<p>Lichen-forming fungi employ a successful mode of nutrition as symbiotic partners with green algae and/or cyanobacteria (the photobiont). Nearly one fifth of all known fungi are obligate lichen formers, yet we know little of how they find compatible partners and establish long-lived symbiotic relationships. The combined growth of these symbionts forms a body (thallus) with emergent properties unlike either of the symbionts individually grown. Based on other well-studied eukaryotic systems, the development of a lichen thallus must rely upon the successful identification and collaboration of these two very different organisms. Identifying the molecular basis of microbe recognition and interactions remains one of the greatest challenges in studying symbiotic systems. </p><p>In this thesis, I determine the stage in which to begin looking for lichen symbiosis specific genes, and then examine mycobiont and photobiont genes that, when compared to the aposymbiotic state, are upregulated in the symbiotic state. Using the symbiosis between the mycobiont <italic>Cladonia grayi</italic> and the photobiont <italic>Asterochloris</italic> sp., as well as scanning electron microscopy observations of the earliest stages of contact between <italic>C. grayi</italic> and <italic>Asterochloris</italic> sp., I determined that the mycobiont undergoes a specific change in phenotypic growth in response to <italic>Asterochloris</italic> sp. This change is particular to the lichen symbiosis, and is not observed with algal shaped inanimate objects or algae other than <italic>Asterochloris</italic>. I then used this phenotypically defined stage that is exclusive to lichen symbiosis to begin studying the the genetic and molecular mechanisms underlying the development of a stratified lichen thallus. Using suppression subtractive hybridization to determine differential gene expression, fungal and algal libraries were made of upregulated genes in the first 2 stages of lichen symbiosis. The symbiotic expression levels of select genes were then verified using quantitative PCR. Lastly, a candidate gene for involvement in lichen symbiosis was transformed into <italic>Saccharomyces cerevisiae</italic> to test for protein function.</p><p>Further results of this study show that the fungal protein products of genes upregulated in lichen symbiosis show significant matches to proteins putatively involved in fungal self and non-self recognition, lipid metabolism, negative regulation of glucose repressible genes, an oxidoreductase, a dioxygenase, and a conserved hypothetical protein. Algal genes that are upregulated in lichen symbiosis include a chitinase-like protein, an amino acid metabolism protein, a dynein related protein, and a protein arginine methyltransferase. Furthermore, genes that are expressed in the early stages of lichen symbiosis are common varying metabolic pathways. Furthermore stages 1 and 2 of development are marked not by a drastic change in transcriptional products, but instead by an overall change in genes that are already expressed. Finally, the <italic>Cladonia~grayi Lip3</italic>was cloned in its entirety from genomic DNA and cDNA, was predicted to be secreted using signal peptide prediction software, and shown to be a functioning secreted extracellular lipase in yeast.</p><p>I conclude that many genes are involved in the interactions of symbionts and the development of a stratified lichen thallus, and that many more genes remain to be discovered. Furthermore, the possibility that genes exist in either symbiont that are specific to lichen symbiosis remains, and that their discovery awaits the creation of better genomic tools for \textit{Cladonia~grayi} and <italic>Asterochloris</italic>.</p> / Dissertation
| Identifer | oai:union.ndltd.org:DUKE/oai:dukespace.lib.duke.edu:10161/1052 |
| Date | January 2009 |
| Creators | Joneson, Suzanne |
| Contributors | Lutzoni, François M. |
| Source Sets | Duke University |
| Language | en_US |
| Detected Language | English |
| Type | Dissertation |
| Format | 3043183 bytes, application/pdf |
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