number of eggs per g of root (EGR). Plants that were homozygous for the Ky21 allele for the most associated marker PZA03172.3 had fewer eggs and fewer EGR than the plants that were homozygous or heterozygous for the B73 allele. Thus, the first QTL for SRKN resistance in maize has been identified and could be incorporated into maize hybrids.
... Plant AMPs has been divided into eight groups, including snakins, cyclotides, thionins, knottins, lipid transfer proteins, heveins, a-hairpinins, and defensins based on their structure (Goyal and Mattoo, 2016;Srivastava et al., 2021). Defensins are large class of AMPs and are mainly found in invertebrates, mammals, and plants (Gerdol et al., 2020;Parisi et al., 2019;Xu and Lu, 2020). Plant defensins are a large and diverse family of plant AMPs. ...
... β-defensin peptides, secreted as components of the innate immune system in various organisms, are one of the best-described groups of antimicrobial peptides from a large number of plants, animals, and fungi for host defense [40,41]. However, the β-defensin peptides subfamily has been converted into neurotoxins in several animals to block ion channels [42]. ...
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The venoms of various sea anemones are rich in diverse toxins, which usually play a dual role in capturing prey and deterring predators. However, the complex components of such venoms have not been well known yet. Here, venomics of integrating transcriptomic and proteomic technologies was applied for the first time to identify putative protein and peptide toxins from different tissues of the representative sea anemone, Heteractis magnifica. The transcriptomic analysis of H. magnifica identified 728 putative toxin sequences, including 442 and 381 from the tentacles and the column, respectively, and they were assigned to 68 gene superfamilies. The proteomic analysis confirmed 101 protein and peptide toxins in the venom, including 91 in the tentacles and 39 in the column. The integrated venomics also confirmed that some toxins such as the ShK-like peptides and defensins are co-expressed in both the tentacles and the column. Meanwhile, a homology analysis was conducted to predict the three-dimensional structures and potential activity of seven representative toxins. Altogether, this venomics study revealed the venom complexity of H. magnifica, which will help deepen our understanding of cnidarian toxins, thereby supporting the in-depth development of valuable marine drugs.
... Что касается регуляторного действия, то применительно к АМП растительного происхождения оно может достигаться посредством опосредованного влияния на организм-мишень, а также через физиолого-биохимические перестройки у растения-реципиента (например, регуляция обменных процессов Рисунок 2. Направления реализации биологических функций АМП растений в аспекте их потенциального применения в качестве средств защиты растений Figure 2. Directions for the biological functions implementation of plant AMPs in case of their potential application as plant protection products На уровне воздействия на патоген речь идет о тех АМП, которые способны к интернализации внутрь клетки-мишени с последующей активацией киназных сигнальных каскадов, активации активных форм кислорода и инициации апоптоза (дефензины) или ингибированием биосинтеза белка (альфа-харпинины) (Citores et al., 2016;Zhang et al., 2019). В случае опосредованного воздействия через само растение для некоторых АМП показана индукция запуска иммунной системы ("systemic acquired resistance") индивидуально или путем совместного применения с некоторыми сигнальными молекулами (салициловая кислота, метилжасмонат), что в большинстве случаев приводит к активации реакции сверхчувствительности и апоптозу (дефензины, тионины, гевеино-подобные пептиды, липид-переносящие белки, циклотиды) (Goyal et al., 2014;Sher Khan et al., 2019;Odintsova et al., 2019;Parisi et al., 2019). Таким образом, данная группа пептидов может позиционироваться в качестве препаратов регуляторного действия. ...
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Antimicrobial peptides (AMPs) are the most important components of plant innate immunity to environmental stress factors and one of the most ancient tools of the defense system. Most of them are synthesized as factors of constitutive plant immunity, but there are also inducible forms belonging to “pathogenesis-related proteins” (PR-proteins from classes 12, 13 and 14). This review provides characteristics of the primary and three-dimensional structures of the main families of plant AMPs. The relationship between the types of spatial arrangement of the polypeptide chain is drawn. The functional analysis of plant AMPs is presented by data on the spectrum and quantitative level of activities against a number of economically significant fungal and bacterial phytopathogens, and related biological effects are indicated. Additionally, current information is provided on the molecular mechanisms of the antimicrobial action of plant AMPs based on defensins as the most studied structural group. In conclusion, aspects of the modes of action for plant AMPs on microorganisms are considered, on the basis of which a variant of the functional classification of these molecules is proposed. Based on these data, the prospects for their use as the basis of biopesticides for plant protection against pathogens were assessed.
... The mechanism of plant disease resistance is the existence of resistance genes in plants, which can resist the invasion and spread of pathogens. These disease resistance genes are divided into physical resistance and chemical resistance factors (Miller et al. 2017;Parisi et al. 2019;Wang et al. 2020;Ninkuu et al. 2022). Therefore, the VQ gene should ultimately trigger at least one of these two disease resistance mechanisms, resulting in changes in plant immunity. ...
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Main conclusion This review provides a detailed description of the function and mechanism of VQ family gene, which is helpful for further research and application of VQ gene resources to improve crops. Abstract Valine-glutamine (VQ) motif-containing proteins are a large class of transcriptional regulatory cofactors. VQ proteins have their own unique molecular characteristics. Amino acids are highly conserved only in the VQ domain, while other positions vary greatly. Most VQ genes do not contain introns and the length of their proteins is less than 300 amino acids. A majority of VQ proteins are predicted to be localized in the nucleus. The promoter of many VQ genes contains stress or growth related elements. Segment duplication and tandem duplication are the main amplification mechanisms of the VQ gene family in angiosperms and gymnosperms, respectively. Purification selection plays a crucial role in the evolution of many VQ genes. By interacting with WRKY, MAPK, and other proteins, VQ proteins participate in the multiple signaling pathways to regulate plant growth and development, as well as defense responses to biotic and abiotic stresses. Although there have been some reports on the VQ gene family in plants, most of them only identify family members, with little functional verification, and there is also a lack of complete, detailed, and up-to-date review of research progress. Here, we comprehensively summarized the research progress of VQ genes that have been published so far, mainly including their molecular characteristics, biological functions, importance of VQ motif, and working mechanisms. Finally, the regulatory network and model of VQ genes were drawn, a precise molecular breeding strategy based on VQ genes was proposed, and the current problems and future prospects were pointed out, providing a powerful reference for further research and utilization of VQ genes in plant improvement.
... It is expected to also reduce the development of resistance because dead cells cannot mutate to become resistant to the antifungal agent. Defensins are thought to act on multiple cellular targets to exert their mechanism of action [12,42,43], further decreasing the likelihood of resistance developing. A pilot study comparing the development of resistance to the plant defensin NaD1 and caspofungin confirmed this hypothesis [44]. ...
Article
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Onychomycosis, or fungal nail infection, causes not only pain and discomfort but can also have psychological and social consequences for the patient. Treatment of onychomycosis is complicated by the location of the infection under the nail plate, meaning that antifungal molecules must either penetrate the nail or be applied systemically. Currently, available treatments are limited by their poor nail penetration for topical products or their potential toxicity for systemic products. Plant defensins with potent antifungal activity have the potential to be safe and effective treatments for fungal infections in humans. The cystine-stabilized structure of plant defensins makes them stable to the extremes of pH and temperature as well as digestion by proteases. Here, we describe a novel plant defensin, Ppdef1, as a peptide for the treatment of fungal nail infections. Ppdef1 has potent, fungicidal activity against a range of human fungal pathogens, including Candida spp., Cryptococcus spp., dermatophytes, and non-dermatophytic moulds. In particular, Ppdef1 has excellent activity against dermatophytes that infect skin and nails, including the major etiological agent of onychomycosis Trichophyton rubrum. Ppdef1 also penetrates human nails rapidly and efficiently, making it an excellent candidate for a novel topical treatment of onychomycosis.
... It was shown that the solution structure of radish defensin (Rs-AFP1) by nuclear magnetic resonance (NMR) consists of three reverse parallel b-folded lamellae and one a-helix, forming a stable babbb structure with four pairs of disulfide bonds acting as support, and the Csab modal sequence (Cysteine stabilized ab motif) was found, fixing a pair of disulfide bonds on the lamellae in the a-helix and b-fold (Vriens et al., 2016). The bfolded lamellar signature sequence (Cys-Xaa-Cys) and the a-helical signature sequence (Cys-Xaa-Xaa-Xaa-Cys) are common to most plant defensins (Parisi et al., 2019). The Cysteine stabilized ab motif indicates that plant defensins are more structurally stable and can be stably expressed in many plants. ...
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Plant defensins are widely distributed in the leaves, fruits, roots, stems, seeds, and tubers. Research shows that defensin in plants play a significant role in physiological metabolism, growth and development. Plant defensins can kill and suppress a variety of pathogenic bacteria. In this study, we understand the phylogenetic relationships, protein characterization, chromosomal localization, promoter and gene structural features of the TaPDFs family through sequence alignment and conserved protein structural domain analysis. A total of 73 PDF gene members in wheat, 15 PDF genes in maize, and 11 PDF genes in rice were identified. A total of 35, 65, and 34 PDF gene members were identified in the genomes of Ae. tauschii, T. urartu, and T. dicoccoides, respectively. TaPDF4.9 and TaPDF2.15 were constructed into pART27 vector with YFP by homologous recombination for subcellular localization analysis. Subcellular localization results showed that TaPDF4.9 and TaPDF2.15 were basically located in the cell membrane and cytoplasm, and TaPDF4.9 was also located in the nucleus. TaPDF4.9 and TaPDF2.15 could inhibit the infection of Phytophthora infestans strain ‘88069’. The results suggest that TaPDFs may be able to improve disease resistance. The study of wheat defensins will be beneficial for improving wheat yield and provides a theoretical basis for research on resistance to wheat diseases.
... Defensins comprise a large family of antifungal peptides expressed in plants. Several of these peptides have been reported to exhibit broad-spectrum antifungal activity against economically important fungal pathogens of crops [11,12,16,33,34]. However, only one plant defensin, RsAFP1, has been reported to be active against this pathogen at 20 µg/mL (3.5 µM) [15]. ...
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White mold disease caused by a necrotrophic ascomycete pathogen Sclerotinia sclerotiorum results in serious economic losses of soybean yield in the USA. Lack of effective genetic resistance to this disease in soybean germplasm and increasing pathogen resistance to fungicides makes white mold difficult to manage. Small cysteine-rich antifungal peptides with multi-faceted modes of action possess potential for development as sustainable spray-on bio-fungicides. We have previously reported that GMA4CG_V6 peptide, a 17-amino acid variant of the MtDef4 defensin-derived peptide GMA4CG containing the active γ-core motif, exhibits potent antifungal activity against the gray mold fungal pathogen Botrytis cinerea in vitro and in planta. GMA4CG_V6 exhibited antifungal activity against an aggressive field isolate of S. sclerotiorum 555 in vitro with an MIC value of 24 µM. At this concentration, internalization of this peptide into fungal cells occurred prior to discernible membrane permeabilization. GMA4CG_V6 markedly reduced white mold disease symptoms when applied to detached soybean leaves, pods, and stems. Its spray application on soybean plants provided robust control of this disease. GMA4CG_V6 at sub-lethal concentrations reduced sclerotia production. It was also non-phytotoxic to soybean plants. Our results demonstrate that GMA4CG_V6 peptide has potential for development as a bio-fungicide for white mold control in soybean.
... The most extensively studied function of plant AMPs is their ability to protect plants from pathogenic microorganisms, making them potential candidates for novel pesticides in agriculture and food preservatives [6]. Whilethe regulatory functions of AMPs have been described, they are not as well-studied except for defensins, which have been examined in the contexts of both plants and pathogens [7]. Plant thionins are recognized as membrane-active peptides that disrupt cell membranes and induce cell death [8]. ...
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This study aimed to obtain a recombinant chimeric protein named trx-NsW2 via theheterologous expression of the multifunctional antimicrobial peptide nigellothionin from black cumin (Nigella sativa L.) seeds in the Escherichia coli system. The protein was purified using a combination of Ni-NTA affinity chromatography and reversed-phase HPLC. Based on the HPLC calibration, the total yield of the protein was calculated to be 650 mg/L of bacterial culture. The fungistatic activity of trx-NsW2 against the food-spoiling fungus Aspergillus niger was demonstrated as itinhibited the maturation of conidiawithout affecting conidial germination or fungal growth. In contrast to mature nigellothionin NsW2, the fusion protein showeda low level of cytotoxicity towards both normal and tumor cell lines at concentrationsof up to 100–200 µM. Interestingly, at lower concentrations, it even stimulated cytokinesis. These findings are of critical importance for applying chimeric antimicrobial proteins obtained via microbiological synthesis in applied science.
... In addition, it lacks a large part of the light chain and the trypsin and chymotrypsin (RCL)-binding site, which explains why CaCPin-II does not inhibit the activity of these enzymes. Plant defensins play a role in both the response against pathogens and the control of plant growth and development [64], and their antifungal activity has been extensively described [19,65]. The defensin signatures are a cysteine-stabilized αβ motif (CSαβ) and a γ-core motif GXC(X3-9)C [66], and peptides that contain variations in this typical structure are called defensin-like [67,68], such as CaCDef-like peptide. ...
Article
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The emergence of resistant microorganisms has reduced the effectiveness of currently available antimicrobials, necessitating the development of new strategies. Plant antimicrobial peptides (AMPs) are promising candidates for novel drug development. In this study, we aimed to isolate, characterize, and evaluate the antimicrobial activities of AMPs isolated from Capsicum annuum. The antifungal potential was tested against Candida species. Three AMPs from C. annuum leaves were isolated and characterized: a protease inhibitor, a defensin-like protein, and a lipid transporter protein, respectively named CaCPin-II, CaCDef-like, and CaCLTP2. All three peptides had a molecular mass between 3.5 and 6.5 kDa and caused morphological and physiological changes in four different species of the genus Candida, such as pseudohyphae formation, cell swelling and agglutination, growth inhibition, reduced cell viability, oxidative stress, membrane permeabilization, and metacaspase activation. Except for CaCPin-II, the peptides showed low or no hemolytic activity at the concentrations used in the yeast assays. CaCPin-II inhibited α-amylase activity. Together, these results suggest that these peptides have the potential as antimicrobial agents against species of the genus Candida and can serve as scaffolds for the development of synthetic peptides for this purpose.
... The subcellular localization of plant defensins thus appears to be crucial in understanding their dual action on plant cells. Although the mechanism of action of plant defensins in fungi growth inhibition is not completely clear, there have been several papers published on the subject (Thevissen et al. 2003;Vriens et al. 2014;Cools et al. 2017;Parisi et al. 2019;Sher Khan et al. 2019;Struyfs et al. Transgenic Res 2021). ...
Article
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Plant defensins are a potential tool in crop improvement programs through biotechnology. Their antifungal action makes them attractive molecules for the production of transgenic plants. Information is currently lacking on what happens to the expression of defense genes in transgenic plants that overexpress a defensin. Here we show the relative expression of four defense-related genes: Mn-sod, PAL1, aos1 and HPL evaluated in two transgenic soybean events (Def1 and Def17) constitutively expressing the NmDef02 defensin gene from Nicotiana megalosiphon. The expression of these defense genes showed a differential profile in the transgenic events, with the increased expression of the aos1 gene and the repression of the Mn-sod gene in both events, when compared to the non-transgenic control. Furthermore, the expression of the PAL1 gene only increased in the Def17 event. The results indicate that although there were some changes in the expression of defense genes in transgenic plants overexpressing the defensin NmDef02; the morphoagronomic parameters evaluated were similar to the non-transgenic control. Understanding the molecular changes that occur in these transgenic plants could be of interest in the short, medium and long term.
... Defensins, in particular, are one of the largest families of AMPs expressed in plants. Because of their potent antifungal activity, diverse amino acid sequences, and multitarget modes of action (MoA), plant defensins have emerged as potential antifungal agents for agriculture (Cools et al., 2017;Leannec-Rialland et al., 2022;Parisi et al., 2019). Plant defensins are 45-to 54-amino-acid, cysteine-rich, cationic peptides characterized by the presence of a cysteine-stabilized αβ motif. ...
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Chemical fungicides have been instrumental in protecting crops from fungal diseases. However, increasing fungal resistance to many of the single-site chemical fungicides calls for the development of new antifungal agents with novel modes of action (MoA). The sequence-divergent cysteine-rich antifungal defensins with multisite MoA are promising starting templates for design of novel peptide-based fungicides. Here, we experimentally tested such a set of 17-amino-acid peptides containing the γ-core motif of the antifungal plant defensin MtDef4. These designed peptides exhibited antifungal properties different from those of MtDef4. Focused analysis of a lead peptide, GMA4CG_V6, showed that it was a random coil in solution with little or no secondary structure elements. Additionally, it exhibited potent cation-tolerant antifungal activity against the plant fungal pathogen Botrytis cinerea, the causal agent of grey mould disease in fruits and vegetables. Its multisite MoA involved localization predominantly to the plasma membrane, permeabilization of the plasma membrane, rapid internalization into the vacuole and cytoplasm, and affinity for the bioactive phosphoinositides phosphatidylinositol 3-phosphate (PI3P), PI4P, and PI5P. The sequence motif RRRW was identified as a major determinant of the antifungal activity of this peptide. While topical spray application of GMA4CG_V6 on Nicotiana benthamiana and tomato plants provided preventive and curative suppression of grey mould disease symptoms, the peptide was not internalized into plant cells. Our findings open the possibility that truncated and modified defensin-derived peptides containing the γ-core sequence could serve as promising candidates for further development of bio-inspired fungicides.
... Peptides also can be arranged parallel to the cell membrane surface causing destruction in a detergent-like manner (the carpet model) (Huan et al., 2020). Non-membrane-targeting peptides enter host cells and control pathogen growth by inhibiting key metabolic processes such as protein translation, nucleic acid biosynthesis, protease activity, and cell division (Huan et al., 2020;Lee et al., 2015;Parisi et al., 2019). ...
Chapter
Human crop production has benefitted from developments in the use of natural and formulated chemical substances to control pathogens and insect herbivores. However, numerous adverse effects of exposure to these chemical substances, pesticides, have been documented in a variety of nontarget organisms. Of particular concern are the negative impacts of pesticide exposure on agriculturally important insect pollinators. While the general effects of pesticides on pollinator health have garnered much interest, the potential role of certain pesticide classes has historically been poorly understood and investigated. Despite their ubiquity in the foraging environment, fungicides were traditionally deemed to be safe for pollinating insects based on low toxicity outcomes in standardized assessments by regulatory agencies. Recently, multiple studies have dispelled this traditional designation by demonstrating numerous sublethal and lethal outcomes for pollinators exposed to various fungicides. Here we provide an overview of the historical underpinnings of fungicide development and application, as well as trends in the implementation of regulatory measures. We discuss exposure routes and the prevalence of fungicides in the environment. Finally, we explore the growing body of literature revealing negative effects of exposure including the specific mechanisms by which fungicides act on non-target pollinators, including fungicide synergisms with other pesticide classes, pests, pathogens and phytochemicals, and fungicide-induced behavioural alterations.
... Plant defensins can cause cellular effects including generation of reactive oxygen species (ROS), which triggers secondary signaling and imbalance of ionic homeostasis, ultimately contributing to cell death 65 . SOD enzyme helps in combating ROS generation 66 . ...
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Lepidopteran insect pest Helicoverpa armigera is one of the most destructive pests of crop plants and several biotechnological approaches are being developed for its control. Plant defensins are small cationic and cysteine-rich peptides that play a role in plant defense. Ingestion of a defensin from Capsicum annuum (CanDef-20) induced a dose-dependent reduction in larval and pupal mass, delayed metamorphosis and also severely reduced fecundity and fertility in H. armigera. To understand the molecular mechanisms of CanDef-20 ingestion-mediated antibiosis in H. armigera larvae, a comparative transcriptomics analysis was carried out. Predominant downregulation of GOs represents serine-type endopeptidases, structural constituents of ribosomes and integral membrane components and differential upregulation of ATP binding, nucleus and translation, while up-regulation of nucleic acid binding represented by transposable elements, were detected. Different isoforms of lipase, serine endopeptidase, glutathione S-transferase, cadherin, alkaline phosphatase and aminopeptidases were found to be upregulated as a compensatory response to CanDef-20 ingestion. In vitro enzyme assays and qPCR analysis of some representative genes associated with vital cellular processes like metamorphosis, food digestion and gut membrane indicated adaptive differential regulations in CanDef-20 fed H. armigera larvae. We conclude that CanDef-20 ingestion affects insect metabolism in a number of ways through its interaction with cell membrane, enzymes, cytoplasmic proteins and triggering transposon mobilization which are linked to growth retardation and adaptive strategies in H. armigera.
... Ribosome inactivating proteins (such as ricin) showing N-glycosidase activity have been identified in several edible plants, including pumpkin, cucumber, beet, and cereals (Dang and Van Damme, 2015;Parisi et al., 2018;Vandenborre et al., 2011). They act as glycosidases that remove a specific adenine residue from an exposed loop of the 28S rRNA (A4324 in mammals), leading to rRNA breakage. ...
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This report is the outcome of an EFSA procurement (NP/EFSA/GMO/2018/01) reviewing relevant scientific information on in silico prediction methods for protein toxicity, that could support the food and feed risk assessment. Several proteins are associated with adverse (toxic) effects in humans and animals, by a variety of mechanisms. These are produced by plants, animals and bacteria to prevail in hostile environments. In the present report, we present an integrated pipeline to perform a comprehensive literature and database search applied to proteins with toxic effects. “Toxin activity” and “toxin‐antitoxin” system strings were used as inputs for this pipeline. UniProtKB was considered as the reference database, and only the UniProtKB curator‐reviewedproteins were considered in the pipeline. Experimentally‐determinedstructures and homology‐based in silico3D models were retrieved from protein structures repositories; family‐, domain‐, motif‐ and other molecular signature‐related information was also obtained from specific databases which are part of the InterPro consortium. Protein aggregation associated with adverse effects was also investigated using different search strategies.This work can serve as the basis for further exploring novel risk assessment strategies for new proteins using in silico predictive methods.
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The Acari Hypothesis posits that acarians, i.e., mites and ticks, are operative agents of allergy. It derived from observations that allergens are molecular elements of acarians or acarian foodstuffs. A corollary of The Hypothesis provides how acarian dietary elements are selected as allergens; namely, a pattern recognition receptor native to the acarian digestive tract complexes with dietary molecules problematic to the acarian. By virtue of its interspecies operability, the receptor then enables not only removal of the dietary elements by the acarian immune system, but also—should such a complex be inoculated into a human—production of an element-specific IgE. Because pattern recognition receptors bind to molecules problematic to the organism from which the receptors originate, it follows that molecules targeted by adaptive IgE, i.e., allergens, must be problematic to acarians. This claim is supported by evidence that host organisms, when infested by acarians, upregulate representative members of allergenic molecular families. Appreciation of the relationship between allergens and acarians provides insight well beyond allergy, shedding light also on the anti-acarian defenses of many living things, especially humans.
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Small cysteine-rich antifungal peptides with multi-site modes of action (MoA) have potential for development as biofungicides. In particular, legumes of the inverted-repeat lacking clade express a large family of nodule-specific cysteine-rich (NCR) peptides that orchestrate differentiation of nitrogen-fixing bacteria into bacteroids. These NCRs can form 2 or 3 intramolecular disulfide bonds and a subset of these peptides with high cationicity exhibits antifungal activity. However, the importance of intramolecular disulfide pairing and MoA against fungal pathogens for most of these plant peptides remains to be elucidated. Our study focused on a highly cationic chickpea NCR13, which has a net charge of +9 and contains six cysteines capable of forming three disulfide bonds. NCR13 expression in Pichia pastoris resulted in formation of two peptide folding variants, NCR13_PFV1 and NCR13_PFV2, that differed in the pairing of two out of three disulfide bonds despite having an identical amino acid sequence. The NMR structure of each PFV revealed a unique three-dimensional fold with the PFV1 structure being more compact but less dynamic. Surprisingly, PFV1 and PFV2 differed profoundly in the potency of antifungal activity against several fungal plant pathogens and their multi-faceted MoA. PFV1 showed significantly faster fungal cell-permeabilizing and cell entry capabilities as well as greater stability once inside the fungal cells. Additionally, PFV1 was more effective in binding fungal ribosomal RNA and inhibiting protein translation in vitro . Furthermore, when sprayed on pepper and tomato plants, PFV1 was more effective in reducing disease symptoms caused by Botrytis cinerea , causal agent of gray mold disease in fruits, vegetables and flowers. In conclusion, our work highlights the significant impact of disulfide pairing on the antifungal activity and MoA of NCR13 and provides structural framework for design of novel, potent antifungal peptides for agricultural use. Author Summary Fungal pathogens cause significant pre-harvest and post-harvest losses of crop yield, making them a serious biological threat to global food security. Chemical fungicides are effective in controlling fungal diseases across various crops. However, rapid evolution of fungal pathogen resistance to single-site chemical fungicides in agriculture has created an urgent need for development of safe, sustainable, and cost-effective multi-site fungicides. Nodule-specific cysteine-rich (NCR) peptides expressed in the inverted-repeat lacking clade legume plants exhibit potent antifungal activity; however, their modes of action (MoA) are poorly understood. Particularly, the specific contribution of disulfide pairing to the potency and spectrum of antifungal activity against fungal plant pathogens and MoA of these peptides remains to be identified. Chickpea NCR13 expressed in P. pastoris generates two peptide variants that differ in their disulfide cross-linking pattern. These variants exhibit striking differences in their three-dimensional structures and potency of antifungal activity against multiple fungal pathogens and MoA. They also differ in their ability to confer resistance to gray mold in pepper and tomato plants. Our study highlights the major impact a specific pattern of disulfide pairing can have on the in vitro and in planta antifungal activity of an NCR peptide.
Article
Unlike early land plants, flowering plants have evolved a pollen tube that transports a pair of non-motile sperm cells to the female gametophyte. This process, known as siphonogamy, was first observed in gymnosperms and later became prevalent in angiosperms. However, the precise molecular mechanisms underlying the male–female interactions remain enigmatic. From the landing of the pollen grain on the stigma to gamete fusion, the male part needs to pass various tests: how does the stigma distinguish between compatible and incompatible pollen? what mechanisms guide the pollen tube towards the ovule? what factors trigger pollen tube rupture? how is polyspermy prevented? and how does the sperm cell ultimately reach the egg? Successful male–female communication is essential for surmounting these challenges, with cysteine-rich peptides (CRPs) playing a pivotal role in this dialogue. In this review, we summarize the characteristics of four distinct classes of CRPs, systematically review recent progress in the role of CRPs in four crucial stages of pollination and fertilization, consider potential applications of this knowledge in crop breeding, and conclude by suggesting avenues for future research.
Chapter
Modern crop production systems that have focused on maximizing yield through the use of synthetic fertilizers and pesticides have led to negative impacts on human health and ecosystems. A major component in any crop production system is crop protection, where chemically synthesized pesticides are routinely and injudiciously applied. More sustainable crop protection systems are desirable. Mother nature has provided nature-based solutions to these problems. There are many documented plant and microbial species currently known to science that we can use for increasing sustainability in crop protection. Many plant species exhibiting potential for disease control are found in the five largest flowering plant families: Asteraceae, Orchidaceae, Fabaceae, Rubiaceae, and Poaceae. Various bacterial members from the Frankia, Bacillus, Pseudomonas, Azospirillum, and Azotobacter genera and fungal members from the Trichoderma, Coniothyrium, Gliocladium, Purpureocillium, and Phlebiopsis genera have been studied for their roles in plant growth promoting and disease resistance. This chapter will explore the potential of utilizing botanical extracts from various plant species and beneficial microorganisms for sustainable crop protection against plant pathogens.
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Plants have evolved extensive defense systems to protect against infections and infestations. Antimicrobial peptides are among the well-known chemical barriers possessed by plants to defend themselves against biotic stresses. Antimicrobial peptides have been shown to be one of the most effective biological defences against micropathogens. They have a lot of chemical and structural varieties to help them with their functions. They are also reported to play a role in antibiotic resistance. Antibiotic peptides can be classified based on their chemical structure, their charge, and their size. The most researched antimicrobial peptides are cyclotides, thionins, hevein-like proteins, defensins, snakins, and lipid transfer proteins. Due to their effectiveness in nature, antimicrobial peptides have gained importance in research for their applications in pharmaceutical sectors. It has been found that they have immunological regulatory activities in addition to their direct antibacterial action. It is also reported that antimicrobial peptides can change human immunogenicity to several diseases by altering physiological signals. They can also control the production of signalling molecules, including mitogen-activated protein kinase (MAPK) signalling, nitric oxide (NO), reactive oxygen species (ROS), and lesion and vasculature healing.
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Using a combination of solid-phase extraction, affinity chromatography, and analytical reverse-phase HPLC, a new linear peptide was isolated from dog-grass (Elytrigia repens) ears, which does not contain cysteine residues. Identification of its primary structure by Edman automated degradation made it possible to reveal the presence of several polyglycine regions, each consisting of 6–8 residues, between which short fragments consisting of polar amino acid residues are localized. The C-terminal fragment of the molecule is a positively charged site enriched in arginine and histidine residues. The structural features of this peptide determine its functionality. Thus, checking the presence of antimicrobial properties in its recombinant analogue, obtained by heterologous expression in a prokaryotic system, made it possible to determine the MIC for the tested fungal cultures only at sufficiently high active concentrations (52–104 μM). However, this compound had regulatory properties: at a concentration of 25 μM, a reactivating effect was noted, which increased the level of survival of Saccharomyces cerevisiae to UV-irradiation. The data obtained expand the understanding of the functional features of plant defense peptides of an unusual structural type.
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The extensive application of cadmium (Cd) in various industrial products results in worldwide contamination. In the first part of this chapter, we mainly focus on cadmium species, mobility, and factors influencing Cd bioavailability in soils. Furthermore, as Cd interacts with essential cellular components adversely affecting microbial biomass and diversity, we also report various sophisticated resistance mechanisms that provide microorganisms tolerance to Cd. Additionally, attention is paid to highlighting rhizodegradation as an interface between microbes and the rhizosphere that can significantly influence the increase of nutrient uptake and decline of metal toxicity. In particular, we further discuss cadmium accumulation, toxicity, and defense mechanisms in plants against Cd toxicity.
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Various proteins introduced into living modified organism (LMO) crops function in plant defense mechanisms against target insect pests or herbicides. This study analyzed the antifungal effects of an introduced LMO protein, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Agrobacterium sp. strain CP4 (CP4-EPSPS). Pure recombinant CP4-EPSPS protein, expressed in Escherichia coli, inhibited the growth of human and plant fungal pathogens (Candida albicans, C. tropicalis, C. krusei, Colletotrichum gloeosporioides, Fusarium solani, F. graminearum, and Trichoderma virens), at minimum inhibitory concentrations (MICs) that ranged from 62.5 to 250 µg/mL. It inhibited fungal spore germination as well as cell proliferation on C. gloeosporioides. Rhodamine-labeled CP4-EPSPS accumulated on the fungal cell wall and within intracellular cytosol. In addition, the protein induced uptake of SYTOX Green into cells, but not into intracellular mitochondrial reactive oxygen species (ROS), indicating that its antifungal action was due to inducing the permeability of the fungal cell wall. Its antifungal action showed cell surface damage, as observed from fungal cell morphology. This study provided information on the effects of the LMO protein, EPSPS, on fungal growth.
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Using a combination of solid-phase extraction, affinity chromatography, and analytical reverse-phase HPLC, a new linear peptide was isolated from dog-grass (Elytrigia repens) ears that does not contain cysteine residues. Identification of its primary structure by Edman automated degradation made it possible to reveal the presence of several polyglycine regions, each consisting of six to eight residues, between which short fragments consisting of polar amino-acid residues are localized. The C-terminal fragment of the molecule is a positively charged site enriched in arginine and histidine residues. The structural features of this peptide determine its functionality. Thus, checking the presence of antimicrobial properties in its recombi-nant analogue, obtained by heterologous expression in a prokaryotic system, made it possible to determine the MIC for the tested fungal cultures only at sufficiently high active concentrations (52-104 μM). However, this compound had regulatory properties: at a concentration of 25 μM, a reactivating effect was noted, which increased the level of survival of Saccharomyces cerevisiae to UV-irradiation. The data we obtained expand the understanding of the functional features of plant defense peptides of an unusual structural type.
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Host defense peptides are expressed in various immune cells, including phagocytic cells and epithelial cells. These peptides selectively alter innate immune pathways in response to infections by pathogens, such as bacteria, fungi, and viruses, and modify the subsequent adaptive immune environment. Consequently, they play a wide range of roles in both innate and adaptive immune responses. These peptides are of increasing importance due to their broad-spectrum antimicrobial activity and their functions as mediators linking innate and adaptive immune responses. This review focuses on the pleiotropic biological functions and related mechanisms of action of human host defense peptides and discusses their potential clinical applications.
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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.
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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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Background: "Invertebrate defensins" belong to the cysteine-stabilized alpha-beta (CS-αβ), also known as the scorpion toxin-like, superfamily. Some other peptides belonging to this superfamily of defensive peptides are indistinguishable from "defensins," but have been assigned other names, making it unclear what, if any, criteria must be met to qualify as an "invertebrate defensin." In addition, there are other groups of defensins in invertebrates and vertebrates that are considered to be evolutionarily unrelated to those in the CS-αβ superfamily. This complicates analyses and discussions of this peptide group. This paper investigates the criteria for classifying a peptide as an invertebrate defensin, suggests a reference cysteine array that may be helpful in discussing peptides in this superfamily, and proposes that the superfamily (rather than the name "defensin") is the appropriate context for studying the evolution of invertebrate defensins with the CS-αβ fold. Methods: CS-αβ superfamily sequences were identified from previous literature and BLAST searches of public databases. Sequences were retrieved from databases, and the relevant motifs were identified and used to create a conceptual alignment to a ten-cysteine reference array. Amino acid sequences were aligned in MEGA6 with manual adjustments to ensure accurate alignment of cysteines. Phylogenetic analyses were performed in MEGA6 (maximum likelihood) and MrBayes (Bayesian). Results: Across invertebrate taxa, the term "defensin" is not consistently applied based on number of cysteines, cysteine spacing pattern, spectrum of antimicrobial activity, or phylogenetic relationship. The analyses failed to reveal any criteria that unify "invertebrate defensins" and differentiate them from other defensive peptides in the CS-αβ superfamily. Sequences from various groups within the CS-αβ superfamily of defensive peptides can be described by a ten-cysteine reference array that aligns their defining structural motifs. Conclusions: The proposed ten-cysteine reference array can be used in addition to current nomenclature to compare sequences in the CS-αβ superfamily and clarify their features relative to one another. This will facilitate analysis and discussion of "invertebrate defensins" in an appropriate evolutionary context, rather than relying on nomenclature.
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Scorpion toxins that block potassium channels and antimicrobial plant defensins share a common structural CSαβ-motif. These toxins contain a toxin signature (K-C4-X-N) in their amino acid sequence, and based on in silico analysis of 18 plant defensin sequences, we noted the presence of a toxin signature (K-C5-R-G) in the amino acid sequence of the Arabidopsis thaliana defensin AtPDF2.3. We found that recombinant (r)AtPDF2.3 blocks Kv1.2 and Kv1.6 potassium channels, akin to the interaction between scorpion toxins and potassium channels. Moreover, rAtPDF2.3[G36N], a variant with a KCXN toxin signature (K-C5-R-N), is more potent in blocking Kv1.2 and Kv1.6 channels than rAtPDF2.3, whereas rAtPDF2.3[K33A], devoid of the toxin signature, is characterized by reduced Kv channel blocking activity. These findings highlight the importance of the KCXN scorpion toxin signature in the plant defensin sequence for blocking potassium channels. In addition, we found that rAtPDF2.3 inhibits the growth of Saccharomyces cerevisiae and that pathways regulating potassium transport and/or homeostasis confer tolerance of this yeast to rAtPDF2.3, indicating a role for potassium homeostasis in the fungal defence response towards rAtPDF2.3. Nevertheless, no differences in antifungal potency were observed between the rAtPDF2.3 variants, suggesting that antifungal activity and Kv channel inhibitory function are not linked.
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Defensins are a well-characterised group of small, disulphide-rich, cationic peptides that are produced by essentially all eukaryotes and are highly diverse in their sequences and structures. Most display broad range antimicrobial activity at low micromolar concentrations, whereas others have other diverse roles, including cell signalling (e.g. immune cell recruitment, self/non-self-recognition), ion channel perturbation, toxic functions, and enzyme inhibition. The defensins consist of two superfamilies, each derived from an independent evolutionary origin, which have subsequently undergone extensive divergent evolution in their sequence, structure and function. Referred to as the cis- and trans-defensin superfamilies, they are classified based on their secondary structure orientation, cysteine motifs and disulphide bond connectivities, tertiary structure similarities and precursor gene sequence. The utility of displaying loops on a stable, compact, disulphide-rich core has been exploited by evolution on multiple occasions. The defensin superfamilies represent a case where the ensuing convergent evolution of sequence, structure and function has been particularly extreme. Here, we discuss the extent, causes and significance of these convergent features, drawing examples from across the eukaryotes.
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The plant defensin NaD1 is a potent antifungal molecule that also targets tumour cells with high efficiency. We examined the features of NaD1 that contribute to these two activities by producing a series of chimeras with NaD2, a defensin that has relatively poor activity on fungiand no activity on tumour cells. All plant defensins have a common tertiary structure known as a cysteine stabilized α-β motif which consists of an α helix and a triple-stranded β-sheet stabilized by four disulphide bonds. The chimeras were produced by replacing loops 1-7, the sequences between each of the conserved cysteine residues on NaD1, with the corresponding loops from NaD2. The loop 5 swap replaced the sequence motif (SKILRR) that mediates tight binding with phosphatidylinositol 4,5 bis-phosphosphate (PI(4,5)P 2 ) and is essential for the potent cytotoxic effect of NaD1 on tumour cells. Consistent with previous reports, there was a strong correlation between PI(4,5)P 2 binding and the tumour cell killing activity of all of the chimeras. However, this correlation did not extend to antifungal activity. Some of the loop swap chimeras were efficient antifungal molecules even though they bound poorly to PI(4,5)P 2 suggesting additional mechanisms operate against fungal cells. Unexpectedly the L1B loop swap chimera was ten times more active than NaD1 on filamentous fungi. This led to the conclusion that defensin loops have evolved as modular components that combine to make antifungal molecules with variable mechanisms of action and that artificial combinations of loops can increase antifungal activity compared to natural variants.
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Defensins play an important role in plant defense against fungal pathogens. The plant defensin, MtDef4, inhibits growth of the ascomycete fungi, Neurospora crassa and Fusarium graminearum, at micromolar concentrations. We have reported that MtDef4 is transported into the cytoplasm of these fungi and exerts its antifungal activity on intracellular targets. Here, we have investigated whether the antifungal mechanisms of MtDef4 are conserved in these fungi. We show that N. crassa and F. graminearum respond differently to MtDef4 challenge. Membrane permeabilization is required for the antifungal activity of MtDef4 against F. graminearum but not against N. crassa. We find that MtDef4 is targeted to different subcellular compartments in each fungus. Internalization of MtDef4 in N. crassa is energy-dependent and involves endocytosis. By contrast, MtDef4 appears to translocate into F. graminearum autonomously using a partially energy-dependent pathway. MtDef4 has been shown to bind to the phospholipid phosphatidic acid (PA). We provide evidence that the plasma membrane localized phospholipase D, involved in the biosynthesis of PA, is needed for entry of this defensin in N. crassa, but not in F. graminearum. To our knowledge, this is the first example of a defensin which inhibits the growth of two ascomycete fungi via different mechanisms. This article is protected by copyright. All rights reserved.
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As global populations continue to increase, agricultural productivity will be challenged to keep pace without overtaxing important environmental resources. A dynamic and integrated approach will be required to solve global food insecurity and position agriculture on a trajectory toward sustainability. Genetically modified (GM) crops enhanced through modern biotechnology represent an important set of tools that can promote sustainable agriculture and improve food security. Several emerging biotechnology approaches were discussed in a recent symposium organized at the 13th IUPAC International Congress of Pesticide Chemistry meeting in San Francisco, CA, USA. This paper summarizes the innovative research and several of the new and emerging technologies within the field of agricultural biotechnology that were presented during the symposium. This discussion highlights how agricultural biotechnology fits within the context of sustainable agriculture and improved food security and can be used in support of further development and adoption of beneficial GM crops.
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Cysteine rich protein families are notoriously difficult to align due to low sequence identity and frequent insertions and deletions. Here we present an alignment method that ensures homologous cysteines align by assigning a unique 10 amino acid barcode to those identified as structurally homologous by the DALI webserver. The free inter-cysteine regions of the barcoded sequences can then be aligned using any standard algorithm. Finally the barcodes are replaced with the original columns to yield an alignment which requires the minimum of manual refinement. Using structural homology information to constrain sequence alignments allows the alignment of highly divergent, repetitive sequences that are poorly dealt with by existing algorithms. Tools are provided to perform this method online using the CysBar web-tool (http://CysBar.science.latrobe.edu.au) and offline (python script available from http://github.com/ts404/CysBar).
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A significant effort is made by the cell to maintain certain phospholipids at specific sites. It is well described that proteins involved in intracellular signaling can be targeted to the plasma membrane and organelles through phospholipid-binding domains. Thus, the accumulation of a specific combination of phospholipids, denoted here as the 'phospholipid code', is key in initiating cellular processes. Interestingly, a variety of extracellular proteins and pathogen-derived proteins can also recognize or modify phospholipids to facilitate the recognition of dying cells, tumorigenesis and host-microbe interactions. In this article, we discuss the importance of the phospholipid code in a range of physiological and pathological processes.Cell Death and Differentiation advance online publication, 9 October 2015; doi:10.1038/cdd.2015.122.
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Plant defensins are small, cysteine-rich peptides with antifungal activity against a broad range of yeast and fungi. In this study we investigated the antibiofilm activity of a plant defensin from coral bells (Heuchera sanguinea), i.e. HsAFP1. To this end, HsAFP1 was heterologously produced using Pichia pastoris as a host. The recombinant peptide rHsAFP1 showed a similar antifungal activity against the plant pathogen Fusarium culmorum as native HsAFP1 purified from seeds. NMR analysis revealed that rHsAFP1 consists of an α-helix and a triple-stranded antiparallel β-sheet stabilised by four intramolecular disulfide bonds. We found that rHsAFP1 can inhibit growth of the human pathogen Candida albicans as well as prevent C. albicans biofilm formation with a BIC50 (i.e. the minimum rHsAFP1 concentration required to inhibit biofilm formation by 50% as compared to control treatment) of 11.00 ± 1.70 μM. As such, this is the first report of a plant defensin exhibiting inhibitory activity against fungal biofilms. We further analysed the potential of rHsAFP1 to increase the activity of the conventional antimycotics caspofungin and amphotericin B towards C. albicans. Synergistic effects were observed between rHsAFP1 and these compounds against both planktonic C. albicans cells and biofilms. Most notably, concentrations of rHsAFP1 as low as 0.53 μM resulted in a synergistic activity with caspofungin against pre-grown C. albicans biofilms. rHsAFP1 was found non-toxic towards human HepG2 cells up to 40 μM, thereby supporting the lack of a general cytotoxic activity as previously reported for HsAFP1. A structure-function study with 24-mer synthetic peptides spanning the entire HsAFP1 sequence revealed the importance of the γ-core and its adjacent regions for HsAFP1 antibiofilm activity. These findings point towards broad applications of rHsAFP1 and its derivatives in the field of antifungal and antibiofilm drug development.
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Defensins are a class of ubiquitously expressed cationic antimicrobial peptides (CAPs) that play an important role in innate defense. Plant defensins are active against a broad range of microbial pathogens and act via multiple mechanisms including cell membrane permeabilization. The cytolytic activity of defensins has been proposed to involve interaction with specific lipid components in the target cell wall or membrane and defensin oligomerization. Indeed, the defensin, Nicotiana alata defensin 1 (NaD1) binds to a broad range of membrane phosphatidylinositol phosphates and forms an oligomeric complex with phosphatidylinositol (4, 5)-bisphosphate (PIP2) that facilitates membrane lysis of both mammalian tumor and fungal cells. Here, we report that the tomato defensin, TPP3, has a unique lipid binding profile that is specific for PIP2 with which it forms an oligomeric complex that is critical for cytolytic activity. Structural characterization of TPP3 by X-ray crystallography and site-directed mutagenesis demonstrated that it forms a dimer in a ‘cationic grip’ conformation that specifically accommodates the head group of PIP2 to mediate cooperative higher order oligomerization and subsequent membrane permeabilization. These findings suggest that certain plant defensins are innate immune receptors for phospholipids and adopt conserved dimeric configurations to mediate PIP2 binding and membrane permeabilization. This mechanism of innate defense may be conserved across defensins from different species.
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Treatment of hyphae of Neurospora crassa with antifungal plant defensins, i.e. Rs-AFP2 and Dm-AMP1 isolated from radish and dahlia seed, respectively, induced a rapid K⁺ efflux, Ca²⁺ uptake, and alkalinization of the incubation medium. The Rs-AFP2-induced alkalinization of the incubation medium could be inhibited with G-protein inhibitors. α-Hordothionin, an antifungal thionin from barley seed, caused a sustained increased Ca²⁺ uptake at subinhibitory concentrations but only a transient increased uptake at inhibitory concentrations. α-Hordothionin also caused increased K⁺ efflux and alkalinization of the medium, but these fluxes occurred more rapidly compared to those caused by plant defensins. Furthermore, α-hordothionin caused permeabilization of fungal hyphae to the non-metabolite α-aminoisobutyric acid and, in addition, altered the electrical properties of artificial lipid bilayers, consistently leading to rupture of the lipid bilayers. The plant defensins did not form ion-permeable pores in artificial membranes and did not exhibit substantial hyphal membrane permeabilization activity. Our results are consistent with the notion that thionins inhibit fungal growth as a result of direct protein-membrane interactions, whereas plant defensins might act via a different, possibly receptor-mediated, mechanism.
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Plant defensins are small, cysteine-rich peptides that possess biological activity towards a broad range of organisms. Their activity is primarily directed against fungi, but bactericidal and insecticidal actions have also been reported. The mode of action of various antifungal plant defensins has been studied extensively during the last decades and several of their fungal targets have been identified to date. This review summarizes the mechanism of action of well-characterized antifungal plant defensins, including RsAFP2, MsDef1, MtDef4, NaD1 and Psd1, and points out the variety by which antifungal plant defensins affect microbial cell viability. Furthermore, this review summarizes production routes for plant defensins, either via heterologous expression or chemical synthesis. As plant defensins are generally considered non-toxic for plant and mammalian cells, they are regarded as attractive candidates for further development into novel antimicrobial agents.
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Cationic antimicrobial peptides (CAPs) such as defensins are ubiquitously found innate immune molecules that often exhibit broad activity against microbial pathogens and mammalian tumor cells. Many CAPs act at the plasma membrane of cells leading to membrane destabilization and permeabilization. In this study, we describe a novel cell lysis mechanism for fungal and tumor cells by the plant defensin NaD1 that acts via direct binding to the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). We determined the crystal structure of a NaD1:PIP2 complex, revealing a striking oligomeric arrangement comprising seven dimers of NaD1 that cooperatively bind the anionic headgroups of 14 PIP2 molecules through a unique 'cationic grip' configuration. Site-directed mutagenesis of NaD1 confirms that PIP2-mediated oligomerization is important for fungal and tumor cell permeabilization. These observations identify an innate recognition system by NaD1 for direct binding of PIP2 that permeabilizes cells via a novel membrane disrupting mechanism. DOI: http://dx.doi.org/10.7554/eLife.01808.001.
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Cationic antifungal peptides (AFPs) act through a variety of mechanisms but share the common feature of interacting with the fungal cell surface. NaD1, a defensin from Nicotiana alata, has potent antifungal activity against a variety of fungi of both hyphal and yeast morphologies. The mechanism of action of NaD1 occurs via three steps: binding to the fungal cell surface, permeabilization of the plasma membrane, and internalization and interaction with intracellular targets to induce fungal cell death. The targets at each of these three stages have yet to be defined. In this study, the screening of a Saccharomyces cerevisiae deletion collection led to the identification of Agp2p as a regulator of the potency of NaD1. Agp2p is a plasma membrane protein that regulates the transport of polyamines and other molecules, many of which carry a positive charge. Cells lacking the agp2 gene were more resistant to NaD1, and this resistance was accompanied by a decreased uptake of defensin. Agp2p senses and regulates the uptake of the polyamine spermidine, and competitive inhibition of the antifungal activity of NaD1 by spermidine was observed in both S. cerevisiae and the plant pathogen Fusarium oxysporum. The resistance of agp2Δ cells to other cationic antifungal peptides and decreased binding of the cationic protein cytochrome c to agp2Δ cells compared to that of wild-type cells have led to a proposed mechanism of resistance whereby the deletion of agp2 leads to an increase in positively charged molecules at the cell surface that repels cationic antifungal peptides.
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Fungal disease is an increasing problem in both agriculture and human health. Treatment of human fungal disease involves the use of chemical fungicides, which generally target the integrity of the fungal plasma membrane or cell wall. Chemical fungicides used for the treatment of plant disease, have more diverse mechanisms of action including inhibition of sterol biosynthesis, microtubule assembly and the mitochondrial respiratory chain. However, these treatments have limitations, including toxicity and the emergence of resistance. This has led to increased interest in the use of antimicrobial peptides for the treatment of fungal disease in both plants and humans. Antimicrobial peptides are a diverse group of molecules with differing mechanisms of action, many of which remain poorly understood. Furthermore, it is becoming increasingly apparent that stress response pathways are involved in the tolerance of fungi to both chemical fungicides and antimicrobial peptides. These signalling pathways such as the cell wall integrity and high-osmolarity glycerol pathway are triggered by stimuli, such as cell wall instability, changes in osmolarity and production of reactive oxygen species. Here we review stress signalling induced by treatment of fungi with chemical fungicides and antifungal peptides. Study of these pathways gives insight into how these molecules exert their antifungal effect and also into the mechanisms used by fungi to tolerate sub-lethal treatment by these molecules. Inactivation of stress response pathways represents a potential method of increasing the efficacy of antifungal molecules.
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Plant defensins are small (45-54 amino acids), basic, cysteine-rich proteins that have a major role in innate immunity in plants. Many defensins are potent antifungal molecules and are being evaluated for their potential to create crop plants with sustainable disease resistance. Defensins are produced as precursor molecules which are directed into the secretory pathway and are divided into two classes based on the absence (class I) or presence (class II) of an acidic C-terminal propeptide (CTPP) of about 33 amino acids. The function of this CTPP had not been defined. By transgenically expressing the class II plant defensin NaD1 with and without its cognate CTPP we have demonstrated that NaD1 is phytotoxic to cotton plants when expressed without its CTPP. Transgenic cotton plants expressing constructs encoding the NaD1 precursor with the CTPP had the same morphology as non-transgenic plants but expression of NaD1 without the CTPP led to plants that were stunted, had crinkled leaves and were less viable. Immunofluorescence microscopy and transient expression of a green fluorescent protein (GFP)-CTPP chimera were used to confirm that the CTPP is sufficient for vacuolar targeting. Finally circular dichroism and NMR spectroscopy were used to show that the CTPP adopts a helical confirmation. In this report we have described the role of the CTPP on NaD1, a class II defensin from Nicotiana alata flowers. The CTPP of NaD1 is sufficient for vacuolar targeting and plays an important role in detoxification of the defensin as it moves through the plant secretory pathway. This work may have important implications for the use of defensins for disease protection in transgenic crops.
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Scorpion K+ channel toxins and insect defensins share a conserved three-dimensional structure and related biological activities (defense against competitors or invasive microbes by disrupting their membrane functions), which provides an ideal system to study how functional evolution occurs in a conserved structural scaffold. Using an experimental approach, we show that the deletion of a small loop of a parasitoid venom defensin possessing the “scorpion toxin signature” (STS) can remove steric hindrance of peptide-channel interactions and result in a neurotoxin selectively inhibiting K+ channels with high affinities. This insect defensin-derived toxin adopts a hallmark scorpion toxin fold with a common cysteine-stabilized α-helical and β-sheet motif, as determined by nuclear magnetic resonance analysis. Mutations of two key residues located in STS completely diminish or significantly decrease the affinity of the toxin on the channels, demonstrating that this toxin binds to K+ channels in the same manner as scorpion toxins. Taken together, these results provide new structural and functional evidence supporting the predictability of toxin evolution. The experimental strategy is the first employed to establish an evolutionary relationship of two distantly related protein families.
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MtDef4 is a 47-amino acid cysteine-rich evolutionary conserved defensin from a model legume Medicago truncatula. It is an apoplast-localized plant defense protein that inhibits the growth of the ascomycetous fungal pathogen Fusarium graminearum in vitro at micromolar concentrations. Little is known about the mechanisms by which MtDef4 mediates its antifungal activity. In this study, we show that MtDef4 rapidly permeabilizes fungal plasma membrane and is internalized by the fungal cells where it accumulates in the cytoplasm. Furthermore, analysis of the structure of MtDef4 reveals the presence of a positively charged γ-core motif composed of β2 and β3 strands connected by a positively charged RGFRRR loop. Replacement of the RGFRRR sequence with AAAARR or RGFRAA abolishes the ability of MtDef4 to enter fungal cells, suggesting that the RGFRRR loop is a translocation signal required for the internalization of the protein. MtDef4 binds to phosphatidic acid (PA), a precursor for the biosynthesis of membrane phospholipids and a signaling lipid known to recruit cytosolic proteins to membranes. Amino acid substitutions in the RGFRRR sequence which abolish the ability of MtDef4 to enter fungal cells also impair its ability to bind PA. These findings suggest that MtDef4 is a novel antifungal plant defensin capable of entering into fungal cells and affecting intracellular targets and that these processes are mediated by the highly conserved cationic RGFRRR loop via its interaction with PA.
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Plant defensins represent a large class of structurally similar peptides found throughout the plant kingdom. Despite a conserved cysteine spacing pattern and three-dimensional structure, their sequences are highly divergent and they display a range of activities including antifungal and antibacterial activities, enzyme inhibitory activities as well as roles in heavy metal tolerance and development. The vast number of sequences along with their diverse range of activities makes it impossible to test the activity and assign function to all plant defensins. However, as the number of characterized defensins increases, in depth sequence analysis may allow us to predict the function of newly identified peptides. In this review, we analyze the sequences of defensins whose activities have been described and group these based on similarity using a maximum-likelihood phylogenetic tree. We also compare the amino acids that have been described as essential for the activity of various plant defensins between these groups. While many more plant defensins will need to be characterized before we can develop rules to predict the activity of novel sequences, this approach may prove useful in identifying structure–function relationships.
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The plant defensin, NaD1, from the flowers of Nicotiana alata, is a member of a family of cationic peptides that displays growth inhibitory activity against several filamentous fungi, including Fusarium oxysporum. The antifungal activity of NaD1 has been attributed to its ability to permeabilize membranes; however, the molecular basis of this function remains poorly defined. In this study, we have solved the structure of NaD1 from two crystal forms to high resolution (1.4 and 1.58 Å, respectively), both of which contain NaD1 in a dimeric configuration. Using protein cross-linking experiments as well as small angle x-ray scattering analysis and analytical ultracentrifugation, we show that NaD1 forms dimers in solution. The structural studies identified Lys4 as critical in formation of the NaD1 dimer. This was confirmed by site-directed mutagenesis of Lys4 that resulted in substantially reduced dimer formation. Significantly, the reduced ability of the Lys4 mutant to dimerize correlated with diminished antifungal activity. These data demonstrate the importance of dimerization in NaD1 function and have implications for the use of defensins in agribiotechnology applications such as enhancing plant crop protection against fungal pathogens.