Mating is important for the survival of Phytophthora infestans, the causal agent of potato and tomato late blight. The mating process increases population fitness and produces thick-walled oospores that can survive harsh environmental conditions. To better understand the mechanisms that regulate of sexual reproduction in P. infestans, the promoters of two mating-induced genes, PITG_002525 and PITG_00483, were studied in order to identify their cis-regulatory elements. PITG_02525 encodes an elicitin-like protein, and PITG_00483 encodes a PUMILIO (Pum) protein. The promoter of PITG_02525 was sequentially deleted and fused with a beta-glucuronidase reporter gene to identify its regulatory motifs. The full length promoter was found to be active in young and mature, and male and female, structures. The deletion analysis identified a potential repressor element and an activator. Similar studies of the pum promoter showed expression both during oosporogenesis and sporulation. One promoter element was found to be essential for the expression of the gene in both stages. Fusing this element with a minimal promoter indicated that it was sufficient for gene expression during sporulation, but not in mating. The RNA binding domain (RBD) of Pum then was used as a model to develop a system for P. infestans protein interaction studies. Expression of the RBD with a tandem affinity purification tag designed with P. infestans codon bias, allowed the successful purification of the Pum RBD with a potential binding partner. This system is adaptable for future studies of other protein interactions. A MudPIT global proteomics comparison of oospores with nonsporulating hyphae (NSH) was then conducted to profile the protein composition of oospores and identify important proteins for germination. It was proposed that the proteins detected in dormant oospores were synthesized during early oosporogenesis and stored for germination. Our study identified proteins that are more abundant in oospores belonging to several functional groups. These included enzymes involved in carbohydrate and fatty acid oxidation to produce energy, and anti-oxidant and anti-stress proteins that may protect the germ tubes, and secreted proteins that could be involved in host infection. This revealed that P. infestans has a very well designed life cycle.

Phytophthora infestans causes the late blight disease of potato and tomato. Spores of Phytophthora are important for dissemination and causing infection. In this thesis, I studied several aspects of Phytophthora spore biology. (1) I performed RNA-seq of sporangia from rye media, potato leaflets, tomato leaflets, and potato tuber slices to investigate whether sporangia from artificial media and from plants are functionally equivalent. I found that sporangia from these different sources had only modest differences in transcriptional profiles, but showed similar infection potential using single-zoospore infection assays. This is an important piece of information indicating that spores from artificial media are good models for the natural situation. (2) I studied a MADS-box transcription factor to understand the regulation of sporangia development in P. infestans. MADS was shown to regulate many sporulation-induced genes, to act before previously known regulators, and to be regulated both at the transcriptional and post-translational levels. (3) I checked for potential artifacts associated with the homology-based gene silencing method that is commonly used to study gene function in Phytophthora. Based on studies of two target genes, the cis-spread of silencing was frequently observed within 500 nt distance of the target gene; however, this did not occur in all transformants generated by the same silencing vector. This suggests that the expression of neighboring genes should be checked to ensure that phenotypes assigned to genes based on silencing data are reliable.

Phytophthora infestans, an oomycete within the phylum Heterokontophyta is one of the most devastating phytopathogens, causing late blight in potato and tomato. Its pathogenic success depends on the formation of different asexual spores such as sporangia and zoospores. Our goal was to identify what regulates transition between each of the five different asexual stages viz. hyphae, spores, cleaving sporangia, swimming zoospores and germinating cysts, by understanding what determines stage-specific transcription. To help accomplish this, we have identified potential binding sites for regulatory elements within the promoters of stage-specific, co-expressed Phytophthora infestans genes, by integrating bioinformatics and traditional molecular biology techniques. Promoter sets, of co-expressed genes identified from expression data, were searched for over-represented motifs using motif discovery algorithms. We were able to detect approximately 20 to 30 over-represented motifs for each of the five stages. Phylogenetic footprinting and positional bias analyses increased the robustness of Transcription Factor Binding Site (TFBS) predictions. Some of the over-represented motifs, which were evolutionarily conserved and showed bias for a certain position within the promoters, were tested for their functionality. Putative TFBSs related to each of the above-mentioned asexual stages, other than the short and unstable swimming zoospore stage, were shown to be biologically active. Molecular biology techniques like promoter-reporter fusion assay, serial deletion, target-specific mutation, RNA-blotting and electromobility shift assay, were used for the functional validation of these potential TFBSs, all of which acted as proximal promoter elements. In addition to these elements, we also looked at the core promoter elements for genes, which had EST evidences for their transcription start sites. A novel core promoter element, specific to the group Pythiales and named DPEpyth, was identified. We have also come up with better Phytophthora specific definitions (consensus sequence), for `FPR' that has been previously detected within the oomycete core promoters and also for the very well known Initiator element (`Inr'). I believe that the identification of these binding sites should lead to a better understanding of signaling pathways regulating spore development and provide insight into new disease control strategies in the future.

Phytophthora infestans, the causal agent of potato late blight, is a significant threat to global food security. Little is known about the metabolism of this oomycete, including how it adapts to different growth and nutrient conditions, while such aspects are one key to understanding host-pathogen interactions. To identify nutrient transporter and metabolic genes involved in pathogenicity, genome and RNA-seq data were mined. One of the major findings was the identification of nitrate assimilation genes that were strongly upregulated at mid time-point tomato leaf tissues compared to late time-point. Mutants silenced for these genes were then generated, which showed a reduction in virulence. This confirmed involvement of the nitrate assimilation genes in pathogenicity. Even though P. infestans can not grow using nitrate as the sole nitrogen source, enzyme assays and isotopic labeling studies indicated that its nitrate assimilation pathway is functional. Enzyme assays revealed few biochemical differences in the activity of nitrate reductase from P. infestans and the enzyme from Phytophthora mirabilis and Pythium ultimum, which are two oomycetes that can grow on nitrate. Isotopic labelling studies indicated that P. infestans and P. mirabilis assimilated little nitrate into amino acids in young cultures, but incorporated substantial nitrate in older cultures. In contrast, Py. ultimum utilized nitrate at both early and late growth stages. Studies of other metabolic pathways revealed the presence of mitochondrial glycolytic pay-off phase in addition to the canonical cytosolic one, and a mitochondrial serine biosynthesis pathway, which is cytoplasmic in other species; both pathways are linked through the intermediate, 3-phosphoglycerate. These novel mitochondrial genes are restricted to stramenopiles. Such novel enzymes could be used as targets for the chemical control of oomycetes. I purpose that manipulation of metabolism is a promising approach for control of P. infestans, one of the most devastating phytopathogens.

Genomic studies on pathogens have given insights to multiple horizons in research that include but are not limited to transcriptional landscaping, host-pathogen interactions, and evolution. These studies have elucidated the role of variation in pathogenic and non-pathogenic strains or species-specific adaptations. We have used genomic and RNA-Seq data to give insight to the genome of Phytophthora infestans, that causes late-blight disease in potato and tomato plants. This study has tried to correctly diagnose P. infestans in the fields, controlling it by elucidating the variation present in different isolates of P. infestans and understanding the mode of evolution among different oomycetes and finally understanding the transcriptional landscape of this pathogen. We have corrected the genome annotation of P. infestans helping us to identify pathogenicity related genes and better functional and structural annotation of the genome. We have also identified regions within the genome of different isolates of P. infestans that show variation and might help explain the differences that exist in terms of pathogenicity and host preferences. Apart from this we have also identified differences and similarities in gene expression levels in different oomycetes in different developmental stages.

Trypanosoma cruzi is the protozoan parasite that causes Chagas disease. Millions of people are infected with the incurable disease and it is estimated that thousands die from Chagas every year. It is thought that all triatomine, which are almost exclusive to the Americas, are capable of being a disease vector and all mammals can be a host of the parasite. Parasites are transmitted from wildlife to man primarily in Latin America, but due to migration, blood transfusions, organ donations, and vertical transmission, the disease now exists worldwide. In addition to Latin America, autochthonous transmission has occurred in North America. Most of the reports of T. cruzi in the United States wildlife are from the southwest where the primary mammalian host is the woodrat. The health of 55 southern California N. fuscipes woodrats was evaluated by PCR using the TCZ1/TCZ2 primer set. None of the woodrats tested were found to be infected with the T. cruzi parasite.

Phytophthora infestans, the causal agent of Irish potato famine (1840), continues to be one of the most devastating plant pathogens of potato and tomato. For completing their life cycle, every pathogen must acquire nutrients from its host organisms. Nutrient acquisition and metabolic adaptations are thus important characteristics of pathogen biology. Information about these aspects in the P. infestans-potato/tomato pathosystems was sought to help us better understand disease progression. To get an idea about the differentially expressed genes on different host tissues and axenically grown P. infestans, an RNA-seq experiment was performed. Several differentially expressed metabolic genes thus identified gave us some clues about the different microenvironments that the pathogen encounters during its in planta growth. Next, to understand how these metabolic genes are expressed in a time-dependent and spatial manner within the host tissues, a confocal microscopy-based approach was used. The proteins of genes coding for invertase activity, participating in sucrose breakdown, were demonstrated to localize within P. infestans haustoria. This is the first report of a metabolic gene being targeted to P. infestans haustoria, thus establishing the role of the latter in nutrient uptake in oomycete plant pathogens. Next, using confocal microscopy, the role of a phosphagen kinase system in energy transport was indirectly provided. The expression of differentially expressed genes depends on timely and coordinated process leading to stage-specific expression, which depends on the binding of specific transcription factors to their cognate transcription factor binding sites (TFBS). A combination of bioinformatics and molecular biology techniques was then used to identify ~100 putative TFBSs regulating the stage-specific expression of developmentally-regulated genes. An understanding of these aspects of pathogen biology may help us devise novel means to control the late blight disease.

Oomycetes, a class in the kingdom Stramenopila, are filamentous eukaryotic microorganisms that are known informally as water molds, but have adapted to many environmental niches. Phytophthora, an oomycete genus of approximately 100 plant-pathogenic species, has specifically adapted to several environmental niches. Environmental conditions play an influential role in the development of Phytophthora, particularly in sporangium formation. Sporangia, the main driver of Phytophthora epidemics, are especially susceptible to damage by environmental conditions, such as light-induced DNA lesions or desiccation by drying. Variable by species, light can stimulate (e.g. P. infestans) or repress (e.g. P. capsici) the production of sporangia. Light may have two developmental roles: an early pre-sporangiophore induction signal, and a later post-sporangiophore repression signal. In this work, I found that although many types of photoreceptors are described in eukaryotes, Phytophthora genomes encode only cryptochrome photoreceptors. The foliar-adapted species P. infestans encodes three cryptochromes, while the soilborne species P. capsici encodes two cryptochromes. Silencing of the P. infestans gene encoding cryptochrome PITG_01718, which lacks an ortholog in P. capsici, inhibits sporulation. While cryptochromes have traditionally been described in other organisms as blue light receptors, each red and blue light represses P. infestans sporulation. In contrast, P. capsici sporulation responds to blue light but not red light. This work also describes the use of RNA-seq to study the genetic control of sporulation by light and humidity. Constant light was found to repress the expression of genes induced in the post-sporangiophore stage by P. infestans, while constant darkness repressed the expression of genes in the pre-sporangiophore stage in P. capsici. Humidity also had a strong effect on repressing gene expression in the pre-sporangiophore stage of sporulation in P. infestans. This work also describes that specific types of nitrogen starvation induce sporulation. In addition to advancing our knowledge of sporulation control in oomycetes, this work also provides a better understanding of the homology-based gene silencing method. While this technique is an effective method for functional genomics in Phytophthora, work in this dissertation shows that the technique must be used with prudence due to the frequency of off-target effects.

The oomycetes are a group of filamentous diploid organisms that superficially resemble true fungi, but instead belong to the kingdom Stramenopiles. Many oomycetes are responsible for devastating diseases of plants, including Phytophthora infestans, the cause of late blight on potato and tomato, and the notorious agent behind the Irish potato famine in the mid 1840s. P. infestans remains as destructive today as it was in the past due to its ability to spread through explosively through susceptible fields given ideal conditions and rapid adaptability to environmental stresses, host defenses, and chemical control. To understand this remarkable adaptability, we examined laboratory- generated sexual population and observed a major genetic locus conferring resistance to the popular fungicide metalaxyl segregating through the progeny. We found that this locus was genetically separate from a previously described locus shown to confer resistance to metalaxyl. Identification of the genes underlying our new major locus was unsuccessful due to the unassembled nature of the currently available reference genome for P. infestans. We sought to construct a new reference genome using a combination of third generation sequencing and scaffolding technology, as well as utilizing two sexual populations of P. infestans to construct genetic maps to assembly of chromosome-sized scaffolds. A large interval of markers strongly associated with metalaxyl resistance was identified at the end of chromosome three. 130 genes were identified in this interval, several of which may play a role in the resistance phenotype. This new reference genome also allowed us to examine the extent of structural variation and genome plasticity within our sexual populations, and we observed considerable plasticity in the forms of whole genome triploidy, tetraploidy, and individual aneuploid chromosomes. Transcriptomic analysis similarly revealed extensive variation among progeny, and our results conclude that sexual reproduction has the potential to induce massive levels of variation in a short period of time in P. infestans.