Enteroviruses Manipulate the Unfolded Protein Response through Multifaceted Deregulation of the Ire1-Xbp1 Pathway.

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.

Defense Peptides From the α-Hairpinin Family Are Components of Plant Innate Immunity.

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.

Defense peptide repertoire of Stellaria media predicted by high throughput next generation sequencing.

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.

Prediction of Leymus arenarius (L.) antimicrobial peptides based on de novo transcriptome assembly.

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.

A novel antifungal peptide from leaves of the weed Stellaria media L.

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.

Novel mode of action of plant defense peptides - hevein-like antimicrobial peptides from wheat inhibit fungal metalloproteases.

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.

Genes and evolution of two-domain toxins from lynx spider venom.

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.

Novel antifungal α-hairpinin peptide from Stellaria media seeds: structure, biosynthesis, gene structure and 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 (C(1)X3C(2)XnC(3)X3C(4)), 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 C(1)-C(4) and C(2)-C(3). 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.

Genes encoding 4-Cys antimicrobial peptides in wheat Triticum kiharae Dorof. et Migush.: multimodular structural organization, instraspecific variability, distribution and role in defence.

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.

Genes encoding hevein-like defense peptides in wheat: distribution, evolution, and role in stress response.

Hevein-like plant defense peptides WAMP-1a/b with a unique 10-Cys motif are found in the wheat Triticum kiharae seeds. Three different wamp genomic and cDNA sequences were derived from T. kiharae; no introns were spotted in the protein-coding regions of the genes. The deduced Wamp precursor proteins consist of a signal peptide, mature peptide (WAMP) and C-terminal prosequence. Origin of WAMPs from class I/IV chitinases via deletion of the catalytic domain is proposed based on homology between their genes. Genome screening of several cereals and goatgrasses from the genera Triticum and Aegilops was performed. No wamp homologues were identified in Triticum monococcum (A(b)A(b)) or Triticum urartu (A(u)A(u)), diploid species with an A genome. To the contrary, highly homologous wamp genes were discovered in hexaploid Triticum aestivum (A(u)A(u)BBDD) and T. kiharae (A(b)A(b)GGDD), and the putative genome donors Triticum timopheevii (A(b)A(b)GG), Aegilops speltoides (BB), and Aegilops tauschii (DD), providing strong evidence for the ancient origin of these genes and their association with the B, D and G genomes. The role of T. kiharae WAMPs in biotic stress is suggested by their antifungal activity and increased accumulation of wamp transcripts in response to phytopathogen challenge. Differential reaction to fungi was demonstrated: Fusarium oxysporum enhanced expression of wamp genes, whereas Aspergillus niger induced transcription reprogramming and alternative polyadenylation. WAMPs participate in plant response also to abiotic stress. Although no changes were noted at elevated or decreased temperatures, high salt concentrations enhanced wamp expression, the first indication of hevein-type peptide participation in salinity stress.

Solution structure of a defense peptide from wheat with a 10-cysteine motif.

Hevein, a well-studied lectin from the rubber tree Hevea brasiliensis, is the title representative of a broad family of chitin-binding polypeptides. WAMP-1a, a peptide isolated from the wheat Triticum kiharae, shares considerable similarity with hevein. The peptide possesses antifungal, antibacterial activity and is thought to play an important role in the defense system of wheat. Importantly, it features a substitution of the conserved serine residue to glycine reducing its carbohydrate-binding capacity. We used NMR spectroscopy to derive the spatial structure of WAMP-1a in aqueous solution. Notably, the mutation was found to strengthen amphiphilicity of the molecule, associated with its mode of action, an indication of the hevein domain multi-functionality. Both primary and tertiary structure of WAMP-1a suggest its evolutionary origin from the hevein domain of plant chitinases.

Isolation, molecular cloning and antimicrobial activity of novel defensins from common chickweed (Stellaria media L.) seeds.

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 (IC(50)≤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.

A novel antifungal hevein-type peptide from Triticum kiharae seeds with a unique 10-cysteine motif.

Two forms of a novel antimicrobial peptide (AMP), named WAMP-1a and WAMP-1b, that differ by a single C-terminal amino acid residue and belong to a new structural type of plant AMP were purified from seeds of Triticum kiharae Dorof. et Migusch. Although WAMP-1a and WAMP-1b share similarity with hevein-type peptides, they possess 10 cysteine residues arranged in a unique cysteine motif which is distinct from those described previously for plant AMPs, but is characteristic of the chitin-binding domains of cereal class I chitinases. An unusual substitution of a serine for a glycine residue in the chitin-binding domain was detected for the first time in hevein-like polypeptides. Recombinant WAMP-1a was successfully produced in Escherichia coli. This is the first case of high-yield production of a cysteine-rich plant AMP from a synthetic gene. Assays of recombinant WAMP-1a activity showed that the peptide possessed high broad-spectrum inhibitory activity against diverse chitin-containing and chitin-free pathogens, with IC(50) values in the micromolar range. The discovery of a new type of AMP active against structurally dissimilar microorganisms implies divergent modes of action and discloses the complexity of plant-microbe interactions.