Browsing by Author "Heitman, Joseph"

Invasive fungal infections are recognized as a global health threat with the World Health Organization listing 4 fungal species as critical pathogens for global study. With the rise of resistance and new pathogenic species continuing to evolve, there is an immediate need for antifungal drug development. Current antifungal therapies are antiquated, have numerous side effects, and consist of only four available classes. The development of novel drugs to treat fungal disease is hindered by the evolutionary conservation between fungi and mammals. With these considerations in mind, this thesis aims to investigate repurposing established therapeutics for use in treating invasive fungal infections.

Chapter 1 focuses on the global impact of fungal disease and highlights Cryptococcus neoformans as a primary pathogen to study. It begins by outlining the current state of fungal pathogenesis and the antifungal therapeutic armament. The development of antifungal agents will additionally be addressed by giving an overview of investigational drug candidates including FKBP12 ligands, Gwt1 inhibitors, and prenylation inhibitors.

In Chapter 2, prenylation inhibition is investigated as a drug development strategy. Novel synthesized compounds and the Food & Drug Administration (FDA) approved carcinoma product Tipifarnib are assessed for their in vitro antifungal activity through microdilution broth minimum inhibitory concentration (MIC) assays. The mechanism of action of Tipifarnib was visualized using fluorescence microscopy before resistance mechanisms were interrogated by isolating fungal colonies with reduced tipifarnib susceptibility and genetically assessed mutations responsible for resistance.

In Chapter 3, the anticancer and immunosuppressive drug rapamycin is investigated for application as an antifungal drug. Treatment with rapamycin mimics starvation and induces cell death through autophagy. Using antifungal susceptibility MIC assays, rapamycin antifungal efficacy is shown to be significantly impacted by the nutritional environment. Additionally, synthesized analogs of rapamycin are investigated for their potential to specifically inhibit the fungal target of FK506 binding protein (FKBP12). Analog specificity is measured by comparing in vitro antifungal activity to immunosuppressive activity. A lead fungal specific analog is tested during an in vivo infection model demonstrating no adverse effects or therapeutic benefit at the concentrations tested.

In Chapter 4, immunosuppressive therapeutic agent FK520 is structurally modified and assayed for fungal specificity. Structure-guided drug design is used to develop two libraries of FK520 analogs. Through in vitro assessment of their antifungal and immunosuppressive activities, the lead compound JH-FK-08 is identified and used as an antifungal therapy during an in vivo cryptococcosis model. JH-FK-08 is shown to significantly reduce fungal burden and extend survival in addition to demonstrating a similar pharmacokinetic profile to parent compound FK506.

In Chapter 5, combination therapies are investigated for FK506 and JH-FK-08. Checkerboard assays are applied to test the interaction of two drugs when inhibiting fungal growth in vitro. JH-FK-08 is shown to have synergistic activity with Gwt1 inhibitors against C. neoformans and Candida albicans. FK506 is shown to have synergistic interactions with rapamycin which is unexpected due to the shared requirement of binding chaperone protein FKBP12. An FKBP12 mutant is shown to have no defect during virulence and competition in vivo experiments. This indicates that an FKBP12 mutation could confer resistance to a combination of FK506 and rapamycin.

Chapter 6 summarizes the results presented throughout this dissertation. The conclusions, impact of findings, and future directions are further considered within the broader context of antifungal therapeutic approaches. In addition, the benefits of repurposing established medications for novel use are highlighted and discussed here. Targeting proteins found in both humans and fungi can be an effective approach especially when designing structurally specific fungal inhibitors. This thesis demonstrates the efficacy of this approach through the development of promising antifungal agent JH-FK-08 shown to significantly extend survival and reduce fungal burden in mice undergoing an invasive fungal infection.

Invasive fungal infections are a major burden on the global healthcare system and represent a significant threat to human health for future generations. The majority of invasive fungal infections in the world are caused by Cryptococcus, Candida, and Aspergillus species. With the changing climate, growing antifungal drug resistance, and an increasing immunocompromised patient population, the need for novel and effective antifungal treatments is higher than ever. Despite this clear unmet need, the FDA has approved no new antifungal drugs for the treatment of life-threatening systemic fungal infection in over 20 years. Moreover, only 4 classes of antifungal drugs are available to treat these invasive fungal infections and all suffer from issues associated with either toxicity, resistance, or bioavailability. Due to the high degree of conservation between the eukaryotic cell targets in fungi and mammals, fungal selectivity and low patient toxicity is a significant issue. In this dissertation, I have taken protein crystal structure-guided design and genetic target elucidation approaches to develop novel, fungal-specific antifungal compounds. I focus primarily on two targets: the serine-threonine specific protein phosphatase, calcineurin, and the putative membrane flippase, Apt1. In Chapter 1, I will detail the global threats faced by opportunistic fungal pathogens and emerging mechanisms of drug resistance. I then summarize the currently available treatment options for invasive mycoses and their associated weaknesses. I conclude by reviewing some of the novel antifungal compounds in development today and explain some of the methods utilized by researchers in modern drug development. In Chapter 2, I summarize our efforts to develop a fungal-specific calcineurin inhibitor called APX879. Both fungal and mammalian calcineurin are inhibited naturally by the small molecule FK506 when bound to its molecular chaperone, FKBP12. Here, I describe how we utilize recently solved protein crystal structures of the fungal calcineurin inhibitory complex (Calcineurin-FK506-FKBP12) from the major fungal pathogens to design an FK506 analog that is increased for its fungal specificity. We identified key structural differences in the 80s loop of FKBP12 that could be exploited by making structural modifications to the C22 of FK506. APX879 is a C22-modified FK506 analog that is reduced for immunosuppressive activity and toxicity as a result of decreased mammalian calcineurin inhibition. Additionally, we show that APX879 still maintains broad-spectrum antifungal activity and is efficacious in an animal model of Cryptococcus infection. In Chapter 3, I describe the development of a second-generation calcineurin inhibitor, JH-FK-05, that is further increased for its fungal selectivity. We began by utilizing protein crystal structures of Aspergillus FKBP12 bound to a natural FK506 analog, FK520 to design additional compounds to test for fungal specificity. These FK520 analogs were screened for a shift in balance of antifungal and immunosuppressive activity, and we found that C22-modified JH-FK-05 maintained broad-spectrum activity and was non-immunosuppressive in vivo. Moreover, when treated with JH-FK-05, mice in two different models of murine cryptococcosis exhibited a significant improvement in survival and tissue fungal burden. We then applied molecular dynamic simulations to JH-FK-05 bound to fungal vs mammalian FKBP12 to identify FK520 residues in the future that could be targeted to increase fungal specificity. In Chapter 4, I briefly illustrate the progress in developing additional FK506 and FK520 analogs. We have synthesized an additional 15 FK506 or FK520 analogs and screened them for their in vitro antifungal activity against Cryptococcus and their in vitro immunosuppressive activity in primary murine T cells. Several of these compounds presented a promising degree of fungal selectivity and have been selected for further study in animal models of fungal infection. In Chapter 5, I provide detailed methods for the elucidation of unknown antifungal targets. In many cases of drug development, a compound is isolated with promising activity before its molecular target is known. An understanding of the mechanism of action for any compound is an essential step before it can be utilized clinically to treat patients. Here, I describe step by step procedures for generating spontaneously resistant mutant strains, testing for antifungal susceptibility, and identification of the causative mutation conferring resistance through genetic crosses and whole-genome sequencing. In Chapter 6, I describe the mechanism of action for Butyrolactol A (ButA), a natural polyketide compound with potent antifungal activity against Cryptococcus. ButA had antifungal activity alone but also could potentiate the activity of caspofungin, which is an antifungal drug that Cryptococcus is naturally resistant to. Although the antifungal activity of ButA had previously been described, the mechanism of action remained unknown. We isolated two resistant mutants to ButA and identified mutations in the gene encoding lipid flippase APT1 by utilizing whole-genome sequencing. ButA had low toxicity in mice at doses as high as 50 mg/kg. However, despite robust in vitro antifungal activity, we detected no therapeutic efficacy of ButA treatment at multiple doses and in combination with caspofungin. Additional pharmacokinetics and pharmacodynamics may shed some light on the discrepancy between the in vitro and in vivo activity of ButA. In Chapter 7, I will summarize the findings presented in this dissertation and provide future directions for each of the conclusions. Additionally, Appendices A and B contain supplementary methods for protein production associated with Chapters 2 and 3, respectively.

Speciation is a central mechanism of biological diversification. While speciation is well studied in plants and animals, in comparison, relatively little is known about speciation in fungi. One fungal model is the Cryptococcus genus, which is best known for the pathogenic Cryptococcus neoformans/Cryptococcus gattii species complex that causes >200,000 new human infections annually. Elucidation of how these species evolved into important human-pathogenic species remains challenging and can be advanced by studying the most closely related nonpathogenic species, Cryptococcus amylolentus and Tsuchiyaea wingfieldii. However, these species have only four known isolates, and available data were insufficient to determine species boundaries within this group. By analyzing full-length chromosome assemblies, we reappraised the phylogenetic relationships of the four available strains, confirmed the genetic separation of C. amylolentus and T. wingfieldii (now Cryptococcus wingfieldii), and revealed an additional cryptic species, for which the name Cryptococcus floricola is proposed. The genomes of the three species are ~ 6% divergent and exhibit significant chromosomal rearrangements, including inversions and a reciprocal translocation that involved intercentromeric ectopic recombination, which together likely impose significant barriers to genetic exchange. Using genetic crosses, we show that while C. wingfieldii cannot interbreed with any of the other strains, C. floricola can still undergo sexual reproduction with C. amylolentus. However, most of the resulting spores were inviable or sterile or showed reduced recombination during meiosis, indicating that intrinsic postzygotic barriers had been established.

A second organism of interest from the Cryptococcus genus is C. depauperatus. This organism is unusual among the genus for its continuously activated sexual cycle. While other members of the genus enter their sexual cycle only under special circumstances, C. depauperatus is a homothallic (self-fertile) species that constantly produces basidiospores when grown on solid media. We sought to discover what genetic elements were responsible for its behavior. We began by analyzing its genome and found homologs of MF, a gene encoding an alpha pheromone, and STE3, a gene encoding a pheromone receptor. A phylogenetic analysis of the two genes shows that they are from opposite mating types and should be compatible with one another. To test whether MF encodes a functional pheromone, we heterologously expressed it as a transgene in MATa and MAT strains of C. neoformans. MATa cells with the transgene became self-filamentous, while MAT cells were unaffected. We then made MATa MF mutants that had a deletion of STE3 or STE6 (the pheromone exporter gene). All of the deletion mutants lost the propensity to self-filament. Therefore, MF encodes a pheromone that can be detected by the Ste3 receptor of MATa cells and exported by Ste6. Additionally, we found that growth in liquid culture suppresses sporulation in C. depauperatus and used this fact to compare mRNA expression levels between cultures that were actively sporulating and those that were not. MF, STE3, and STE6 were all upregulated under conditions that permit sporulation. Thus, we have shown that one factor contributing to the constant sexual cycle of C. depauperatus is that it produces a pheromone and a pheromone receptor that are compatible with each other.

Sexual reproduction in fungi is governed by a specialized genomic region called the mating-type locus (MAT). The ascomycetes, the largest phylum of fungi, primarily possess a bipolar mating system while the basidiomycetes, the second largest group, are mostly tetrapolar. The human fungal pathogen and basidiomycetous yeast Cryptococcus neoformans has evolved a bipolar mating system that encodes homeodomain (HD) and pheromone/receptor (P/R) genes. The MAT locus of C. neoformans is unusually large, spans greater than 100 kb, and encodes more than 20 genes. To understand how the pathogenic Cryptococcus species complex evolved this unique bipolar mating system, we investigated the evolution of MAT in closely and distantly related species and discovered an extant sexual cycle in Cryptococcus amylolentus.

Phylogenetic analysis using a six-gene multi-locus sequencing (MLS) approach identified the most closely related species to the pathogenic Cryptococcus species complex that are currently known. The two non-pathogenic sibling species, Tsuchiyaea wingfieldii and Cryptococcus amylolentus, and the more distantly related species Filobasidiella depauperata define the Filobasidiella clade. We also resolved the phylogeny of the species located in the sister clade, Kwoniella. A comprehensive tree dendrogram revealed that the 15 Tremellales species examined suggests a common saprobic ancestor. Moreover, the pathogenic Cryptococcus species have a saprobic origin but later emerged as pathogens. We further characterized the mating-type locus for T. wingfieldii and C. amylolentus by cloning and sequencing two unlinked genomic loci encoding the HD and P/R genes. Interestingly, linked and likely divergently transcribed homologs for SXI1 and SXI2 are present in T. wingfieldii and C. amylolentus, while the P/R alleles contain many genes also found in the MAT locus of the pathogenic Cryptococcus species. Also, hypothetical genes present in C. neoformans MAT are also MAT-linked in both species and indicate a possible translocation event between chromosomes 4 and 5 of C. neoformans. Our analysis of MAT in the sibling species indicates that T. wingfieldii is likely tetrapolar, and the C. amylolentus sequence comparison of the dimorphic SXI1 and SXI2 region and the pheromone receptor, STE3, suggests that C. amylolentus is also tetrapolar. The examination of MAT in these sibling species confirms the model for MAT evolution previously proposed in which this structure in C. neoformans and C. gattii evolved from an ancestral tetrapolar mating system. Moreover, the organization of MAT in these sibling species mirrors key aspects of the proposed intermediates in the evolution of MAT in the pathogenic Cryptococcus species, and for sex chromosomes in plants, animals, and alga in general.

We discovered an extant sexual cycle for C. amylolentus, a species previously thought to be asexual. Matings between two strains of opposite mating-types produce dikaryotic hyphae with fused clamp connections and uni- and bi-nucleate basidiospores. Genotyping of basidiospores using markers linked and unlinked to MAT revealed that genetic exchange (recombination) occurs during the sexual cycle of C. amylolentus, and it is likely that either aneuploids are generated during sex or more than one meiosis event occurs within each basidium. This is in contrast to C. neoformans, where only one meiotic event per basidium has been observed. Uniparental mitochondrial inheritance has also been observed in C. amylolentus progeny; similar to the pathogenic Cryptococcus species, mtDNA is inherited from the C. amylolentus MATa parent. Analysis of sex in C. amylolentus has provided insight into the mechanisms that phylogenetically related fungi employ in orchestrating sexual reproduction.

We also extended our analysis to include the distantly related tetrapolar basidiomycete Tremella mesenterica. We completed comparisons of MAT-specific genes between five strains of T. mesenterica and identified the regions that define its mating-type system. The HD locus is limited to the SXI1- and SXI2-like genes while the P/R locus is defined by STE3, STE12, STE20, and the pheromone gene, tremerogen a-13. Interestingly, many of the genes associated with the MAT locus of the pathogenic Cryptococcus species flank the HD and P/R locus and are not incorporated in MAT in T. mesenterica. The MAT region includes transposons and C. neoformans hypothetical genes also present in T. wingfieldii and C. amylolentus. The mating-type system in T. mesenterica reflects an ancestral intermediate in the evolution of the MAT locus in the pathogenic Cryptococcus species. In conclusion, this study provides an in-depth analysis on the structure, function, and evolution of an unusual mating-type locus with broader implications for the transitions in modes of sexual reproduction in fungi that impact gene flow in populations.

Cryptococcus gattii has been actively emerging and adapting to the climates and environment in western North America, bringing with it life-threatening disease to humans and animals. Through systematic investigations of the dynamics of this pathogen in the United States, by studying outbreaks and individual cases, our studies have aimed to increase the understanding of the expansion, emergence, pathogenicity, molecular epidemiology, population structure, and speciation dynamics of this organism that had previously been largely restricted to tropical and sub-tropical climates of the world.

Molecular sequence typing has revealed that there are four distinct C. gattii molecular types (VGI-VGIV). A major focus of our efforts to examine this pathogen in the United States surrounds the unprecedented C. gattii outbreak that emerged in British Columbia, Canada in 1999 and has since expanded throughout the Pacific Northwest region of the United States (Chapters 2, 3). This outbreak has resulted in a large number of infections in both humans and animals, including a high percentage of otherwise healthy individuals. The outbreak isolates are primarily molecular type VGIIa (the major genotype), VGIIb (the minor genotype), or VGIIc, a novel genotype that emerged in Oregon in approximately 2005.

The North American Pacific Northwest harbors one of the highest incidences of C. gattii infections. In an expansion of molecular epidemiology and population analysis of both MLST and VNTR markers, we show that the VGIIc group is clonal and hypothesize it arose recently. The VGIIa/c outbreak lineages are sexually fertile and studies support ongoing recombination in the global VGII population. This illustrates two hallmarks of emerging outbreaks: high clonality and the emergence of novel genotypes via recombination. In macrophage and murine infections, the novel VGIIc genotype and VGIIa/major isolates from the United States are highly virulent compared to similar non-outbreak VGIIa/major-related isolates. Molecular analysis distinguishes clonal expansion of the VGIIa/major outbreak genotype from related but distinguishable less-virulent genotypes isolated from other geographic regions. Our evidence documents emerging hypervirulent genotypes in the United States that may expand further and provides insight into the possible molecular and geographic origins of the outbreak.

While the outbreak is a significant public health concern, an overlooked but considerable disease burden attributable to C. gattii among HIV/AIDS patients in Southern California has also occurred (chapter 4). In our studies, we examined the molecular epidemiology, population structure, and virulence attributes of C. gattii isolates collected from a cohort of HIV/AIDS patients in Los Angeles County, California. We show that these isolates consist almost exclusively of VGIII molecular type (>93%), in contrast to the vast majority of VGII molecular type isolates found in the outbreak region. Based on molecular phylogenetic analysis, the global VGIII population structure can be divided into two groups, VGIIIa and VGIIIb. We show that isolates from the Californian patients are virulent in murine and macrophage models of infection, with VGIIIa significantly more virulent than VGIIIb. Several VGIII isolates are highly fertile and able to produce large numbers of spores that may serve as infectious propagules. Based on molecular analysis, the a and α VGIII MAT locus alleles are largely syntenic with limited rearrangements compared to the known VGI (a/α) and VGII (α) MAT loci, but each has unique characteristics including a distinct deletion flanking the 5' VGIII MAT a alleles. Fingerprinting analyses of the MAT locus shows that the α allele is more heterogeneous than the aallele. Our studies indicate that C. gattii VGIII is endemic in Southern California, with other isolates originating from the neighboring regions of Mexico, and in some rarer cases from Oregon and Washington state. Given that >1,000,000 cases of cryptococcal infection and >620,000 attributable mortalities occur annually in the context of the global AIDS pandemic, our findings suggest a significant burden of C. gattii infection in AIDS patients may be unrecognized, with potential prognostic and therapeutic implications. These results signify the need to classify pathogenic Cryptococcus cases and highlight possible host differences among the C. gattii molecular types, influencing infection of immunocompetent (VGI/VGII) vs. immunocompromised (VGIII/VGIV) hosts.

In 2007, the first confirmed case of Cryptococcus gattii was reported in the state of North Carolina, USA (chapter 5). An otherwise healthy HIV- male patient presented with a large upper thigh cryptococcoma in February, which was surgically removed and the patient was started on long-term high-dose fluconazole treatment. In May of 2007, the patient presented to the emergency room with seizures. Magnetic resonance imaging revealed two large CNS lesions found to be cryptococcomas based on brain biopsy. Prior chest CT imaging had revealed small lung nodules indicating that C. gattii spores or desiccated yeast were likely inhaled into the lungs and dissemination occurred to both the leg and CNS. The patient's travel history included a visit throughout the San Francisco, California region in September-October of 2006, consistent with acquisition during this time period. Cultures from both the leg and brain biopsies were subjected to analysis. Both isolates were C. gattii, VGI molecular type. Based on molecular studies and virulence in a heterologous host model, the leg and brain isolates are identical, but the two differed in mating fertility. Two clinical isolates, one from a transplant recipient in San Francisco and the other from Australia, were identical to the North Carolina isolate at all markers tested. Closely related isolates that differ at only one or a few noncoding markers are present in the Australian environment. Our findings support a model in which C. gattii VGI was transferred from Australia to California, possibly though an association with its common host plant E. camaldulensis, and the patient was exposed in San Francisco and returned to present with disease in North Carolina.

To elucidate the speciation dynamics between molecular types VGII and VGIII and what influence nuclear and mitochondrial genomes have on intracellular proliferation and hyoervirulence, we conducted a comprehensive analysis of progeny sets between these molecular types, including progeny sets of VGIIa x VGIIIα and VGIIα x VGIIIa crosses (chapter 6). Our analysis reveals that spore viability is exceedingly low, supporting that these are distinct species. We also found that the mitochondrial genome of virulent strains may be necessary but not fully sufficient to confer virulence characteristics. These studies show that each molecular type is likely a distinct species, which was further supported by high levels of diploid or aneuploid progeny, and also shed light into the possible control that both the mitochondrial and nuclear genomes may play in hypervirulence of C. gattii outbreak genotypes. Future analyses of both the regions regulating the virulence and also the generation of progeny sets between other species will further address the roles of both speciation and virulence evolution in C. gattii.

Overall, the studies documented in this dissertation have increased the understanding of molecular epidemiology, population structures, fertility, phenotypic characteristics, virulence characterizations, and speciation of this expanding and emerging fungal pathogen in the United States. This dissertation adds a foundation to the studies of C. gattii in the United States and enables future research to be conducted in several critical areas to better understand and ultimately influence surveillance, prognosis, and treatment of patients and animals in future years.

One of the key challenges of the 21st century is the emergence and reemergence of pathogens. Fungal pathogens represent an important portion of this problem, as the cohort of immunocompromised patients susceptible to common fungal pathogens rapidly expands in the developed world, and HIV/AIDS continues to present substantial challenges in the developing world. Understanding the processes by which pathogens emerge is critical, both for treating current outbreaks and for preventing future outbreaks. Here I have focused on the human fungal pathogen Cryptococcus deuterogattii, an emerging pathogen responsible for an ongoing outbreak in the Pacific Northwest region of the United States and Canada over the past approximately 15 years. To do so, I have taken comparative genomic, population genomic, and classic genetic approaches to understanding the origin of the outbreak, and the evolution of virulence at three different levels. I will first compare C. deuterogattii to the rest of the Cryptococcus pathogenic species complex, followed by comparisons of clonal clusters within C. deuterogattii, and finally I will compare individual strains within the VGIIa cluster.

I will begin in Chapter 1 by introducing genetic and non-genetic drivers of phenotypic diversity. I will then introduce the diversity of the fungal RNAi pathway, as well as discuss the frequent losses of the RNAi pathway throughout the eukaryotic kingdom.

In Chapter 2, I will examine the differences between the C. deuterogattii species responsible for the Pacific Northwest outbreak and the non-outbreak members of the Cryptococcus pathogenic species complex. I began by comparing the R265 reference genome of C. deuterogattii to the C. neoformans, C. deneoformans, and C. gattii reference genomes. Here we discovered that the core components of the RNAi pathway, including both argonautes, one of the two Dicers, and the only RNA-dependent RNA polymerase had been lost in the C. deuterogattii genome. These gene losses are conserved across the entire species and are defining characteristics of the species. We utilized this information to conduct a comparative genomics screen and identified a total of 14 conserved genes that were lost in the C. deuterogattii genome. We tested these for function in the RNAi pathway and discovered that a number of previously uncharacterized genes are novel RNAi components. In total, 9 of the 14 genes have been shown to play a role in the RNAi pathway. We demonstrated that the sex-induced (SIS) and mitotic-induced (MIS) silencing pathways share core components, and that SIS requires additional pathway components not required by MIS, suggesting that SIS may be a related and more specialized version of MIS. Finally, we showed that SIS appears to be induced through the pheromone signaling and MAP kinase cascade.

In Chapter 3, I will describe a whole genome resequencing project where we sequenced and analyzed clonal outbreak strains from the Pacific Northwest, related strains from outside the outbreak, as well as representatives of diverse global isolates. We utilized phylogenomic inference to provide evidence that the three clonal subgroups of the outbreak had distinct proximal origins: VGIIa in South America, VGIIb in Australia, and VGIIc with no identified origin outside the United States. We also demonstrated that the C. deuterogattii population shows patterns consistent with ancestral mating, but shows little evidence of more recent mating events, meaning that the population is characterized primarily by long periods of clonal growth with only intermittent episodes of sexual recombination.

In Chapter 4, I will examine variation within the VGIIa and neighboring VGIIa-like groups of C. deuterogattii uncovered through our resequencing study. We identified an msh2 nonsense allele ancestral to the VGIIa-like group. Here we demonstrate that this allele is responsible for a mutator phenotype that is particularly severe in genes containing homopolymer runs. Mutator strains are uncommon in eukaryotic microbes, and this lineage may represent a rare stable and successful environmental hypermutator lineage. However, I will also present evidence that the mutator state leads to high mutational burden and eventually loss of virulence, and argue that the mutator allele did not play a role in the expansion of the Pacific Northwest Outbreak in the VGIIa group.

In Chapter 5, I will conclude this thesis and provide some thought towards future directions that emerge from this work. Finally, in Appendices A and B I will discuss a pair of unfinished projects focusing on identifying novel mycoviruses in RNAi deficient lineages and the genetic basis of 5-FC resistance.

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