2. Sub-cellular membrane proteomics

Our second task was aimed at developing different sub-cellular proteomic analyses in order to have access to membrane proteins of the fully established AM symbiosis.

One of the most important morphological changes occurring in arbuscular mycorrhizal (AM) roots happens when the plant plasma membrane (PM) invaginates around the fungal arbuscular structures resulting in the periarbuscular membrane formation. To investigate whether AM symbiosis-specific proteins accumulate at this stage, 2 complementary mass spectrometric approaches, targeting the root PM from the model legume Medicago truncatula were designed. Membrane extracts were first enriched in PM using a discontinuous sucrose gradient method. The resulting PM fractions were further analyzed with (i) an automated two-dimensional liquid chromatography coupled to tandem mass spectrometry (2-D LC-MS/MS) using a strong cation exchange and a reversed-phase chromatography and (ii) SDS-PAGE combined with a systematic LC-MS/MS analysis. Seventy-eight proteins, including hydrophobic ones, could be reproducibly identified in the PM fraction from non-inoculated roots, representing the first survey of the M. truncatula root PM proteome. Comparison between non-inoculated and Glomus intraradices-inoculated roots revealed two proteins different in the mycorrhizal root PM fraction. They corresponded to an H+-ATPase (Mtha1) and a predicted glycosylphosphatidylinositol (GPI) -anchored blue copper-binding protein (MtBcp1), both potentially located on the periarbuscular membrane. The exact role of MtBcp1 in AM symbiosis remains to be investigated.