Graduate Theses, Dissertations, and Problem Reports 2017 Elucidating Function in Early Diverging Non-Flagellated Fungi Using Metabolomics and Proteomics Gregory R. Boyce Follow this and additional works at: https://researchrepository.wvu.edu/etd Recommended Citation Boyce, Gregory R., "Elucidating Function in Early Diverging Non-Flagellated Fungi Using Metabolomics and Proteomics" (2017). Graduate Theses, Dissertations, and Problem Reports. 5245. https://researchrepository.wvu.edu/etd/5245 This Thesis is protected by copyright and/or related rights. It has been brought to you by the The Research Repository @ WVU with permission from the rights-holder(s). You are free to use this Thesis in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you must obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Thesis has been accepted for inclusion in WVU Graduate Theses, Dissertations, and Problem Reports collection by an authorized administrator of The Research Repository @ WVU. For more information, please contact [email protected]. Elucidating function in early diverging non-flagellated fungi using metabolomics and proteomics Gregory R. Boyce Dissertation submitted to the Davis College of Agriculture, Natural Resources and Design at West Virginia University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Genetics and Developmental Biology Matthew T. Kasson, Ph.D., Co-Chair ` Joseph Morton, Ph.D., Co-Chair Daniel Panaccione, Ph.D. Teiya Kijimoto, Ph.D. Greg Kilby, Ph.D. Division of Plant and Soil Sciences Morgantown, West Virginia 2017 Keywords: Metabolomics, Mucoromycota, Zoopagomycota Copyright 2017 Gregory R. Boyce Abstract Exploring Metabolic Capacity in Two Distinct Early Diverging Fungal Symbioses Gregory Boyce 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. iii Table of Contents Abstract ......................................................................................................................................................... ii Table of Contents ......................................................................................................................................... iii Chapter 1 Introduction .............................................................................................................................. 1 Chapter 2 Rhizophagus dimorphicus sp. nov., a dimorphic species in Glomeraceae (Glomeromycota), produces glomoid spores with two contrasting phenotypes and a partitioning of divergent metabolisms 7 Abstract ..................................................................................................................................................... 7 Introduction .............................................................................................................................................. 8 Materials and Methods ............................................................................................................................. 9 Morphological analysis ....................................................................................................................... 10 DNA extraction, amplification and sequencing .................................................................................. 11 Phylogenetic analyses ......................................................................................................................... 12 Global proteome analyses .................................................................................................................. 12 Global metabolome analyses .............................................................................................................. 14 Taxonomy ................................................................................................................................................ 15 Dark synanomorph .............................................................................................................................. 16 White synanomorph ........................................................................................................................... 17 Global Proteomics Results .................................................................................................................. 18 Global Metabolomics Results ............................................................................................................. 19 Discussion................................................................................................................................................ 20 Chapter 3 Applied metabolomics reveals host and environmental influence on metabolism in Rhizophagus clarus ................................................................................................................................. 30 Abstract ................................................................................................................................................... 30 Introduction ............................................................................................................................................ 31 Materials and Methods ........................................................................................................................... 33 Experimental Material ........................................................................................................................ 33 Experimental Design ........................................................................................................................... 33 iv Spore metabolite extraction ............................................................................................................... 34 Normalization of samples by spore protein content .......................................................................... 35 Analytical procedure and LC-MS Conditions ....................................................................................... 35 Data Processing and Metabolite identification