Perennial ryegrass (Lolium perenne L.) is one of the most important species for temperate pastoral agriculture, forming associations with genetically diverse groups of mutualistic fungal endophytes. ...However, only two taxonomic groups (E. festucae var. lolii and LpTG-2) have so far been described. In addition to these two well-characterised taxa, a third distinct group of previously unclassified perennial ryegrass-associated endophytes was identified as belonging to a putative novel taxon (or taxa) (PNT) in a previous analysis based on simple sequence repeat (SSR) marker diversity. As well as genotypic differences, distinctive alkaloid production profiles were observed for members of the PNT group.
A detailed phylogenetic analysis of perennial ryegrass-associated endophytes using components of whole genome sequence data was performed using complete sequences of 7 nuclear protein-encoding genes. Three independently selected genes (encoding a DEAD/DEAH box helicase Sbp4, a glycosyl hydrolase family 92 protein and a MEAB protein), none of which have been previously used for taxonomic studies of endophytes, were selected together with the frequently used 'house-keeping' genes tefA and tubB (encoding translation elongation factor 1-α and β-tubulin, respectively). In addition, an endophyte-specific gene (perA for peramine biosynthesis) and the fungal-specific MT genes for mating-type control were included. The results supported previous phylogenomic inferences for the known species, but revealed distinctive patterns of diversity for the previously unclassified endophyte strains, which were further proposed to belong to not one but two distinct novel taxa. Potential progenitor genomes for the asexual endophytes among contemporary teleomorphic (sexual Epichloë) species were also identified from the phylogenetic analysis.
Unique taxonomic status for the PNT was confirmed through comparison of multiple nuclear gene sequences, and also supported by evidence from chemotypic diversity. Analysis of MT gene idiomorphs further supported a predicted independent origin of two distinct perennial ryegrass-associated novel taxa, designated LpTG-3 and LpTG-4, from different members of a similar founder population related to contemporary E. festucae. The analysis also provided higher resolution to the known progenitor contributions of previously characterised perennial ryegrass-associated endophyte taxa.
A number of pasture and turf grass species form mutually beneficial symbiotic associations with endophytic fungal species. Within the fescue grasses, diploid meadow fescue (Festuca pratensis Huds.) ...interacts with Neotyphodium uncinatum while allohexaploid tall fescue (Festuca arundinacea Schreb.) has been reported to associate with Neotyphodium coenophialum and two other morphologically distinct taxa (Festuca arundinacea taxonomic groups 2 and 3 FaTG-2 and FaTG-3). The evolutionary history of hexaploid tall fescue is complex, as part of a species group with varying ploidy levels and exhibiting distinct ecogeographical morphotypes. To evaluate both naturally occurring variation and host grass taxon specificity, diversity was determined in collections representing multiple meadow fescue and tall fescue accessions. Initial screening with a minimal set of endophyte-specific simple sequence repeat (SSR) genetic markers detected endophyte incidence in 33% of 701 tested accessions. Subsequent analysis identified N. coenophialum genotypes within Continental and rhizomatous hexaploid and octoploid tall fescue F. arundinacea subsp. atlantigena (St.-Yves) Auquier accessions. Festuca arundinacea taxonomic group 2 and FaTG-3 endophytes appeared to be restricted to Mediterranean hexaploid and decaploid tall fescue F. arundinacea cirtensis (St.-Yves) Gamisans hosts. Endophytes of meadow fescue were confirmed as belonging to N. uncinatum. This study has elucidated host specificity of fescue endophyte taxa and supported models for host-symbiont coevolution. A substantial number of candidate novel endophytes have been identified that are suitable for metabolic characterization and deployment by inoculation in fescue breeding programs.
Symbiotic associations between tall fescue grasses and asexual Epichloë fungal endophytes exhibit biosynthesis of alkaloid compounds causing both beneficial and detrimental effects. Candidate novel ...endophytes with favourable chemotypic profiles have been identified in germplasm collections by screening for genetic diversity, followed by metabolite profile analysis in endogenous genetic backgrounds. A subset of candidates was subjected to genome survey sequencing to detect the presence or absence and structural status of known genes for biosynthesis of the major alkaloid classes. The capacity to produce specific metabolites was directly predictable from metabolic data. In addition, study of duplicated gene structure in heteroploid genomic constitutions provided further evidence for the origin of such endophytes. Selected strains were inoculated into meristem-derived callus cultures from specific tall fescue genotypes to perform isogenic comparisons of alkaloid profile in different host backgrounds, revealing evidence for host-specific quantitative control of metabolite production, consistent with previous studies. Certain strains were capable of both inoculation and formation of longer-term associations with a nonhost species, perennial ryegrass (Lolium perenne L.). Discovery and primary characterisation of novel endophytes by DNA analysis, followed by confirmatory metabolic studies, offers improvements of speed and efficiency and hence accelerated deployment in pasture grass improvement programs.
Epoxy-janthitrems are a class of indole diterpenes with structural similarity to lolitrem B. Two taxa of asexual
endophytes have been reported to produce epoxy-janthitrems,
TG-3 (
Taxonomic Group 3; ...e.g., NEA12) and
TG-4 (e.g., E1).
epoxy-janthitrems are not well understood, the biosynthetic pathway and associated gene complement have not been described and while the literature suggests they are associated with superior protection against pasture insect pests and are tremorgenic in grazing mammals, these properties have not been confirmed using isolated and purified compounds. Whole genome sequence analysis was used to identify candidate genes for epoxy-janthitrem biosynthesis that are unique to epoxy-janthitrem producing strains of
. A gene,
, was identified with homology to aromatic prenyl transferases involved in synthesis of indole diterpenes. The location of the epoxy-janthitrem biosynthesis gene cluster (JTM locus) was determined in the assembled nuclear genomes of NEA12 and E1. The JTM locus contains cluster 1 and cluster 2 of the lolitrem B biosynthesis gene cluster (LTM locus), as well as four genes
, and
that are unique to
spp. that produce epoxy-janthitrems. Expression of each of the genes identified was confirmed using transcriptome analysis of perennial ryegrass-NEA12 and perennial ryegrass-E1 symbiota. Sequence analysis confirmed the genes are functionally similar to those involved in biosynthesis of related indole diterpene compounds. RNAi silencing of
and in planta assessment in host-endophyte associations confirms the role of
in epoxy-janthitrem production. Using LCMS/MS technologies, a biosynthetic pathway for the production of epoxy-janthitrems I-IV in
endophytes is proposed.
Methods for the identification and localisation of endophytic fungi are required to study the establishment, development, and progression of host-symbiont interactions, as visible reactions or ...disease symptoms are generally absent from host plants. Fluorescent proteins have proved valuable as reporter gene products, allowing non-invasive detection in living cells. This study reports the introduction of genes for two fluorescent proteins, green fluorescent protein (GFP) and red fluorescent protein, DsRed, into the genomes of two distinct perennial ryegrass (
L.)-associated
endophyte strains using
-mediated transformation. Comprehensive characterisation of reporter gene-containing endophyte strains was performed using molecular genetic, phenotypic, and bioinformatic tools. A combination of long read and short read sequencing of a selected transformant identified a single complex T-DNA insert of 35,530 bp containing multiple T-DNAs linked together. This approach allowed for comprehensive characterisation of T-DNA integration to single-base resolution, while revealing the unanticipated nature of T-DNA integration in the transformant analysed. These reporter gene endophyte strains were able to establish and maintain stable symbiotum with the host. In addition, the same endophyte strain labelled with two different fluorescent proteins were able to cohabit the same plant. This knowledge can be used to provide the basis to develop strategies to gain new insights into the host-endophyte interaction through independent and simultaneous monitoring in planta throughout its life cycle in greater detail.
Tall fescue and meadow fescue are important as temperate pasture grasses, forming mutualistic associations with asexual Neotyphodium endophytes. The most frequently identified endophyte of ...Continental allohexaploid tall fescue is Neotyphodium coenophialum, while representatives of two other taxa (FaTG-2 and FaTG-3) have been described as colonising decaploid and Mediterranean hexaploid tall fescue, respectively. In addition, a recent study identified two other putatively novel endophyte taxa from Mediterranean hexaploid and decaploid tall fescue accessions, which were designated as uncharacterised Neotyphodium species (UNS) and FaTG-3-like respectively. In contrast, diploid meadow fescue mainly forms associations with the endophyte taxon Neotyphodium uncinatum, although a second endophyte taxon, termed N. siegelii, has also been described.
Multiple copies of the translation elongation factor 1-a (tefA) and β-tubulin (tub2) 'house-keeping' genes, as well as the endophyte-specific perA gene, were identified for each fescue-derived endophyte taxon from whole genome sequence data. The assembled gene sequences were used to reconstruct evolutionary relationships between the heteroploid fescue-derived endophytes and putative ancestral sub-genomes derived from known sexual Epichloë species. In addition to the nuclear genome-derived genes, the complete mitochondrial genome (mt genome) sequence was obtained for each of the sequenced endophyte, and phylogenetic relationships between the mt genome protein coding gene complements were also reconstructed.
Complex and highly reticulated evolutionary relationships between Epichloë-Neotyphodium endophytes have been predicted on the basis of multiple nuclear genes and entire mitochondrial protein-coding gene complements, derived from independent assembly of whole genome sequence reads. The results are consistent with previous studies while also providing novel phylogenetic insights, particularly through inclusion of data from the endophyte lineage-specific gene, as well as affording evidence for the origin of cytoplasmic genomes. In particular, the results obtained from the present study imply the possible occurrence of at least two distinct E. typhina progenitors for heteropoid taxa, as well the ancestral contribution of an endophyte species distinct from (although related to) contemporary E. baconii to the extant hybrid species. Furthermore, the present study confirmed the distinct taxonomic status of the newly identified fescue endophyte taxa, FaTG-3-like and UNS, which are consequently proposed to be renamed FaTG4 and FaTG5, respectively.
Asexual fungal endophytes of the genus Epichloë form mutually beneficial associations with cool-season pasture grasses such as ryegrasses (Lolium spp.). Alkaloid production by the fungus confers both ...beneficial (deterrence of invertebrate herbivory) and detrimental (toxicity to mammalian livestock) attributes. A few novel strains with desirable metabolite profiles have been advanced into commercial production by inoculation of perennial ryegrass cultivars. In the present study, an integrated process for discovery of novel endophytes based on exploitation of genotypic information has been designed and implemented. A survey of genetic diversity was performed on a large-scale, customised germplasm collection (containing 244 accessions) in order to identify previously uncharacterised endophyte genotypes. Preliminary qualitative metabolic profiling in the endogenous genetic background permitted elimination of undesirable combinations, and definition of a subset of priority candidates. A novel method was developed for inoculation of endophytes into meristem-culture-derived callus tissue of single genotypes from multiple perennial ryegrass cultivars, in order to allow isogenic comparisons with respect to both host and endophyte genotype. Beneficial toxin profiles were confirmed for associations formed with the grass genotypic panel, and semi-quantitative metabolite analysis provided evidence for genotype-specific effects of both host and genotype on levels of alkaloid production. Vegetative stability was also assessed over both shorter and longer terms. A final set of three prioritised candidates was obtained, two of which (belonging to the known taxa E. festucae var. lolii and LpTG-2) produce the alkaloids ergovaline and peramine. The third candidate endophyte, belonging to a putative novel taxon, solely produces representatives of an additional alkaloid class, the epoxy-janthitrems.
Symbiotic associations between tall fescue grasses and asexual Epichloee fungal endophytes exhibit biosynthesis of alkaloid compounds causing both beneficial and detrimental effects. Candidate novel ...endophytes with favourable chemotypic profiles have been identified in germplasm collections by screening for genetic diversity, followed by metabolite profile analysis in endogenous genetic backgrounds. A subset of candidates was subjected to genome survey sequencing to detect the presence or absence and structural status of known genes for biosynthesis of the major alkaloid classes. The capacity to produce specific metabolites was directly predictable from metabolic data. In addition, study of duplicated gene structure in heteroploid genomic constitutions provided further evidence for the origin of such endophytes. Selected strains were inoculated into meristem-derived callus cultures from specific tall fescue genotypes to perform isogenic comparisons of alkaloid profile in different host backgrounds, revealing evidence for host-specific quantitative control of metabolite production, consistent with previous studies. Certain strains were capable of both inoculation and formation of longer-term associations with a nonhost species, perennial ryegrass (Lolium perenne L.). Discovery and primary characterisation of novel endophytes by DNA analysis, followed by confirmatory metabolic studies, offers improvements of speed and efficiency and hence accelerated deployment in pasture grass improvement programs.Original Abstract: Les associations symbiotiques entre la fetuque elevee et les champignons endophytes asexues Epichloee se caracterisent par la synthese d'alcaloides qui ont des effets a la fois benefiques et nuisibles. Des endophytes candidats inedits presentant des chimiotypes favorables ont ete identifies au sein de collections de ressources genetiques en criblant d'abord pour la diversite genetique, suivi d'une analyse des profils metaboliques au sein de fonds genetiques endogenes. Un sous-ensemble de candidats a ete assujetti a un sequencage partiel afin de detecter la presence ou absence ainsi que l'integrite structurale de genes connus comme etant impliques dans la synthese des principales classes d'alcaloides. La capacite a produire des metabolites specifiques etait previsible sur la base des donnees metaboliques. De plus, une etude sur la duplication genique au sein de genomes heteroploides a apporte des evidences additionnelles concernant l'origine de tels endophytes. Les souches selectionnees ont ete inoculees chez des cultures de cals derivees de meristemes chez tous les genotypes de fetuque elevee de maniere a realiser des comparaisons isogeniques des profils d'alcaloides au sein de differents fonds genetiques de l'hote et cela a permis de reveler un effet de l'hote sur la quantite de metabolites produits conformement aux etudes anterieures. Certaines souches etaient competentes a l'inoculation et a l'etablissement de la formation d'associations durables avec une espece non-hote, le raygrass vivace (Lolium perenne L.). L'identification et la caracterisation preliminaire de nouveaux endophytes au moyen d'analyses d'ADN, suivies d'etudes metaboliques de confirmation, offrent une amelioration dans la rapidite et l'efficacite des programmes d'amelioration genetique des graminees fourrageres, et consequemment dans leur deploiement. Traduit par la Redaction
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