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1.
Ecol Lett ; 25(8): 1879-1888, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35810320

RESUMO

Plants interacting with mutualistic fungi (MF) or antagonistic fungi (AF) can form associations with bacteria. We assessed whether the performance gain conferred by mutualistic bacteria to fungal-associated plants is affected by the interaction between symbiont traits, type of bacterial-protective traits against AF and abiotic/biotic stresses. Results showed that (A) performance gain conferred by bacteria to MF-associated plants was greater when symbionts promoted distinct rather than similar plant functions, (B) bacterial-based alleviation of the AF's negative effect on plants was independent of the type of protective trait, (C) bacteria promoted a greater performance of symbiotic plants in presence of biotic, but not abiotic, stress compared to stress-free situations. The plant performance gain was not affected by any fungal-bacterial trait combination but optimised when bacteria conferred resistance traits in biotic stress situations. The effects of bacteria on fungal-associated plants were controlled by the interaction between the symbionts' functional traits and the relationship between bacterial traits and abiotic/biotic stresses.


Assuntos
Plantas , Simbiose , Bactérias , Fungos , Fenômenos Fisiológicos Vegetais , Plantas/microbiologia
2.
Environ Microbiol ; 23(4): 2116-2131, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33350014

RESUMO

Recent studies have identified key genes that control the symbiotic interaction between Epichloë festucae and Lolium perenne. Here we report on the identification of specific E. festucae genes that control host infection. Deletion of setB, which encodes a homologue of the H3K36 histone methyltransferase Set2/KMT3, reduced histone H3K36 trimethylation and led to severe defects in colony growth and hyphal development. The E. festucae ΔclrD mutant, which lacks the gene encoding the homologue of the H3K9 methyltransferase KMT1, displays similar developmental defects. Both mutants are completely defective in their ability to infect L. perenne. Alleles that complement the culture and plant phenotypes of both mutants also complement the histone methylation defects. Co-inoculation of either ΔsetB or ΔclrD with the wild-type strain enables these mutants to colonize the host. However, successful colonization by the mutants resulted in death or stunting of the host plant. Transcriptome analysis at the early infection stage identified four fungal candidate genes, three of which encode small-secreted proteins, that are differentially regulated in these mutants compared to wild type. Deletion of crbA, which encodes a putative carbohydrate binding protein, resulted in significantly reduced host infection rates by E. festucae.


Assuntos
Epichloe , Epichloe/genética , Epichloe/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Histonas/genética , Metiltransferases/genética , Poaceae , Simbiose/genética
3.
Mol Microbiol ; 112(3): 837-853, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31165508

RESUMO

Studies on the regulation of fungal secondary metabolism highlight the importance of histone H3K4 methylation regulators Set1, CclA (Ash2) and KdmB (KDM5), but it remains unclear whether these proteins act by direct modulation of H3K4me3 at the target genes. In filamentous fungi, secondary metabolite genes are frequently located near telomeres, a site where H3K4 methylation is thought to have a repressive role. Here we analyzed the role of CclA, KdmB and H3K4me3 in regulating the subtelomeric EAS and LTM cluster genes in Epichloë festucae. Depletion of H3K4me3 correlated with transcriptional activation of these genes in ΔcclA, similarly enrichment of H3K4me3 correlated with transcriptional repression of the genes in ΔkdmB which was accompanied by significant reduction in the levels of the agriculturally undesirable lolitrems. These transcriptional changes could only be explained by the alterations in H3K4me3 and not in the subtelomerically-important marks H3K9me3/K27me3. However, H3K4me3 changes in both mutants were not confined to these regions but occurred genome-wide, and at other subtelomeric loci there were inconsistent correlations between H3K4me3 enrichment and gene repression. Our study suggests that CclA and KdmB are crucial regulators of secondary metabolite genes, but these proteins likely act via means independent to, or in conjunction with the H3K4me3 mark.


Assuntos
Epichloe/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Histonas/metabolismo , Epichloe/genética , Proteínas Fúngicas/genética , Genoma Fúngico , Histonas/genética , Mutação , Metabolismo Secundário
4.
Mol Plant Microbe Interact ; 32(10): 1324-1335, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31107632

RESUMO

In ascomycetes and basidiomycetes, iron-responsive GATA-type transcriptional repressors are involved in regulating iron homeostasis, notably to prevent iron toxicity through control of iron uptake. To date, it has been unknown whether this iron regulator contributes toward mutualistic endosymbiosis of microbes with plants, a system where the endophyte must function within the constraints of an in-host existence, including a dependency on the host for nutrient acquisition. Functional characterization of one such protein, SreA from Epichloë festucae, a fungal endosymbiont of cool-season grasses, indicates that regulation of iron homeostasis processes is important for symbiotic maintenance. The deletion of the sreA gene (ΔsreA) led to iron-dependent aberrant hyphal growth and the gradual loss of endophyte hyphae from perennial ryegrass. SreA negatively regulates the siderophore biosynthesis and high-affinity iron uptake systems of E. festucae, similar to other fungi, resulting in iron accumulation in mutants. Our evidence suggests that SreA is involved in the processes that moderate Epichloë iron acquisition from the plant apoplast, because overharvesting of iron in ΔsreA mutants was detected as premature chlorosis of the host using a hydroponic plant growth assay. E. festucae appears to have a tightly regulated iron management system, involving SreA that balances endophyte growth with its survival and prevents overcompetition with the host for iron in the intercellular niche, thus promoting mutualistic associations. Mutations that interfere with Epichloë iron management negatively affect iron-dependent fungal growth and destabilize mutualistic Epichloë -ryegrass associations.


Assuntos
Epichloe , Fatores de Transcrição GATA , Lolium , Simbiose , Epichloe/genética , Proteínas Fúngicas/genética , Fatores de Transcrição GATA/genética , Fatores de Transcrição GATA/metabolismo , Ferro/metabolismo , Lolium/microbiologia , Mutação , Simbiose/genética
5.
Fungal Genet Biol ; 125: 71-83, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30731202

RESUMO

Epichloë festucae forms mutualistic symbiotic interactions with grasses of the Lolium and Festuca genera. Protection from insect and mammalian herbivory are the best-documented host benefits of these associations. The two main classes of anti-mammalian alkaloids synthesized by E. festucae are the ergot alkaloids and indole diterpenes, of which ergovaline and lolitrems are the principal terminal products. Synthesis of both metabolites require multiple gene products encoded by clusters of 11 genes located at the subtelomeric regions of chromosomes I and III respectively. These loci are essentially unexpressed in axenic culture but among the most highly expressed genes in planta. We show here that heterochromatin 1 protein (HepA) is an important component of the regulatory machinery that maintains these loci in a silent state in culture. Deletion of this gene led to derepression of eas and ltm gene expression under non-symbiotic culture conditions. Although there was no obvious culture phenotype, RNAseq analysis revealed that around 1000 genes were differentially expressed in the ΔhepA mutant compared to wild type with just one-third upregulated. Inoculation of the ΔhepA mutants into seedlings of Lolium perenne led to a severe host interaction phenotype characterized by a reduction in tiller length but an increase in tiller number. Hyphae within the leaves of these associations were much more abundant in the intercellular spaces of the leaves and aberrantly colonized the vascular bundles. This physiological change was accompanied by a dramatic change in the transcriptome with around 900 genes differentially expressed, with two thirds of these upregulated. This major physiological change was accompanied by a decrease in ltm gene expression and loss of the ability to synthesize lolitrems. These results show that HepA has an important role in controlling the chromatin state of these sub-telomeric secondary metabolite genes, including their symbiosis-specific regulation.


Assuntos
Proteínas Cromossômicas não Histona/genética , Epichloe/genética , Alcaloides de Claviceps/genética , Simbiose/genética , Homólogo 5 da Proteína Cromobox , Diterpenos/metabolismo , Endófitos/genética , Endófitos/crescimento & desenvolvimento , Epichloe/crescimento & desenvolvimento , Epigênese Genética , Alcaloides de Claviceps/biossíntese , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/genética , Heterocromatina/genética , Hifas/genética , Hifas/crescimento & desenvolvimento , Lolium/genética , Lolium/microbiologia
6.
Fungal Genet Biol ; 111: 60-72, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29155067

RESUMO

The symbiosis between Epichloë festucae and its host perennial ryegrass (Lolium perenne) is a model system for mutualistic interactions in which the fungal endophyte grows between plant shoot cells and acquires host nutrients to survive. E. festucae synthesises the siderophore epichloënin A (EA) via SidN, a non-ribosomal peptide synthetase (NRPS). EA is involved in the acquisition of iron, an essential micronutrient, as part of the process of maintaining a stable symbiotic interaction. Here, we mutated a different NRPS gene sidC and showed that it is required for production of a second siderophore ferricrocin (FC). Furthermore mutations in sidA, encoding an l-ornithine N5-monooxygenase, abolished both EA and FC production. Axenic growth phenotypes of the siderophore mutants were altered relative to wild-type (WT) providing insights into the roles of E. festucae siderophores in iron trafficking and consequently in growth and morphogenesis. During iron-limitation, EA is the predominant siderophore and in addition to its role in iron acquisition it appears to play roles in intracellular iron sequestration and oxidative stress tolerance. FC in contrast is exclusively located intracellularly and is the dominant siderophore under conditions of iron sufficiency when it is likely to have roles in iron storage and iron transport. Intriguingly, EA acts to promote but may also moderate E. festucae growth (depending on the amount of available iron). We therefore hypothesise that coordinated cellular iron sequestration through FC and EA may be one of the mechanisms that E. festucae employs to manage and restrain its growth in response to iron fluxes and ultimately persist as a controlled symbiont.


Assuntos
Epichloe/fisiologia , Ferro/metabolismo , Peptídeo Sintases/fisiologia , Sideróforos/fisiologia , Epichloe/enzimologia , Epichloe/genética , Genes Fúngicos , Homeostase , Lolium/microbiologia , Mutagênese , Estresse Oxidativo , Peptídeo Sintases/biossíntese , Peptídeo Sintases/genética , Sideróforos/biossíntese , Sideróforos/genética
7.
Fungal Genet Biol ; 85: 14-24, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26519220

RESUMO

Fungal endophytes belonging to the genus Epichloë form associations with temperate grasses belonging to the sub-family Poöideae that range from mutualistic through to pathogenic. We previously identified a novel endophyte gene (designated gigA for grass induced gene) that is one of the most abundantly expressed fungal transcripts in endophyte-infected grasses and which is distributed and highly expressed in a wide range of Epichloë grass associations. Molecular and biochemical analyses indicate that gigA encodes a small secreted protein containing an imperfect 27 amino acid repeat that includes a kexin protease cleavage site. Kexin processing of GigA liberates within the plant multiple related products, named here as epichloëcyclins, which we have demonstrated by MS/MS to be cyclic peptidic in nature. Gene deletion of gigA leads to the elimination of all epichloëcyclins with no conspicuous phenotypic impact on the host grass, suggesting a possible bioactive role. This is a further example of a ribosomal peptide synthetic (RiPS) pathway operating within the Ascomycetes, and is the first description of such a pathway from a mutualistic symbiotic fungus from this Phylum.


Assuntos
Endófitos/genética , Epichloe/genética , Proteínas Fúngicas/genética , Poaceae/microbiologia , Endófitos/fisiologia , Epichloe/fisiologia , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Peptídeos Cíclicos/genética , Peptídeos Cíclicos/metabolismo , Biossíntese de Proteínas , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Simbiose , Espectrometria de Massas em Tandem
8.
PLoS Pathog ; 9(5): e1003332, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23658520

RESUMO

We have identified from the mutualistic grass endophyte Epichloë festucae a non-ribosomal peptide synthetase gene (sidN) encoding a siderophore synthetase. The enzymatic product of SidN is shown to be a novel extracellular siderophore designated as epichloënin A, related to ferrirubin from the ferrichrome family. Targeted gene disruption of sidN eliminated biosynthesis of epichloënin A in vitro and in planta. During iron-depleted axenic growth, ΔsidN mutants accumulated the pathway intermediate N(5)-trans-anhydromevalonyl-N(5)-hydroxyornithine (trans-AMHO), displayed sensitivity to oxidative stress and showed deficiencies in both polarized hyphal growth and sporulation. Infection of Lolium perenne (perennial ryegrass) with ΔsidN mutants resulted in perturbations of the endophyte-grass symbioses. Deviations from the characteristic tightly regulated synchronous growth of the fungus with its plant partner were observed and infected plants were stunted. Analysis of these plants by light and transmission electron microscopy revealed abnormalities in the distribution and localization of ΔsidN mutant hyphae as well as deformities in hyphal ultrastructure. We hypothesize that lack of epichloënin A alters iron homeostasis of the symbiotum, changing it from mutually beneficial to antagonistic. Iron itself or epichloënin A may serve as an important molecular/cellular signal for controlling fungal growth and hence the symbiotic interaction.


Assuntos
Epichloe/metabolismo , Ferro/metabolismo , Lolium/microbiologia , Sideróforos/biossíntese , Simbiose/fisiologia , Epichloe/genética , Epichloe/ultraestrutura , Deleção de Genes , Genes Fúngicos/fisiologia , Lolium/genética , Lolium/metabolismo , Lolium/ultraestrutura , Sideróforos/genética
9.
Mol Plant Pathol ; 24(11): 1430-1442, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37477276

RESUMO

Epichloë festucae uses a siderophore-mediated system to acquire iron, which is important to maintain endophyte-grass symbioses. Here we investigate the roles of the alternative iron acquisition system, reductive iron assimilation (RIA), via disruption of the fetC gene, which encodes a multicopper ferroxidase, either alone (i.e., ΔfetC) or in combination with disruption of the gene sidA, which encodes a siderophore biosynthesis enzyme (i.e., ΔfetC/ΔsidA). The phenotypic characteristics of these mutants were compared to ΔsidA and wild-type (WT) strains during growth under axenic culture conditions (in culture) and in symbiosis with the host grass, perennial ryegrass (in planta). Under iron deficiency, the colony growth rate of ΔfetC was slightly slower than that of WT, while the growth of ΔsidA and ΔfetC/ΔsidA mutants was severely suppressed. Siderophore analyses indicated that ΔfetC mutants hyperaccumulate ferriepichloënin A (FEA) at low iron concentrations and ferricrocin and FEA at higher iron concentrations. When compared to WT, all mutant strains displayed hyperbranching hyphal structures and a reduced ratio of Epichloë DNA to total DNA in planta. Furthermore, host colonization and vertical transmission through infection of the host seed were significantly reduced in the ΔfetC/ΔsidA mutants, confirming that high-affinity iron uptake is a critical process for Epichloë transmission. Thus, RIA and siderophore iron uptake are complementary systems required for the maintenance of iron metabolism, fungal growth, and symbiosis between E. festucae and perennial ryegrass.


Assuntos
Epichloe , Lolium , Lolium/microbiologia , Sideróforos/metabolismo , Epichloe/metabolismo , Simbiose/genética , Endófitos , Ferro/metabolismo , Sementes/metabolismo , DNA/metabolismo
10.
Microbiol Resour Announc ; 12(6): e0014023, 2023 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-37154770

RESUMO

We report a preliminary survey of the endophytic bacterial microbiota of seed from wild carrot (Daucus carota) using 16S rRNA gene amplicon sequencing. Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria were the most abundant phyla detected, while Bacillus, Massilia, Paenibacillus, Pantoea, Pseudomonas, Rhizobium, Sphingomonas, and Xanthomonas were the most abundant genera.

11.
J Agric Food Chem ; 71(38): 13965-13978, 2023 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-37704203

RESUMO

The various grass-induced epichloëcyclins of the Epichloë spp. are ribosomally synthesized and post-translationally modified peptides (RiPPs), produced as small, secreted cyclopeptides from a single gene, gigA. Here, four clustered and coregulated genes (gigA, gigB, gigC, and kexB) with predicted roles in epichloëcyclin production in Epichloë festucae were evaluated through gene disruption. Subsequent chemical analysis indicates that GigB is a DUF3328 domain-containing protein associated with cyclization of epichloëcyclins; GigC is a methyltransferase enzyme responsible for N-methylation of desmethylepichloëcyclins; and KexB is a subtilisin-like enzyme, partly responsible for the propeptide cleavage of epichloëcyclin intermediates. Symbiotic effects on the host phenotype were not observed for gigA, gigC, or kexB mutants, although ΔgigB infection correlated with increased host tiller height and biomass, while only ΔkexB exhibited an effect on endophyte morphology. Disrupting epichloëcyclin biosynthesis showed negligible influence on the biosynthesis of E. festucae-associated alkaloids. Epichloëcyclins may perform other secondary metabolism functions in Epichloë and other fungi.


Assuntos
Epichloe , Lolium , Lolium/metabolismo , Epichloe/genética , Epichloe/metabolismo , Peptídeos Cíclicos/genética , Peptídeos Cíclicos/metabolismo , Proteínas Fúngicas/metabolismo , Simbiose , Família Multigênica
12.
Microorganisms ; 10(7)2022 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-35889134

RESUMO

Genotyping by sequencing (GBS) was used to reveal the inherent genetic variation within the haploid fungi Sarocladium zeae isolated from diverse Zea germplasm, including modern Zea mays and its wild progenitors-the teosintes. In accordance with broad host relationship parameters, GBS analysis revealed significant host lineages of S. zeae genetic diversity, indicating that S. zeae genetic variation may associate with different evolutionary histories of host species or varieties. Based on a recently identified PKS-NRPS gene responsible for pyrrocidine biosynthesis in S. zeae fungi, a novel PCR assay was developed to discriminate pyrrocidine-producing S. zeae strains. This molecular method for screening bioactive strains of S. zeae is complementary to other approaches, such as chemical analyses. An eGFP-labelled S. zeae strain was also developed to investigate the endophytic transmission of S. zeae in Z. mays seedlings, which has further improved our understanding of the transmission modes of S. zeae endophytes in maize tissues.

13.
Fungal Biol ; 126(1): 35-46, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34930557

RESUMO

Apple scab, caused by the fungal pathogen Venturia inaequalis, is the most economically important disease of apple (Malus x domestica) worldwide. To develop durable control strategies against this disease, a better understanding of the genetic mechanisms underlying the growth, reproduction, virulence and pathogenicity of V. inaequalis is required. A major bottleneck for the genetic characterization of V. inaequalis is the inability to easily delete or disrupt genes of interest using homologous recombination. Indeed, no gene deletions or disruptions in V. inaequalis have yet been published. Using the melanin biosynthesis pathway gene trihydroxynaphthalene reductase (THN) as a target for inactivation, which has previously been shown to result in a light-brown colony phenotype when transcriptionally silenced using RNA interference, we show, for the first time, that the CRISPR-Cas9 gene editing system can be successfully applied to the apple scab fungus. More specifically, using a CRISPR-Cas9 single guide RNA (sgRNA) targeted to the THN gene, delivered by a single autonomously replicating Golden Gate-compatible plasmid, we were able to identify six of 36 stable transformants with a light-brown phenotype, indicating an ∼16.7% gene inactivation efficiency. Notably, of the six THN mutants, five had an independent mutation. As part of our pipeline, we also report a high-resolution melting (HRM) curve protocol for the rapid detection of CRISPR-Cas9 gene-edited mutants of V. inaequalis. This protocol identified a single base pair deletion mutation in a sample containing only 5% mutant genomic DNA, indicating high sensitivity for mutant screening. In establishing CRISPR-Cas9 as a tool for gene editing in V. inaequalis, we have provided a strong starting point for studies aiming to decipher gene function in this fungus. The associated HRM curve protocol will enable CRISPR-Cas9 transformants to be screened for gene inactivation in a high-throughput and low-cost manner, which will be particularly powerful in cases where the CRISPR-Cas9-mediated gene inactivation efficiency is low.


Assuntos
Ascomicetos , Malus , Ascomicetos/genética , Sistemas CRISPR-Cas , Fungos do Gênero Venturia , Edição de Genes , Malus/genética , Doenças das Plantas
14.
Front Plant Sci ; 13: 1025698, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36340377

RESUMO

Asexual Epichloë are endophytic fungi that form mutualistic symbioses with cool-season grasses, conferring to their hosts protection against biotic and abiotic stresses. Symbioses are maintained between grass generations as hyphae are vertically transmitted from parent to progeny plants through seed. However, endophyte transmission to the seed is an imperfect process where not all seeds become infected. The mechanisms underpinning the varying efficiencies of seed transmission are poorly understood. Host gene expression in response to Epichloë sp. LpTG-3 strain AR37 was examined within inflorescence primordia and ovaries of high and low endophyte transmission genotypes within a single population of perennial ryegrass. A genome-wide association study was conducted to identify population-level single nucleotide polymorphisms (SNPs) and associated genes correlated with vertical transmission efficiency. For low transmitters of AR37, upregulation of perennial ryegrass receptor-like kinases and resistance genes, typically associated with phytopathogen detection, comprised the largest group of differentially expressed genes (DEGs) in both inflorescence primordia and ovaries. DEGs involved in signaling and plant defense responses, such as cell wall modification, secondary metabolism, and reactive oxygen activities were also abundant. Transmission-associated SNPs were associated with genes for which gene ontology analysis identified "response to fungus" as the most significantly enriched term. Moreover, endophyte biomass as measured by quantitative PCR of Epichloë non-ribosomal peptide synthetase genes, was significantly lower in reproductive tissues of low-transmission hosts compared to high-transmission hosts. Endophyte seed-transmission efficiency appears to be influenced primarily by plant defense responses which reduce endophyte colonization of host reproductive tissues.

15.
Front Fungal Biol ; 3: 944234, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-37746172

RESUMO

Epichloë festucae var. lolii and Epichloë sp. LpTG-3 are filamentous fungal endophytes of perennial ryegrass (Lolium perenne) that have a substantial impact on New Zealand's agricultural economy by conferring biotic advantages to the host grass. Overall, Epichloë endophytes contribute NZ$200 million to the economy annually, with strain AR37 estimated to contribute NZ$3.6 billion to the New Zealand economy over a 20-year period. This strain produces secondary metabolites, including epoxyjanthitrems, which are a class of indole diterpenes, associated with the observed effects of AR37 on livestock and insect pests. Until very recently, AR37 was intractable to genetic modification but this has changed with the application of CRISPR-Cas9 based gene editing techniques. In this paper, gene inactivation by CRISPR-Cas9 was used to deconvolute the genetic basis for epoxyjanthitrem biosynthesis, including creating an AR37 strain that has been edited to remove the biosynthesis of all indole diterpenes. We show that gene editing of Epichloë can be achieved without off-target events or introduction of foreign DNA (footprint-less) through an AMA1-based plasmid that simultaneously expresses the CRISPR-Cas9 system and selectable marker. Genetic modification events in these transformants were investigated through genome sequencing and in planta chemistry.

16.
G3 (Bethesda) ; 12(4)2022 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-35191483

RESUMO

Fungi from the genus Epichloë form systemic endobiotic infections of cool season grasses, producing a range of host-protective natural products in return for access to nutrients. These infections are asymptomatic during vegetative host growth, with associations between asexual Epichloë spp. and their hosts considered mutualistic. However, the sexual cycle of Epichloë spp. involves virulent growth, characterized by the envelopment and sterilization of a developing host inflorescence by a dense sheath of mycelia known as a stroma. Microscopic analysis of stromata revealed a dramatic increase in hyphal propagation and host degradation compared with asymptomatic tissues. RNAseq was used to identify differentially expressed genes in asymptomatic vs stromatized tissues from 3 diverse Epichloë-host associations. Comparative analysis identified a core set of 135 differentially expressed genes that exhibited conserved transcriptional changes across all 3 associations. The core differentially expressed genes more strongly expressed during virulent growth encode proteins associated with host suppression, digestion, adaptation to the external environment, a biosynthetic gene cluster, and 5 transcription factors that may regulate Epichloë stroma formation. An additional 5 transcription factor encoding differentially expressed genes were suppressed during virulent growth, suggesting they regulate mutualistic processes. Expression of biosynthetic gene clusters for natural products that suppress herbivory was universally suppressed during virulent growth, and additional biosynthetic gene clusters that may encode production of novel host-protective natural products were identified. A comparative analysis of 26 Epichloë genomes found a general decrease in core differentially expressed gene conservation among asexual species, and a specific decrease in conservation for the biosynthetic gene cluster expressed during virulent growth and an unusual uncharacterized gene.


Assuntos
Epichloe , Animais , Epichloe/genética , Estágios do Ciclo de Vida , Poaceae/genética , Simbiose/genética , Transcriptoma
17.
J Biol Chem ; 285(4): 2415-27, 2010 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-19923209

RESUMO

Nonribosomal peptide synthetases (NRPSs) are large, multidomain proteins that are involved in the biosynthesis of an array of secondary metabolites. We report the structure of the third adenylation domain from the siderophore-synthesizing NRPS, SidN, from the endophytic fungus Neotyphodium lolii. This is the first structure of a eukaryotic NRPS domain, and it reveals a large binding pocket required to accommodate the unusual amino acid substrate, N(delta)-cis-anhydromevalonyl-N(delta)-hydroxy-L-ornithine (cis-AMHO). The specific activation of cis-AMHO was confirmed biochemically, and an AMHO moiety was unambiguously identified as a component of the fungal siderophore using mass spectroscopy. The protein structure shows that the substrate binding pocket is defined by 17 amino acid residues, in contrast to both prokaryotic adenylation domains and to previous predictions based on modeling. Existing substrate prediction methods for NRPS adenylation domains fail for domains from eukaryotes due to the divergence of their signature sequences from those of prokaryotes. Thus, this new structure will provide a basis for improving prediction methods for eukaryotic NRPS enzymes that play important and diverse roles in the biology of fungi.


Assuntos
Proteínas Fúngicas/metabolismo , Ácidos Hidroxâmicos/metabolismo , Neotyphodium/enzimologia , Peptídeo Sintases/metabolismo , Sideróforos/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Proteínas Fúngicas/química , Dados de Sequência Molecular , Ornitina/análogos & derivados , Ornitina/metabolismo , Peptídeo Sintases/química , Poliadenilação/fisiologia , Estrutura Terciária de Proteína , Sideróforos/biossíntese , Especificidade por Substrato
18.
Front Microbiol ; 12: 786156, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35237240

RESUMO

Agriculture is fundamental for food production, and microbiomes support agriculture through multiple essential ecosystem services. Despite the importance of individual (i.e., niche specific) agricultural microbiomes, microbiome interactions across niches are not well-understood. To observe the linkages between nearby agricultural microbiomes, multiple approaches (16S, 18S, and ITS) were used to inspect a broad coverage of niche microbiomes. Here we examined agricultural microbiome responses to 3 different nitrogen treatments (0, 150, and 300 kg/ha/yr) in soil and tracked linked responses in other neighbouring farm niches (rumen, faecal, white clover leaf, white clover root, rye grass leaf, and rye grass root). Nitrogen treatment had little impact on microbiome structure or composition across niches, but drastically reduced the microbiome network connectivity in soil. Networks of 16S microbiomes were the most sensitive to nitrogen treatment across amplicons, where ITS microbiome networks were the least responsive. Nitrogen enrichment in soil altered soil and the neighbouring microbiome networks, supporting our hypotheses that nitrogen treatment in soil altered microbiomes in soil and in nearby niches. This suggested that agricultural microbiomes across farm niches are ecologically interactive. Therefore, knock-on effects on neighbouring niches should be considered when management is applied to a single agricultural niche.

19.
J Fungi (Basel) ; 6(4)2020 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-33261217

RESUMO

The relationship between Epichloë endophytes found in a wide range of temperate grasses spans the continuum from antagonistic to mutualistic. The diversity of asexual mutualistic types can be characterised by the types of alkaloids they produce in planta. Some of these are responsible for detrimental health and welfare issues of ruminants when consumed, while others protect the host plant from insect pests and pathogens. In many temperate regions they are an essential component of high producing resilient tall fescue and ryegrass swards. This obligate mutualism between fungus and host is a seed-borne technology that has resulted in several commercial products being used with high uptake rates by end-user farmers, particularly in New Zealand and to a lesser extent Australia and USA. However, this has not happened by chance. It has been reliant on multi-disciplinary research teams undertaking excellent science to understand the taxonomic relationships of these endophytes, their life cycle, symbiosis regulation at both the cellular and molecular level, and the impact of secondary metabolites, including an understanding of their mammalian toxicity and bioactivity against insects and pathogens. Additionally, agronomic trials and seed biology studies of these microbes have all contributed to the delivery of robust and efficacious products. The supply chain from science, through seed companies and retailers to the end-user farmer needs to be well resourced providing convincing information on the efficacy and ensuring effective quality control to result in a strong uptake of these Epichloë endophyte technologies in pastoral agriculture.

20.
Microbiol Resour Announc ; 9(27)2020 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-32616631

RESUMO

Here, we report a 29-Mb draft genome sequence of strain 72-15.1 of Simplicillium aogashimaense (Cordycipitaceae, Hypocreales). Strain 72-15.1 was a unique fungal isolate obtained from asymptomatic tillers of the tropical grass Brachiaria brizantha.

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