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Charpentier, Myriam (2008): Functional Characterisation of Two Channels Proteins Involved in Leguminous Symbiosis. Dissertation, LMU München: Fakultät für Biologie



Legume-rhizobial symbiosis results in the formation of a new organ, the nitrogen-fixing root nodule. A chemical communication between both partners accompanies the invasion of plant host cells by bacteria and the development of the root nodule. In response to plant-released flavonoids, rhizobia produce lipo-chito-oligosaccharide signalling molecules, so called Nod factors. Early signal transduction in legumes such as Lotus japonicus and Medicago truncatula, is associated with a succession of tightly orchestrated ion fluxes across different membrane systems of the host cell. The Nod factor perception at the plasma membrane triggers Ca2+ oscillations that are associated with the nucleus. CASTOR and POLLUX are required for Ca2+ spiking. Homology modeling suggested CASTOR and POLLUX might be ion channels. However, experimental confirmation was lacking. Therefore we performed biochemical and electrophysiological analysis to define their role. Here we show that CASTOR and POLLUX form two independent homocomplexes in the nuclear rim in planta. We reconstituted CASTOR in planar lipid bilayers and electrophysiological measurements revealed that CASTOR is a cation channel preferentially permeable to potassium. The permeability of the sequence-related POLLUX for cation could be as well demonstrated through expression of POLLUX in different yeast mutants. Furthermore, we demonstrate that a voltage-dependent magnesium blocking mechanism contributes to reduce the conductance of CASTOR at negative membrane potential. By screening a L. japonicus roots cDNA library using yeast-two-hybrid system, a SNF7 protein interacting with CASTOR was found which acts as positive regulator in the nodulation pathway. Collectively the data demonstrate that both CASTOR and POLLUX are nuclear localized cation channels. Therefore, we propose that CASTOR and POLLUX may act as counter ion channels to facilitate a rapid efflux of charge associated with the calcium efflux. Alternatively and not mutually exclusive, they may catalyze a nuclear membrane depolarization leading to the activation of calcium channels responsible for calcium spiking