Loline alkaloids (LA) are secondary metabolites produced by Epichloandamp;euml; (anamorph, Neotyphodium) grass endophytes. They are toxic and deterrent to a broad range of herbivorous insects but not to livestock. This protective bioactivity has spurred considerable research into the LA biosynthetic pathway. LOL, the gene cluster containing nine genes, is required for LA biosynthesis. The regulation of LOL genes during LA production in culture and in symbio is of interest. In this study, coordinate regulation between LOL gene expression and LA production level was investigated in both MM culture and symbiota. Results showed that expression of LOL genes in N. uncinatum MM culture were tightly correlated with each other (p andamp;lt; 0.0005), and all presented a significant temporal quadratic pattern during LA production. Gene expression started before LA were detectable, and increased while LA accumulated. The highest gene expression level was reached before the highest amounts of LA were detected, and gene expression level declined to a very low level after amounts of LA plateaued. Observations suggested that the hierarchical clusters based on the correlation coefficient could help to predict the roles of LOL genes in the LA pathway. In symbiota, coordinate coregulation of LOL gene expression with LA was found in E. festucae-meadow fescue inflorescences and stromata, whereby lower LOL gene expression corresponded with the lower LA level in stromata. In N. uncinatum (or N. siegelii)-meadow fescue vegetative tissues, dramatically higher LA levels were found in younger leaf tissue than in older leaf tissue, yet no evidence was found to relate this difference to LOL gene expression differences. Instead, substrate availability may regulate the LA level. In particular, asparagine was more than 10-fold higher in young leaf tissue than in old tissue, although proline was significantly lower in young tissue. Therefore, different regulatory mechanisms underlie LOL gene expression and LA production in different circumstances. The GUS activity of Pro-lolC2-GUS and Pro-lolA2-GUS in Neotyphodium species was almost undetectable in culture, though the activity could be detected in symbiota. The mRNA of GUS did not exhibit the same pattern as lolC2 or lolA2 in culture during LA production time course. A Pro-lolC2-cre transgene was expressed in complex medium, in which lolC2 mRNA was not detectable. These results suggest that proper regulation of LOL genes in culture or symbiota is dependent on the LOL cluster.

Mutualistic relationships between endophytic fungi and grasses have shown to improve the hardiness of the host. This relationship is common in grasses, including North American native grasses that are important in both forage and grassland ecosystems. Elymus species, such as Canada wildrye (CWR), commonly host the endophytic fungi, Epichloё, while southeastern wildrye (SEWR) may not. In this study, seed of CWR and seed and leaves from local SEWR were assessed for endophyte infection. Infection status was confirmed via histological analysis of seed squashes and leaf peels. Presence/absence of endophyte was determined by scanning for mycelia within seed and between leaf epidermal cells. Following assessment, all SEWR germplasm were endophyte-free (E-), while CWR was endophyte-infected (E+). Endophyte- infected CWR seed were used to isolate and culture the symbiotic endophyte. Isolated endophyte was used to infect E- SEWR. Infection status of SEWR was determined using leaf peels and PCR. This research will help determine if artificial endophyte infection can be performed in SEWR.

Clavicipitaceous fungal endophytes of the genera Epichloë and Neotyphodium form symbioses with grasses of the family Pooideae in which they can synthesise an array of bioprotective alkaloids. Some strains produce the ergot alkaloid ergovaline, which is implicated in livestock toxicoses caused by ingestion of endophyteinfected grasses. Cloning and analysis of a plant-induced non-ribosomal peptide synthetase (NRPS) gene from Neotyphodium lolii and analysis of the E. festucae E2368 genome sequence revealed a complex gene cluster for ergot alkaloid biosynthesis. The EAS cluster contained a single-module NRPS gene, lpsB, and other genes orthologous to genes in the ergopeptine gene cluster of Claviceps purpurea and the clavine cluster of Aspergillus fumigatus. Functional analysis of lpsB confirmed its role in ergovaline synthesis and bioassays with the lpsB mutant unexpectedly suggested that ergovaline was not required for black beetle (Heteronychus arator) feeding deterrence from epichloë-infected grasses. Southern analysis showed the cluster was linked with previously identified ergot alkaloid biosynthetic genes, dmaW and lpsA, at a subtelomeric location. The ergovaline genes are closely associated with transposon relics, including retrotransposons, autonomous DNA transposons and miniature inverted-repeat transposable elements (MITEs), which are very rare in other fungi. All genes in the cluster were highly expressed in planta but expression was very low or undetectable in mycelia from axenic culture, including under nitrogen-, carbonor phosphate-limited conditions. Comparative analysis of the EAS gene cluster in four different epichloë strains showed marked differences in gene expression and ergot alkaloid synthesis. Gene order is conserved in each strain although evidence for recombination between two MITEs and expansion or reduction of a simple sequence repeat (SSR) at a single intergenic region was observed. Heterologous expression of a candidate regulatory gene, laeA, from Aspergillus nidulans, which is a global regulator of secondary metabolism in aspergilli, did not affect eas gene expression. This, along with phylogeny and microsynteny analysis, suggests there is not an orthologue of this gene in epichloë. This work provides a genetic foundation for elucidating biochemical steps in the ergovaline pathway, the ecological role of individual ergot alkaloid compounds, and the regulation of their synthesis in planta.

LOL is a novel secondary metabolite gene cluster associated with the production of loline alkaloids (saturated 1-aminopyrrolizidine alkaloids with an oxygen bridge) exclusively in closely related grass-endophyte species in the genera Epichloë and Neotyphodium. In this study I characterize the LOL cluster in E. festucae, including the presentation of sequence corresponding to 10 individual lol genes as well as defining the boundaries of the cluster and evaluation of the genomic DNA region flanking LOL in E. festucae. In addition to characterizing the LOL cluster in E. festucae, I present LOL sequence from two additional species, Neotyphodium coenophialum and Neotyphodium sp. PauTG-1. Together with two recently published LOL clusters from N. uncinatum, these data allow for a powerful phylogenetic comparison of five clusters from four closely related species. There is a high degree of microsynteny (conserved gene order and orientation) among the five LOL clusters, allowing us to predict potential transcriptional co-regulatory binding motifs in lol promoter regions. The relatedness of LOL clusters is especially interesting in light of the history of interspecific hybridizations that generated the asexual, Neotyphodium lineages. In fact, three of the clusters appear to have been introduced to different Neotyphodium species by the same ancestral Epichloë species, for which present day isolates are no longer able to produce lolines. To address the evolutionary origins of the cluster we have investigated the phylogenetic relationships of particular lol ORFs to their paralogous primary metabolism genes (and gene families) from endophytes, other fungi and even other kingdoms. I present extensive evidence that at least two individual lol genes have evolved from primary metabolism genes within the fungal ancestors of endophytes, rather than being introduced via horizontal gene transfer. I also present complementation studies in Neurospora crassa exploring the functional divergence of one lol gene from its primary metabolism paralog. While it is clear that these insecticidal compounds should convey a selective advantage to the fungus and its host, thus explaining preservation of the trait, this analysis provides an exploration into the evolutionary origin and maintenance of the genes that comprise the LOL and the cluster itself.

LOL is a novel secondary metabolite gene cluster associated with the production of loline alkaloids (saturated 1-aminopyrrolizidine alkaloids with an oxygen bridge) exclusively in closely related grass-endophyte species in the genera Epichloë and Neotyphodium. In this study I characterize the LOL cluster in E. festucae, including the presentation of sequence corresponding to 10 individual lol genes as well as defining the boundaries of the cluster and evaluation of the genomic DNA region flanking LOL in E. festucae. In addition to characterizing the LOL cluster in E. festucae, I present LOL sequence from two additional species, Neotyphodium coenophialum and Neotyphodium sp. PauTG-1. Together with two recently published LOL clusters from N. uncinatum, these data allow for a powerful phylogenetic comparison of five clusters from four closely related species. There is a high degree of microsynteny (conserved gene order and orientation) among the five LOL clusters, allowing us to predict potential transcriptional co-regulatory binding motifs in lol promoter regions. The relatedness of LOL clusters is especially interesting in light of the history of interspecific hybridizations that generated the asexual, Neotyphodium lineages. In fact, three of the clusters appear to have been introduced to different Neotyphodium species by the same ancestral Epichloë species, for which present day isolates are no longer able to produce lolines. To address the evolutionary origins of the cluster we have investigated the phylogenetic relationships of particular lol ORFs to their paralogous primary metabolism genes (and gene families) from endophytes, other fungi and even other kingdoms. I present extensive evidence that at least two individual lol genes have evolved from primary metabolism genes within the fungal ancestors of endophytes, rather than being introduced via horizontal gene transfer. I also present complementation studies in Neurospora crassa exploring the functional divergence of one lol gene from its primary metabolism paralog. While it is clear that these insecticidal compounds should convey a selective advantage to the fungus and its host, thus explaining preservation of the trait, this analysis provides an exploration into the evolutionary origin and maintenance of the genes that comprise the LOL and the cluster itself.

Loline alkaloids, found in many grass-Epichloë symbiota, are toxic or feeding deterrent to invertebrates. The loline alkaloids all share a saturated pyrrolizidine ring with a 1-amine group and an ether bridge linking C2 and C7. The steps in biosynthesis of loline alkaloids are catalyzed by enzymes encoded by a gene cluster, designated LOL, in the Epichloë genome. This dissertation addresses the enzymatic, genetic and evolutionary basis for diversification of these alkaloids, focusing on ether bridge formation and the subsequent modifications of the 1-amine to form different loline alkaloids. Through gene complementation of a natural lolO mutant and comparison of LOL clusters in strains with different loline alkaloid profiles, I found that lolO, predicted to encode a 2-oxoglutarate-dependent nonheme iron (2OG/Fe) dioxygenase, is required in formation of the ether bridge. Through application of isotopically labeled compound to Epichloë uncinata culture, I established that exo-1-acetamidopyrrolizidine (AcAP) and N-acetylnorloline (NANL) are true pathway intermediates. Application of AcAP to yeast expressing lolO resulted in production of NANL, establishing that LolO is sufficient to catalyze this unusual oxygenation reaction. After ether formation, modifications on the 1-amino group give loline, N-methylloline (NML), N-formylloline (NFL) and N-acetylloline (NAL). A double knockout of lolN, predicted to encode an acetamidase, and lolM, predicted to encode a methyltransferase, produced only NANL. Complementation of the double knockout with wild-type lolN and lolM restored the loline alkaloid profile. These results indicate that LolN is involved in deacetylating NANL to produce norloline, which is then modified to form the other lolines. Crude protein extract of a yeast transformant expressing LolM converted norloline to loline and NML, and loline to NML, supporting the hypothesis that LolM functions as a methyltransferase in the loline-alkaloid biosynthesis pathway. The alkaloid NAL was observed in some but not all plants symbiotic with Epichloë siegelii, and when provided with exogenous loline, asymbiotic meadow fescue (Lolium pratense) plants produced N-acetylloline (NAL), indicating that a plant acetyltransferase converts loline to NAL. I further analyzed the basis for loline alkaloid diversity by comparing the LOL clusters in the Epichloë and Atkinsonella species with different loline alkaloid profiles, and found that LOL clusters changed position, orientation and gene content over their evolutionary history. Frequent, independent losses of some or all late pathway genes, lolO, lolN, lolM and lolP, resulted in diverse loline alkaloid profiles. In addition, phylogenetic analyses demonstrated transspecies polymorphism of the LOL clusters. Based on my findings, I established that in Epichloë and Atkinsonella species the ether bridge is formed on acetamidopyrrolizidine. My study of the loline alkaloid profile of Adenocarpus decorticans (Fabaceae) suggests that these plants probably use a similar strategy at least with respect to ether-bridge formation. Further diversification steps of loline alkaloids in grass-Clavicipitaceae symbiota are carried out by enzymes of both Epichloë species and the host plant. Finally, I present evidence that LOL clusters have evolved by balancing selection for chemical diversity.