Differentially expressed genes in cotyledon of ewes fed mycotoxins.

BACKGROUND: Ergot alkaloids (E+) are mycotoxins produced by the endophytic fungus, Epichloë coenophiala, in tall fescue that are associated with ergotism in animals. Exposure to ergot alkaloids during gestation reduces fetal weight and placental mass in sheep. These reductions are related to vasoconstrictive effects of ergot alkaloids and potential alterations in nutrient transport to the fetus. Cotyledon samples were obtained from eight ewes that were fed E+ (n = 4; E+/E+) or E- (endophyte-free without ergot alkaloids; n = 4; E-/E-) seed during both mid (d 35 to 85) and late (d 85-133) gestation to assess differentially expressed genes associated with ergot alkaloid induced reductions in placental mass and fetal weight, and discover potential adaptive mechanisms to alter nutrient supply to fetus. RESULTS: Ewes fed E+/E+ fescue seed during both mid and late gestation had 20% reduction in fetal body weight and 33% reduction in cotyledon mass compared to controls (E-/E-). Over 13,000 genes were identified with 110 upregulated and 33 downregulated. Four genes had a |log2FC| > 5 for ewes consuming E+/E+ treatment compared to controls: LECT2, SLC22A9, APOC3, and MBL2. REViGO revealed clusters of upregulated genes associated glucose, carbohydrates, lipid, protein, macromolecular and cellular metabolism, regulation of wound healing and response to starvation. For downregulated genes, no clusters were present, but all enriched GO terms were associated with anion and monocarboxylic acid transport. The complement and coagulation cascade and the peroxisome proliferator-activated receptor signaling pathway were found to be enriched for ewes consuming E+/E+ treatment. CONCLUSIONS: Consumption of ergot alkaloids during gestation altered the cotyledonary transcriptome specifically related to macronutrient metabolism, wound healing and starvation. These results show that ergot alkaloid exposure upregulates genes involved in nutrient metabolism to supply the fetus with additional substrates in attempts to rescue fetal growth.

Toxic tall fescue grazing increases susceptibility of the Angus steer fecal microbiota and plasma/urine metabolome to environmental effects.

Impaired thermoregulation and lowered average daily gains (ADG) result when livestock graze toxic endophyte (Epichloë coenophialum)-infected tall fescue (E+) and are hallmark signs of fescue toxicosis (FT), a disease exacerbated by increased temperature and humidity (+temperature-humidity index; +THI). We previously reported FT is associated with metabolic and microbiota perturbations under thermoneutral conditions; here, we assessed the influence of E+ grazing and +THI on the microbiota:metabolome interactions. Using high-resolution metabolomics and 16S rRNA gene sequencing, plasma/urine metabolomes and the fecal microbiota of Angus steers grazing non-toxic or E+ tall fescue were evaluated in the context of +THI. E+ grazing affected the fecal microbiota profile; +THI conditions modulated the microbiota only in E+ steers. E+ also perturbed many metabolic pathways, namely amino acid and inflammation-related metabolism; +THI affected these pathways only in E+ steers. Integrative analyses revealed the E+ microbiota correlated and co-varied with the metabolomes in a THI-dependent manner. Operational taxonomic units in the families Peptococcaceae, Clostridiaceae, and Ruminococcaceae correlated with production parameters (e.g., ADG) and with multiple plasma/urine metabolic features, providing putative FT biomarkers and/or targets for the development of FT therapeutics. Overall, this study suggests that E+ grazing increases Angus steer susceptibility to +THI, and offers possible targets for FT interventions.

Aboveground Epichloë coenophiala-Grass Associations Do Not Affect Belowground Fungal Symbionts or Associated Plant, Soil Parameters.

Cool season grasses host multiple fungal symbionts, such as aboveground Epichloë endophytes and belowground arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSEs). Asexual Epichloë endophytes can influence root colonization by AMF, but the type of interaction-whether antagonistic or beneficial-varies. In Schedonorus arundinaceus (tall fescue), Epichloë coenophiala can negatively affect AMF, which may impact soil properties and ecosystem function. Within field plots of S. arundinaceus that were either E. coenophiala-free (E-), infected with the common, mammal-toxic E. coenophiala strain (CTE+), or infected with one of two novel, non-toxic strains (AR542 NTE+ and AR584 NTE+), we hypothesized that (1) CTE+ would decrease AMF and DSE colonization rates and reduce soil extraradical AMF hyphae compared to E- or NTE+, and (2) this would lead to E- and NTE+ plots having greater water stable soil aggregates and C than CTE+. E. coenophiala presence and strain did not significantly alter AMF or DSE colonization, nor did it affect extraradical AMF hypha length, soil aggregates, or aggregate-associated C and N. Soil extraradical AMF hypha length negatively correlated with root AMF colonization. Our results contrast with previous demonstrations that E. coenophiala symbiosis inhibits belowground AMF communities. In our mesic, relatively nutrient-rich grassland, E. coenophiala symbiosis did not antagonize belowground symbionts, regardless of strain. Manipulating E. coenophiala strains within S. arundinaceus may not significantly alter AMF communities and nutrient cycling, yet we must further explore these relationships under different soils and environmental conditions given that symbiont interactions can be important in determining ecosystem response to global change.

ABC transporter and metallothionein expression affected by NI and Epichloe endophyte infection in tall fescue.

Epichloe endophytes are symbiotic fungi which unlike mycorrhiza grow within aerial parts of host plants. The fungi may increase host tolerance to both biotic and abiotic stresses. In this study, the effect of endophyte infection on growth and tolerance, carbohydrate contents and ABC (ABC transporter) and MET (metallothionein) expression in the leaves of tall fescue (Festuca arundinacea) plants cultivated in Ni polluted soil were evaluated. The endophyte infected (E+) and non-infected (E-) fescue plants were cultivated in soil under different Ni concentrations (30, 90 and 180mgkg(-1)). Growth parameters including root, shoot, total biomass, tiller number and total chlorophyll content of plants and H2O2 content of shoots were measured at the end of experiment. Ni translocation to the shoots, carbohydrate contents in roots and expression of ABC and MET of the leaves were also measured after 10 weeks of growth. Results demonstrated the beneficial effect of endophyte association on growth and Ni tolerance of tall fescue under Ni stress through an avoidance mechanism (reduction of Ni accumulation and translocation to the shoots). Endophyte infected plants showed less ABC and MET expression compared to the endophyte free plants. In endophyte free plants, H2O2 production had a significant positive correlation with genes expression, indicating that an increase in H2O2 might be involved in the up-regulation of ABC and MET under Ni stress.

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.

What triggers grass endophytes to switch from mutualism to pathogenism?

Symbioses between cool season grasses and fungi of the family Clavicipitaceae are an integral component of both natural and agricultural ecosystems. An excellent experimental model is the association between the biotrophic fungus Epichloë festucae and Lolium perenne (perennial ryegrass). The fungal partner produces a suite of secondary metabolites that protect the host from various biotic and abiotic stresses. The plant host provides a source of nutrients and a mechanism of dissemination via seed transmission. Crucial mechanisms that maintain a stable mutualistic association include signaling through the stress activated MAP kinase pathway and production of reactive oxygen species by the fungal NADPH oxidase (Nox) complex. Disruption of components of the Nox complex (NoxA, NoxR and RacA), or the stress-activated MAP kinase (SakA), leads to a breakdown in this finely balanced association, resulting in pathogenic infection instead of mutualism. Hosts infected with fungi lacking a functional Nox complex, or the stress-activated MAP kinase, display a stunted phenotype and undergo premature senescence, while the fungus switches from restricted to proliferative growth. To gain insight into the mechanisms that underlie these physiological changes, high throughput mRNA sequencing has been used to analyze the transcriptomes of both host and symbiont in wild-type and a mutant association. In the ΔsakA mutant association, a dramatic up-regulation of fungal hydrolases and transporters was observed, changes consistent with a switch from restricted symbiotic to proliferative pathogenic growth. Analysis of the plant transcriptome revealed dramatic changes in expression of host genes involved in pathogen defense, transposon activation and hormone biosynthesis and response. This review highlights how finely tuned grass-endophyte associations are, and how interfering with the signaling pathways involved in maintenance of these associations can trigger a change from mutualistic to pathogenic interaction.