The molecular and biochemical basis of nitrogen transfer by the model arbuscular mycorrhizal fungus Rhizophagus irregularis

Plants can increase their effective root length and surface area by investing in symbioses with soil fungi, forming mycorrhizal associations. In mutualistic mode, the plant provides photosynthate (fixed carbon) to the fungus, whereas the fungus provides nutrients to the plant. Arbuscular mycorrhizal (AM) associations are found in 80% of vascular plant families and as a consequence, the AM symbiosis is of tremendous significance to life on this planet, in both natural and agricultural ecosystems. Work in recent years has substantially increased our understanding of nitrogen nutrition in the AM symbiosis. At the molecular level, a working model for nitrogen uptake, metabolism, and transfer has emerged. In this dissertation, mechanisms and genes believed to be responsible for nitrogen flows in the AM symbiosis are described and open questions about the pathway and its regulation are highlighted. Molecular and biochemical experimental approaches were used to investigate these unresolved questions. A compartmented microcosm was developed for aseptic and leakage-free whole-plant mycorrhizal experiments. This was used to monitor S and N uptake by the fungal extraradical mycelium (ERM) and its transfer to host plants. Our results show rapid S and N transfer by ERM to the host plants. Using growth parameter measurements, chlorophyll contents as well as 15N labeling, we conclude that nitrogen transfer from an arbuscular mycorrhizal fungus confers growth benefits on the host plant under nitrogen limiting conditions and that the microcosm system developed will be useful for future work on AM nutrition and metabolism under physiologically relevant conditions. Isotopic labeling time course experiments using different 15N and 13C labeled substrates as well as expression analysis of the expression of key genes were performed using microcosms and an in vitro monoxenic culture system. The results demonstrated the operation of a new pathway of N transfer by AMF to the host via nitrate translocation from the extraradical mycelium to plant roots and shoots. The results also indicate that ornithine is made in the ERM via pyrroline-5-carboxylate and that some of it is broken down in the IRM to glutamate and to a lesser extent to putrescine. Labeling analysis strongly suggests that ornithine is also translocated from the intraradical mycelium to the ERM and is used to make arginine there. Changes in gene expression are consistent with the labeling data on N uptake, metabolism and movement. Gene expression analysis of glutamate dehydrogenase suggests a potential dissimilatory role in the IRM.