Transcriptome analysis of Epichloë strains in tall fescue in response to drought stress.

Epichloë coenophiala, a systemic fungal symbiont (endophyte) of tall fescue (Lolium arundinaceum), has been documented to confer to this grass better persistence than plants lacking the endophyte, especially under stress conditions such as drought. The response, if any, of the endophyte to imposition of stress on the host plant has not been characterized previously. Therefore, we investigated effects on gene expression by E. coenophiala and a related endophyte when plant-endophyte symbiota were subjected to acute water-deficit stress. Plants harboring different endophyte strains were grown in sand in the greenhouse, then half were deprived of water for 48 h and the other half were watered controls. RNA was isolated from different plant tissues, and mRNA sequencing (RNA-seq) was conducted to identify genes that were differentially expressed comparing stress treatment with control. We compared two different plants harboring the common toxic E. coenophiala strain (CTE) and two non-ergot-alkaloid-producing Epichloë strains in tall fescue pseudostems, and in a second experiment we compared responses of E. coenophiala CTE in plant pseudostem and crown tissues. The endophytes responded to the stress with increased expression of genes involved in oxidative stress response, oxygen radical detoxification, C-compound carbohydrate metabolism, heat shock, and cellular transport pathways. The magnitude of fungal gene responses during stress varied among plant-endophyte symbiota. Responses in pseudostems and crowns involved some common pathways as well as some tissue-specific pathways. The fungal response to water-deficit stress involved gene expression changes in similar pathways that have been documented for plant stress responses, indicating that Epichloë spp. and their host plants either coordinate stress responses or separately activate similar stress response mechanisms that work together for mutual protection.

Differential gene expression in tall fescue tissues in response to water deficit.

Tall fescue (Festuca arundinacea Schreb.) is a popular pasture and turf grass particularly known for drought resistance, allowing for its persistence in locations that are unfavorable for other cool-season grasses. Also, its seed-borne fungal symbiont (endophyte) Epichloë coenophiala, which resides in the crown and pseudostem, can be a contributing factor in its drought tolerance. Because it contains the apical meristems, crown survival under drought stress is critical to plant survival as well as the endophyte. In this study, we subjected tall fescue plants with their endophyte to water-deficit stress or, as controls with normal watering, then compared plant transcriptome responses in four vegetative tissues: leaf blades, pseudostem, crown, and roots. A transcript was designated a differentially expressed gene (DEG) if it exhibited at least a twofold expression difference between stress and control samples with an adjusted p value of .001. Pathway analysis of the DEGs across all tissue types included photosynthesis, carbohydrate metabolism, phytohormone biosynthesis and signaling, cellular organization, and a transcriptional regulation. While no specific pathway was observed to be differentially expressed in the crown, genes encoding auxin response factors, nuclear pore anchors, structural maintenance of chromosomes, and class XI myosin proteins were more highly differentially expressed in crown than in the other vegetative tissues, suggesting that regulation in expression of these genes in the crown may aid in survival of the meristems in the crown.

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.

Transcriptome Analysis and Differential Expression in Tall Fescue Harboring Different Endophyte Strains in Response to Water Deficit.

Two tall fescue [Lolium arundinaceum (Schreb.) Darbysh. = Schedonorus arundinaceus (Schreb.) Dumort. = Festuca arundinacea var. arundinacea Schreb.] plant genotypes with an Epichloë coenophiala (Morgan-Jones & W. Gams) C.W. Bacon & Schardl common toxic endophyte (CTE), one with a nontoxic strain (NTE19) and one with another Epichloë species (FaTG-4) were evaluated and compared with their respective endophyte-free clones for responses to water-deficit stress in the greenhouse. One of the plant genotypes (P27) showed a positive effect of its CTE strain on tiller production after stress and resumed watering. In transcriptome analysis of the pseudostems (leaf sheath whorls), differentially expressed genes (DEGs) were defined as having at least twofold expression difference and false discovery rate (FDR) < 0.05 in comparisons of water treatment (stressed or watered), endophyte presence or absence, or both. Stress affected 38% of the plant transcripts including those for the expected stress-response pathways. The DEGs affected by endophyte in stressed plants were unique to individual plant genotypes. In unstressed plants, endophyte presence tended to reduce expression of genes putatively for defense against fungi, but in unstressed P27 endophyte presence there was enhanced expression of dehydrin and heat shock protein genes. Our results indicated subtle and variable effects of endophytes on tall fescue gene expression; where the endophyte confers protection, its effects on plant gene expression may help prime the plant for stress resistance.

Infection Rates and Alkaloid Patterns of Different Grass Species with Systemic Epichloë Endophytes.

Symbiotic Epichloë species are fungal endophytes of cool-season grasses that can produce alkaloids with toxicity to vertebrates and/or invertebrates. Monitoring infections and presence of alkaloids in grasses infected with Epichloë species can provide an estimate of possible intoxication risks for livestock. We sampled 3,046 individuals of 13 different grass species in three regions on 150 study sites in Germany. We determined infection rates and used PCR to identify Epichloë species diversity based on the presence of different alkaloid biosynthesis genes, then confirmed the possible chemotypes with high-performance liquid chromatography (HPLC)/ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) measurements. Infections of Epichloë spp. were found in Festuca pratensis Huds. (81%), Festuca ovina L. aggregate (agg.) (73%), Lolium perenne L. (15%), Festuca rubra L. (15%) and Dactylis glomerata L. (8%). The other eight grass species did not appear to be infected. For the majority of Epichloë-infected L. perenne samples (98%), the alkaloids lolitrem B and peramine were present, but ergovaline was not detected, which was consistent with the genetic evaluation, as dmaW, the gene encoding the first step of the ergot alkaloid biosynthesis pathway, was absent. Epichloë uncinata in F. pratensis produced anti-insect loline compounds. The Epichloë spp. observed in the F. ovina agg. samples showed the greatest level of diversity, and different intermediates of the indole-diterpene pathway could be detected. Epichloë infection rates alone are insufficient to estimate intoxication risks for livestock, as other factors, like the ability of the endophyte to produce the alkaloids, also need to be assessed.IMPORTANCE Severe problems of livestock intoxication from Epichloë-infected forage grasses have been reported from New Zealand, Australia, and the United States, but much less frequently from Europe, and particularly not from Germany. Nevertheless, it is important to monitor infection rates and alkaloids of grasses with Epichloë fungi to estimate possible intoxication risks. Most studies focus on agricultural grass species like Lolium perenne and Festuca arundinacea, but other cool-season grass species can also be infected. We show that in Germany, infection rates and alkaloids differ between grass species and that some of the alkaloids can be toxic to livestock. Changes in grassland management due to changing climate, especially with a shift toward grasslands dominated with Epichloë-infected species such as Lolium perenne, may result in greater numbers of intoxicated livestock in the near future. We therefore suggest regular monitoring of grass species for infections and alkaloids and call for maintaining heterogenous grasslands for livestock.

Transcriptomics of Epichloë-Grass Symbioses in Host Vegetative and Reproductive Stages.

Epichloë species are fungal symbionts (endophytes) of cool-season grasses that transmit vertically via inflorescence primordia (IP), ovaries (OV), and ultimately, embryos. Epichloë coenophiala, an endophyte of tall fescue (Schedonorus arundinaceus), provides multiple protective benefits to the grass. We conducted transcriptome analysis of the tall fescue-E. coenophiala symbiosis, comparing IP, OV, vegetative pseudostems (PS), and the lemma and palea (LP) (bracts) of the young floret. Transcriptomes of host OV and PS exhibited almost no significant differences attributable to endophyte presence or absence. Comparison of endophyte gene expression in different plant parts revealed numerous differentially expressed genes (DEGs). The 150 endophyte DEGs significantly higher in PS over OV included genes for alkaloid biosynthesis and sugar or amino acid transport. The 277 endophyte DEGs significantly higher in OV over PS included genes for protein chaperones (including most heat-shock proteins), trehalose synthesis complex, a bax inhibitor-1 protein homolog, the CLC chloride ion channel, catalase, and superoxide dismutase. Similar trends were apparent in the Brachypodium sylvaticum-Epichloë sylvatica symbiosis. Gene expression profiles in tall fescue IP and LP indicated that the endophyte transcriptome shift began early in host floral development. We discuss possible roles of the endophyte DEGs in colonization of reproductive grass tissues.

Molecular identification and characterization of endophytes from uncultivated barley.

Epichloë species (Clavicipitaceae, Ascomycota) are endophytic symbionts of many cool-season grasses. Many interactions between Epichloë and their host grasses contribute to plant growth promotion, protection from many pathogens and insect pests, and tolerance to drought stress. Resistance to insect herbivores by endophytes associated with Hordeum species has been previously shown to vary depending on the endophyte-grass-insect combination. We explored the genetic and chemotypic diversity of endophytes present in wild Hordeum species. We analyzed seeds of Hordeum bogdanii, H. brevisubulatum, and H. comosum obtained from the US Department of Agriculture's (USDA) National Plant Germplasm System (NPGS), of which some have been reported as endophyte-infected. Using polymerase chain reaction (PCR) with primers specific to Epichloë species, we were able to identify endophytes in seeds from 17 of the 56 Plant Introduction (PI) lines, of which only 9 lines yielded viable seed. Phylogenetic analyses of housekeeping, alkaloid biosynthesis, and mating type genes suggest that the endophytes of the infected PI lines separate into five taxa: Epichloë bromicola, Epichloë tembladerae, and three unnamed interspecific hybrid species. One PI line contained an endophyte that is considered a new taxonomic group, Epichloë sp. HboTG-3 (H. bogdanii Taxonomic Group 3). Phylogenetic analyses of the interspecific hybrid endophytes from H. bogdanii and H. brevisubulatum indicate that these taxa all have an E. bromicola allele but the second allele varies. We verified in planta alkaloid production from the five genotypes yielding viable seed. Morphological characteristics of the isolates from the viable Hordeum species were analyzed for their features in culture and in planta. In the latter, we observed epiphyllous growth and in some cases sporulation on leaves of infected plants.

Epichloë festucae endophytic growth in florets, seeds, and seedlings of perennial ryegrass (Lolium perenne).

Many symbiotic Epichloë species are seed-transmitted in their grass hosts. For a detailed investigation of Epichloë festucae colonization throughout the life cycle of its host, the authors transformed strain Fl1 with a fungal-active gene for enhanced cyan-fluorescent protein (eCFP), introduced it into perennial ryegrass (Lolium perenne), and used confocal microscopy to track its growth in the shoot apex, floral primordium, floral organs, seeds, and seedlings. Hyphae intercellularly colonized leaf sheaths, blades, true stems, and leaf primordia, and among floral primordia the endophyte exhibited different levels of colonization. In preanthesis florets, E. festucae colonized the pistil and stamen, but not pollen grains, and ramified throughout the ovule nucellus, but not the integument or embryo sac. Generally, only a single hypha was observed extended from the ovary placenta into the ovule. Within 4 d after anthesis, fungal hyphae had ramified throughout the developing seed and embryo. As the embryo matured, fungal hyphae became abundant between the testa and aleurone layer, and around the shoot apex and radical of the embryonic axis. During germination, hyphae accumulated in the mesocotyl and invaded the newly formed shoot apex near the meristem. In this host-fungus symbiosis, transmission to seedlings averaged 41% in 2010 and 76% in 2011. Each year, the frequency of ovary infection was similar to the frequency of infecting embryos and seedlings, indicating that colonization of the ovary and embryo was required for seed transmission.

Transcriptome response of Lolium arundinaceum to its fungal endophyte Epichloë coenophiala.

Tall fescue (Lolium arundinaceum) is one of the primary forage and turf grasses in temperate regions of the world. A number of favourable characteristics of tall fescue are enhanced by its seed-transmissible fungal symbiont (endophyte) Epichloë coenophiala. Our approach was to assemble the tall fescue transcriptome, then identify differentially expressed genes (DEGs) for endophyte-symbiotic (E+) vs endophyte-free (E-) clones in leaf blades, pseudostems, crowns and roots. RNA-seq reads were used to construct a tall fescue reference transcriptome and compare gene expression profiles. Over all tissues examined, 478 DEGs were identified between the E+ and E- clones for at least one tissue (more than two-fold; P < 0.0001, 238 E+ > E- and 240 E- > E+), although no genes were differentially expressed in all four tissues. Gene ontology (GO) terms, GO:0010200 (response to chitin), GO:0002679 (respiratory burst during defence response) and GO:0035556 (intracellular signal transduction) were significantly overrepresented among 25 E- > E+ DEGs in leaf blade, and a number of other DEGs were associated with defence and abiotic response. In particular, endophyte effects on various WRKY transcription factors may have implications for symbiotic stability, endophyte distribution in the plant, or defence against pathogens.

Enzymes from fungal and plant origin required for chemical diversification of insecticidal loline alkaloids in grass-Epichloë symbiota.

The lolines are a class of bioprotective alkaloids that are produced by Epichloë species, fungal endophytes of grasses. These alkaloids are saturated 1-aminopyrrolizidines with a C2 to C7 ether bridge, and are structurally differentiated by the various modifications of the 1-amino group: -NH2 (norloline), -NHCH3 (loline), -N(CH3)2 (N-methylloline), -N(CH3)Ac (N-acetylloline), -NHAc (N-acetylnorloline), and -N(CH3)CHO (N-formylloline). Other than the LolP cytochrome P450, which is required for conversion of N-methylloline to N-formylloline, the enzymatic steps for loline diversification have not yet been established. Through isotopic labeling, we determined that N-acetylnorloline is the first fully cyclized loline alkaloid, implying that deacetylation, methylation, and acetylation steps are all involved in loline alkaloid diversification. Two genes of the loline alkaloid biosynthesis (LOL) gene cluster, lolN and lolM, were predicted to encode an N-acetamidase (deacetylase) and a methyltransferase, respectively. A knockout strain lacking both lolN and lolM stopped the biosynthesis at N-acetylnorloline, and complementation with the two wild-type genes restored production of N-formylloline and N-acetylloline. These results indicated that lolN and lolM are required in the steps from N-acetylnorloline to other lolines. The function of LolM as an N-methyltransferase was confirmed by its heterologous expression in yeast resulting in conversion of norloline to loline, and of loline to N-methylloline. One of the more abundant lolines, N-acetylloline, was observed in some but not all plants with symbiotic Epichloë siegelii, and when provided with exogenous loline, asymbiotic meadow fescue (Lolium pratense) plants produced N-acetylloline, suggesting that a plant acetyltransferase catalyzes N-acetylloline formation. We conclude that although most loline alkaloid biosynthesis reactions are catalyzed by fungal enzymes, both fungal and plant enzymes are responsible for the chemical diversification steps in symbio.

Ether bridge formation in loline alkaloid biosynthesis.

Lolines are potent insecticidal agents produced by endophytic fungi of cool-season grasses. These alkaloids are composed of a pyrrolizidine ring system and an uncommon ether bridge linking carbons 2 and 7. Previous results indicated that 1-aminopyrrolizidine was a pathway intermediate. We used RNA interference to knock down expression of lolO, resulting in the accumulation of an alkaloid identified as exo-1-acetamidopyrrolizidine based on high-resolution MS and NMR. Genomes of endophytes differing in alkaloid profiles were sequenced, revealing that those with mutated lolO accumulated exo-1-acetamidopyrrolizidine but no lolines. Heterologous expression of wild-type lolO complemented a lolO mutant, resulting in the production of N-acetylnorloline. These results indicated that the non-heme iron oxygenase, LolO, is required for ether bridge formation, probably through oxidation of exo-1-acetamidopyrrolizidine.

Tall fescue endophyte effects on tolerance to water-deficit stress.

BACKGROUND: The endophytic fungus, Neotyphodium coenophialum, can enhance drought tolerance of its host grass, tall fescue. To investigate endophyte effects on plant responses to acute water deficit stress, we did comprehensive profiling of plant metabolite levels in both shoot and root tissues of genetically identical clone pairs of tall fescue with endophyte (E+) and without endophyte (E-) in response to direct water deficit stress. The E- clones were generated by treating E+ plants with fungicide and selectively propagating single tillers. In time course studies on the E+ and E- clones, water was withheld from 0 to 5 days, during which levels of free sugars, sugar alcohols, and amino acids were determined, as were levels of some major fungal metabolites. RESULTS: After 2-3 days of withholding water, survival and tillering of re-watered plants was significantly greater for E+ than E- clones. Within two to three days of withholding water, significant endophyte effects on metabolites manifested as higher levels of free glucose, fructose, trehalose, sugar alcohols, proline and glutamic acid in shoots and roots. The fungal metabolites, mannitol and loline alkaloids, also significantly increased with water deficit. CONCLUSIONS: Our results suggest that symbiotic N. coenophialum aids in survival and recovery of tall fescue plants from water deficit, and acts in part by inducing rapid accumulation of these compatible solutes soon after imposition of stress.

The epichloae: alkaloid diversity and roles in symbiosis with grasses.

Epichloae (Epichloë and Neotyphodium species; Clavicipitaceae) are fungi that live in systemic symbioses with cool-season grasses, and many produce alkaloids that are deterrent or toxic to herbivores. The epichloae colonize much of the aerial plant tissues, and most benignly colonize host seeds to transmit vertically. Of their four chemical classes of alkaloids, the ergot alkaloids and indole-diterpenes are active against mammals and insects, whereas peramine and lolines specifically affect insects. Comparative genomic analysis of Clavicipitaceae reveals a distinctive feature of the epichloae, namely, large repeat blocks in their alkaloid biosynthesis gene loci. Such repeat blocks can facilitate gene losses, mutations, and duplications, thus enhancing diversity of alkaloid structures within each class. We suggest that alkaloid diversification is selected especially in the vertically transmissible epichloae.

Currencies of mutualisms: sources of alkaloid genes in vertically transmitted epichloae.

The epichloae (Epichloë and Neotyphodium species), a monophyletic group of fungi in the family Clavicipitaceae, are systemic symbionts of cool-season grasses (Poaceae subfamily Poöideae). Most epichloae are vertically transmitted in seeds (endophytes), and most produce alkaloids that attack nervous systems of potential herbivores. These protective metabolites include ergot alkaloids and indole-diterpenes (tremorgens), which are active in vertebrate systems, and lolines and peramine, which are more specific against invertebrates. Several Epichloë species have been described which are sexual and capable of horizontal transmission, and most are vertically transmissible also. Asexual epichloae are mainly or exclusively vertically transmitted, and many are interspecific hybrids with genomic contributions from two or three ancestral Epichloë species. Here we employ genome-scale analyses to investigate the origins of biosynthesis gene clusters for ergot alkaloids (EAS), indole-diterpenes (IDT), and lolines (LOL) in 12 hybrid species. In each hybrid, the alkaloid-gene and housekeeping-gene relationships were congruent. Interestingly, hybrids frequently had alkaloid clusters that were rare in their sexual ancestors. Also, in those hybrids that had multiple EAS, IDT or LOL clusters, one cluster lacked some genes, usually for late pathway steps. Possible implications of these findings for the alkaloid profiles and endophyte ecology are discussed.

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.

Are loline alkaloid levels regulated in grass endophytes by gene expression or substrate availability?

Many cool-season grasses (Poaceae, subfam. Pooideae) possess seedborne fungal symbionts, the epichloae, known for their bioprotective properties, and especially for production of anti-insect alkaloids such as lolines. Asexual epichloae (Neotyphodium species) are primarily or entirely transmitted vertically, whereas the sexual structures (stromata) of the related Epichloë species give rise to horizontally transmissible spores (ascospores). In certain grass-Neotyphodium species symbiota, levels of lolines are extremely high and apparently limited by availability of precursor amino acids, whereas sexual epichloae generally produce much lower levels. This may reflect the inherent conflict between the vertical and horizontal transmission; although the plant and seeds may be protected by the alkaloids, the sexual cycle depends on anthomyiid flies for cross-fertilization. Given this insect role, we predicted that loline biosynthesis would be down-regulated in the stromata relative to the corresponding asymptomatic tissues (inflorescences) of the same symbiota. This prediction was substantiated, and RNA-seq and RT-qPCR analysis indicated that the loline biosynthesis genes are dramatically upregulated in asymptomatic inflorescences compared to stromata. The fundamental difference between asexual and sexual epichloae in regulation of loline alkaloid levels is in keeping with evolutionary trends for greater host control on metabolism of their vertically transmitted symbionts compared to contagious symbionts.

Chemotaxis disruption in Pratylenchus scribneri by tall fescue root extracts and alkaloids.

Tall fescue (Festuca arundinacea) forms a symbiotic relationship with the clavicipitalean fungal endophyte Neotyphodium coenophialum. Endophyte-infected grass is tolerant to nematode, but the factors responsible are unknown. One objective of this work was to determine if root extracts of tall fescue effected chemoreceptor activity of Pratylenchus scribneri by using an in vitro chemoreception bioassay. Another objective was to determine if specific ergot alkaloids (ergovaline, ergotamine, a-ergocryptine, ergonovine), and loline alkaloids, all produced by the fungal endophyte, altered chemotaxis with this bioassay. Methanolic extract from roots altered chemotaxis activities in this nematode but only from roots of plants cultured 45 > or = d, which repelled nematodes. Extracts prepared from noninfected grasses were attractants. This assay indicated that the alkaloids were either repellents or attractants. N-formylloline was an attractant at concentrations of 20 microg/ml and lower, while at higher concentrations it was a repellent. Ergovaline, the major ergot alkaloid produced by the endophyte, was repellent at both high and low concentrations and caused complete death of the nematodes.

Regulation of a chemical defense against herbivory produced by symbiotic fungi in grass plants.

Neotyphodium uncinatum and Neotyphodium siegelii are fungal symbionts (endophytes) of meadow fescue (MF; Lolium pratense), which they protect from insects by producing loline alkaloids. High levels of lolines are produced following insect damage or mock herbivory (clipping). Although loline alkaloid levels were greatly elevated in regrowth after clipping, loline-alkaloid biosynthesis (LOL) gene expression in regrowth and basal tissues was similar to unclipped controls. The dramatic increase of lolines in regrowth reflected the much higher concentrations in young (center) versus older (outer) leaf blades, so LOL gene expression was compared in these tissues. In MF-N. siegelii, LOL gene expression was similar in younger and older leaf blades, whereas expression of N. uncinatum LOL genes and some associated biosynthesis genes was higher in younger than older leaf blades. Because lolines are derived from amino acids that are mobilized to new growth, we tested the amino acid levels in center and outer leaf blades. Younger leaf blades of aposymbiotic plants (no endophyte present) had significantly higher levels of asparagine and sometimes glutamine compared to older leaf blades. The amino acid levels were much lower in MF-N. siegelii and MF-N. uncinatum compared to aposymbiotic plants and MF with Epichloë festucae (a closely related symbiont), which lacked lolines. We conclude that loline alkaloid production in young tissue depleted these amino acid pools and was apparently regulated by availability of the amino acid substrates. As a result, lolines maximally protect young host tissues in a fashion similar to endogenous plant metabolites that conform to optimal defense theory.

Loline alkaloids: Currencies of mutualism.

Several species of Lolium and other cool-season grasses (Poaceae subfamily Pooideae) tend to harbor symbiotic, seed-transmitted, fungi that enhance their fitness by various means. These fungal endophytes--species of Neotyphodium or Epichloë (Clavicipitaceae)--are known for production of antiherbivore metabolites such as the bioprotective loline alkaloids. Lolines are saturated pyrrolizidines with an exo-1-amine and an ether bridge between C-2 and C-7. The ether bridge is an unusual feature for a biogenic compound in that it links two bridgehead carbon atoms. Much of the loline-biosynthetic pathway has been elucidated by administering isotopically labeled precursors to fungal cultures and by comparisons of loline biosynthesis genes to known gene families. The first step appears to be an unusual gamma-substitution reaction involving an enzyme related to O-acetylhomoserine (thiol) lyase, but which uses the secondary amine of L-proline rather than a sulfhydryl group as the nucleophile. The strained ether bridge is added after formation of the pyrrolizidine rings. Lolines with dimethylated or acylated 1-amines have insect antifeedant and insecticidal activities comparable to nicotine, but little or no toxicity to mammals. Considering the surprising abundance of lolines in some grass-endophyte symbiota, possible additional effects on plant stress tolerance and physiology are worth future consideration. In this review, we discuss the history of loline discovery, methods of analysis, biological activities and distribution in nature, as well as progress on the genetics and biochemistry of their biosynthesis, and on the chemical synthesis of these alkaloids.