Saville, Barry J

Cytokinin biosynthesis in organisms aside from plant species has often been viewed as a byproduct of tRNA degradation. Recent evidence suggests that these tRNA degradation products may actually have a role in the development of these organisms, particularly fungi. This thesis examines the importance of cytokinins, a group of phytohormones involved in plant cell division and differentiation as well as the phytohormone abscisic acid, involved in plant response to environmental factors, and their presence and role in fungi.

An initial survey was conducted on 20 temperate forest fungi of differing nutritional modes. Using HPLC-ESI MS/MS, cytokinin and abscisic acid were detected in all fungi regardless of their mode of nutrition or phylogeny. The detection of the same seven CKs across all fungi suggested the existence of a common CK biosynthetic pathway and dominance of the tRNA pathway in fungi. Further, the corn smut fungus Ustilago maydis is capable of producing CKs separate from its host and different U. maydis strains induce disease symptoms of differing severity. To determine if CK production during infection alters disease development a disease time course was conducted on cob tissue infected with U. maydis dikaryotic and solopathogenic strains. Dramatic changes in phytohormones including an increase in ABA followed by increases in cisZCKs were detected in tumour tissue particularity in the more virulent dikaryon infection, suggesting a role for CKs in strain virulence. Mining of the U. maydis genome identified a sole tRNA-isopentenyltransferase, a key enzyme in CK biosynthesis. Targeted gene deletion mutants were created in U. maydis which halted U. maydis CK production and decreased pathogenesis and virulence in seedling and cob infections. CK and ABA profiling carried out during disease development found that key changes in these hormones were not found in deletion mutant infections and cob tumour development was severely impaired. These findings suggested that U. maydis CK production is necessary for tumour development in this pathosystem. The research presented in this thesis highlights the importance of fungal CKs, outlines the dominant CK pathway in fungi, identifies a key enzyme in U. maydis CK biosynthesis and reveals the necessity of CK production by U. maydis in the development of cob tumours.

Author Keywords: abscisic acid, cytokinins, high performance liquid chromatography-electrospray ionization tandem mass spectrometry, tRNA degradation pathway, Ustilago maydis, Zea mays

Ustilago maydis (DC) Corda is the causal agent of 'common smut of corn'. Completion of the U. maydis lifecycle is dependent on development inside its host, Zea mays. Symptoms of U. maydis infection include chlorosis and the formation of tumours on all aerial corn tissues. Within the tumours, thick-walled diploid teliospores form; these are the reproductive and dispersal agent for the fungus. U. maydis is the model to study basidiomycete biotrophic plant-pathogen interactions. It holds this status in part because of the completely sequenced 20.5 Mb genome; however, thorough genome annotation is required to fully realize the value of this resource. The research presented here improved U. maydis genome annotation through the analysis of cDNA library sequences and comparative genomics. These analyses identified and characterized pathogenesis-related genes, and identified putative meiosis genes. This enabled the use of U. maydis as a model for investigating 'host-induced' meiosis. Further, the cDNA library analyses identified non-coding RNAs (ncRNAs) and natural antisense transcripts (NATs). NATs are endogenous RNA molecules with regions complementary to a protein-coding transcript. Although NATs have been identified in a wide variety of mammals, plants, and fungi, very few have been functionally characterized. Over 200 U. maydis NATs were annotated by analyzing full-length cDNA sequences. NAT structural features were characterized. Strand-specific RT-PCR was used to detect NATs in U. maydis and in a related smut fungus, U. hordei. The data supported a common role for NATs in smut teliospore development, independent of the RNA interference pathway. Analysis of the expression of one U. maydis NAT, as-um02151, in haploid cells, led to a model for NAT function in U. maydis during teliospore dormancy. This model proposed NATs facilitate the maintenance of stored mRNAs through the formation of double-stranded RNA. In testing this model, it was determined that the deletion of two separate upstream regulatory regions, one of which contained a ncRNA (ncRNA1), altered NAT levels and decreased pathogenesis. These studies strengthened U. maydis as a model organism, and began the functional investigation of NATs in U. maydis, which identified a new class of fungal pathogenesis genes.

Author Keywords: cDNA library analysis, genome annotation, mRNA stability, natural antisense transcripts, pathogenesis, Ustilago maydis

Interactions between hosts and pathogens play a crucial role in their adaptation, evolution and persistence. These interactions have been extensively studied in model organisms, yet it is unclear how well they represent mechanisms of disease response in primary vectors in natural settings. The objective of my thesis was to investigate host-pathogen interactions in natural host populations exposed to raccoon rabies virus (RRV). RRV is endemic to North America, that causes acute encephalopathies in mammals and is commonly regarded as 100% lethal if untreated; however variable immune responses have been noted in natural reservoirs. In order to further understand variable immune responses to RRV, my thesis examined (i) potential immunogenetic associations to RRV using genes intimately associated with an immune response, (ii) the nature of immune responses triggered in the host after infection, and (iii) viral expression and genetic variation, to provide insight into factors that may influence RRV virulence.

Immunogenetic variation of RRV vectors was assessed using major histocompatibility complex (MHC) DRB alleles. Associations were found between specific MHC alleles, RRV status, and viral lineages. Further, similarities at functionally relevant polymorphic sites in divergent RRV vector species, raccoons and skunks, suggested that both species recognize and bind a similar suite of peptides, highlighting the adaptive significance of MHC and contemporary selective pressures.

To understand mechanisms of disease spread and pathogenesis, I screened for variation and expression of genes indicative of innate immune response and patterns of viral gene expression. RRV activated components of the innate immune system, with transcript levels correlated with the presence of RRV. These data indicate that timing of the immune response is crucial in pathogenesis. Expression patterns of viral genes suggest they are tightly controlled until reaching the central nervous system (CNS), where replication increases significantly. These results suggest previous molecular mechanisms for rabies host response derived from mouse models do not strictly apply to natural vector populations. Overall my research provides a better understanding of the immunological factors that contribute to the pathogenesis of RRV in a natural system.

Author Keywords: immune response, major histocompatibility complex, rabies, raccoons, skunks, virus

Glycine max (soybean) is an economically important plant species that registers a relatively low yield/seed weight compared to other food and oil seed crops due to higher rates of flower and pod abortion. Alleviation of this abortion rate can be achieved by altering the sink strength of the reproductive organs of soybeans. Cytokinin (CK) plays a fundamental role in promoting growth of sink organ (flowers and seeds) by increasing the assimilate demand. Cytokinin oxidase/dehydrogenase (CKX) is an enzyme that catalyses the irreversible breakdown of active CKs and hence reduce the cytokinin content. The current thesis uncovers the members of CKX gene family in soybeans and the natural variations among CKX genes within soybean varieties with different yield characteristics. The identification of null variants of OsCKX2 that resulted in large yield increases by Ashikari et al. (2005) provided a rationale for current thesis. The soybean CKX genes along with the ones from Arabidopsis, Rice and Maize were used to construct a phylogenetic tree. Using comparative phylogeny, protein properties and bioinformatic programs, the potential effect of the identified natural variations on soybean yield was predicted. Five genes among the seventeen soybean CKXs identified, showed polymorphisms. One of the natural variations, A159G, in the gene GmCKX16 occurred close to the active site of the protein and was predicted to affect the activity of enzyme leading to higher accumulation of CKs and hence increased seed weight. Use of such natural variations in marker assisted breeding could lead to the development of higher yielding soybean varieties.

Author Keywords: CKX, Cytokinins, Seed weight, Seed Yield, SNPs, Soybeans

Ustilago maydis (D.C.) Corda is a biotrophic pathogen that secretes effectors to establish and maintain a relationship with its host, Zea mays. In this pathosystem, the molecular function of effectors is well-studied, but the regulation of effector gene expression remains largely unknown. This study characterized Zfp1, a putative U. maydis Zn(II)2Cys6 transcription factor, as a modulator of effector gene expression. The amino acid sequence of Zfp1 indicated the presence of a GAL4-like zinc binuclear cluster as well as a fungal specific transcription factor domain. Nuclear localization was confirmed by tagging Zfp1 with enhanced green fluorescent protein. Deletion of zfp1 resulted in attenuated hyphal growth, reduced infection frequency, an arrest in pathogenic development, and decreased anthocyanin production. This phenotype can be attributed to the altered transcript levels of genes encoding predicted and confirmed U. maydis effectors in the zfp1 deletion strain during pathogenic growth. Complementation of zfp1 deletion strain with tin2, an effector involved in anthocyanin induction, suggested this effector is downstream of Zfp1 and its expression is influenced by this transcription factor during in planta growth. When wild-type zfp1 was ectopically inserted in the zfp1 deletion strain, pathogenesis and virulence were partially restored. This, coupled with zfp1 over-expression strains having a similar phenotype as the deletion strains, suggested Zfp1 may interact with other proteins for full function. These findings show that Zfp1, in conjunction with one or more binding partners, contributes to U. maydis pathogenesis, virulence, and anthocyanin production through the regulation of effector gene expression.

Author Keywords: effector, pathogenesis, transcription factor, Ustilago maydis, Zea mays, zinc finger

Ustilago maydis is a biotrophic fungal plant pathogen that causes 'common smut of corn' disease. During infection, U. maydis develops a metabolic dependency on its host, relying on uptake of the carbon molecules provided within Zea mays tissues. The research presented indicated a requirement for metabolism of the pentose sugar D-xylose through functional investigation of a U. maydis xylitol dehydrogenase (uxm1), an enzyme involved in the bioconversion of D-xylose. This work is the first to outline the importance of pentose metabolism during biotrophic plant pathogenesis, as U. maydis haploid cells lacking this gene were impaired in their ability to cause disease and grow on medium containing only D-xylose. This thesis also explored the possibility that expression of this carbon-related gene is controlled by antisense RNAs (asRNAs), endogenous molecules with complementarity to mRNAs. Previous investigation of U. maydis asRNAs identified some that are exclusively expressed in the dormant teliospore, suggesting they have a functional role within this cell-type. A subset of these asRNAs at the uxm1 locus were investigated, with the purpose of identifying the mechanism(s) by which they influence U. maydis pathogenesis. This investigation involved the creation and functional analysis of a series of U. maydis deletion and expression strains. Together, these findings provided additional knowledge regarding the possible functions of U. maydis asRNAs, and their involvement in controlling important cellular processes, such as carbon metabolism and pathogenesis.

Author Keywords: antisense transcripts, fungal carbon metabolism, non-coding RNAs, pathogenesis, Ustilago maydis, xylitol dehydrogenase

Puccinia triticina, wheat leaf rust (WLR), is the most economically damaging fungal rust of wheat on a global scale. This study identified transcriptome changes in a recently emerged race of WLR in Ontario with a new virulence type relative to a possible ancestor race. Also, this study focused on detecting variation in candidate virulence genes and uncovering novel insight into WLR virulence evolution. Various race-by-variety interactions were evaluated using RNA-seq experiments. A list of genes with statistically significant expression changes in each comparison was prepared and predicted effectors were retained for further analysis. Proteins with nonsynonymous substitutions were run through BLASTx to identify potential orthologs. Over 100 candidate effectors with a 2-fold or higher change in transcript level were identified. Seven of these candidate effector genes were recognized to contain single nucleotide polymorphisms (SNPs) which altered the amino acid sequence of the resulting protein. The information gained may aid in targeted breeding programs to combat new WLR races as well as provide the basis for functional analysis of WLR using potential orthologs in a model basidiomycete.

Author Keywords: effectors, RNA-seq, rust fungi, SNPs, transcriptome, wheat leaf rust

The aim of this work was to identify the DNA sequences recognized by the Giardia TBP (gTBP) in vivo by using a chromatin immunoprecipitation assay (ChIP). Since a specific antibody for the protein of interest is required for this assay, a company was contracted to produce and purify a custom polyclonal antibody from the immunization of rabbits. Recombinant GST-gTBP was produced at a suitable yield and purity and used as the immunogen. The antibody was then tested for reactivity to the native protein in our laboratory. By Western blot analysis, it was possible to observe the enrichment of the gTBP within the nuclear fraction compared to a cytoplasmic fraction extracted from Giardia cells. However, the antibody could not be successfully used in an immunoprecipitation assay - suggesting that the antibody is unable to bind to the native structure of gTBP. Therefore, the focus of this work was changed to analyse gTBP via multiple sequence alignments, homology modelling and BLAST to identify any unique regions that may contribute to its unusual binding characteristics. These techniques were also used to identify specific regions of gTBP that may be used to generate synthetic peptides as immunogens for future antibody production.

Author Keywords: ChIP, Giardia intestinalis, Homology modelling, Immunoprecipitation, TATA-binding protein, Western Blotting

Ustilago maydis is a basidiomycete smut fungus and the causal agent of common smut of corn. Disease progression and fungal development in this pathogen occur in planta, terminating in the production of dormant teliospores. Dormant spores of many fungi are characterized by reduced metabolic activity, which is restored during spore germination. The transition out of dormancy requires the rapid translation of stored mRNAs, which may be stabilized through natural antisense transcript (NAT)-mediated mechanisms. Transcript analysis revealed that as-ssm1, a NAT to the mitochondrial seryl-tRNA synthetase (ssm1), is detected in the dormant teliospore and absent in haploid cells. Disruption of ssm1 leads to cell lysis, indicating it is essential for cellular viability. Presented data supports the hypothesis that as-ssm1 has a role in facilitating teliospore dormancy through stabilizing ssm1 transcripts, which reduces mitochondrial function. as-ssm1 expression during in planta development begins 10 days post-infection, coinciding with the first appearance of dormant teliospores. To assess the impact of as-ssm1 expression on cell division, virulence and mitochondrial function, as-ssm1 was ectopically expressed in haploid cells, leading to increased ssm1 transcript levels and the formation of double-stranded RNA. These expression mutants are characterized by attenuated growth rate, virulence, mitochondrial membrane potential and oxygen consumption. Together, these findings support a role for NATs in moderating mitochondrial function during the onset of teliospore dormancy.

Author Keywords: Dormant teliospore, Mitochondria, mRNA stability, Natural antisense transcripts, Non-coding RNA, Ustilago maydis

Giardia intestinalis is a protozoan parasite that causes waterborne diarrheal disease in animals and humans. It is an unusual eukaryote as it lacks the capacity for heme biosynthesis; nonetheless it encodes heme proteins, including three cytochrome b5 isotypes (gCYTB5s) of similar size. Homology modelling of their structures predicts increased heme pocket polarity compared to mammalian isotypes, which would favour the oxidized state

and lower their reduction potentials (E°'). This was confirmed by spectroelectrochemical experiments, which measured E°' of -171 mV, -140 mV and -157 mV for gCYTB5-I, II, III respectively, compared to +7 mV for bovine microsomal cytochrome b5. To explore the influence of heme pocket polarity in more detail, five gCYTB5-I mutants in which polar residues were replaced by nonpolar residues at one of three positions were investigated.

While these substitutions all increased the reduction potential, replacement of a conserved tyrosine residue at position-61 with phenylalanine had the most significant effect, raising E°' by 106 mV. This tyrosine residue occurs in all gCYTB5s and is likely the greatest contributor to their low reduction potentials. Finally, complementary substitutions were made into a bovine microsomal cytochrome b5 triple mutant to lower its reduction potential. These not only lowered the E°' by more than 140 mV but also weakened the interaction of heme with the protein. The lower reduction potentials of the gCYTB5s may indicate that these proteins have different roles from their more well-known mammalian counterparts.