Thesis (Ph.D.)--Brown University, 2008. / Vita. Adviser: Wen-Ming Chu. Includes bibliographical references.

<p> Agriculture in the Midwest United States (Illinois, Indiana, Iowa, Michigan, Minnesota, Ohio, and Wisconsin) is a critically important component of the United States economy and also for world exports of food grain. This is well reflected in the 2012 Census of Agriculture which showed that these states had a market value of crop and livestock products sold in excess of $80,000,000,000 (USDA, 2012). Within the U.S. the three Midwest states, Illinois, Iowa, and Minnesota are ranked 2nd, 3rd, and 4th for the economic value of crops sold. This economic value of agriculture in the Midwest encompasses not only corn, soybeans, livestock, vegetables, fruits, tree nuts, and berries but also nursery and many greenhouse plants. Soil is the one common underlying platform for agriculture and if agriculture has to remain profitable and sustainable, a scientific understanding of soils and their relationship to plant productivity is critical. </p><p> Soils harbor probably the most diverse microbial ecosystems on Earth (Delmont et al., 2011) and we are just beginning to understand the full extent of this diversity and how it influences agricultural productivity and how in turn agricultural practices influence the microbial diversity. Estimations indicate that approximately 1,000 Giga base pairs (Gbp) of microbial genomic sequences exist per gram of soil (Vogel et al., 2009). Microorganisms occupy almost every available niche on Earth and directly affect the environment and agricultural systems by a range of mechanisms that include biological nitrogen fixation (Hungria, Franchini, Campo, &amp; Graham, 2005), suppression of diseases (Mendes et al., 2011), decomposition of organic components (Schmidt et al., 2011), plant growth promotion (Bhattacharya &amp; Jha, 2012), soil nutrient cycling (Brussard, 2012) and bioremediation (Ali et al., 2012). Soil microbial community structure and its associated and interdependent biological processes can be affected by the way land is used and managed. Since a vast majority of soil microorganisms do not respond to "traditional" culturing techniques (Delmont et al., 2011), it has been difficult to study and characterize the functional and phylogenetic diversity of these important ecosystems until recent advances in next-generation DNA sequencing which have begun to unravel what is beneath our feet (Caporaso et al., 2010). According to Food and Agricultural Organization (FAO), the amount of land used for agriculture is about 11% (http://www.fao.org/docrep/005/y4252e/y4252e06.htm) and the emissions which can have serious environmental and health effects from agricultural food production far outweigh the total emissions from all the other industries combined (Bauer, Tsigardis, &amp; Miller, 2016). Thus, any steps to fine-tune the management practices and the way the agricultural land is utilized can go a long way in sustaining our way of life while maintaining a healthy environment. </p><p> The purpose of this study is to examine the shifts in the taxonomic diversity of bacteria in soils at phylum, class and order level between two distinct agricultural practices&ndash;Conventional Tillage (CT) and Conservation Tillage (NT) in Southern Illinois along with changes in soil compaction and soil phosphatase activity. The larger idea, based on results reported here and elsewhere, is to encourage conservative tillage practices using a combination of diverse cover crop systems and continuous soil cover which seem to enhance functional microbial diversity in the soil (Ajay &amp; Ngouajio, 2012; Verzeaux et al., 2016). Research also indicates the presence of higher numbers of bacteria of varied trophic groups, as well as increased species richness in bacteria in well-managed soils with minimal tilling and this, may correspond to more resilience to drying and rewetting disturbances in the soil (Anne et al., 2006). </p><p> This research may be the first to reconstruct the entire soil bacterial community in agricultural fields of Southern Illinois and will also hopefully be a precursor for more studies aimed at not only understanding soil from a biological bacterial perspective but also in deciphering interesting patterns that can help correlate changes in land management practices and how they impact bacterial communities. It may help us in developing a methodology to use bacterial taxa as indicators of soil management practices. The study will also detect previously unreported rare bacterial taxa-specific for this region and regional geochemistry.</p><p>

The mitochondrion contains its own genome. Replication of the mitochondrial DNA (mtDNA) is an essential process that, in most organisms, occurs through the cell cycle with no known mechanism to ensure spatial or temporal constrain. Failures to maintain mtDNA copy number affects cellular functions causing several human disorders. However, it is not clear how the cells control the mtDNA copy number. The mtDNA of trypanosomes, known as kinetoplast DNA (kDNA), is a structurally complex network of topologically interlocked DNA molecules (minicircles and maxicircles). The replication mechanism of the kDNA differs greatly with all other eukaryotic systems. Key features of the kDNA replication mechanism include defined regions for main replication events, coordination of a large number of proteins to drive the replication process, and replication once per cell cycle in near synchrony with nuclear S phase. Two main regions known as the kinetoflagellar zone (KFZ) and the antipodal sites are where main kDNA replication events are known to occur (i.e, initiation, DNA synthesis and Okazaki fragment processing). So far, the localization of the proteins involved in kDNA replication is restricted to two main regions: the KFZ and the antipodal sites. Three mechanisms that directly regulate kDNA replication proteins and serve to control kDNA replication have been proposed: (1) Reduction and oxidation status of the universal minicircle sequence binding protein (UMSBP) controls its binding to the origin sequence, (2) Trans-acting factors regulate the stability of mRNA encoding mitochondrial Topoisomerase II during the cell cycle and, (3) Regulation of TbPIF2 helicase protein levels by a HslVU-like protease to control maxicircle copy number. These mechanisms seem to be protein specific and it appears that a combination rather than a single mechanism regulates kDNA replication. In this study we used Trypanosoma brucei to understand how mitochondrial DNA replication is controlled. We investigated the mechanism of how proteins transiently localize to the sites of DNA synthesis during cell cycle stages. Our data provides a comprehensive analysis of the first two examples of T. brucei kDNA replication proteins that have a cell cycle dependent localization (Ch. 2 and 3). The localization of two of the three essential mitochondrial DNA polymerases (TbPOLIC and TbPOLID) is under tight cell cycle control and not regulated by proteolysis. TbPOLIC and TbPOLID localize to the antipodal sites during kDNA S phase, however, at other cell cycle stages TbPOLIC becomes undetectable by immunofluorescent analysis and TbPOLID disperses through the mitochondrial matrix. In agreement with this data, TbPOLIC and TbPOLID replication complexes were not detected using affinity purification presumably because only a fraction of these proteins are participating in replication at a given time (Ch. 4). We propose that spatial and temporal changes in the dynamic localization of essential kDNA replication proteins provide a novel mechanism to control kDNA replication.

My dissertation explores several aspects of the relationship between morphological and molecular evolution in amoeboid lineages: Chapter 1—General Introduction. This chapter provides an overview of the most pressing issues in Amoebozoa phylogeny that are dealt with in the remainder of the thesis. Chapter 2—Reducing the impact of PCR-mediated recombination in molecular evolution and environmental studies using a new generation high fidelity DNA polymerase. This chapter addresses the methodological difficulty in the study of large gene families, the generation of artifactual sequences by recombination during PCR Chapter 3—Evolution of the actin gene family in testate lobose amoebae (Arcellinida) is characterized by two distinct clades of paralogs and recent independent expansions. This chapter explores intriging patterns of evolution in the actin gene families of testate amoebae. Chapter 4—Comprehensive phylogenetic reconstruction of Amoebozoa based on concatenated analysis of SSU-rDNA and actin genes. A deep phylogenetic analyses of the Amoebozoa, enables exploration of well supported taxonomic units within the group. Chapter 5—Interpreting the evolutionary history of the Tubulinea (Amoebozoa), in light of a multigene phylogeny. This chapter explores a more restrict taxonomic unit within the Amoebozoa—the Tubulinea—based on an expanded sample of genes and taxa. Chapter 6—The chastity of amoebae: re-evaluating evidence for sex in amoeboid organisms. This chapter asks whether the null-hypothesis that amoebae are asexual is consistent with current phylogenetic evidence.

<p> Photosystem II (PSII) is recognized as the main site for high light induced damage. One of the core subunits of PSII, D1 protein encoded by the <i> psbA</i> gene, is identified as a high turnover protein that undergoes degradation and replacement as a part of the repair process in PSII. Studies on D1 repair have shown the synchronous nature of D1 degradation and synthesis. FtsH a AAA protease, is known to cause D1 degradation. Therefore, it is widely speculated that damaged D1 is replaced by newly synthesized D1. Although, there is no direct evidence suggesting the removal of damaged subunits only. Alternatively, an induction of the general increase in the turnover rate of D1 could trigger the replacement of all D1 subunits in a random fashion. In this work, I have addressed these two alternate hypotheses by developing a genetic system involving dual D1 expression. Strains were constructed with the parallel expression of a WT <i>psbA2</i> in the ectopic and a damage prone mutant <i>psbA2</i> at the native locus. Firstly, a WT ectopic strain (eWT) was constructed by transforming a <i>psbA</i> deletion strain with a WT <i>psbA2</i>, which was synthesized using synthetic biology and fusion PCR. Secondly, mutant <i>psbA2</i> was introduced into the native locus of the eWT strain. All the dual D1 strains showed high PSII activity. Higher PSII levels represent increased proportion of PSII with WT D1 incorporated, as the mutant does not correspond to high PSII levels. Immunoblot analysis of a dual strain nS345P: eWT against D1 did not show an accumulation of the S345P form, which exists as pre-D1. However, deletion of the FtsH protease in nS345P: eWT showed an accumulation of pre-D1 along with mature D1. This indicates that S345P mutant forms are targeted for repair in the dual strain. Additionally, <i>psbA2</i> gene dosage effect was also studied by expressing two WT <i>psbA2</i>. Similar levels of PSII and D1 were observed in the single and double WT strain. This suggests that level of PSII is being regulated and that D1 is not the regulating factor. Therefore, PSII homeostasis is maintained by some other protein factor. </p>

<p> The ascomycete fungus <i>Magnaporthe oryzae,</i> causative agent of rice blast disease, poses a threat to global food security, destroying enough rice to feed 60 million people each year. Characterization of the host-pathogen interaction between rice and <i>M. oryzae</i> is critical, as better understanding of the system may lead to better disease control strategies. The sequenced genome and repertoire of molecular tools available have made <i> M. oryzae</i> an ideal model system for understanding general plant-pathogen interactions as well. </p><p> The objective of this dissertation was to characterize the <i>M. oryzae</i> homologs of <i>Histoplasma capsulatum RYP</i> (<i><u> R</u>equired for <u>Y</u>east <u>P</u>hase </i>) genes that are required for transition to the parasitic phase. <i> H. capsulatum</i> is a human pathogen that undergoes a dimorphic switch from filamentous to yeast cell growth at 37&deg;C, the host body temperature. Four<i>H. capsulatum RYP</i> genes were identified in a forward genetic screen to identify genes required for entry into the yeast phase. <i> RYP1</i> is a member of the Gti1_Pac2 family, which contains previously characterized regulators of dimorphic switching. <i>RYP2</i> and <i> RYP3</i> are homologs of <i>vosA</i> and <i>velB,</i> members of the Velvet family, best characterized in <i>Aspergillus nidulans, </i> where they coordinate morphological differentiation with secondary metabolism. <i>RYP4</i> is a zinc binuclear cluster protein, a main class in the zinc finger transcription factor family. Deletion of the <i> M. oryzae RYP1</i> homolog, <i>RIG1</i> (<i><u> R</u>equired for <u>I</u>nfectious <u>G</u>rowth </i>), resulted in a non-pathogenic mutant on susceptible rice cultivars, even upon removal of the host penetration barrier. <i>&Delta;rig1</i> was blocked in the transition to infectious hyphal growth, similar to <i> H. capsulatum ryp1,</i> which could not transition to the yeast phase. Deletion mutants of <i>M. oryzae RYP2, RYP3,</i> and <i>RYP4 </i> homologs were similar to the wild type in somatic growth and pathogenicity indicating that although <i>RIG1</i> is a pathogenicity factor conserved in plant and animal pathogens, such conservation does not apply to all of the <i>RYP</i> pathogenicity genes identified in <i>H. capsulatum. </i> </p><p> <i>&Delta;rig1</i> is the first <i>M. oryzae</i> mutant known to be blocked in production of primary infection hyphae. Overall, the study suggests limited parallels exist in phase transition of fungal pathogens of plants and animals.</p>

<p>RNA interference (RNAi) acts as an antiviral defense mechanism in fungi, plants, nematodes, insects, and mammals. In antiviral RNAi, virus-specific double-stranded RNA is processed into small interfering RNAs (siRNAs) to guide specific viral RNA degradation by the RNAi machinery. Although antiviral RNAi is non cell-autonomous in plants, it is unknown if antiviral RNAi is also systemic in animals. In this dissertation, I characterized the nematode <i> Caenorhabditis elegans</i> mutants defective in systemic RNAi in their antiviral RNAi response induced by either the replication of a Flock house virus-derived replicon or the infection of Orsay virus. The results from these genetic studies provided evidence for the first time to support an antiviral function of systemic RNAi in animals. Comparison of the population of viral siRNAs by deep sequencing further revealed that <i>C. elegans</i> mutants with strong defects in systemic antiviral RNAi were all partially defective in the biogenesis of the viral secondary siRNAs. A possible role for the viral siRNAs in systemic antiviral RNAi is discussed. </p>

<p> Env7 is a vacuole membrane-localized protein kinase that is orthologous to the human serine/threonine protein kinase, STK16. It is evolutionarily well-conserved throughout Eukarya, and it has one ortholog in Bacteria. Phylogenetic analyses of sequences homologous to Env7 revealed clades that are inconsistent with established eukaryotic phylogeny, suggesting that both horizontal and vertical gene transmission are responsible for their conservation. Conserved amino acid residues and motifs that are potentially important to Env7's catalytic activity, localization, and interactions with other proteins were also identified and assessed. Additionally, one such conserved motif&mdash;the glycine-rich loop&mdash;was mutated in an effort to affect ATP binding in Env7. The phenotype resulting from this mutation was a slightly increased number of mutant cells exhibiting multi-lobed vacuoles under normal conditions.</p>

<p> Fungi are thought to be one of the most diverse groups of organisms in the Arctic. They drive mineral and energy cycles and influence the occurrence of other organisms as mutualists (mycorrhizae, endophytes, lichens), decomposers and pathogens. Nevertheless, information on fungal biodiversity and distribution patterns in relation to environments across the Arctic is still sparse. Molecular methods were used to examine the diversity and community structures of ectomycorrhizal fungi (EMF) associated with two principal arctic host plants, <i> Salix arctica</i> and <i>Dryas integrifolia,</i> as well as total soil fungal communities of adjacent disturbed and undisturbed areas of patterned-ground features across the five bioclimatic subzones (A-E) of the North American Arctic. Key findings include the following: (1) More diverse fungal communities had been observed than previously known. These communities encompass nearly all fungal phyla and included all fungal guilds. However, a few species-rich fungal families dominated these fungal communities. (2) Surprisingly, species richness did not decline with latitude. (3) The most abundant fungal taxa were widely distributed in and beyond the Arctic. Yet root (EMF) and soil fungal communities showed niche preferences in regard to bioclimatic subzones. Furthermore, disturbed and undisturbed patterned ground features harbored different soil fungal communities with the exception of the coldest subzone A. In contrast, EMF community composition was not affected by host plant identity. (4) Fungal communities in the warmest subzone E were distinct from the other arctic subzones and the majority of taxa matched fungi from the boreal forest. (5) Key drivers of fungal community and guild composition along the bioclimatic gradient included regional climate, pH as well as vegetation composition and productivity across the subzones. At the local scale of patterned-ground features, fungal communities were correlated with vegetation composition and microclimate. With a warming climate, I would expect an enhanced colonization of patterned-ground features by vascular plants that would then affect fungal community structure not only at the species level, but also at the level of fungal guilds. In particular I would expect increases in fungi that are symbiotic with plants and a northward shift of both plant and fungal taxa.</p>

Thesis (Ph.D.)--University of Hawai'i at Manoa, 2005. / (UnM)AAI3198363. Source: Dissertation Abstracts International, Volume: 66-12, Section: B, page: 6430. Chair: Dulal Borthakur.