The intracellular motility seen in the cytoplasm of angiosperm plant pollen tubes is known as reverse fountain cytoplasmic streaming (i.e., cyclosis). This effect occurs when organelles move anterograde along the cortex of the cell and retrograde down the center of the cell. The result is a displacement of cytoplasmic volume causing a cyclic motion of organelles and bulk liquid. Visually, the organelles appear to be traveling in a backwards fountain hence the name. The use of light microscopy bioimaging in this study has documented reverse fountain cytoplasmic streaming for the first time in fungal hyphae of Rhizopus oryzae and other members in the order Mucorales (Mucoromycota). This is a unique characteristic of the mucoralean fungi, with other fungal phyla (e.g., Ascomycota, Basidiomycota) exhibiting unidirectional cytoplasmic behavior that lacks rhythmic streaming (i.e., sleeve-like streaming). The mechanism of reverse fountain cytoplasmic streaming in filamentous fungi is currently unknown. However, in angiosperm plant pollen tubes it’s correlated with the arrangement and activity of the actin cytoskeleton. Thus, the current work assumes that filamentous actin and associated proteins are directly involved with the cytoplasmic behavior in Mucorales hyphae. From an evolutionary perspective, fungi in the Mucorales may have developed reverse fountain cytoplasmic streaming as a method to transport various organelles over long and short distances. In addition, the mechanism is likely to facilitate driving of polarized hyphal growth.

Here I document the breadth of the CAP (Cysteine-RIch Secretory Proteins (CRISP), Antigen 5 (Ag5), and the Pathogenesis-Related 1 (PR)) protein superfamily and trace some of the major events in the evolution of this family with particular focus on vertebrate CRISP proteins. Specifically, I sought to study the origin of these CAP subfamilies using both amino acid sequence data and gene structure data, more precisely the positions of exon/intron borders within their genes. Counter to current scientific understanding, I find that the wide variety of CAP subfamilies present in mammals, where they were originally discovered and characterized, have distinct homologues in the invertebrate phyla contrary to the common assumption that these are vertebrate protein subfamilies. In addition, I document the fact that primitive eukaryotic CAP genes contained only one exon, likely inherited from prokaryotic SCP-domain containing genes which were, by nature, free of introns. As evolution progressed, an increasing number of introns were inserted into CAP genes, reaching 2 to 5 in the invertebrate world, and 5 to 15 in the vertebrate world. Lastly, phylogenetic relationships between these proteins appear to be traceable not only by amino acid sequence homology but also by preservation of exon number and exon borders within their genes.

The remarkable conservation of molecular and intra-/inter-cellular pathways underpinning the fundamental aspects of sugar partitioning in two evolutionarily divergent organisms – a non-vascular moss Physcomitrella patens and a vascular cereal crop Oryza sativa (rice) – forms the basis of this manuscript. Much of our current knowledge pertaining to sugar partitioning in plants mainly comes from studies in thale cress, Arabidopsis thaliana, but how photosynthetic sugar is loaded into the phloem in a crop as important as rice is still debated. Even less is known about the mechanistic aspects of sugar movement in mosses. In plants, sugar either moves passively via intercellular channels called plasmodesmata, or through the cell wall spaces in an energy-consuming process. As such, I first investigated the structure of plasmodesmata in rice leaf minor vein using electron tomography to create as of yet unreported 3D models of these channels in both simple and branched conformations. Contrary to generally held belief, I report two different 3D morphotypes of simple plasmodesmata in rice. Furthermore, the complementary body of evidence in arabidopsis implicates plasma membrane localized Proton Pyrophosphatase (H+-PPase) in the energy-dependent movement of sugar. Within this wider purview, I studied the in situ ultrastructural localization patterns of H+-PPase orthologs in high-pressure frozen tissues of rice and physcomitrella. Were H+-PPases neo-functionalized in the vascular tissues of higher plants? Or are there evolutionarily conserved roles of this protein that transcend the phylogenetic diversity of land plants? I show that H+-PPases are distinctly expressed in the actively growing regions of both rice and physcomitrella. As expected, H+-PPases were also localized in the vascular tissues of rice. But surprisingly, H+-PPase orthologs were also prominently expressed at the gametophyte-sporophyte junction of physcomitrella. Upon immunogold labeling, H+-PPases were found to be predominantly localized at the plasma membrane of the phloem complexes of rice source leaves, and both the vacuoles and plasma membrane of the transfer cells in the physcomitrella haustorium, linking H+-PPases in active sucrose loading in both plants. As such, these findings suggest that the localization and presumably the function of H+-PPases are conserved throughout the evolutionary history of land plants.

The distinguishing feature of the filamentous fungi is the hyphae - tube-like microscopic cells that exhibit polarized growth via apical extension and allow the fungus to interact with its environment. Fungi elongate at the hyphal apex, through the localized construction of new plasma membrane and cell wall through the exocytosis of secretory vesicles. One population of these vesicles have been identified as chitosomes, containing chitin synthase isoenzymes, which are responsible for the polymerization of N-acetylglucosamine from UDP N-acetylglucosamine into chitin, the primary fibrillar component of the fungal cell wall. The chitosomes, in addition to other vesicles, can be observed aggregating in the hyphal tip in most filamentous fungi. In the Ascomycota and Basidiomycota, this collection of vesicles exhibits discrete organization and has been termed a Spitzenkörper. Although accumulations of vesicles can be observed in the hyphal tip of many growing filamentous fungi, some debate continues as to what precisely defines a Spitzenkörper. This study reports the details of three separate projects: first, to document the effects of deleting a single chitin synthase, CHS-1 and CHS-6 in Neurospora crassa with regards to hyphal ultrastructure, cytoplasmic organization, and growth in comparison to the wild-type. Given the importance of chitin synthesis in fungal cell growth, deletion of a critical chitin synthase presumably impacts cell wall structure, fungal growth and cytoplasmic organization. Second, an examination of the ultrastructure of four zygomycetous fungi - Coemansia reversa, Mortierella verticillata, Mucor indicus, and Gilbertella persicaria has been conducted. Utilization of cryofixation and freeze-substitution techniques for electron microscopy has produced improved preservation of cytoplasmic ultrastructure, particularly at the hyphal apex, allowing detailed analysis of vesicle size, contents, and organization. Lastly, hyphal tip organization was reviewed in a broad range of fungi. Previous studies had either focused on a few select fungi or representative groups. Vesicle organization, composition and size do appear to vary among the classes of fungi, but some trends, like the vesicle crescent in the zygomycetous fungi have been documented.

Coronaviruses are a medically significant group of viruses that cause respiratory and enteric infections in humans and a broad range of animals. Coronaviruses assemble at the internal membranes of the endoplasmic reticulum- Golgi intermediate compartment (ERGIC). While there is a basic understanding of how viruses assemble at these membranes, the full mechanistic details are not understood. The coronavirus envelope (E) protein is a small multifunctional viroporin protein that plays a role in virus assembly but its function is unknown. The two goals of this study were : 1. To identify and analyze the localization of MHV E and 2. To identify the functions of conserved residues in the tail of the E protein. This study closely examined the localization, dynamics and mobility of the mouse hepatitis virus (MHV) E protein to gain insight into its functions. The results from the first aim of this study showed that the MHV E protein localizes at the site of assembly in the ERGIC-Golgi region based on analysis by immunofluorescence and correlative electron microscopy. A novel tetra-cysteine tagged MHV E protein was used to study the dynamics of the protein in cells. A recombinant MHV E Lumio virus was used to study the trafficking and mobility of the E protein. Live cell imaging and surface biotinylation confirmed that the E protein does not traffic to the cell surface. Fluorescence recovery after photo-bleaching (FRAP) analyses revealed that the E protein is mobile at the site of localization. As a part of the second aim, conserved prolines and tyrosine in the tail of the protein were targeted by site directed mutagenesis and analyzed for functionality. While none of the residues were absolutely essential for localization or virus production, the mutations had varying degrees of effect on envelope formation, protein stability and virus release. Differential scanning calorimetry data suggests that the proline and tyrosine residues enhance interaction with lipids. A wild type (WT) peptide contained the conserved residues was also able to significantly reduce the hexagonal phase transition temperature of lipids, whereas a mutant peptide with alanine substitutions for the residues did not cause a temperature shift. This suggests that the peptide can induce a negative curvature in lipids. The E protein may be playing a role as a scaffold to allow membrane bending to initiate budding or possibly scission. This data, along with the localization data, suggests that the E protein plays a mechanistic role at the site of virus assembly possibly by remodeling the membrane thereby allowing virus budding and/or scission.

In the United States, Escherichia coli O157:H7 (E. coli O157:H7) is the most frequent cause of hemolytic uremic syndrome (HUS) and it is also the primary cause of acute renal failure in children. The most common route of the infection is ingestion of contaminated meat or dairy product originating from cattle or vegetables contaminated with bovine manure. Since cattle are the main reservoir for human infection with E. coli O157:H7, the reduction of intestinal colonization by these bacteria in cattle is the best approach to prevent human infections. Intimin is an outer membrane protein of E. coli O157:H7 that plays an important role in adhesion of the bacteria to the host cell. Hence, I proposed to express intimin protein in tomato plants to use it as a vaccine candidate to reduce or prevent intestinal colonization of cattle with E. coli O157:H7. I expressed His-tagged intimin protein in tomato plants and tested the purified plant-derived intimin as a vaccine candidate in animal trials. I demonstrated that mice immunized intranasally with purified tomato-derived intimin produced intimin-specific serum IgG1and IgG2a, as well as mucosal IgA. I further demonstrated that mice immunized with intimin significantly reduced time of the E. coli O157:H7 shedding in their feces after the challenge with these bacteria, as compared to unimmunized mice. Shiga toxin is the major virulence factor that contributes to HUS. Since Shiga toxin B subunit has an important role in the attachment of the toxin to its receptor, I fused intimin to Shiga toxin B subunit to create multivalent subunit vaccine and tested the effects upon immunization of mice with the B subunit when combined with intimin. His-tagged intimin, Shiga toxin B subunit, and Shiga toxin-intimin fusion proteins were expressed in E. coli and purified. I demonstrated that this multivalent fusion protein vaccine candidate elicited intimin- and Shiga toxin B-specific IgG1, IgG2a, and IgA antibodies in mice. I also showed a reduction in the duration of the bacterial shedding after the challenge compared to the control sham-immunized groups.