A new presymbiotic recognition mechanism from cereals enabling root invasion by arbuscular mycorrhizal fungi

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Maize and rice offer highly attractive plant system for an integrated combination of biochemical, molecular, cell biological, genetic and metabolomic techniques. The equivalent str2 mutant phenotype obtained for both plant species permits the coordinated design of suitable experimental approaches in both of them. The methodological expertise required for all the proposed experimental work is available across the Paszkowski, Eastmond and Wallington labs.
Transformation of rice and Medicago truncatula is efficiently run by joint-applicant Emma Wallington, which is particularly important for this proposal as each of the three objectives relies on the swift and reliable generation of the transgenic plant material. We produce all our constructs by Golden Gate cloning and base the selection on hygromycin or kanamycin.
The Paszkowski lab has extensive experience with live laser scanning confocal microscopy of rice roots, including BiFC. At the Crop Science Centre, the newly installed microscopy suite enables cutting edge laser scanning confocal microscopy on site.
For the unbiased identification of STR2 interactors from rice, an estradiol-inducible system is preferred to achieve control of induction and high quantities of the GFP-tagged STR2 protein. In our hands, the presence of the estradiol does not interfere with AM symbiosis development. Immunoprecipitation follows routine protocols and the pulled-down proteins will be analysed at the Cambridge Centre for Proteomics via spectrometry.
For metabolomic analysis of str2 root exudate and surface lipids (objective 3), both polar and nonpolar metabolites will be analysed from exudate by high res. LC-MS/MS, and surface lipids by GC-MS, using pipelines that are well established at Rothamsted Research. Most metabolites of interest can be identified using accurate mass data and MS/MS fragmentation patterns, but expertise in purification and 1H-NMR will also be available should further structural analysis be required.