Plant immunity represents a sophisticated system, including both basal and inducible mechanisms, to prevent pathogen infection. Antimicrobial peptides (AMPs) are among the innate immunity components ...playing a key role in effective and rapid response against various pathogens. This review is devoted to a small family of defense peptides called α-hairpinins. The general characters of the family, as well as the individual features of each member, including biological activities, structures of precursor proteins, and spatial structures, are described. Possible applications of α-hairpinin peptides in drug design are discussed.
Many viruses are known to trigger endoplasmic reticulum (ER) stress in host cells, which in turn can develop a protective unfolded protein response (UPR). Depending on the conditions, the UPR may ...lead to either cell survival or programmed cell death. One of three UPR branches involves the upregulation of Xbp1 transcription factor caused by the unconventional cytoplasmic splicing of its mRNA. This process is accomplished by the phosphorylated form of the endoribonuclease/protein kinase Ire1/ERN1. Here, we show that the phosphorylation of Ire1 is up-regulated in HeLa cells early in enterovirus infection but down-regulated at later stages. We also find that Ire1 is cleaved in poliovirus- and coxsackievirus-infected HeLa cells 4-6 h after infection. We further show that the Ire1-mediated Xbp1 mRNA splicing is repressed in infected cells in a time-dependent manner. Thus, our results demonstrate the ability of enteroviruses to actively modulate the Ire1-Xbp1 host defensive pathway by inducing phosphorylation and proteolytic cleavage of the ER stress sensor Ire1, as well as down-regulating its splicing activity. Inactivation of Ire1 could be a novel mode of the UPR manipulation employed by viruses to modify the ER stress response in the infected cells.
Leymus arenarius is a unique wild growing Poaceae plant exhibiting extreme tolerance to environmental conditions. In this study we for the first time performed whole-transcriptome sequencing of ...lymegrass seedlings using Illumina platform followed by de novo transcriptome assembly and functional annotation. Our goal was to identify transcripts encoding antimicrobial peptides (AMPs), one of the key components of plant innate immunity. Using the custom software developed for this study that predicted AMPs and classified them into families, we revealed more than 160 putative AMPs in lymegrass seedlings. We classified them into 7 families based on their cysteine motifs and sequence similarity. The families included defensins, thionins, hevein-like peptides, snakins, cyclotide, alfa-hairpinins and LTPs. This is the first communication about the presence of almost all known AMP families in trascriptomic data of a single plant species. Additionally, cysteine-rich peptides that potentially represent novel families of AMPs were revealed. We have confirmed by RT-PCR validation the presence of 30 transcripts encoding selected AMPs in lymegrass seedlings. In summary, the presented method of pAMP prediction developed by us can be applied for relatively fast and simple screening of novel components of plant immunity system and is well suited for whole-transcriptome or genome analysis of uncharacterized plants.
The multilayered plant immune system relies on rapid recognition of pathogen‐associated molecular patterns followed by activation of defense‐related genes, resulting in the reinforcement of plant ...cell walls and the production of antimicrobial compounds. To suppress plant defense, fungi secrete effectors, including a recently discovered Zn‐metalloproteinase from Fusarium verticillioides, named fungalysin Fv‐cmp. This proteinase cleaves class IV chitinases, which are plant defense proteins that bind and degrade chitin of fungal cell walls. In this study, we investigated plant responses to such pathogen invasion, and discovered novel inhibitors of fungalysin. We produced several recombinant hevein‐like antimicrobial peptides named wheat antimicrobial peptides (WAMPs) containing different amino acids (Ala, Lys, Glu, and Asn) at the nonconserved position 34. An additional Ser at the site of fungalysin proteolysis makes the peptides resistant to the protease. Moreover, an equal molar concentration of WAMP‐1b or WAMP‐2 to chitinase was sufficient to block the fungalysin activity, keeping the chitinase intact. Thus, WAMPs represent novel protease inhibitors that are active against fungal metalloproteases. According to in vitro antifungal assays WAMPs directly inhibited hyphal elongation, suggesting that fungalysin plays an important role in fungal development. A novel molecular mechanism of dynamic interplay between host defense molecules and fungal virulence factors is suggested.
Being perfectly adapted to diverse environments, chickweed (Stellaria media (L.) Vill), a ubiquitous garden weed, grows widely in Europe and North America. As opposed to the model plants, many weeds, ...and S. media in particular, have been poorly studied, although they are likely to contain promising components of immunity and novel resistance genes. In this study, for the first time RNA-seq analysis of healthy and infected with Fusarium oxysporum chickweed seedlings, as well as de novo transcriptome assembly and annotation, are presented. Note, this research is focused on antimicrobial peptides (AMPs), the major components of plant immune system. Using custom software developed earlier, 145 unique putative AMPs (pAMPs) including defensins, thionins, hevein-like peptides, snakins, alpha-hairpinins, LTPs, and cysteine-rich peptides with novel cysteine motifs were predicted. Furthermore, changes in AMP expression profile in response to fungal infection were traced. In addition, the comparison of chickweed AMP repertoire with those of other Caryophyllaceae plants whose transcriptomes are presently available is made. As a result, alpha-hairpinins and hevein-like peptides which display characteristic modular structure appear to be specific AMPs distinguishing S. media from Dianthus caryophyllus, Silene vulgaris, and Silene latifolia. Finally, revealing several AMPs with proven antimicrobial activity gives opportunity to conclude that the presented method of AMP repertoire analysis reveals highly active AMPs playing vital role in plant immunity.
•RNA-seq analysis of healthy and infected chickweed seedlings is presented.•145 unique putative antimicrobial peptides (pAMPs) belonging to different families are predicted.•Comparison of chickweed pAMP repertoire with other Caryophyllaceae plants is made.•α-hairpinins, hevein-like pAMPs distinguish S. media from Caryophyllaceae plants.•The method presented reveals highly active pAMPs playing vital role in plant defense system.
A novel peptide named SmAMP3 was isolated from leaves of common chickweed (Stellaria media L.) by a combination of acidic extraction and a single-step reversed-phase HPLC and sequenced. The peptide ...is basic and cysteine-rich, consists of 35 amino acids, and contains three disulphide bridges. Homology search revealed that SmAMP3 belongs to the family of hevein-like antimicrobial peptides carrying a conserved chitin-binding site. Efficient binding of chitin by SmAMP3 was proved by in vitro assays. Molecular modeling confirmed conservation of the chitin-binding module in SmAMP3 locating the variable amino acid residues to the solvent-exposed loops of the molecule. The peptide exhibits potent antifungal activity against important plant pathogens in the micromolar range, although it is devoid of antibacterial activity at concentrations below 10 μM. As judged by chromatographic behavior and mass spectrometric data, the peptide is constitutively expressed in above-ground organs and seeds of S. media plants, thus representing an important player in the preformed branch of the plant immune system.
•A novel peptide named SmAMP3 was isolated from leaves of common chickweed (Stellaria media L.).•SmAMP3 belongs to the family of hevein-like AMPs carrying a conserved chitin-binding site.•The peptide exhibits potent antifungal activity against plant pathogens in the micromolar range.•This peptide is constitutively expressed in above-ground organs and seeds of S. media plants.
A novel family of antifungal peptides was discovered in the wheat Triticum kiharae Dorof. et Migusch. Two members of the family, designated Tk‐AMP‐X1 and Tk‐AMP‐X2, were completely sequenced and ...shown to belong to the α‐hairpinin structural family of plant peptides with a characteristic C1XXXC2‐X(n)‐C3XXXC4 motif. The peptides inhibit the spore germination of several fungal pathogens in vitro. cDNA and gene cloning disclosed unique structure of genes encoding Tk‐AMP‐X peptides. They code for precursor proteins of unusual multimodular structure, consisting of a signal peptide, several α‐hairpinin (4‐Cys) peptide domains with a characteristic cysteine pattern separated by linkers and a C‐terminal prodomain. Three types of precursor proteins, with five, six or seven 4‐Cys peptide modules, were found in wheat. Among the predicted family members, several peptides previously isolated from T. kiharae seeds were identified. Genes encoding Tk‐AMP‐X precursors have no introns in the protein‐coding regions and are upregulated by fungal pathogens and abiotic stress, providing conclusive evidence for their role in stress response. A combined PCR‐based and bioinformatics approach was used to search for related genes in the plant kingdom. Homologous genes differing in the number of peptide modules were discovered in phylogenetically‐related Triticum and Aegilops species, including polyploid wheat genome donors. Association of the Tk‐AMP‐X genes with A, B/G or D genomes of hexaploid wheat was demonstrated. Furthermore, Tk‐AMP‐X‐related sequences were shown to be widespread in the Poaceae family among economically important crops, such as barley, rice and maize.
Database
Nucleotide sequence data have been deposited in the EMBL database under accession numbers: HF562347, HF562348, HF562349, HF562350, HF562351, HF562352, HF562353, HF562354, HF562355, HF562356, HF562357, HF562358, HF562359, HF562360, HF562361, HF562362, HF562363 and HF562364.
Genes encoded new wheat peptides code for precursor proteins of unusual multimodular structure consisting of a signal peptide, several α‐hairpinin (4‐Cys) peptide domains with a characteristic cysteine pattern separated by linkers and a C‐terminal prodomain. Three types of precursor proteins, with five, six or seven 4‐Cys peptide modules were found.
•Spiderines are two-domain toxins comprising cytolytic and neurotoxic domains.•20 spiderines are found in lynx spiders Oxyopes takobius and Oxyopes lineatus.•Spiderine genes do not contain ...introns.•Negative selection acts on both spiderine domains.•Spiderines evolved from single-domain knottin toxins affecting ion channels.
Spiderines are comparatively long polypeptide toxins (∼110 residues) from lynx spiders (genus Oxyopes). They are built of an N-terminal linear cationic domain (∼40 residues) and a C-terminal knottin domain (∼60 residues). The linear domain empowers spiderines with strong cytolytic activity. In the present work we report 16 novel spiderine sequences from Oxyopes takobius and Oxyopes lineatus classified into two subfamilies. Strikingly, negative selection acts on both linear and knottin domains. Genes encoding Oxyopes two-domain toxins were sequenced and found to be intronless. We further discuss a possible scenario of lynx spider modular toxin evolution.
Plant defense against disease is a complex multistage system involving initial recognition of the invading pathogen, signal transduction and activation of specialized genes. An important role in ...pathogen deterrence belongs to so-called plant defense peptides, small polypeptide molecules that present antimicrobial properties. Using multidimensional liquid chromatography, we isolated a novel antifungal peptide named Sm-AMP-X (33 residues) from the common chickweed (Stellaria media) seeds. The peptide sequence shows no homology to any previously described proteins. The peculiar cysteine arrangement (C¹X₃C²XₙC³X₃C⁴), however, allocates Sm-AMP-X to the recently acknowledged α-hairpinin family of plant defense peptides that share the helix-loop-helix fold stabilized by two disulfide bridges C¹–C⁴ and C²–C³. Sm-AMP-X exhibits high broad-spectrum activity against fungal phytopathogens. We further showed that the N- and C-terminal “tail” regions of the peptide are important for both its structure and activity. The truncated variants Sm-AMP-X1 with both disulfide bonds preserved and Sm-AMP-X2 with only the internal S–S-bond left were progressively less active against fungi and presented largely disordered structure as opposed to the predominantly helical conformation of the full-length antifungal peptide. cDNA and gene cloning revealed that Sm-AMP-X is processed from a unique multimodular precursor protein that contains as many as 12 tandem repeats of α-hairpinin-like peptides. Structure of the sm-amp-x gene and two related pseudogenes sm-amp-x-ψ1 and sm-amp-x-ψ2 allows tracing the evolutionary scenario that led to generation of such a sophisticated precursor protein. Sm-AMP-X is a new promising candidate for engineering disease resistance in plants.
Two novel highly homologous defensins, Sm-AMP-D1 and Sm-AMP-D2, were isolated from seeds of common chickweed Stellaria media L. (family Cariophyllaceae). They show sequence homology to defensins of ...the Brassicaceae plants and display strong inhibitory activity against phytopathogenic fungi and oomycetes in the micromolar range (IC50≤1μM). The cDNA sequences coding for Sm-AMP-D1 and Sm-AMP-D2 were obtained. They code for highly homologous precursor proteins, consisting of a signal peptide of 32 amino acid residues and the mature peptide domain of 50 amino acid residues. The Sm-AMP-D1 and Sm-AMP-D2 precursors differ by two amino acids: one in the signal peptide region, and the other, in the mature peptide domain. Two Sm-D1-encoding genes were identified in S. media genome by PCR amplification from the genomic DNA using Sm-D1-specific primers. They contain a single 599-bp intron in the signal peptide domain and differ from each other by nucleotide substitutions in the intron and 3′-untranslated regions, while the coding sequences are well conserved. One of the genes matched perfectly the sm-D1 cDNA sequence. The sm-D genes show promise for engineering pathogen resistance in crops and expand our knowledge on weed genomics.
► Two highly homologous defensins from common chickweed were isolated and sequenced. ► Defensins exhibit strong antifungal activity against important plant pathogens. ► Three defensin-encoding genes were identified, two of which are expressed in seeds. ► Defensins precursors consist of a signal peptide and a mature peptide domains. ► The genomic DNA sequences contain an intron in the signal peptide region.
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