Transcriptome-Wide Identification and Expression Analysis of Genes Encoding Defense-Related Peptides of Filipendula ulmaria in Response to Bipolaris sorokiniana Infection.

Peptides play an essential role in plant development and immunity. Filipendula ulmaria, belonging to the Rosaceae family, is a medicinal plant which exhibits valuable pharmacological properties. F. ulmaria extracts in vitro inhibit the growth of a variety of plant and human pathogens. The role of peptides in defense against pathogens in F. ulmaria remains unknown. The objective of this study was to explore the repertoire of antimicrobial (AMPs) and defense-related signaling peptide genes expressed by F. ulmaria in response to infection with Bipolaris sorokiniana using RNA-seq. Transcriptomes of healthy and infected plants at two time points were sequenced on the Illumina HiSeq500 platform and de novo assembled. A total of 84 peptide genes encoding novel putative AMPs and signaling peptides were predicted in F. ulmaria transcriptomes. They belong to known, as well as new, peptide families. Transcriptional profiling in response to infection disclosed complex expression patterns of peptide genes and identified both up- and down-regulated genes in each family. Among the differentially expressed genes, the vast majority were down-regulated, suggesting suppression of the immune response by the fungus. The expression of 13 peptide genes was up-regulated, indicating their possible involvement in triggering defense response. After functional studies, the encoded peptides can be used in the development of novel biofungicides and resistance inducers.

The γ-Core Motif Peptides of AMPs from Grasses Display Inhibitory Activity against Human and Plant Pathogens.

Antimicrobial peptides (AMPs) constitute an essential part of the plant immune system. They are regarded as alternatives to conventional antibiotics and pesticides. In this study, we have identified the γ-core motifs, which are associated with antimicrobial activity, in 18 AMPs from grasses and assayed their antimicrobial properties against nine pathogens, including yeasts affecting humans, as well as plant pathogenic bacteria and fungi. All the tested peptides displayed antimicrobial properties. We discovered a number of short AMP-derived peptides with high antimicrobial activity both against human and plant pathogens. For the first time, antimicrobial activity was revealed in the peptides designed from the 4-Cys-containing defensin-like peptides, whose role in plant immunity has remained unknown, as well as the knottin-like peptide and the C-terminal prodomain of the thionin, which points to the direct involvement of these peptides in defense mechanisms. Studies of the mode of action of the eight most active γ-core motif peptides on yeast cells using staining with propidium iodide showed that all of them induced membrane permeabilization leading to cell lysis. In addition to identification of the antimicrobial determinants in plant AMPs, this work provides short candidate peptide molecules for the development of novel drugs effective against opportunistic fungal infections and biopesticides to control plant pathogens.

The γ-Core Motif Peptides of Plant AMPs as Novel Antimicrobials for Medicine and Agriculture.

The γ-core motif is a structural element shared by most host antimicrobial peptides (AMPs), which is supposed to contribute to their antimicrobial properties. In this review, we summarized the available data on the γ-core peptides of plant AMPs. We describe γ-core peptides that have been shown to exhibit inhibitory activity against plant and human bacterial and fungal pathogens that make them attractive scaffolds for the development of novel anti-infective agents. Their advantages include origin from natural AMP sequences, broad-spectrum and potent inhibitory activity, and cost-effective production. In addition, some γ-core peptides combine antimicrobial and immunomodulatory functions, thus broadening the spectrum of practical applications. Some act synergistically with antimycotics and fungicides, so combinations of peptides with conventionally used antifungal agents can be suggested as an effective strategy to reduce the doses of potentially harmful chemicals. The presented information will pave the way for the design of novel antimicrobials on the basis of γ-core motif peptides, which can find application in medicine and the protection of crops from diseases.

Synthetic Oligopeptides Mimicking γ-Core Regions of Cysteine-Rich Peptides of Solanum lycopersicum Possess Antimicrobial Activity against Human and Plant Pathogens.

Plant cysteine-rich peptides (CRPs) represent a diverse group of molecules involved in different aspects of plant physiology. Antimicrobial peptides, which directly suppress the growth of pathogens, are regarded as promising templates for the development of next-generation pharmaceuticals and ecologically friendly plant disease control agents. Their oligopeptide fragments are even more promising because of their low production costs. The goal of this work was to explore the antimicrobial activity of nine short peptides derived from the γ-core-containing regions of tomato CRPs against important plant and human pathogens. We discovered antimicrobial activity in peptides derived from the defensin-like peptides, snakins, and MEG, which demonstrates the direct involvement of these CRPs in defense reactions in tomato. The CRP-derived short peptides appeared particularly active against the gram-positive bacterium Clavibacter michiganensis, which causes bacterial wilt-opening up new possibilities for their use in agriculture to control this dangerous disease. Furthermore, high inhibitory potency of short oligopeptides was demonstrated against the yeast Cryptococcus neoformans, which causes serious diseases in humans, making these peptide molecules promising candidates for the development of next-generation pharmaceuticals. Studies of the mode of action of the two most active peptides indicate fungal membrane permeabilization as a mechanism of antimicrobial action.

Molecular Insights into the Role of Cysteine-Rich Peptides in Induced Resistance to Fusarium oxysporum Infection in Tomato Based on Transcriptome Profiling.

Cysteine-rich peptides (CRPs) play an important role in plant physiology. However, their role in resistance induced by biogenic elicitors remains poorly understood. Using whole-genome transcriptome sequencing and our CRP search algorithm, we analyzed the repertoire of CRPs in tomato Solanum lycopersicum L. in response to Fusarium oxysporum infection and elicitors from F. sambucinum. We revealed 106 putative CRP transcripts belonging to different families of antimicrobial peptides (AMPs), signaling peptides (RALFs), and peptides with non-defense functions (Major pollen allergen of Olea europaea (Ole e 1 and 6), Maternally Expressed Gene (MEG), Epidermal Patterning Factor (EPF)), as well as pathogenesis-related proteins of families 1 and 4 (PR-1 and 4). We discovered a novel type of 10-Cys-containing hevein-like AMPs named SlHev1, which was up-regulated both by infection and elicitors. Transcript profiling showed that F. oxysporum infection and F. sambucinum elicitors changed the expression levels of different overlapping sets of CRP genes, suggesting the diversification of functions in CRP families. We showed that non-specific lipid transfer proteins (nsLTPs) and snakins mostly contribute to the response of tomato plants to the infection and the elicitors. The involvement of CRPs with non-defense function in stress reactions was also demonstrated. The results obtained shed light on the mode of action of F. sambucinum elicitors and the role of CRP families in the immune response in tomato.

Defensins of Grasses: A Systematic Review.

The grass family (Poaceae) is one of the largest families of flowering plants, growing in all climatic zones of all continents, which includes species of exceptional economic importance. The high adaptability of grasses to adverse environmental factors implies the existence of efficient resistance mechanisms that involve the production of antimicrobial peptides (AMPs). Of plant AMPs, defensins represent one of the largest and best-studied families. Although wheat and barley seed γ-thionins were the first defensins isolated from plants, the functional characterization of grass defensins is still in its infancy. In this review, we summarize the current knowledge of the characterized defensins from cultivated and selected wild-growing grasses. For each species, isolation of defensins or production by heterologous expression, peptide structure, biological activity, and structure-function relationship are described, along with the gene expression data. We also provide our results on in silico mining of defensin-like sequences in the genomes of all described grass species and discuss their potential functions. The data presented will form the basis for elucidation of the mode of action of grass defensins and high adaptability of grasses to environmental stress and will provide novel potent molecules for practical use in medicine and agriculture.

Non-Specific Lipid Transfer Proteins in Triticum kiharae Dorof. et Migush.: Identification, Characterization and Expression Profiling in Response to Pathogens and Resistance Inducers.

Non-specific lipid-transfer proteins (nsLTPs) represent a family of plant antimicrobial peptides (AMPs) implicated in diverse physiological processes. However, their role in induced resistance (IR) triggered by non-pathogenic fungal strains and their metabolites is poorly understood. In this work, using RNA-seq data and our AMP search pipeline, we analyzed the repertoire of nsLTP genes in the wheat Triticum kiharae and studied their expression in response to Fusarium oxysporum infection and treatment with the intracellular metabolites of Fusarium sambucinum FS-94. A total of 243 putative nsLTPs were identified, which were classified into five structural types and characterized. Expression analysis showed that 121 TkLTPs including sets of paralogs with identical mature peptides displayed specific expression patters in response to different treatments pointing to their diverse roles in resistance development. We speculate that upregulated nsLTP genes are involved in protection due to their antimicrobial activity or signaling functions. Furthermore, we discovered that in IR-displaying plants, a vast majority of nsLTP genes were downregulated, suggesting their role as negative regulators of immune mechanisms activated by the FS-94 elicitors. The results obtained add to our knowledge of the role of nsLTPs in IR and provide candidate molecules for genetic engineering of crops to enhance disease resistance.

Defensin-like peptides in wheat analyzed by whole-transcriptome sequencing: a focus on structural diversity and role in induced resistance.

Antimicrobial peptides (AMPs) are the main components of the plant innate immune system. Defensins represent the most important AMP family involved in defense and non-defense functions. In this work, global RNA sequencing and de novo transcriptome assembly were performed to explore the diversity of defensin-like (DEFL) genes in the wheat Triticum kiharae and to study their role in induced resistance (IR) mediated by the elicitor metabolites of a non-pathogenic strain FS-94 of Fusarium sambucinum. Using a combination of two pipelines for DEFL mining in transcriptome data sets, as many as 143 DEFL genes were identified in T. kiharae, the vast majority of them represent novel genes. According to the number of cysteine residues and the cysteine motif, wheat DEFLs were classified into ten groups. Classical defensins with a characteristic 8-Cys motif assigned to group 1 DEFLs represent the most abundant group comprising 52 family members. DEFLs with a characteristic 4-Cys motif CX{3,5}CX{8,17}CX{4,6}C named group 4 DEFLs previously found only in legumes were discovered in wheat. Within DEFL groups, subgroups of similar sequences originated by duplication events were isolated. Variation among DEFLs within subgroups is due to amino acid substitutions and insertions/deletions of amino acid sequences. To identify IR-related DEFL genes, transcriptional changes in DEFL gene expression during elicitor-mediated IR were monitored. Transcriptional diversity of DEFL genes in wheat seedlings in response to the fungus Fusarium oxysporum, FS-94 elicitors, and the combination of both (elicitors + fungus) was demonstrated, with specific sets of up- and down-regulated DEFL genes. DEFL expression profiling allowed us to gain insight into the mode of action of the elicitors from F. sambucinum. We discovered that the elicitors up-regulated a set of 24 DEFL genes. After challenge inoculation with F. oxysporum, another set of 22 DEFLs showed enhanced expression in IR-displaying seedlings. These DEFLs, in concert with other defense molecules, are suggested to determine enhanced resistance of elicitor-pretreated wheat seedlings. In addition to providing a better understanding of the mode of action of the elicitors from FS-94 in controlling diseases, up-regulated IR-specific DEFL genes represent novel candidates for genetic transformation of plants and development of pathogen-resistant crops.

An Extract Purified from the Mycelium of a Tomato Wilt-Controlling Strain of Fusarium sambucinum Can Protect Wheat against Fusarium and Common Root Rots.

An approach to manage seed-transmitted Fusarium crown-foot-root rot (FCR, Fusarium spp.) and common root rot (CRR, Bipolaris sorokiniana) on wheat, avoiding environmental risks of chemicals, is seed treatments with microbial metabolites. F. sambucinum strain FS-94 that induces resistance to tomato wilt was shown by this study to be a source of non-fungitoxic wheat-protecting metabolites, which were contained in a mycelium extract purified by gel-chromatography and ultrafiltration. Plant-protecting effect of the purified mycelial extract (PME) was demonstrated in vegetation experiments using a rolled-towel assay and by small-plot field trials. To elucidate mechanisms putatively underlying PME protective activity, tests with cultured Triticum aestivum and T. kiharae cells, particularly the extracellular alkalinization assay, as well as gene expression analysis in germinated wheat seeds were used. Pre-inoculation treatments of seeds with PME significantly decreased the incidence (from 30 to 40%) and severity (from 37 to 50%) of root rots on seedlings without any inhibition of the seed germination and potentiation of deoxynivalenol (DON), DON monoacetylated derivatives and zearalenon production in FCR agents. In vegetation experiments, reductions in the DON production were observed with doses of 0.5 and 1 mg/mL of PME. Pre-sowing PME application on seeds of two spring wheat cultivars naturally infected with FCR and CRR provided the mitigation of both diseases under field conditions during four growing seasons (2013⁻2016). PME-induced ion exchange response in cultured wheat cells, their increased survivability, and up-regulated expression of some defensins' genes in PME-exposed seedlings allow the suggestion of the plant-mediated character of disease-controlling effect observed in field.

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.