Antimicrobial peptaibols induce defense responses and systemic resistance in tobacco against tobacco mosaic virus

Trichoderma spp. are well-known biocontrol agents because of their antimicrobial activity against bacterial and fungal phytopathogens. However, the biochemical mechanism of their antiviral activity remains largely unknown. In this study, we found that Trichokonins, antimicrobial peptaibols isolated from Trichoderma pseudokoningii SMF2, could induce defense responses and systemic resistance in tobacco (Nicotiana tabacum var. Samsun NN) against tobacco mosaic virus (TMV) infection. Local Trichokonin (100 nM) treatment led to 54% lesion inhibition, 57% reduction in average lesion diameter and 30% reduction in average lesion area in systemic tissue of tobacco compared with control, indicating that Trichokonins induced resistance in tobacco against TMV infection. Trichokonin treatment increased the production of reactive oxygen species and phenolic compounds in tobacco. Additionally, application of Trichokonins significantly increased activities of pathogenesis-related enzymes PAL and POD, and upregulated the expression of several plant defense genes. These results suggested that multiple defense pathways in tobacco were involved in Trichokonin-mediated TMV resistance. We report on the antivirus mechanism of peptaibols, which sheds light on the potential of peptaibols in plant viral disease control.     

Solid-state fermentation for Trichokonins production from Trichoderma koningii SMF2 and preparative purification of Trichokonin VI by a simple protocol

Trichokonins are peptaibols produced by Trichoderma koningii SMF2. The main isoforms are Trichokonin VI, Trichokonin VII and Trichokonin VIII. The solid-state fermentation (SSF) was applied for the production of Trichokonin VI. The fermentation factors, which included inoculum size, incubation temperature, initial moisture content and initial pH, were investigated and optimized by response surface methodology. The maximum Trichokonin VI production (4.07mg/g dry substrate) was achieved by employing inoculum size of 18%, incubation temperature at 24.3 degrees C, initial moisture content of 77.5% and initial pH at 5.0. Furthermore, gel filtration and preparative HPLC were used for separation of Trichokonin VI from a crude extract of the T. koningii SMF2 culture. With this preparative purification protocol under optimized fermentation conditions, 146.20mg Trichokonin VI was obtained from 1kg solid cultures. It has been shown that the obtained Trichokonin VI is more than 95% in purity. This is the first report on optimization of peptaibols production in SSF with high content. An efficient method for the preparative purification of Trichokonin VI is also proposed.     

The antimicrobial peptaibol trichokonin IV promotes plant growth and induces systemic resistance against Botrytis cinerea infection in moth orchid

Trichokonins (TKs) are antimicrobial peptaibols that are extracted from Trichoderma pseudokoningii strain SMF2. We discovered that TK VI, the primary active constituent of TKs, not only promotes growth, but also induces systemic resistance against grey mould caused by Botrytis cinerea in moth orchid (Phalaenopsis). Firstly, following treatment with TK VI, the growth of several varieties of Phalaenopsis increased relative to the control treatment. Both the aboveground and belowground biomass increased, particularly the length, superficial area, volume and root branching. Secondly, treatment with TK VI by either root or foliar application controlled grey mould on the moth orchids. Following irrigation with TK VI, the activities of defence‐related enzymes, including peroxidase, polyphenol oxidase and phenylalanine ammonia‐lyase, and the resistance‐related enzymes, including superoxide dismutase and catalase, all increased, while those of harmful substance malondialdehyde decreased. These findings indicated that induced systemic resistance was the primary mechanism of control.     

Longibramides A–E,Peptaibols Isolated from a Mushroom Derived Fungus Trichoderma longibrachiatum Rifai DMG-3-1-1

Five new peptaibols, longibramides A–E ( 1 – 5 ) with 11 amino acid residues, were isolated from a fungus Trichoderma longibrachiatum Rifai DMG-3-1-1, which was isolated from a mushroom Clitocybe nebularis (Batsch) P. Kumm collected from coniferous forest in the subboreal area of northeast China. The structures of longibramides A–E were determined by their spectroscopic data (NMR and MS-MS spectra), their absolute configurations were determined by X-ray diffractions and Marfey's analyses. The X-ray diffractions of longibramides A, B, and the similar CD spectra of A–E showed that they all had α-helix conformations. Longibramides B and E showed moderate cytotoxicities against BV2 and MCF-7 cells and also showed some inhibitory effects against methicillin-resistant Staphylococcus aureus MRSA T144. L-trans-Hyp was not commonly found in natural peptaibols, which was the 6^th or 10^th amino acid residue in longibramides C–E. The X-ray diffractions of longibramides A and B afforded the accuracy conformations of their secondary structures, which maybe help to interpret the structure-activity relationships of the family of peptaibols in the future.     

Antimicrobial Peptaibols,Trichokonins, Inhibit Mycelial Growth and Sporulation and Induce Cell Apoptosis in the Pathogenic Fungus <Emphasis Type="Italic">Botrytis cinerea</Emphasis>

Trichokonins (TKs) are antimicrobial peptaibols extracted from Trichoderma pseudokoningii strain SMF2. In this paper, it was discovered that TK VI, the main active ingredient of TKs, had a profound inhibitory effect on the growth and sporulation of the moth orchid gray mold, Botrytis cinerea. In addition, TK VI increased the cell membrane permeability of the pathogen. Further investigation of nuclear DNA fragmentation, subcellular structure disintegration, and mitochondrial membrane potential depolarization, as well as the appearance of reactive oxygen species, indicated that TK VI could induce programmed cell death in the necrotrophic pathogenic fungus B. cinerea.     

Diversity Profile and Dynamics of Peptaibols Produced by Green Mould Trichoderma Species in Interactions with Their Hosts Agaricus bisporus and Pleurotus ostreatus

Certain Trichoderma species are causing serious losses in mushroom production worldwide. Trichoderma aggressivum and Trichoderma pleuroti are among the major causal agents of the green mould diseases affecting Agaricus bisporus and Pleurotus ostreatus, respectively. The genus Trichoderma is well‐known for the production of bioactive secondary metabolites, including peptaibols, which are short, linear peptides containing unusual amino acid residues and being synthesised via non‐ribosomal peptide synthetases (NRPSs). The aim of this study was to get more insight into the peptaibol production of T. aggressivum and T. pleuroti. HPLC/MS‐based methods revealed the production of peptaibols closely related to hypomurocins B by T. aggressivum, while tripleurins representing a new group of 18‐residue peptaibols were identified in T. pleuroti. Putative NRPS genes enabling the biosynthesis of the detected peptaibols could be found in the genomes of both Trichoderma species. In vitro experiments revealed that peptaibols are potential growth inhibitors of mushroom mycelia, and that the host mushrooms may have an influence on the peptaibol profiles of green mould agents.     

In vitro antagonism of Thielaviopsis paradoxa by Trichoderma longibrachiatum

Seventy-nine Trichoderma strains were isolated from soil taken from 28 commercial plantations of Agave tequilana cv. ‘Azul’ in the State of Jalisco, Mexico. Nine of these isolates produced nonvolatile metabolites that completely inhibited the growth of Thielaviopsis paradoxa on potato dextrose agar plates. These isolates were identified as Trichoderma longibrachiatum on the basis of their morphology and DNA sequence analysis of two genes (ITS rDNA and translation elongation factor EF-1α). Mycoparasitism of Th. paradoxa by T. longibrachiatum strains in dual cultures was examined by scanning electron microscopy. The Trichoderma hyphae grew alongside the Th. paradoxa hyphae, but penetration of Thielaviopsis hyphae by Trichoderma was no apparent. Aleurioconidia of Th. paradoxa were parasitized by Trichoderma. Both hyphae and aleurioconidia of Th. paradoxa lost turgor pressure, wrinkled, collapsed and finally disintegrated. In liquid cultures, all nine Trichoderma isolates produced proteases, β-1,3-glucanases and chitinases that would be responsible for the degradation of Thielaviopsis hyphae. These results demonstrate that the modes of action of T. longibrachiatum involved against Th. paradoxa in vitro experiments are mycoparasitism and the production of nonvolatile toxic metabolites.     

Production of antifungal metabolites by the ectomycorrhizal fungusPisolithus tinctorius strain SMF

Summary An ectomycorrhizal fungus,Pisolithus tinctorius strain SMF, isolated from a basidiocarp removed from the roots of a recently fallen old growth fir in the Smoky Mountains of Tennessee, was characterized for its in vitro production of antifungal metabolites. On solid mediumP. tinctorius SMF strongly inhibited growth of strains ofFusarium solani, Geotrichum candidum, Phanerochaete chrysosporium, andVerticillium dahliae, all species known to be plant pathogens. Evidence from paired colony growth inhibition studies on agar plates indicated that production of antifungal agents byP. tinctorius SMF may be enhanced by close physical contact with other fungi. The antifungal activity ofP. tinctorius SMF was much greater than that of several culture collection strains ofP. tinctorius. The culture collection strains either showed no or very limited activity. The antifungal activity was associated with an apparently inducible metabolism ofP. tinctorius SMF and with the production of darkly colored water soluble phenolic metabolites. Small scale fermentation studies showed that the phenolics are readily producible by submerged culture fermentation. This is the first report of submerged culture production of antifungal metabolites by an ectomycorrhizal fungus.     

The Production of Multiple Small Peptaibol Families by Single 14‐Module Peptide Synthetases in Trichoderma/Hypocrea

The most common sequences of peptaibiotics are 11‐residue peptaibols found widely distributed in the genus Trichoderma/Hypocrea. Frequently associated are 14‐residue peptaibols sharing partial sequence identity. Genome sequencing projects of three Trichoderma strains of the major clades reveal the presence of up to three types of nonribosomal peptide synthetases with 7, 14, or 18–20 amino acid‐adding modules. Here, we provide evidence that the 14‐module NRPS type found in T. virens, T. reesei (teleomorph Hypocrea jecorina), and T. atroviride produces both 11‐ and 14‐residue peptaibols based on the disruption of the respective NRPS gene of T. reesei, and bioinformatic analysis of their amino acid‐activating domains and modules. The sequences of these peptides may be predicted from the gene sequences and have been confirmed by analysis of families of 11‐ and 14‐residue peptaibols from the strain 618, termed hypojecorins A (23 sequences determined, 4 new) and B (3 sequences determined, 2 new), and the recently established trichovirins A from T. virens. The distribution of 11‐ and 14‐residue products is strain‐specific and depends on growth conditions as well. Possible mechanisms of module skipping are discussed.     

Foreign Peptides Expressed in Engineered Chimeric Self Molecules

Quorum sensing (QS) has received significant attention in the past few decades. QS describes population density dependent cell to cell communication in bacteria using diffusible signal molecules. These signal molecules produced by bacterial cells, regulate various physiological processes important for social behavior and pathogenesis. One such process regulated by quorum sensing molecules is the production of a biosurfactant, rhamnolipid. Rhamnolipids are important microbially derived surface active agents produced by Pseudomonas spp. under the control of two interrelated quorum sensing systems; namely las and rhl. Rhamnolipids possess antibacterial, antifungal and antiviral properties. They are important in motility, cell to cell interactions, cellular differentiation and formation of water channels that Currently, biosurfactants are unable to compete economically with chemically synthesized compounds in the market due to high production costs. Once the genes required for biosurfactant production have been identified, they can be placed under the regulation of strong promoters in nonpathogenic, heterologous hosts to enhance production. The production of rhamnolipids could be increased by cloning both the rhlAB rhamnosyltransferase genes and the rhlRI quorum sensing system into a suitable bacterium such as E. coli or P. putida and facilitate rhamnolipid production. Biosurfactants can also be genetically engineered for different industrial applications assuming there is a strong understanding of both the genetics and the structure-function relationships of each component of the molecule. Genetic engineering of surfactin has already been reported, with recent papers describing the creation of novel peptide structures from the genetic recombination of several peptide synthetases. Recent application of dynamic metabolic engineering strategies for controlled gene expression could lower the cost of fermentation processes by increasing the product formation. Therefore, by integrating a genetic circuit into applications of metabolic engineering the biochemical production can be optimized. Furthermore, novel strategies could be designed on the basis of information obtained from the studies of quorum sensing and biosurfactants produced suggesting enormous practical applications.     

Food deprivation is not a prerequisite for the amoebal to plasmodial transition in Physarum polycephalum

The effect of food supply on the onset of asexual and sexual plasmodium formation in Physarum polycephalum was studied. Asexual differentiation occurs readily in amoebae carrying the matAh mating type allele. The density at which these amoebae begin to differentiate is influenced by the ind locus, which controls the production of a diffusible inducer. The alleles ind-1 and ind-2 are known. Strains carring the ind-1 allele begin plasmodium formation at a low amoebal density (rapid differentiation), while strains carring the ind-2 allele differentiate at a higher amoebal density (slow differentiation). The onset of differentiation is characteristic of the strain and did not change with a 20-fold variation in the number of food bacteria available. Sexual differentiation occurs between compatible amoebal strains. For a given pair of amoebal strains the onset of plasmodium formation occurs at a characteristic cell density that is determined by the genetic backgrounds of the strains. The ind locus is one of the genes that influences this cell density. Plasmodia are formed at a lower cell density in crosses involving compatible amoebae carrying the ind-1 allele than they are in crosses with strains carrying the ind-2 allele. As was found for asexual differentiation, an approximate 20-fold variation in the food supply did not affect the initiation of sexual plasmodium formation. These results suggest that in most cases starvation does not trigger the differentiation of amoebae into plasmodia. The time of onset of plasmodium formation is determined largely by genetic factors.     

Functional characterization of a three-component regulatory system involved in quorum sensing-based regulation of peptide antibiotic production in Carnobacterium maltaromaticum

Background  

Quorum sensing is a form of cell-to-cell communication that allows bacteria to control a wide range of physiological processes in a population density-dependent manner. Production of peptide antibiotics is one of the processes regulated by quorum sensing in several species of Gram-positive bacteria, including strains of Carnobacterium maltaromaticum. This bacterium and its peptide antibiotics are of interest due to their potential applications in food preservation. The molecular bases of the quorum sensing phenomenon controlling peptide antibiotic production in C. maltaromaticum remain poorly understood. The present study was aimed at gaining a deeper insight into the molecular mechanism involved in quorum sensing-mediated regulation of peptide antibiotic (bacteriocin) production by C. maltaromaticum. We report the functional analyses of the CS (autoinducer)-CbnK (histidine protein kinase)-CbnR (response regulator) three-component regulatory system and the three regulated promoters involved in peptide antibiotic production in C. maltaromaticum LV17B.     

Lysine Overproduction Mutations in the YeastSaccharomyces cerevisiae Are Introduced into Industrial Yeast Strains

Yeast mutants resistant to a toxic lysine analog, thialysine were obtained by a method described in the literature [1]. A strain excreting the maximum amount of lysine (0.45 g/l) was selected from these mutants. The intracellular content of lysine was also increased by 30%. The genetic nature of lysine overproduction was studied in this strain. An increase in the amount of excreted lysine was shown to be determined by at least two genes, one of which carries a mutation of thialysine resistance manifesting the pleiotropic effect of lysine overproduction (Th1 ^R) and the other is involved in the regulation of lysine production (PRL). Linkage groups of these genes were determined: the first gene was mapped to the IV chromosome and the second, to the XV chromosome. Both genetic characters were introduced into industrial baker's yeast strains via a series of backcrosses. The stabilization of the genome in the newly derived strains was confirmed by electrokaryotyping.