Intracellular Accommodation of Mycorrhiza and Rhizobia by Plants: Molecular Mechanisms and Evolution

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Genetic and biochemical approaches will be used to identify the molecular mechanisms that control the intracellular accommodation of arbuscular mycorrhizal (AM) fungi and rhizobial bacteria (Sinorhizobium meliloti) by Medicago truncatula. The project will in part focus on a novel plant gene, Hermes, which is essential for both rhizobial and AM invasion. The HERMES protein features an N-terminal major sperm protein (MSP)/vesicle associated membrane protein (VAMP) domain and a C-terminal series of ankyrin repeats and which suggest it may be involved in membrane trafficking. One goal of the proposed research is directed at identifying where this protein is found within the cell using confocal microscopy, using GFP-fusions in combination with Agrobacterium rhizogenes hairy root transformation, as well as immunolocalization approaches. Another aim is to find how it might interact with other plant proteins which will be done by searching for protein-protein interactions by screening a yeast-two hybrid library. An extension of this goal is to identify additional genes that are involved in this process. To do this, forward and reverse genetic approaches, including TILLING and screening an existing collection of transposon insertion mutants will be used to identify other related mutants. An additional goal is to identify genes that are specifically expressed within the tissues accommodating rhizobia and AMF by using the technique of laser capture microdissection (LCM) in combination with deep sequencing (Illumina, 454 technologies). Lastly, the project will explore whether Hermes is involved in the intracellular accommodation of biotrophic fungi which form feeding structures (haustoria) analogous to arbuscules by testing the phenotype of the hermes mutant in response to attack by various pathogens.

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