The growth promotion in endophyte symbiotic plants does not penalise the resistance to herbivores and bacterial microbiota.

A trade-off between growth and defence against biotic stresses is common in plants. Fungal endophytes of the genus Epichloë may relieve this trade-off in their host grasses since they can simultaneously induce plant growth and produce antiherbivore alkaloids that circumvent the need for host defence. The Epichloë ability to decouple the growth-defence trade-off was evaluated by subjecting ryegrass with and without Epichloë endophytes to an exogenous treatment with gibberellin (GA) followed by a challenge with Rhopalosiphum padi aphids. In agreement with the endophyte-mediated trade-off decoupling hypothesis, the GA-derived promotion of plant growth increased the susceptibility to aphids in endophyte-free plants but did not affect the insect resistance in endophyte-symbiotic plants. In line with the unaltered insect resistance, the GA treatment did not reduce the concentration of Epichloë-derived alkaloids. The Epichloë mycelial biomass was transiently increased by the GA treatment but at the expense of hyphal integrity. The response of the phyllosphere bacterial microbiota to both GA treatment and Epichloë was also evaluated. Only Epichloë, and not the GA treatment, altered the composition of the phyllosphere microbiota and the abundance of certain bacterial taxa. Our findings clearly demonstrate that Epichloë does indeed relieve the plant growth-defence trade-off.

Harnessing cold adaptation for postglacial colonisation: Galactinol synthase expression and raffinose accumulation in a polyploid and its progenitors.

Allotetraploid white clover (Trifolium repens) formed during the last glaciation through hybridisation of two European diploid progenitors from restricted niches: one coastal, the other alpine. Here, we examine which hybridisation-derived molecular events may have underpinned white clover's postglacial niche expansion. We compared the transcriptomic frost responses of white clovers (an inbred line and an alpine-adapted ecotype), extant descendants of its progenitor species and a resynthesised white clover neopolyploid to identify genes that were exclusively frost-induced in the alpine progenitor and its derived subgenomes. From these analyses we identified galactinol synthase, the rate-limiting enzyme in biosynthesis of the cryoprotectant raffinose, and found that the extant descendants of the alpine progenitor as well as the neopolyploid white clover rapidly accumulated significantly more galactinol and raffinose than the coastal progenitor under cold stress. The frost-induced galactinol synthase expression and rapid raffinose accumulation derived from the alpine progenitor likely provided an advantage during early postglacial colonisation for white clover compared to its coastal progenitor.

Epichloë fungal endophyte interactions in perennial ryegrass (Lolium perenne L.) modified to accumulate foliar lipids for increased energy density.

BACKGROUND: Commercial cultivars of perennial ryegrass infected with selected Epichloë fungal endophytes are highly desirable in certain pastures as the resulting mutualistic association has the capacity to confer agronomic benefits (such as invertebrate pest deterrence) largely due to fungal produced secondary metabolites (e.g., alkaloids). In this study, we investigated T2 segregating populations derived from two independent transformation events expressing diacylglycerol acyltransferase (DGAT) and cysteine oleosin (CO) genes designed to increase foliar lipid and biomass accumulation. These populations were either infected with Epichloë festucae var. lolii strain AR1 or Epichloë sp. LpTG-3 strain AR37 to examine relationships between the introduced trait and the endophytic association. Here we report on experiments designed to investigate if expression of the DGAT + CO trait in foliar tissues of perennial ryegrass could negatively impact the grass-endophyte association and vice versa. Both endophyte and plant characters were measured under controlled environment and field conditions. RESULTS: Expected relative increases in total fatty acids of 17-58% accrued as a result of DGAT + CO expression with no significant difference between the endophyte-infected and non-infected progeny. Hyphal growth in association with DGAT + CO expression appeared normal when compared to control plants in a growth chamber. There was no significant difference in mycelial biomass for both strains AR1 and AR37, however, Epichloë-derived alkaloid concentrations were significantly lower on some occasions in the DGAT + CO plants compared to the corresponding null-segregant progenies, although these remained within the reported range for bioactivity. CONCLUSIONS: These results suggest that the mutualistic association formed between perennial ryegrass and selected Epichloë strains does not influence expression of the host DGAT + CO technology, but that endophyte performance may be reduced under some circumstances. Further investigation will now be required to determine the preferred genetic backgrounds for introgression of the DGAT + CO trait in combination with selected endophyte strains, as grass host genetics is a major determinant to the success of the grass-endophyte association in this species.

Can Aphid Herbivory Induce Intergenerational Effects of Endophyte-conferred Resistance in Grasses?

Plants have evolved mechanisms to survive herbivory. One such mechanism is the induction of defences upon attack that can operate intergenerationally. Cool-season grasses (sub-family Pooideae) obtain defences via symbiosis with vertically transmitted fungal endophytes (genus Epichloë) and can also show inducible responses. However, it is unknown whether these herbivore-induced responses can have intergenerational effects. We hypothesized that herbivory by aphids on maternal plants induces the intergenerational accumulation of endophyte-derived defensive alkaloids and resistance intensification in the progeny. We subjected mother plants symbiotic or not with Epichloë occultans, a species known for its production of anti-insect alkaloids known as lolines, to the aphid Rhopalosiphum padi. Then, we evaluated the progeny of these plants in terms of loline alkaloid concentration, resistance level (through herbivore performance), and shoot biomass. Herbivory on mother plants did not increase the concentration of lolines in seeds but it tended to affect loline concentration in progeny plants. There was an overall herbivore-induced intergenerational effect increasing the endophyte-conferred defence and resistance. Symbiotic plants were more resistant to aphids and had higher shoot biomass than their non-symbiotic counterparts. Since maternal herbivory did not affect the loline concentrations in seeds, the greater resistance of the progeny could have resulted from an inherited mechanism of epigenetic regulation. It would be interesting to elucidate the origin of this regulation since it could come from the host or the fungal symbiont. Thus, endophyte-driven differential fitness between symbiotic and non-symbiotic plants might be higher as generations pass on in presence of herbivores.

Efficient nonenzymatic cyclization and domain shuffling drive pyrrolopyrazine diversity from truncated variants of a fungal NRPS.

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.

Regulation of subtelomeric fungal secondary metabolite genes by H3K4me3 regulators CclA and KdmB.

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.

Ontogenetic and trans-generational dynamics of a vertically transmitted fungal symbiont in an annual host plant in ozone-polluted settings.

Tropospheric ozone is an abiotic stress of increasing importance in the context of global climate change. This greenhouse gas is a potent phytotoxic molecule with demonstrated negative effects on crop yield and natural ecosystems. Recently, oxidative stress has been proposed as a mechanism that could regulate the interaction between cool-season grasses and Epichloë endophytes. We hypothesized that exposure of Lolium multiflorum plants, hosting endophytes to an ozone-polluted environment at different ontogenetic phases, would impact the trans-generational dynamics of the vertically transmitted fungal symbiont. Here, we found that the ozone-induced stress on the mother plants did not affect the endophyte vertical transmission but it impaired the persistence of the fungus in the seed exposed to artificial ageing. Endophyte longevity in seed was reduced by exposure of the mother plant to ozone. Although ozone exposure did not influence either the endophyte mycelial concentration or their compound defences (loline alkaloids), a positive correlation was observed between host fitness and the concentration of endophyte-derived defence compounds. This suggests that fungal defences in grass seeds were not all produced in situ but remobilized from the vegetative tissues. Our study reveals ozone trans-generational effects on the persistence of a beneficial symbiont in a host grass.

Fungal Alkaloid Occurrence in Endophyte-Infected Perennial Ryegrass during Seedling Establishment.

The symbiotic Epichloë festucae var. lolii endophyte produces alkaloids which can provide its host grass, perennial ryegrass (Lolium perenne L), with a selective advantage in both natural and agricultural managed ecosystems. This study focuses on understanding the alkaloid concentrations that occur in endophyte-infected perennial ryegrass during the early establishment phase. In a glasshouse experiment fungal alkaloid concentrations (peramine, lolitrem B, ergovaline, and epoxy-janthitrems) were measured in perennial ryegrass seedlings infected with E. festucae var. lolii proprietary strains AR1, AR37, NEA2, and NZ common toxic for 69 days after sowing. The endophyte becomes metabolically active, starting alkaloid production, as early as 6 days after sowing. Alkaloid concentrations peaked in 8- to 10- day-old seedlings due to a seedling growth slowdown. This study provides data showing that the loss of insect protection in endophyte-infected seedlings is linked to a reduction in chemical defence after seed-stored, maternally synthesised alkaloids are diluted by seedling dry matter accumulation.

Correction: Fungal Alkaloid Occurrence in Endophyte-Infected Perennial Ryegrass during Seedling Establishment.

The original version of this article unfortunately contained a mistake. There is a mistake in the affiliation section and Figure 6. The correct affiliation and Figure 6 are shown here.

Orthologous peramine and pyrrolopyrazine-producing biosynthetic gene clusters in Metarhizium rileyi, Metarhizium majus and Cladonia grayi.

Peramine 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.

The plant hormone salicylic acid interacts with the mechanism of anti-herbivory conferred by fungal endophytes in grasses.

The plant hormone salicylic acid (SA) is recognized as an effective defence against biotrophic pathogens, but its role as regulator of beneficial plant symbionts has received little attention. We studied the relationship between the SA hormone and leaf fungal endophytes on herbivore defences in symbiotic grasses. We hypothesize that the SA exposure suppresses the endophyte reducing the fungal-produced alkaloids. Because of the role that alkaloids play in anti-herbivore defences, any reduction in their production should make host plants more susceptible to herbivores. Lolium multiflorum plants symbiotic and nonsymbiotic with the endophyte Epichloë occultans were exposed to SA followed by a challenge with the aphid Rhopalosiphum padi. We measured the level of plant resistance to aphids, and the defences conferred by endophytes and host plants. Symbiotic plants had lower concentrations of SA than did the nonsymbiotic counterparts. Consistent with our prediction, the hormonal treatment reduced the concentration of loline alkaloids (i.e., N-formyllolines and N-acetylnorlolines) and consequently decreased the endophyte-conferred resistance against aphids. Our study highlights the importance of the interaction between the plant immune system and endophytes for the stability of the defensive mutualism. Our results indicate that the SA plays a critical role in regulating the endophyte-conferred resistance against herbivores.

Complex Interactions among Sheep, Insects, Grass, and Fungi in a Simple New Zealand Grazing System.

Epichloë fungi (Ascomycota) live within aboveground tissues of grasses and can have important implications for natural and managed ecosystems through production of alkaloids. Nonetheless, vertebrate herbivores may possess traits, like oral secretions, that mitigate effects of alkaloids. We tested if sheep saliva mitigates effects of Epichloë alkaloids on a beetle pest of perennial ryegrass (Lolium perenne L.) in a New Zealand pasture setting. Plants with one of several fungal isolates were clipped with scissors, grazed by sheep, or clipped with sheep saliva applied to cut ends of stems. We then assessed feeding damage by Argentine stem weevils on blade segments collected from experimental plants. We found that clipping plants induced synthesis of an alkaloid that reduces feeding by beetles and that sheep saliva mitigates this effect. Unexpectedly, the alkaloid (perloline) that explains variation in beetle feeding is one produced not by the endophyte, but rather by the plant. Yet, these effects depended upon fungal isolate. Such indirect, complex interactions may be much more common in both managed and natural grassland systems than typically thought and could have implications for managing grazing systems.

Defining the pathways of symbiotic Epichloë colonization in grass embryos with confocal microscopy.

Asexual cool-season grass endophytes of the genus Epichloë (Ascomycota: Clavicipitaceae) are strictly vertically disseminated. The hosts of these mutualistic fungi express no symptoms during the fungal lifecycle that takes place entirely within the plant, while their hosts receive beneficial outcomes. These fungi are distributed in two major locations within the mature seeds of their hosts; namely, within the embryo (including the scutellum, coleoptile, plumule, radicle, and coleorhiza tissues) and between the aleurone and pericarp layers, with the latter hyphae playing no role in transmission of the fungus to the next plant generation. Conflicting evidence remains in the literature on the timing of embryo colonization. In a detailed investigation, utilizing confocal microscopy to observe the distribution of Epichloë coenophiala strain AR601 in tall fescue (Lolium arundinaceum), we tracked endophyte hyphal colonization in the ovary (pre-fertilization) through to the fully mature seed stage. Confocal microscopy images revealed that at the early and mature developmental stages of the embryo sac, before host grass fertilization, there were large quantities of endophyte mycelium present, especially around the antipodal cells, indicating that this endophyte enters the embryo sac before the fertilization stage. After host fertilization, fungal hyphae could be seen in the true embryo and early nonstarchy endosperm. Understanding the mechanisms of transmission to the seed is important for commercial seed producers and end users.

Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the clavicipitaceae reveals dynamics of alkaloid loci.

The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some-including the infamous ergot alkaloids-have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses.

The Effect of N-Carbamylglutamate Supplementation during the Last Third of Gestation on the Growth and Development of Fetuses Born to Nutrient-Restricted Twin-Bearing Ewes.

N-carbamylglutamate (NCG) is postulated to improve fetal growth in nutrient-restricted gestations when supplemented from day 35 to 110 of gestation, but the effects of supplementation from 100 days of gestation to birth have not been evaluated. The aim of this study was to evaluate the effect of oral NCG supplementation from 100 days of gestation (dga) to term in naturally nutrient-restricted grazing twin-bearing ewes, on the maternal body weight (BW), body condition score (BCS), placental morphology, fetal body and organ weights and blood biochemistry and antioxidant status in the ewe and fetuses. Eighteen twin-bearing ewes maintained under grazing management were randomly allocated to either a treatment group (NCG; n = 10), orally dosed once daily with 60 mg/kg of NCG from day 100 until 140 dga, or an unsupplemented control group (CON; n = 8). At 140 dga, blood gases, redox status, maternal and fetal plasma and fetal biometrics were obtained after caesarian section. The serum concentration of NCG was increased 15-fold in the NCG ewes compared to the CON. No major effects on dam or fetal body weight nor on blood biochemistry or antioxidant parameters were observed. These results indicate that NCG supplementation in mid-to-late gestation to grazing ewes was unable to rescue the negative production effects of severe natural nutritional restriction on both the dam and fetuses.

Translocation of Loline Alkaloids in Epichloë-Infected Cereal and Pasture Grasses: What the Insects Tell Us.

Aphids are major pests of cereal and pasture grasses throughout the world, vectoring disease and reducing plant production. There are few control options other than insecticides. Epichloë endophytes that produce loline alkaloids in their hosts provide a possible mechanism of control, with both meadow fescue and tall fescue naturally infected with loline-producing endophytes showing a resistance to Rhopalosiphum padi. We screened Elymus spp. naturally infected with endophytes that produced loline alkaloids at concentrations known to affect aphids on fescue but found no effect on these insects infesting Elymus. A synthetic loline-producing endophyte association with rye also had no effect on the aphids. After hypothesizing that the lolines were being translocated in the xylem in Elymus and rye rather than the phloem, we tested the rye and meadow fescue infected with loline-producing endophytes against a xylem feeding spittlebug. The endophyte in rye inhibited the feeding of the insect and reduced its survival, whereas the endophyte-infected meadow fescue had no effect on the spittlebug but reduced the number of aphids. Lolines applied to the potting medium of endophyte-free and endophyte-infected rye, ryegrass, and tall fescue resulted in a decrease in the aphid populations on the endophyte-free pasture grasses relative to the untreated controls but had no effect on aphid numbers on the rye. We tentatively conclude that lolines, produced in both natural and synthetic association with Elymus and rye, are partitioned in the xylem rather than the phloem, where they are inaccessible to aphids.

Characterization of the Biosynthetic Gene Cluster for the Ribosomally Synthesized Cyclic Peptide Epichloëcyclins in Epichloë festucae.

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.

Identification of Genetic Diversity, Pyrrocidine-Producing Strains and Transmission Modes of Endophytic Sarocladium zeae Fungi from Zea Crops.

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.

Dissection of the epoxyjanthitrem pathway in Epichloë sp. LpTG-3 strain AR37 by CRISPR gene editing.

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.

Cross-species transcriptomics identifies core regulatory changes differentiating the asymptomatic asexual and virulent sexual life cycles of grass-symbiotic Epichloë fungi.

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.