<p>Pap-Pit is a low affinity phosphate transporter found in <em>S. meliloti</em> and many other microorganisms. Pit is the transporter and Pap is the Pit accessory protein. Pap has been shown to be required for the function of Pap-Pit system in <em>S. meliloti</em>. In this study, <em>pap-pit</em> or <em>pit</em> alone from three species of bacteria have been expressed <em>in trans</em> in the <em>E. coli</em> Pi uptake mutants to check their ability to complement the Pi uptake deficiency of the hosts. A visualization tag, SNAP-tag, has been fused to <em>S. meliloti</em> Pap to help determine the subcellular localization of Pap. Here we show that there is an optimal level of Pap-Pit in the cells, and Pap appears to modulate this level to optimize the function of the system. We also demonstrate that Pap is probably localized intracellularly along the cell membrane. In addition, a <em>S. meliloti pap-pit </em>deletion strain has been prepared and to be used as the background strain for site-directed mutagenesis in Pap. The highly conserved surface amino acids in Pap have been identified to be the candidates for the site-directed mutagenesis.</p> / Master of Science (MSc)

1,25-dihydroxyvitamin D3 [1,25(0H)2D3] has been shown to promote phosphate transport rapidly in the perfused duodenal loop, relative to controls, reaching treated/av basal at T = 40 min = 1.82 ± 0.42 and 1.11 ± 0.21, respectively. By using isolated chick enterocytes, studies were undertaken to determine whether 1,25(0H)2D3 has a direct effect on isolated intestinal cells that is manifested by either enhanced uptake or extrusion of phosphate. In time course studies, with 4- to 8-wk-old chicks, 32P uptake in enterocytes at 10 min after addition of test substance was 0%, 130%, 151%, and 123% of controls for 10 pM, 50 pM, 130 pM, and 300 pM 1,25(0H)2D3, respectively. The metabolite 24,25- dihydroxyvitamin D3 [24,25(0H)2D3] exerted an inhibitory effect on phosphate uptake by 1,25(0H)2D3 at a concentration of 130 pM. This result was in agreement with perfusion studies and supports the physiological relevance of isolated cell studies. For signal transduction studies, isolated enterocytes were incubated with 20 µM forskolin (an activator of protein kinase A), 100 nM phorbol ester (an activator of protein kinase C), or 2 µM BAY K 8644 (a calcium channel activator). Enhanced 32P levels relative to controls were found for phorbol ester (126% of controls at T = 7 min, P < 0.05) and BAY K 8644 (150% of controls at T = 7 min, P < 0.05) but not for forskolin, suggesting involvement of protein kinase C and calcium channel signal transduction pathways in uptake. These results paralleled those observed for the perfused duodenal loop. For aging studies, white leghorn roosters were raised for 7, 14, and 28 wk prior to experiments. These studies showed a 1,25(0H)2D3-mediated increase in 32P uptake in isolated cells at 7 wk, but not at 14 or 28 wk. Further analysis of isolated basal lateral membrane (BLM) on SDS-PAGE followed by Western analysis with a well characterized antibody (Ab099) showed a decreased expression of the putative membrane receptor for 1,25(0H)2D3 with increasing age, paralleling the results obtained for 32P uptake in isolated intestinal cell studies. Analyses of 1,25(0H)2D3 effect on protein kinase C activity likewise revealed hormone-mediated stimulation in cells from 7-wk- old chicks, with decreasing responsiveness at a later age. The combined results indicate a physiologically important role for 1,25(0H)2D3 membrane-initiated phosphate uptake in enterocytes of young, rapidly growing animals. Furthermore, these studies validate the use of isolated intestinal cells for further studies on ribozyme-mediated ablation of the 1,25(0H)2D3 membrane receptor function.

Die arbuskuläre Mykorrhiza (AM) ist eine mutualistische Symbiose, die die Phosphataufnahme und Pathogenresistenz von Pflanzen verbessern kann. In der vorliegenden Doktorarbeit wurde die Rolle der Protonen-pumpenden ATPase MtHA1 für die AM Symbiose in Medicago truncatula untersucht. In MtHA1 Mutanten konnten AM Pilze nur noch verkürzte Arbuskel ohne typische Verzweigungen ausbilden. Dies zeigte sich auch in Expressionsmustern von Genen, die für Proteine in verschiedenen Bereichen der periarbuskulären Membran kodieren. Außerdem waren AM Pilzbesiedelung, die verbesserte Nährstoffaufnahme und die Wachstumsförderung in MtHA1 mutierten Pflanzen reduziert. Die Mykorrhiza-induzierte Resistenz (MIR) wurde näher in M. truncatula Pflanzen untersucht, die von Aphanomyces euteiches infiziert waren, dem Erreger einer Wurzelfäule in Leguminosen. In einem geteilten Wurzelsystem, das eine hohe Expression von Verteidigungsgenen aufwies, unterdrückte ein AM Pilz diese Expression und erhöhte in Folge die Empfindlichkeit für das Pathogen. In Wurzeln von Topfkulturen dagegen konnte eine typische MIR beobachtet werden, die wahrscheinlich auf erhöhter Aktivität der Jasmonat/Ethylen-regulierten Verteidigungsantwort beruht, verursacht durch eine Unterdrückung der Salizylsäuresynthese. Im Ergebnis zeigt diese Arbeit die bedeutende Rolle des Gens MtHA1 für die Bildung und Funktion der arbuskelhaltigen Zellen. Die Mutation des Gens führt zur verminderten Arbuskelverzweigung, reduzierter Phosphataufnahme und Wachstumsförderung in der Mykorrhiza und schließlich zu einer geringeren Gesamtbesiedelung durch den AM Pilz. Genexpressionsanalysen weisen darauf hin, dass unterschiedliche Mechanismen den lokalen und systemischen Wechselwirkungen zwischen AM Pilzen und Pathogenen in der Wurzel zu Grunde liegen. Verschieden physiologische Zustände von geteilten Wurzelsystemen und Wurzeln in Topfkulturen erschweren allerdings einen direkten Vergleich der beiden experimentellen Ansätze. / Arbuscular mycorrhiza (AM) is a wide spread mutualistic symbiosis, which can improve phosphate acquisition and pathogen resistance of plants. In the current Ph.D. thesis the role of a proton pumping ATPase (MtHA1) for the AM symbiosis in Medicago truncatula was investigated. In MtHA1 mutant plants, different AM fungi only developed truncated arbuscules without forming typical hyphal branches, and this phenotype was mirrored by expression patterns of genes for proteins located in different areas of the periarbuscular membrane. AM fungal colonization, improved phosphate uptake and plant growth promotion were reduced in MtHA1 mutant plants. Mycorrhiza-induced resistance (MIR) and the nodule symbiosis were, however, not affected. MIR was further analyzed in the M. truncatula infected with Aphanomyces euteiches which causes a root-rot disease in legumes. In a split root system showing high levels of defense-gene expression, colonization of an AM fungus reduced this expression and in consequence increased susceptibility of the roots for the pathogen. In roots of pot cultures, however, a typical MIR was observed and could be based on the higher activity of jasmonate/ethylene-regulated defense responses due to suppression of salicylic acid biosynthesis. In conclusion, this work shows that the gene MtHA1 encoding a proton pumping-ATPase plays a critical role in the formation and function of arbuscule-containing cells. Expression of the mutated gene results in reduced formation of arbuscule branches. This in turn negatively influences mycorrhizal phosphate uptake, plant growth promotion and overall mycorrhizal colonization of the roots. Gene expression analyses indicate that different mechanisms underlay local and systemic interactions between the mycorrhizal fungus and the root pathogen. The different physiological stages of pot culture and split root system make a comparison of the two experimental approaches, however, difficult.

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