Genome defense likely evolved to prevent the spread of transposable elements and invading viruses. Effective genome defense mechanisms have limited colonization of the Neurospora crassa genome by transposable elements. A novel DNA transposon named Sly1-1 was discovered in the genome of the most widely used lab "wildtype" strain FGSC 2489 (OR74A). Meiotic silencing by unpaired DNA (MSUD), also simply called "meiotic silencing", prevents the expression of regions of the genome that are unpaired during karyogamy. This mechanism is posttranscriptional and involves the production of small RNA, so called "masiRNAs" by proteins homologous to those involved in RNAi silencing pathways in animals, fungi and plants. Here, I demonstrate production of small RNAs when Sly1-1 was unpaired in a cross between two wild type strains. These small RNAs are dependent on SAD-1, an RNA-dependent RNA polymerase necessary for meiotic silencing. I present the first case of endogenously produced masiRNA from a novel N. crassa DNA transposable element during the sexual development. By comparing genome sequences of two mating partners, I also identified (1) unpaired regions undergoing meiotic silencing, some of which contained the repetitive sequences and transposon relics. Small RNA produced from such regions shared the same features as masiRNAs; (2) unpaired regions immune to meiotic silencing with genes contained protected from meiotic silencing via abolishing small RNA production; (3) common regions shared by two mating patterns but with small RNAs production during karyogamy. The dissection of these regions revealed the role of meiotic silencing on resisting repetitive sequences and on compromising the unpaired genes essential in sexual development.

Species belonging to the basal fungal lineages (Blastocladiomycota, Chytridiomycota, Cryptomycota, and Neocallimastigomycota) reproduce via motile zoospores and are found in both aquatic and terrestrial environments. These organisms, colloquially referred to as "chytrids", are traditionally understudied, despite being active decomposers, parasites, a nd symbionts with other organisms in the ecosystem. This dissertation research uses a comparative genomics approach to answer questions about these fungi and their interactions with their environment and other fungi. Chapters 2 and 3 examine the details of putative rhodopsin-mediated photoreception in basal lineages using structural mechanics and genome-wide gain-loss analyses. Chapter 4 provides a transcriptome analysis of one member of the genus Coelomomyces, the only known entomopathic chytrid genus. Chapter 5 describes surprising observations regarding competitive and inhibitory behavior in one member of the Chytridiomycota. Finally, Appendix A briefly looks at gain-loss analysis of molecular aspects of the evolutionary transition from aquatic motile single cells to terrestrial multicellullar organisms.

Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis responsible for worldwide decline in amphibian populations. Previous analysis of the Bd genome revealed a unique expansion of the carbohydrate-binding module family 18 (CBM18) predicted to be a sub-class of chitin recognition domains. CBM expansions have been linked to the evolution of pathogenicity in a variety of fungal species by protecting the fungus from the host. Based on phylogenetic analysis and presence of additional protein domains, the gene family can be classified into 3 classes: Tyrosinase-, Deacetylase-, and Lectin-like. Examination of the mRNA expression levels from sporangia and zoospores of nine of the cbm18 genes found that the Lectin-like genes had the highest expression while the Tyrosinase-like genes showed little expression, especially in zoospores. Heterologous expression of GFP-tagged copies of four CBM18 genes in Saccharomyces cerevisiae demonstrated that two copies containing secretion signal peptides are trafficked to the cell boundary. The Lectin-like genes cbm18-ll1 and cbm18-ll2 co-localized with the chitinous cell boundaries visualized by staining with calcofluor white. In vitro assays of the full length and single domain copies from CBM18-LL1 demonstrated chitin binding and no binding to cellulose or xylan. Expressed CBM18 domain proteins were demonstrated to protect the fungus, Trichoderma reeseii, in vitro against hydrolysis from exogenously added chitinase, likely by binding and limiting exposure of fungal chitin. These results demonstrate that cbm18 genes can play a role in fungal defense and expansion of their copy number may be an important pathogenicity factor of this emerging infectious disease of amphibians.

Understanding the various processes/pathways necessary for the biogenesis and maintenance of the cell wall is of immense value as that knowledge can be used for developing antifungals. This dissertation attempts to make a significant contribution in furthering our understanding of the fungal cell wall and its various components. I have identified and characterized genes involved in cell wall growth and maintenance in the model fungus Neurospora crassa by employing a functional screen. I utilized comparative genomics approaches to reconstruct the evolutionary history of cell wall polysaccharides chitin and glucan in the early diverging fungal clades- Microsporidia, Cryptomycota, Chytridiomycota, Blastocladiomycota and Mucormycota. Using a combination of biochemical and comparative genomics I attempted to compare and contrast the cell wall composition of the early diverging fungal clades Chytridiomycota, Blastocladiomycota and Mucormycota to the recently diverged Dikarya fungi. The research presented in this dissertation should lead to the characterization of novel cell wall genes in the filamentous fungi that eventually will lead to development of better drug targets for designing anti-fungal drugs. Also it will lead to better understanding of the cell walls of evolutionarily distinct fungi, which will enable us to combat the pathogenic fungi in a more powerful way. In terms of broader impact this research will be an important contribution to the knowledge of cell wall in the fungal community.

Micro-colonial fungi (MCF) are an extremotolerant group of polyphyletic fungi found in different extremes of this planet, from Southern Californian deserts to Antarctic tundras, and even in the lungs of cystic fibrosis patients. MCF are found in the Eurotiomycetes and Dothideomycetes classes, two lineages that diverged millions of years ago, yet MCF from both these clades have developed nearly identical features. The work described in this thesis encompasses a wide range of MCF, uncovering their unusual niches, and understanding their genomic structure. The first chapter describes three new species of MCF isolated from biological soil crusts collected from the deserts of southern California. Two MCF, respectfully, use traditional Cahuilla tribal terms for nomenclature. We introduce to you Neophaeococcomyces mojaviensis, Coniosporium tulheliwenetii, and Taxawa tesnikishii. The second chapter has us travel to Antarctica where 25 strains of an endemic MCF, Friedmanniomyces endolithicus, were collected for 40 years and analyzed for geologic spatial placement, ploidy, and mating types. Within our population of 25, we received an interesting mix of ploidies and it may be the way F. endolithicus survives the extremes of Antarctica. We were also able to place the time of F. endolithicus divergence from other Dothideomycetes to 48 million years ago, near to the time predicted when Antarctica split from Pangea and drifted to the region it is found in today. Our final chapter takes us into the lungs of a cystic fibrosis patient to find a slow-growing MCF, Exophiala dermatitidis, persisting for over three years. Twenty-four specimens of E. dermatitidis were isolated from this patient in the span of three years with countless antibiotics to clear other persistent infections. Population structure showed three different groups with varying mutational rates, phenotypic filamentation, and chromosomal structures as possible main culprits for persistence. Heterogenous phenotypes were observed in the three groups, and we could not make ties to melanin pigmentation and itraconazole antifungal therapy. Through this work, it has become increasingly clear there needs to be more effort, whether in the clinical cases or environmental, to consider the slow-growing MCFs as possible causative agents or pivotal functional characters in different extreme habitats.

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