A mass spectrometric approach to identify arbuscular mycorrhiza-related proteins in root plasma membrane fractions

Abstract

One of the most important morphological changes occurring in arbuscular mycorrhizal (AM) roots takes place 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, two complementary MS 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 analysed with (i) an automated 2-D LC-MS/MS using a strong cation exchange and RP chromatography, and (ii) SDS-PAGE combined with a systematic LC-MS/MS analysis. Seventy-eight proteins, including hydrophobic ones, were 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 that differed in the mycorrhizal root PM fraction. They corresponded to an H(+)-ATPase (Mtha1) and a predicted glycosylphosphatidylinositol-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.

The plasma membrane proteome of Medicago truncatula roots as modified by arbuscular mycorrhizal symbiosis

“…In contrast, unlike SUT2, in PMenriched fractions we could not detect any peptide specific to MtPT4, the AM-inducible phosphate transporter that localizes on the branch domain of the periarbuscular membrane even though one might have expected the partitioning of the periarbuscular membrane with the plasmalemma because of the connectivity between the two. Because the cells containing arbuscules (and periarbuscular membranes) are diluted within total root tissues, it is possible that periarbuscular membrane proteins may fall below the detection limit (Valot et al 2006). We cannot exclude, however, that proteins belonging to the branch domain may not have been enriched to a similar extent as other PM proteins.…”

“…After centrifugation at 16,000×g for 20 min, the supernatant was collected, filtered through two successive meshes (63 and 38 μm), and centrifuged at 96,000×g for 1 h. The microsomal pellet was resuspended in 10 mM Tris-MES, pH 7.3, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 15% sucrose (w/w). PM enrichment from the microsomal fractions of AM and NM roots was initially performed using the sucrose gradient centrifugation protocol described in (Valot et al 2006). Microsomes (15% sucrose fraction) were loaded on a discontinuous sucrose gradient consisting of 3.5 ml of 38% (w/w) and 3 ml of 33% sucrose in 1 mM Tris-MES, pH 7.2, and 1 mM MgSO 4 .…”

“…After microsome purification, separation of root membranes according to their buoyant density was first performed using a 15/33/38% sucrose gradient (later referred to as gradient I) according to Valot et al (2006). This method allowed recovery, both from AM and NM root microsomal fractions, of the highest PM marker enzyme activity, at the 33/38% interface, while the SW26-insensitive ATPase activity, reflecting contamination by other membranes, was the lowest.…”

“…Despite the economic importance of this legume, however, a comprehensive characterization of the root PM proteome as modified by AM fungi has not been performed. Previous attempts to characterize the PM proteomes of mycorrhizal and nonmycorrhizal M. truncatula roots have been hindered by the lack of protein abundance measurement and incomplete genome resources (Valot et al 2006). Although, because of their hydrophobicity and relatively low abundance, the inventory of membrane proteins has lagged behind the survey of soluble proteomes, membrane proteomics has benefitted in recent years from technical advances in mass spectrometry (MS), bioinformatics resources, and label-free quantification methods (Abdallah et al 2012(Abdallah et al , 2014.…”

Live-Cell Imaging Reveals Periarbuscular Membrane Domains and Organelle Location in Medicago truncatula Roots during Arbuscular Mycorrhizal Symbiosis

“…In a comparison of membrane proteins from AM and non-AM M. truncatula roots, Valot et al (2006) identified two candidates present exclusively in mycorrhizal roots: a H + -ATPase, MtHA1, and a blue copper-binding protein, MtBcp1, which is predicted to be posttranslationally modified with a glycosylphosphatidylinositol (GPI) moiety. GPI anchors, which are added to secreted proteins in the ER after cleavage of a C-terminal peptide signal, result in localization of modified proteins to the extracellular leaflet of the plasma membrane (Eisenhaber et al, 2003).…”