Ectopic Expression of the Rhizobium etli amtB Gene Affects the Symbiosome Differentiation Process and Nodule Development

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Abstract
Under conditions of nitrogen limitation, soil bacteria of the genus Rhizobium are able to induce the development of symbiotic nodules on the roots of leguminous plants. During nodule organogenesis, bacteria are released endocytotically inside the invaded plant cells where they differentiate into their endosymbiotic form called bacteroids. Bacteroids surrounded by a plant-derived peribacteroid membrane are nondividing, organelle-like structures, called symbiosomes, that use nitrogenase to reduce N2 to ammonia. Experiments performed in vitro with isolated symbiosomes have previously led to the suggestion that the NH3 produced by the bacteroids is released as NH4+ into the plant cytosol. Furthermore, it was observed that the bacterial amtB (ammonium/methylammonium transport B) gene is switched off very early during symbiosis, just when bacteria are released into the host cells. We report here that the ectopic expression of amtB in bacteroids alters the ability of bacteria to invade the host cells and the symbiosome differentiation process. Both the NtrC protein, which controls the expression of the bacterial genes involved in NH4+ assimilation, and the nitrogenase activity are essential to observe the amtB-mediated effect. Our results support the idea that in vivo bacteroids do not take up NH4+ and demonstrate that the transcriptional down-regulation of the amtB gene is essential for an effective symbiotic interaction.