This proposal will use an innovative multidisciplinary approach to investigate complex invader-mediated reprogramming of eukaryotic organisms in an ancient microbe-host association, arbuscular mycorrhiza (AM) symbiosis. We aim to pioneer the application of in planta time-lapse live-cell imaging using multi-photon confocal microscopy (MPCM) combined with ultrathin (2-D) TEM stereology and high-resolution three dimensional (3-D) Serial Block-Face Scanning Electron Microscopy (SBFSEM). Data sets generated will provide a high resolution quantitative 3-D reconstruction of membrane surfaces intimately involved in the intracellular plant-fungal dialogue.
To date, time-lapse live-cell imaging using confocal laser scanning microscopy (CLSM) in inner cortical root cell layers has been hampered by low resolution and photo-bleaching that impairs cell viability. Deep-tissue imaging at high resolution with minimal tissue damage is permitted by state-of-the-art multi-focal MPCM recently acquired at the Cambridge Advanced Imaging Centre (CAIC). MPCM of rice lines expressing fluorescently labelled membrane marker-proteins will provide a first quantitative 4-D account of membrane dynamics during intracellular colonization by AM fungi. In addition, 2-D ultrathin TEM will allow quantitative stereological estimations of the volume fraction of the host occupied by the microbe and surface area per unit volume of host and fungal membranes. SBFSEM imaging with 3-D reconstruction at a resolution of 5nm will provide a first structural atlas of 'naïve' and colonized cells at unprecedented resolution. MPCM and SBFSEM phenotyping of rice mutants compromised in arbuscule development will provide a novel insight into the complex molecular mechanisms that underpin cellular morphogenesis during plant endosymbiosis. Significantly, this data will provide a structural framework for 3-D modelling of complex membrane dynamics during the establishment of endosymbioses.
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