RESUMO
Fluorescent dyes were assessed for their ability to stain viable hyphae of the fungi Neotyphodium lolii and N. coenophialum, symbiotic endophytes of the Pooideae grasses Lolium perenne and Festuca arundinacea, respectively. The fluorescein-based fluorophores; fluorescein diacetate (FDA), 5(6)-carboxy-fluorescein diacetate (CFDA), 5-chloromethylfluorescein diacetate (CMFDA) and the chitin-binding stain, Calcofluor while M2R, were assessed for staining of endophyte hyphae in vitro from axenic fungal cultures and in planta, including epidermal leaf sheath peels, nodes, ovaries, embryos and meristems. CMFDA produced the greatest intensity of staining of fungal hyphae and gave excellent contrast in planta compared to the plant cells. Compared to the other dyes, CMFDA was also the least affected by photo bleaching and continued to fluoresce up to 2 h after initial excitation. None of the fluorescent dyes stained fungal hyphae in seed.
Assuntos
Endófitos/química , Fluoresceínas/química , Corantes Fluorescentes/química , Neotyphodium/química , Poaceae/microbiologia , Endófitos/fisiologia , Neotyphodium/fisiologia , Poaceae/fisiologia , Coloração e Rotulagem , SimbioseRESUMO
Epoxyjanthitrems I-IV (1-4) and epoxyjanthitriol (5) were isolated from seed of perennial ryegrass (Lolium perenne) infected with the endophytic fungus Epichloë festucae var. lolii. Although structures for epoxyjanthitrems I-IV have previously been proposed in the literature, this is the first report of a full structural elucidation yielding NMR (Nuclear magnetic resonance) assignments for all five epoxyjanthitrem compounds, and additionally, it is the first isolation of epoxyjanthitriol (5). Epoxyjanthitrem I induced tremors in mice and gave a dose dependent reduction in weight gain and feeding for porina (Wiseana cervinata), a common pasture pest in New Zealand. These data suggest that epoxyjanthitrems are involved in the observed effects of the AR37 endophyte on livestock and insect pests.
Assuntos
Endófitos/química , Epichloe/química , Insetos/efeitos dos fármacos , Lolium/microbiologia , Micotoxinas/química , Micotoxinas/farmacologia , Tremor/induzido quimicamente , Animais , Modelos Animais de Doenças , Feminino , Interações entre Hospedeiro e Microrganismos , Camundongos , Nova ZelândiaRESUMO
Bioprotective alkaloids produced by Epichloë and closely related asexual Neotyphodium fungal endophytes protect their grass hosts from insect and mammalian herbivory. One class of these compounds, known for antimammalian toxicity, is the indole-diterpenes. The LTM locus of Neotyphodium lolii (Lp19) and Epichloë festuce (Fl1), required for the biosynthesis of the indole-diterpene lolitrem, consists of 10 ltm genes. We have used PCR and Southern analysis to screen a broad taxonomic range of 44 endophyte isolates to determine why indole-diterpenes are present in so few endophyte-grass associations in comparison to that of the other bioprotective alkaloids, which are more widespread among the endophtyes. All 10 ltm genes were present in only three epichloë endophytes. A predominance of the asexual Neotyphodium spp. examined contained 8 of the 10 ltm genes, with only one N. lolii containing the entire LTM locus and the ability to produce lolitrems. Liquid chromatography-tandem mass spectrometry profiles of indole-diterpenes from a subset of endophyte-infected perennial ryegrass showed that endophytes that contained functional genes present in ltm clusters 1 and 2 were capable of producing simple indole-diterpenes such as paspaline, 13-desoxypaxilline, and terpendoles, compounds predicted to be precursors of lolitrem B. Analysis of toxin biosynthesis genes by PCR now enables a diagnostic method to screen endophytes for both beneficial and detrimental alkaloids and can be used as a resource for screening isolates required for forage improvement.
Assuntos
Diterpenos/metabolismo , Epichloe/genética , Epichloe/metabolismo , Proteínas Fúngicas/genética , Indóis/metabolismo , Southern Blotting , Cromatografia Líquida , DNA Fúngico/genética , Diterpenos/farmacologia , Indóis/farmacologia , Lolium/química , Espectrometria de Massas , Família Multigênica , Micotoxinas/metabolismo , Micotoxinas/farmacologia , Neotyphodium/genética , Neotyphodium/metabolismo , Neurotoxinas/metabolismo , Neurotoxinas/farmacologia , Reação em Cadeia da Polimerase/métodosRESUMO
Many grasses live in association with asymptomatic fungi (Neotyphodium spp. endophytes), which grow in the intercellular spaces of the grass. These endophytes produce a range of alkaloids that protect the grass against grazing by mammals and insects. One of these alkaloids is an unusual pyrrolopyrazine, peramine. Peramine appears to be continuously produced by the endophyte, but does not progressively accumulate. No mechanism for the removal of peramine by its further metabolism or any other process has been reported. Our aim was to detect peramine or peramine metabolites in plant fluids to determine if peramine is mobilized, metabolized or excreted by the plant. We also wanted to determine if other fungal metabolites are mobilized by the plant, as has been proposed for the loline alkaloids. We developed a highly sensitive method for the analysis of peramine, using a linear ion trap mass spectrometer. We studied the fragmentation pathway of peramine using ESI MSn and ESI FTICRMS. Based on these results we developed a single reaction monitoring method using the fragmentation of the guanidinium moiety. Cut leaf fluid and guttation fluid of different grass endophyte associations (Lolium perenne with Neotyphodium lolii, Festuca arundinacea with Neotyphodium coenophialum, and Elymus sp. with Epichloë sp.) were analysed. Peramine was detected in the cut leaf fluid of all grass-endophyte associations, but not in the guttation fluid of all associations. In some associations we also detected lolines and ergot peptide alkaloids. This is the first report showing the mobilization of fungal alkaloids into plant fluids by the host plant in grass-endophyte associations.
Assuntos
Alcaloides/metabolismo , Fungos/metabolismo , Compostos Heterocíclicos com 2 Anéis/metabolismo , Lolium/metabolismo , Poliaminas/metabolismo , Lolium/microbiologia , Espectrometria de Massas por Ionização por Electrospray , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Endophytes of the genus Epichloë (Clavicipitaceae, Ascomycota) frequently occur within cool-season grasses and form interactions with their hosts that range from mutualistic to antagonistic. Many Epichloë species have arisen via interspecific hybridization, resulting in species with two or three subgenomes that retain all or nearly all of their original parental genomes, a process termed allopolyploidization. Here, we characterize Epichloë hybrida, sp. nov., a mutualistic species that has increasingly become a model system for investigating allopolyploidy in fungi. The Epichloë species so far identified as the closest known relatives of the two progenitors of E. hybrida are E. festucae var. lolii and E. typhina. We confirm that the nuclear genome of E. hybrida contains two homeologs of most protein-coding genes from E. festucae and E. typhina, with genome-wide gene expression analysis indicating a slight bias in overall gene expression from the E. typhina subgenome. Mitochondrial DNA is detectable only from E. festucae, whereas ribosomal DNA is detectable only from E. typhina. Inheriting ribosomal DNA from just one parent might be expected to preferentially favor interactions with ribosomal proteins from the same parent, but we find that ribosomal protein genes from both parental subgenomes are nearly all expressed equally in E. hybrida. Finally, we provide a comprehensive set of resources for this model system that are intended to facilitate further study of fungal hybridization by other researchers.
Assuntos
Diploide , Endófitos/classificação , Endófitos/genética , Epichloe/classificação , Epichloe/genética , Quimera , DNA Fúngico/genética , DNA Mitocondrial/genética , DNA Ribossômico/genética , Perfilação da Expressão GênicaRESUMO
Three grass host species--tall fescue, Festuca arundinacea Schreber; meadow fescue, Festuca pratensis Hudson; and perennial ryegrass, Lolium perenne L.--each infected with a number of different Neotyphodium endophyte isolates, were investigated for their effects on fall armyworm, Spodoptera frugiperda (J.E. Smith). Alkaloid profiles varied among associations. Choice and no-choice tests comparing feeding and early development of S. frugiperda larvae on endophyte-infected and endophyte-free leaf blade material were performed. Endophyte-mediated resistance to S. frugiperda was greatest in meadow fescue and weakest in tall fescue. Some endophyte isolates, particularly in perennial ryegrass and meadow fescue, had a major effect on feeding and development of S. frugiperda, whereas others had no effect or were only weakly efficacious. In tall fescue, some associations deterred S. frugiperda from feeding in choice tests but had no effect on development, whereas larvae reared on other associations weighed significantly more than control larvae fed endophyte-free grass. It was concluded that the deleterious consequences of endophyte infection were easily masked by other factors in tall fescue. Relative leaf age had no effect on feeding preferences in the three host species. Chemical analysis of herbage from the plants used, and results from a no-choice study using spiked artificial diets, failed to individually implicate any of the major known alkaloids (peramine, lolitrem B, ergovaline, and lolines) in the observed effects on S. frugiperda. Hypotheses explaining these observations, and their impact on creating desirable grass-endophyte associations for use in pastures, are discussed.
Assuntos
Alcaloides/farmacologia , Larva/efeitos dos fármacos , Mariposas/efeitos dos fármacos , Poaceae/parasitologia , Animais , Interações Hospedeiro-Parasita , Poaceae/microbiologiaRESUMO
The penitremane and janthitremane families of indole-diterpenes are abundant natural products synthesized by Penicillium crustosum and P. janthinellum. Using a combination of PCR, cosmid library screening, and Illumina sequencing we have identified gene clusters encoding enzymes for the synthesis of these compounds. Targeted deletion of penP in P. crustosum abolished the synthesis of penitrems A, B, D, E, and F, and led to accumulation of paspaline, a key intermediate for paxilline biosynthesis in P. paxilli. Similarly, deletion of janP and janD in P. janthinellum abolished the synthesis of prenyl-elaborated indole-diterpenes, and led to accumulation in the latter of 13-desoxypaxilline, a key intermediate for the synthesis of the structurally related aflatremanes synthesized by Aspergillus flavus. This study helps resolve the genetic basis for the complexity of indole-diterpene natural products found within the Penicillium and Aspergillus species. All indole-diterpene gene clusters identified to date have a core set of genes for the synthesis of paspaline and a suite of genes encoding multi-functional cytochrome P450 monooxygenases, FAD dependent monooxygenases, and prenyl transferases that catalyse various regio- and stereo- specific oxidations that give rise to the diversity of indole-diterpene products synthesized by this group of fungi.
Assuntos
Diterpenos/metabolismo , Genes Fúngicos , Indóis/metabolismo , Micotoxinas/metabolismo , Penicillium/genética , Penicillium/metabolismo , Sequência de Bases , Clonagem Molecular , DNA Fúngico/análise , Proteínas Fúngicas/genética , Dados de Sequência Molecular , Família Multigênica , Oxigenases/genética , Análise de Sequência de DNA , Transferases/genéticaRESUMO
Neotyphodium sp. Lp1, an endophytic fungus from perennial ryegrass (Lolium perenne), produces the mycotoxin ergovaline in infected grasses, whereas a mutant in which a particular peptide synthetase gene is knocked out does not. We examined the impact of this knockout on other constituents of the ergot alkaloid pathway. Two simple lysergic acid amides, ergine and a previously undescribed amide, were eliminated by the knockout. Lysergic acid accumulated in the knockout endophyte, but quantities were only 13% of the total lysergic acid derivatives accumulated in the wild type. Concentrations of several clavines were not substantially affected. However, a novel clavine accumulated to higher concentrations in perennial ryegrass containing the knockout strain. The results indicate that production of simple lysergic acid amides requires the activity or products of the ergovaline-associated peptide synthetase and that the regulation of ergot alkaloid production is modified in response to the relatively late block in the pathway.
Assuntos
Ascomicetos/genética , Ascomicetos/metabolismo , Alcaloides de Claviceps/metabolismo , Lolium/microbiologia , Ascomicetos/enzimologia , Alcaloides de Claviceps/análise , Alcaloides de Claviceps/química , Ácido Lisérgico/metabolismo , Mutagênese , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismoRESUMO
An improved extraction and cleanup procedure for quantitative analysis of ergovaline in Neotyphodium-infected grass tissues by high-performance liquid chromatography was developed, utilizing aqueous 2-propanol-lactic acid as extraction solvent. Losses of sample material and time requirements were significantly reduced, handling procedures simplified, and ergovaline and internal standard ergotamine recovered with similar efficiency from extracts. Analyses can be carried out on very small amounts (2-5 mg of dry weight) of samples and another endophyte-alkaloid, peramine, determined in the same extracts. Calibration curves with 2-propanol-lactic acid were linear over the range 0.004-0.938 microM ergovaline (= 2-500 ng/mL) in extracts, corresponding to 0.04-10 microg/g in samples. The distribution of ergovaline in the plant was extremely heterogeneous, indicating low in-planta mobility and strong regulation of accumulation by the internal plant environment. In contrast, peramine was much more uniformly distributed. These results clearly demonstrate very large differences in the tissue specificities of ergovaline and peramine.
Assuntos
Acremonium/metabolismo , Alcaloides/análise , Ergotaminas/análise , Lolium/química , Doenças das Plantas , Poaceae/química , 2-Propanol , Ácido Láctico , Lolium/microbiologia , Poaceae/microbiologia , Distribuição TecidualRESUMO
Turnip (Brassica rapa ssp. rapa) and rape (Brassica napus ssp. biennis) and other brassica forage crops are regarded as "safe" feed for cattle during late summer and fall in the North Island of New Zealand when high Pithomyces chartarum spore counts in pastures frequently lead to sporidesmin toxicity (facial eczema). Sporadic acute severe cases of turnip photosensitization in dairy cows characteristically exhibit high γ-glutamyl transferase and glutamate dehydrogenase serum enzyme activities that mimic those seen in facial eczema. The two diseases can, however, be distinguished by histopathology of the liver, where lesions, in particular those affecting small bile ducts, differ. To date, the hepato-/cholangiotoxic phytochemical causing liver damage in turnip photosensitization in cattle is unknown. Of the hydrolysis products of the various glucosinolate secondary compounds found in high concentrations in turnip and rape, work has shown that nitriles and epithionitriles can be hepatotoxic (and nephro- or pancreatotoxic) in rats. These derivatives include ß-hydroxy-thiiranepropanenitrile and 3-hydroxy-4-pentenenitrile from progoitrin; thiiranepropanenitrile and 4-pentenenitrile from gluconapin; thiiranebutanenitrile and 5-hexenenitrile from glucobrassicanapin; phenyl-3-propanenitrile from gluconasturtiin; and indole-3-acetonitrile from glucobrassicin. This perspective explores the possibility of the preferential formation of such derivatives, especially the epithionitriles, in acidic conditions in the bovine rumen, followed by absorption, hepatotoxicity, and secondary photosensitization.
Assuntos
Brassica napus/química , Brassica rapa/química , Glucosinolatos/toxicidade , Indóis/toxicidade , Nitrilas/toxicidade , Animais , Bovinos , Doença Hepática Induzida por Substâncias e Drogas/veterinária , Modelos Animais de Doenças , Glucosinolatos/química , Indóis/química , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Nova Zelândia , Nitrilas/química , RatosRESUMO
The indole-diterpene paxilline is an abundant secondary metabolite synthesized by Penicillium paxilli. In total, 21 genes have been identified at the PAX locus of which six have been previously confirmed to have a functional role in paxilline biosynthesis. A combination of bioinformatics, gene expression and targeted gene replacement analyses were used to define the boundaries of the PAX gene cluster. Targeted gene replacement identified seven genes, paxG, paxA, paxM, paxB, paxC, paxP and paxQ that were all required for paxilline production, with one additional gene, paxD, required for regular prenylation of the indole ring post paxilline synthesis. The two putative transcription factors, PP104 and PP105, were not co-regulated with the pax genes and based on targeted gene replacement, including the double knockout, did not have a role in paxilline production. The relationship of indole dimethylallyl transferases involved in prenylation of indole-diterpenes such as paxilline or lolitrem B, can be found as two disparate clades, not supported by prenylation type (e.g., regular or reverse). This paper provides insight into the P. paxilli indole-diterpene locus and reviews the recent advances identified in paxilline biosynthesis.
Assuntos
Deleção de Genes , Expressão Gênica , Genes Fúngicos , Indóis/metabolismo , Família Multigênica , Penicillium/genética , Sequência de Aminoácidos , Clonagem Molecular , Biologia Computacional , Diterpenos/metabolismo , Escherichia coli , Loci Gênicos , Alcaloides Indólicos/metabolismo , Dados de Sequência Molecular , Micotoxinas/metabolismo , Penicillium/metabolismo , Análise de Sequência de DNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Epichloë festucae Fl1 in association with Lolium perenne synthesizes a diverse range of indole-diterpene bioprotective metabolites, including lolitrem B, a potent tremorgen. The ltm genes responsible for the synthesis of these metabolites are organized in three clusters at a single sub-telomeric locus in the genome of E. festucae. Here we resolve the genetic basis for the remarkable indole-diterpene diversity observed in planta by analyzing products that accumulate in associations containing ltm deletion mutants of E. festucae and in cells of Penicillium paxilli containing copies of these genes under the control of a P. paxilli biosynthetic gene promoter. We propose a biosynthetic scheme to account for this metabolic diversity.
Assuntos
Regulação Fúngica da Expressão Gênica , Indóis/química , Família Multigênica , Micotoxinas/biossíntese , Poaceae/metabolismo , Cromatografia Líquida/métodos , Epichloe , Deleção de Genes , Alcaloides Indólicos , Espectrometria de Massas/métodos , Modelos Químicos , Modelos Genéticos , Micotoxinas/química , Penicillium/metabolismo , Poaceae/microbiologia , Regiões Promotoras Genéticas , Fatores de TempoRESUMO
A fast method was developed to directly infuse raw plant extracts into a linear ion trap mass spectrometer, using the ion trap to isolate and fragment as many ions as possible from the extract. The full mass spectra can be analysed by multivariate statistics to determine discriminating ions, and the fragmentation data allows rapid classification or identification of these ions. The methodology was used to screen a wide range of strains of endophytic fungi in perennial ryegrass seeds for differences in metabolic profiles. The results show that this newly developed methodology is able to determine discriminating ions that can be present in very low concentrations. It also yielded sufficient fragmentation data to classify or identify the discriminating ions.
Assuntos
Ascomicetos/metabolismo , Análise de Injeção de Fluxo/métodos , Proteínas Fúngicas/análise , Lolium/microbiologia , Mapeamento de Peptídeos/métodos , Sementes/microbiologia , Espectrometria de Massas por Ionização por Electrospray/métodos , Ascomicetos/isolamento & purificaçãoRESUMO
While much is known about the biosynthesis of secondary metabolites by filamentous fungi their biological role is often less clear. The assumption is these pathways have adaptive value to the organism but often the evidence to support this role is lacking. We provide the first genetic evidence that the fungal produced secondary metabolite, peramine, protects a host plant from insect herbivory. Peramine is a potent insect feeding deterrent synthesized by Epichloë/Neotyphodium mutualistic endophytes in association with their grass hosts. The structure of peramine, a pyrrolopyrazine, suggests that it is the product of a reaction catalysed by a two-module non-ribosomal peptide synthetase (NRPS). Candidate sequences for a peramine synthetase were amplified by reverse transcription polymerase chain reaction. Four unique NRPS products were identified, two of which were preferentially expressed in planta. One of these hybridized to known peramine producing strains. This clone was used to isolate an Epichloë festucae cosmid that contained a two-module NRPS, designated perA. Nine additional genes, which show striking conservation of microsynteny with Fusarium graminearum and other fungal genomes, were identified on the perA-containing cosmid. Associations between perennial ryegrass and an E. festucae mutant deleted for perA lack detectable levels of peramine. A wild-type copy of perA complemented the deletion mutant, confirming that perA is a NRPS required for peramine biosynthesis. In a choice bioassay, plant material containing the perA mutant was as susceptible to Argentine stem weevil (ASW) (Listronotus bonariensis) feeding damage as endophyte-free plants confirming that peramine is the E. festucae metabolite responsible for ASW feeding deterrent activity.