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Epichloë spp. often form mutualistic interactions with cool-season grasses, such as Lolium perenne . However, the molecular mechanisms underlying this interaction remain poorly understood. In this study, we employed reduced representation bisulfite sequencing method (epiGBS) to investigate the impact of the Epichloë sp. Lp TG-3 strain AR37 on the methylome of L. perenne across multiple grass generations and under drought stress conditions. Our results showed that the presence of the endophyte leads to a decrease in DNA methylation across genomic features, with differentially methylated regions primarily located in intergenic regions and CHH contexts. The presence of the endophyte was consistently associated with hypomethylation in plants across generations. This research sheds new light on the molecular mechanisms governing the mutualistic interaction between Epichloë sp. Lp TG-3 strain AR37 and L. perenne . It underscores the role of methylation changes associated with endophyte infection and suggests that the observed global DNA hypomethylation in L. perenne may be influenced by factors such as the duration of the endophyte-plant association and the accumulation of genetic and epigenetic changes over time.

[This corrects the article DOI: 10.3389/fpls.2023.1258100.].[This corrects the article DOI: 10.3389/fpls.2023.1258100.].

Elymus dahucirus is an essential plant for ecological restoration in fragile ecological areas and mining area restoration. As lawn grass, it can quickly cover soil and prevent soil erosion, so it is commonly used as a pioneer grass for lawn greening and slope protection. In recent years, with the development of mineral resources, Qinghai–Tibet Plateau soil is facing the threat of heavy metal cadmium (Cd) pollution. E. dahuricus can host the filamentous fungus Epichloë bromicola. To make better use of the advantages that Epichloë bring to host plants to alleviate heavy metal pollution in soil, plant growth and antioxidant capacity effects on E. bromicola infected (E+) and uninfected (E−) E. dahuricus were determined under Cd stress. During Cd treatment, plant growth was decreased by Cd stress, while E+ plants exhibited equal or better growth compared to E− plants. Cd treatment induces a proline and antioxidant enzyme burst in infected plants, while malondialdehyde (MDA) increases. E. bromicola improved plant growth and antioxidant capacity. E. dahuricus breeding strategies could use the information here in efforts to improve the performance of E. dahuricus in both environmental protection and agronomic contexts.

The relationship between cool-season grasses and fungal endophytes is widely regarded as mutualistic, but there is growing uncertainty about whether changes in resource supply and environment benefit both organisms to a similar extent. Here, we infected two perennial ryegrass (Lolium perenne) cultivars (AberDove, Fennema) that differ in carbohydrate content with three strains of Neotyphodium lolii (AR1, AR37, common strain) that differ intrinsically in alkaloid profile. We grew endophyte-free and infected plants under high and low nitrogen (N) supply and used quantitative PCR (qPCR) to estimate endophyte concentrations in harvested leaf tissues. Endophyte concentration was reduced by 40% under high N supply, and by 50% in the higher sugar cultivar. These two effects were additive (together resulting in 75% reduction). Alkaloid production was also reduced under both increased N supply and high sugar cultivar, and for three of the four alkaloids quantified, concentrations were linearly related to endophyte concentration. The results stress the need for wider quantification of fungal endophytes in the grassland-foliar endophyte context, and have implications for how introducing new cultivars, novel endophytes or increasing N inputs affect the role of endophytes in grassland ecosystems.

The inoculation of Epichloë endophytes into modern cereals, resulting in systemic infection, depends on the genetics of both the host and the endophyte strain deployed. Until very recently, the only modern cereal to have been infected with Epichloë, in which normal phenotype seed-transmitted associations were achieved, is rye (Secale cereale). Whilst minor in-roads have been achieved in infecting hexaploid wheat (Triticum aestivum), the phenotypes of these associations have all been extremely poor, including host death and stunting. To identify host genetic factors that may impact the compatibility of Epichloë infection in wheat, wheat–alien chromosome addition/substitution lines were inoculated with Epichloë, and the phenotypes of infected plants were assessed. Symbioses were identified whereby infected wheat plants were phenotypically like uninfected controls. These plants completed their full lifecycle, including the vertical transmission of Epichloë into the next generation of grain, and represent the first ever compatible wheat–Epichloë associations to be created.

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.

Infection of the pasture grass Lolium perenne with the seed-transmitted fungal endophyte Epichloë festucae enhances its resilience to biotic and abiotic stress. Agricultural benefits of endophyte infection can be increased by generating novel symbiotic associations through inoculating L. perenne with selected Epichloë strains. Natural symbioses have coevolved over long periods. Thus, artificial symbioses will probably not have static properties, but symbionts will coadapt over time improving the fitness of the association. Here we report for the first time on temporal changes in a novel association of Epichloë strain AR37 and the L. perenne cultivar Grasslands Samson. Over nine generations, a seed maintenance program had increased the endophyte seed transmission rates to > 95% (from an initial 76%). We observed an approximately fivefold decline in endophyte biomass concentration in vegetative tissues over time (between generations 2 and 9). This indicates strong selection pressure toward reducing endophyte-related fitness costs by reducing endophyte biomass, without compromising the frequency of endophyte transmission to seed. We observed no obvious changes in tillering and only minor transcriptomic changes in infected plants over time. Functional analysis of 40 plant genes, showing continuously decreasing expression over time, suggests that adaptation of host metabolism and defense mechanisms are important for increasing the fitness of this association, and possibly fitness of such symbioses in general. Our results indicate that fitness of novel associations is likely to improve over time and that monitoring changes in novel associations can assist in identifying key features of endophyte-mediated enhancement of host fitness.

Endophytes of the genus Epichloë (Clavicipitaceae, Ascomycota) frequently occur within cool-season grasses and form interactions with their hosts that range from mutualistic to antagonistic. Many Epichloë species have arisen via interspecific hybridization, resulting in species with two or three subgenomes that retain all or nearly all of their original parental genomes, a process termed allopolyploidization. Here, we characterize Epichloë hybrida, sp. nov., a mutualistic species that has increasingly become a model system for investigating allopolyploidy in fungi. The Epichloë species so far identified as the closest known relatives of the two progenitors of E. hybrida are E. festucae var. lolii and E. typhina. We confirm that the nuclear genome of E. hybrida contains two homeologs of most protein-coding genes from E. festucae and E. typhina, with genome-wide gene expression analysis indicating a slight bias in overall gene expression from the E. typhina subgenome. Mitochondrial DNA is detectable only from E. festucae, whereas ribosomal DNA is detectable only from E. typhina. Inheriting ribosomal DNA from just one parent might be expected to preferentially favor interactions with ribosomal proteins from the same parent, but we find that ribosomal protein genes from both parental subgenomes are nearly all expressed equally in E. hybrida. Finally, we provide a comprehensive set of resources for this model system that are intended to facilitate further study of fungal hybridization by other researchers.

Secreted proteins, those involved in cell wall biogenesis, are likely to play a role in communication in the symbiotic interaction between the fungal endophyte Epichloë festucae with perennial ryegrass (Lolium perenne), particularly given the close association between fungal hyphae and the plant cell wall. Our hypothesis was that secreted proteins are likely to be responsible for establishing and maintaining a normal symbiotic relationship. We analyzed an endophyte EST database for genes with predicted signal peptide sequences. Here, we report the identification and characterization of rhgA; a gene involved in the regulation of hyphal growth in planta. In planta analysis of ΔrhgA mutants showed that disruption of rhgA resulted in extensive unregulated hyphal growth. This phenotype was fully complemented by insertion of the rhgA gene and suggests that rhgA is important for maintaining normal hyphal growth during symbiosis. Identification and characterization of rhgA, a gene important for maintaining a symbiotic relationship between beneficial fungal endophytes and their plant hosts.

The seed-transmitted fungal symbiont, , colonizes grasses by infecting host tissues as they form on the shoot apical meristem (SAM) of the seedling. How this fungus accommodates the complexities of plant development to successfully colonize the leaves and inflorescences is unclear. Since adenosine 3', 5'-cyclic monophosphate (cAMP)-dependent signaling is often essential for host colonization by fungal pathogens, we disrupted the cAMP cascade by insertional mutagenesis of the adenylate cyclase gene ( ). Consistent with deletions of this gene in other fungi, mutants had a slow radial growth rate in culture, and hyphae were convoluted and hyper-branched suggesting that fungal apical dominance had been disrupted. Nitro blue tetrazolium (NBT) staining of hyphae showed that cAMP disruption mutants were impaired in their ability to synthesize superoxide, indicating that cAMP signaling regulates accumulation of reactive oxygen species (ROS). Despite significant defects in hyphal growth and ROS production, Δ mutants were infectious and capable of forming symbiotic associations with grasses. Plants infected with Δ were marginally less robust than the wild-type (WT), however hyphae were hyper-branched, and leaf tissues heavily colonized, indicating that the tight regulation of hyphal growth normally observed in maturing leaves requires functional cAMP signaling.