Fungi from the genus Epichloë form systemic and seed-transmissible endophytic infections of grasses in a symbiosis that is considered predominately mutualistic. During asymptomatic growth these Epichloë fungi produce a variety of secondary metabolites that benefit their host by providing protection against herbivory and disease. However, the Epichloë sexual cycle is antagonistic to its host, characterised by the proliferation of hyphae on reproductive tillers to form fungal stromata that encase and sterilise developing host inflorescences. Production of known host-protective Epichloë-derived secondary metabolites is suppressed in these stromata; however, we describe here the pyrrolodiazipic acids, a novel class of fungal alkaloids that are specifically produced in Epichloë stromata. Transcriptomic analysis identified the five-gene PZA cluster that, unlike other Epichloë biosynthetic gene clusters, is specifically expressed in stromata. Reconstruction of this PZA cluster in a heterologous host enabled the function of the encoded enzymes to be defined, key biosynthetic intermediates to be identified, and the final product characterised – stromatine, a pyrrolodiazepine with carboxylic acid functionality. Analysis of material from many different Epichloë-host associations revealed that stromatine was universally and exclusively found in stroma tissues. Stromatine was also produced in exceptionally large amounts, contributing almost 1% of the total stroma dry mass in some grass-endophyte associations. PZA gene deletion analyses across three different Epichloë species yielded interesting results; stromatine appears to be required for host infection by some Epichloë species, while the accumulation of PZA pathway intermediates appears to be detrimental to symbiosis. Genomic analyses reveal the PZA cluster is conserved among sexual Epichloë species but is dispensable for asexual strains. Combined with the tissue specificity and high levels of production, this suggests that stromatine plays a critical role in the Epichloë sexual cycle, with potential bioactivities and functions to be dissected further.

Terpene cyclases (TCs) catalyze the generation of complex polycyclic terpenoids through elaborate mechanistic cascades. Very little is known about the unusual type II TCs responsible for generation of a large diverse family of meroterpenoids known as indole diterpenoids (IDTs). The radarins are insecticidal and cytotoxic IDTs that possess an unusual core architecture for which the catalytic machinery is unknown. Here, we interrogate the genome of Aspergillus fresenii to reveal the biosynthetic machinery responsible for delivering the specific radarin scaffold. We demonstrate that a bespoke regioselective IDT epoxidase (RadM) acts at the terminal olefin of precursor 3 '-geranylgeranylindole, providing access to this distinct class of IDT architectures. Coexpression of this epoxidase with a noncanonical type II TC RadB led to the biosynthesis of radarin C, in a complex skeletal rearrangement that proceeds via rare alcoholic deprotonation and ketone formation. Our modeling of RadB reveals that catalysis by RadB is enabled by a unique His residue that acts as both the proton donor and acceptor. Intriguingly, coexpression of RadM with an alternative A. fresenii IDT cyclase gene, which does not share this catalytic His, revealed an alternate partial cyclization of 14,15-epoxy-3 '-GGI, producing a new decalin IDT. This work solves the final piece of the biosynthetic puzzle to access the full suite of known IDT architectures, informs type II cyclase engineering efforts, and expands the repertoire of impressive chemistry catalyzed by noncanonical TCs.

PpzF, a sulfotransferase encoded in the PPZ biosynthetic gene cluster from the fungal insect pathogen Metarhizium majus, was shown to catalyze the formation of peramine-8-O-sulfate from 8-hydroxyperamine. Both the substrate and the product were detected in Penicillium paxilli expressing ppzA-ppzG, supporting their roles as pathway intermediates. Gene ppzF is the second fungal sulfotransferase gene to be functionally characterized and the first whose endogenous role has been revealed. The discovery of peramine-8-O-sulfate represents a significant step toward identifying the elusive final product(s) of the PPZ pathway.

Indole diterpenes (IDTs) are a large class of highly complex fungal natural products that possess a wide array of intriguing bioactivities. While IDTs are structurally diverse, the first four steps of IDT biosynthesis are highly conserved and result typically in the formation of a tetrahydropyran (THP)-ring containing structure, most commonly paspaline. The biosynthetic genes responsible for these steps are the most extensively studied of all IDT genes and collectively define the core biosynthetic pathway. Here we show that the fourth fundamental step, formation of the THP ring, is catalysed by a terpene cyclase encoded by an overlooked and uncharacterised fifth gene, idtA. All previously delineated biosynthetic routes have incorrectly attributed this step to the terpene cyclase IdtB, leading to imprecise pathway reconstructions and ignoring the fully evolved biosynthetic solution for core IDT generation. Moreover, while IdtA terpene cyclases are found in Eurotiomycetes fungi, in Sordariomycetes fungi this step is catalysed by the unrelated protein IdtS, demonstrating that two distinct solutions to this chemistry exist. All biosynthetic gene clusters known to specify production of THP-containing IDTs include an idtA or idtS gene. These findings reset the paradigm for core IDT biosynthesis and support accurate heterologous biosynthesis of these complex natural products.

Entomopathogenic fungus Metarhizium majus contains the nine-gene PPZ cluster, with ppzA, encoding a peramine-producing nonribosomal peptide synthetase, as the central component. In this work, the roles of two α-ketoglutarate, iron-dependent oxygenases encoded by the PPZ genes ppzC and ppzD were elucidated. PpzD was found to produce both trans-4-hydroxy-l-proline and trans-3-hydroxy-l-proline in a 13.1:1 ratio, yielding a key precursor for peramine biosynthesis. PpzC was found to act directly on peramine, yielding the novel analogue 8-hydroxyperamine.

The significant structural diversity and potent bioactivity of the fungal indole diterpenes (IDTs) has attracted considerable interest in their biosynthesis. Although substantial skeletal diversity is generated by the action of noncanonical terpene cyclases, comparatively little is known about these enzymes, particularly those involved in the generation of the subgroup containing emindole SA and DA, which show alternate terpenoid skeletons. Here, we describe the IDT biosynthetic machinery generating these unusual IDT architectures from Aspergillus striatus and Aspergillus desertorum. The function of four putative cyclases was interrogated via heterologous expression. Two specific cyclases were identified that catalyze the formation of epimers emindole SA and DA from A. striatus and A. desertorum, respectively. These cyclases are both clustered along with all the elements required for basic IDT biosynthesis yet catalyze an unusual Markovnikov-like cyclization cascade with alternate stereochemical control. Their identification reveals that these alternate architectures are not generated by mechanistically sloppy or promiscuous enzymes, but by cyclases capable of delivering precise regio- and stereospecificities.

Abstract 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.

Nodulisporic acids (NAs) are structurally complex potent antiinsectan indole diterpenes. We previously reported the biosynthetic gene cluster for these metabolites in Hypoxylon pulicicidum and functionally characterised the first five steps of the biosynthetic pathway. Here we reveal a highly complex biosynthetic array, furnishing multiple end products through expression of cluster components in Penicillium paxilli. We show that seven additional cluster-encoded gene products comprise the biosynthetic machinery that elaborate precursor NAF in this highly branched pathway. The combined action of these enzymes delivers 37 NA congeners including four major end products, NAA, NAA(1), NAA(2) and NAA(4). The plethora of intermediates arises due to modification of the carboxylated prenyl tail by a single promiscuous P450 monooxygenase, NodJ, a pivotal branchpoint enzyme which produces four distinct biosynthetic products giving rise to the complex metabolic grid that characterises NA biosynthesis.

Summary Epichloë festucae is an endophytic fungus that forms a symbiotic association with Lolium perenne . Here we analysed how the metabolome of the ryegrass apoplast changed upon infection of this host with sexual and asexual isolates of E. festucae . A metabolite fingerprinting approach was used to analyse the metabolite composition of apoplastic wash fluid from uninfected and infected L. perenne . Metabolites enriched or depleted in one or both of these treatments were identified using a set of interactive tools. A genetic approach in combination with tandem MS was used to identify a novel product of a secondary metabolite gene cluster. Metabolites likely to be present in the apoplast were identified using MarVis in combination with the BioCyc and KEGG databases, and an in‐house Epichloë metabolite database. We were able to identify the known endophyte‐specific metabolites, peramine and epichloëcyclins, as well as a large number of unknown markers. To determine whether these methods can be applied to the identification of novel Epichloë ‐derived metabolites, we deleted a gene encoding a NRPS ( lgsA ) that is highly expressed in planta . Comparative MS analysis of apoplastic wash fluid from wild‐type‐ vs mutant‐infected plants identified a novel Leu/Ile glycoside metabolite present in the former.

SummaryPeramine is a non‐ribosomal peptide‐derived pyrrolopyrazine (PPZ)‐containing molecule with anti‐insect properties. Peramine is known to be produced by fungi from genus Epichloë, which form mutualistic endophytic associations with cool‐season grass hosts. Peramine biosynthesis has been proposed to require only the two‐module non‐ribosomal peptide synthetase (NRPS) peramine synthetase (PerA), which is encoded by the 8.3 kb gene perA, though this has not been conclusively proven. Until recently, both peramine and perA were thought to be exclusive to fungi of genus Epichloë; however, a putative perA homologue was recently identified in the genome of the insect‐pathogenic fungus Metarhizium rileyi. We use a heterologous expression system and a hydrophilic interaction chromatography‐based analysis method to confirm that PerA is the only pathway‐specific protein required for peramine biosynthesis. The perA homologue from M. rileyi (MR_perA) is shown to encode a functional peramine synthetase, establishing a precedent for distribution of perA orthologs beyond genus Epichloë. Furthermore, perA is part of a larger seven‐gene PPZ cluster in M. rileyi, Metarhizium majus and the stalked‐cup lichen fungus Cladonia grayi. These PPZ genes encode proteins predicted to derivatize peramine into more complex PPZ metabolites, with the orphaned perA gene of Epichloë spp. representing an example of reductive evolution.

Nonribosomal peptide synthetases (NRPSs) generate the core peptide scaffolds of many natural products. These include small cyclic dipeptides such as the insect feeding deterrent peramine, which is a pyrrolopyrazine (PPZ) produced by grass-endophytic Epichloë fungi. Biosynthesis of peramine is catalyzed by the 2-module NRPS, PpzA-1, which has a C-terminal reductase (R) domain that is required for reductive release and cyclization of the NRPS-tethered dipeptidyl-thioester intermediate. However, some PpzA variants lack this R domain due to insertion of a transposable element into the 3' end of ppzA We demonstrate here that these truncated PpzA variants utilize nonenzymatic cyclization of the dipeptidyl thioester to a 2,5-diketopiperazine (DKP) to synthesize a range of novel PPZ products. Truncation of the R domain is sufficient to subfunctionalize PpzA-1 into a dedicated DKP synthetase, exemplified by the truncated variant, PpzA-2, which has also evolved altered substrate specificity and reduced N-methyltransferase activity relative to PpzA-1. Further allelic diversity has been generated by recombination-mediated domain shuffling between ppzA-1 and ppzA-2, resulting in the ppzA-3 and ppzA-4 alleles, each of which encodes synthesis of a unique PPZ metabolite. This research establishes that efficient NRPS-catalyzed DKP biosynthesis can occur in vivo through nonenzymatic dipeptidyl cyclization and presents a remarkably clean example of NRPS evolution through recombinant exchange of functionally divergent domains. This work highlights that allelic variants of a single NRPS can result in a surprising level of secondary metabolite diversity comparable to that observed for some gene clusters.

Epichloë festucae is an endophytic fungus that forms a mutualistic symbiotic association with Lolium perenne. Here we analysed how the metabolome of the ryegrass apoplast changed upon infection of this host with sexual and asexual isolates of E. festucae. A metabolite fingerprinting approach was used to analyse the metabolite composition of apoplastic wash fluid from non-infected and infected L. perenne. Metabolites enriched or depleted in one or both of these treatments were identified using a set of interactive tools. A genetic approach in combination with tandem mass spectrometry was used to identify a novel product of a secondary metabolite gene cluster. Metabolites likely to be present in the apoplast were identified using the MarVis Pathway in combination with the BioCyc and KEGG databases, and an in-house Epichloë metabolite database. We were able to identify the known endophyte-specific metabolites, peramine and epichloëcyclins, as well as a large number of unknown markers. To determine whether these methods can be applied to the identification of novel Epichloë-derived metabolites, we deleted a gene encoding a NRPS (lgsA) that is highly expressed in planta. Comparative mass spectrometric analysis of apoplastic wash fluid from wild-type- versus mutant- infected plants identified a novel Leu/Ile glycoside metabolite present in the former.

SummaryEpichloë festucaeis an endophytic fungus that forms a mutualistic symbiotic association withLolium perenne. Here we analysed how the metabolome of the ryegrass apoplast changed upon infection of this host with sexual and asexual isolates ofE. festucae. A metabolite fingerprinting approach was used to analyse the metabolite composition of apoplastic wash fluid from non-infected and infectedL. perenne. Metabolites enriched or depleted in one or both of these treatments were identified using a set of interactive tools. A genetic approach in combination with tandem mass spectrometry was used to identify a novel product of a secondary metabolite gene cluster. Metabolites likely to be present in the apoplast were identified using the MarVis Pathway in combination with the BioCyc and KEGG databases, and an in-houseEpichloëmetabolite database. We were able to identify the known endophyte-specific metabolites, peramine and epichloëcyclins, as well as a large number of unknown markers. To determine whether these methods can be applied to the identification of novelEpichloë-derived metabolites, we deleted a gene encoding a NRPS (lgsA) that is highly expressedin planta. Comparative mass spectrometric analysis of apoplastic wash fluid from wild-type- versus mutant- infected plants identified a novel Leu/Ile glycoside metabolite present in the former.

Calcineurin is a conserved calcium/calmodulin-dependent protein phosphatase, consisting of a catalytic subunit A and a regulatory subunit B, which is involved in calcium-dependent signalling and regulation of various important cellular processes. In this study, we functionally characterized the catalytic subunit A (CnaA) of the endophytic fungus Epichloë festucae which forms a symbiotic association with the grass host Lolium perenne. We deleted the CnaA-encoding gene cnaA in E. festucae and examined its role in hyphal growth, cell wall integrity and symbiosis. This ΔcnaA strain had a severe growth defect with loss of radial growth and hyper-branched hyphae. Transmission electron microscopy and confocal microscopy analysis of the mutant revealed cell wall defects, aberrant septation and the formation of intrahyphal hyphae, both in culture and in planta. The mutant strain also showed a reduced infection rate in planta. The fluorescence of mutant hyphae stained with WGA-AF488 was reduced, indicating reduced chitin accessibility. Together, these results show that E. festucae CnaA is required for fungal growth, maintaining cell wall integrity and host colonization.

Epichloe endophytes form mutualistic symbiotic associations with aerial tissues of temperate grasses. Intercalary growth of hyphae within the leaves enables fungal growth to be synchronized with host leaf growth, leading to formation of a highly structured and tightly regulated symbiotic network. Mutations in fungal genes that disrupt cell cell fusion and other key signalling pathways lead to an antagonistic interaction characterized by unregulated growth of endophytic hyphae and detrimental effects on host growth. Transcriptome analysis of these mutant associations provides key insights into the regulation of the symbiosis. In nature a similar switch in growth occurs when hyphae transition into the sexual cycle forming stromata that abort host inflorescences. Endophyte infection of the grass host leads to a major reprogramming of host metabolism and alters host development. Changes in endophyte cell wall structure and the repertoire of effectors secreted into the host apoplast accompany establishment of a mutualistic interaction within the leaves.

Structural features of genomes, including the three-dimensional arrangement of DNA in the nucleus, are increasingly seen as key contributors to the regulation of gene expression. However, studies on how genome structure and nuclear organisation influence transcription have so far been limited to a handful of model species. This narrow focus limits our ability to draw general conclusions about the ways in which three-dimensional structures are encoded, and to integrate information from three-dimensional data to address a broader gamut of biological questions. Here, we generate a complete and gapless genome sequence for the filamentous fungus, Epichloë festucae. We use Hi-C data to examine the three-dimensional organisation of the genome, and RNA-seq data to investigate how Epichloë genome structure contributes to the suite of transcriptional changes needed to maintain symbiotic relationships with the grass host. Our results reveal a genome in which very repeat-rich blocks of DNA with discrete boundaries are interspersed by gene-rich sequences that are almost repeat-free. In contrast to other species reported to date, the three-dimensional structure of the genome is anchored by these repeat blocks, which act to isolate transcription in neighbouring gene-rich regions. Genes that are differentially expressed in planta are enriched near the boundaries of these repeat-rich blocks, suggesting that their three-dimensional orientation partly encodes and regulates the symbiotic relationship formed by this organism.

Penicillium paxilli ATCC 26601 is an asexual filamentous fungal species known for its production of the mycotoxin paxilline. We present here the 35-Mb draft genome sequence for this organism.

ABSTRACT Peramine is an insect-feeding deterrent produced by Epichloë species in symbiotic association with C 3 grasses. The perA gene responsible for peramine synthesis encodes a two-module nonribosomal peptide synthetase. Alleles of perA are found in most Epichloë species; however, peramine is not produced by many perA -containing Epichloë isolates. The genetic basis of these peramine-negative chemotypes is often unknown. Using PCR and DNA sequencing, we analyzed the perA genes from 72 Epichloë isolates and identified causative mutations of perA null alleles. We found nonfunctional perA -ΔR* alleles, which contain a transposon-associated deletion of the perA region encoding the C-terminal reductase domain, are widespread within the Epichloë genus and represent a prevalent mutation found in nonhybrid species. Disparate phylogenies of adjacent A2 and T2 domains indicated that the deletion of the reductase domain (R*) likely occurred once and early in the evolution of the genus, and subsequently there have been several recombinations between those domains. A number of novel point, deletion, and insertion mutations responsible for abolishing peramine production in full-length perA alleles were also identified. The regions encoding the first and second adenylation domains (A1 and A2, respectively) were common sites for such mutations. Using this information, a method was developed to predict peramine chemotypes by combining PCR product size polymorphism analysis with sequencing of the perA adenylation domains.