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Development of grass-endophyte associations with minimal or no detrimental effects in combination with beneficial characteristics is important for pastoral agriculture. The feasibility of enhancing production of an endophyte-derived beneficial alkaloid through introduction of an additional gene copy was assessed in a proof-of-concept study. Sexual and asexual Epichloë species that form symbiotic associations with cool-season grasses of the Poaceae sub-family Pooideae produce bioactive alkaloids that confer resistance to herbivory by a number of organisms. Of these, peramine is thought to be crucial for protection of perennial ryegrass (Lolium perenne L.) from the Argentinian stem weevil, an economically important exotic pest in New Zealand, contributing significantly to pasture persistence. A single gene (perA) has been identified as solely responsible for peramine biosynthesis and is distributed widely across Epichloë taxa. In the present study, a functional copy of the perA gene was introduced into three recipient endophyte genomes by Agrobacterium tumefaciens-mediated transformation. The target strains included some that do not produce peramine, and others containing different perA gene copies. Mitotically stable transformants generated from all three endophyte strains were able to produce peramine in culture and in planta at variable levels. In summary, this study provides an insight into the potential for artificial combinations of alkaloid biosynthesis in a single endophyte strain through transgenesis, as well as the possibility of using novel genome editing techniques to edit the perA gene of non-peramine producing strains.

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.

Background: 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., Results: 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., Conclusions: 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.

Background: 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., Results: 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., Conclusions: 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. An integrated process for discovery of novel endophytes based on exploitation of genotypic information has been designed and implemented. Genetic and chemotypic diversity analysis identified candidate strains, which were further characterised using a novel inoculation method that allows isogenic comparisons with respect to both host and endophyte genotype. Three prioritised candidates with desirable alkaloid profiles were obtained, one of which belongs to a putative novel taxon distinct from previously known groupings. [ABSTRACT FROM AUTHOR]

Fungal species of the Neotyphodiumand Epichloë genera are endophytes of pasture grasses showing complex differences of life-cycle and genetic architecture. Simple sequence repeat (SSR) markers have been developed from endophyte-derived expressed sequence tag (EST) collections. Although SSR array size polymorphisms are appropriate for phenetic analysis to distinguish between taxa, the capacity to resolve phylogenetic relationships is limited by both homoplasy and heteroploidy effects. In contrast, nonrepetitive sequence regions that flank SSRs have been effectively implemented in this study to demonstrate a common evolutionary origin of grass fungal endophytes. Consistent patterns of relationships between specific taxa were apparent across multiple target loci, confirming previous studies of genome evolution based on variation of individual genes. Evidence was obtained for the definition of endophyte taxa not only through genomic affinities but also by relative gene content. Results were compatible with the current view that some asexual Neotyphodiumspecies arose following interspecific hybridisation between sexual Epichloë ancestors. Phylogenetic analysis of SSR-flanking regions, in combination with the results of previous studies with other EST-derived SSR markers, further permitted characterisation of Neotyphodium isolates that could not be assigned to known taxa on the basis of morphological characteristics. [ABSTRACT FROM AUTHOR]

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 (Lolium perenne L.)-associated Epichloë endophyte strains using A. tumefaciens-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. [ABSTRACT FROM AUTHOR]

Epoxy-janthitrems are a class of indole diterpenes with structural similarity to lolitrem B. Two taxa of asexual Epichloë endophytes have been reported to produce epoxy-janthitrems, LpTG-3 (Lolium perenne Taxonomic Group 3; e.g., NEA12) and LpTG-4 (e.g., E1). Epichloë 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 Epichloë. A gene, jtmD, 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 jtmD, jtmO, jtm01, and jtm02 that are unique to Epichloë 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 jtmD and in planta assessment in host-endophyte associations confirms the role of jtmD in epoxy-janthitrem production. Using LCMS/MS technologies, a biosynthetic pathway for the production of epoxy-janthitrems I–IV in Epichloë endophytes is proposed. [ABSTRACT FROM AUTHOR]