Elucidating Function in Early Diverging Non-Flagellated Fungi Using Metabolomics and Proteomics

The obligate lifestyle of many of the early diverging non-flagellated fungi have long precluded them from functional studies reserved primarily for their easily cultured, more derived counterparts. Modern --omics--based tools including transciptomics, proteomics and metabolomics have facilitated a better understanding of many fungi but have yet to be employed in the study of early diverging non-flagellated fungi with few exceptions. Using global and targeted metabolomics and prototeomics across the symbiotic spectrum, a series of studies were undertaken to better understand metabolism of three symbiotic interactions: two AM fungus-plant associations including Rhizophagus dimorphicus -- grass, R. clarus -- legume, and one fungus-insect interaction, Massospora -- cicada. For Rhizophagus dimorphicus, a previously undescribed dimorphic AM fungus, a multi --omics approach was taken to better understand the metabolic differences underlying phenotypic differences. The results of the multi --omics approach revealed a truncated proteome and metabolome being expressed in the white morphotype when compared to the dark morphotype. These differences in the proteome and the metabolome of these synanamorphs suggest a potential compartmentalization of metabolism in R. dimorphicus. However further investigation is required to fully understand this phenomenon. To better understand the interactions among AMF fungi, plant host, and environment, an applied metabolomics approach was used to elucidate the impact of each variable individually and combined on the primary metabolism of Rhizophagus clarus, another well described AMF member. Two leguminous plant species, Robinia pseudoacia (black locust) and Trifolium pretense (red clover) were inoculated and maintained at two different pH (pH 4.5 and 7.5). Individual hosts and both soil pH yielded distinct metabolite profiles from fungal spores, with soil pH having a greater influence on overall metabolism. Pathway analysis showed significant changes in intermediates and end products of the glutamate catabolism pathway as a function of change in medium pH. The results of this study provide insight into how AMF fungi regulate the pH of their host tissues, the result of which may facilitate widespread colonization across suboptimal soil conditions. Finally, global and targeted metabolomics were used to better understand obligate entomopathogenic fungi, Massospora species, and compare two contemporary Massospora -- cicada consortia, M. cicadina-infected periodical cicadas (Magicicada spp.) and M. levispora comb nov.-infected banger-wing cicadas (Platypedia putnami) to better help elucidate factors influencing host colonization and putative behavioral modification. Results of the global metabolomics uncovered a diverse assortment of secondary metabolites including psilocybin from M. levispora comb. nov. and cathinone from M. cicadina. These psychotropic compounds may enhance aggressiveness/stamina of infected cicadas to ensure continued spore dispersal despite debilitating infections that likely result in fatigue, decreased muscle strength and general malaise. Such discoveries as presented here have only begun to scratch the surface of the vast metabolic capacity of these two groups of enigmatic fungi. Using a systems biology approach has been shown to be an effective strategy to investigate the complex functionality of these two symbiotic systems