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Fungal proteins in the extra‐radical phase of arbuscular mycorrhiza: a shotgun proteomic picture

2008; Wiley; Volume: 181; Issue: 2 Linguagem: Inglês

10.1111/j.1469-8137.2008.02659.x

1469-8137

Ghislaine Recorbet, Hélène Rogniaux, V. Gianinazzi-Pearson, Eliane Dumas‐Gaudot,

Fungal Biology and Applications

The mycelial network that develops outside the roots in arbuscular mycorrhiza (AM) is considered as the most functionally diverse component of this symbiosis. Extra-radical mycelia (ERM) not only provide extensive pathways for nutrient fluxes through the soil, but also have strong influences upon biogeochemical cycling and agro-ecosystem functioning (Purin & Rillig, 2008). Despite the recognized importance of ERM in AM symbiosis ecology, the mechanisms by which fungal networks extend and function remain poorly characterized at a large scale, with most of the studies performed so far being devoted to the analysis of fungal morphological features and nutrient metabolism (Fortin et al., 2002). The functioning of ERM presumably relies on the existence of a complex regulation of fungal gene expression with regard to nutrient sensing, production of specific enzymes and resource partitioning between host roots and microsymbionts (Leake et al., 2004). In this respect, proteomics is likely to be one of the best methodologies to decipher some key functions of mycelial networks. However, so far, only one study based on two-dimensional electrophoresis (2D-PAGE) has been conducted to monitor the fungal proteins that accumulate in the extra-radical phase of mycorrhiza (Dumas-Gaudot et al., 2004). Although large-scale protein-profiling experiments have been dominated by the use of 2D-PAGE because it features high protein separation capacity, this method was reported to under-represent proteins with extreme physicochemical properties (size, isoelectric point, transmembrane domains) and those of low abundance (Haynes & Roberts, 2007). Such limitations of 2D-PAGE for analytical protein profiling have led to the more recent development of shotgun proteomic approaches designed to optimize proteome coverage, including one-dimensional (1D)-PAGE-nanoscale capillary liquid chromatography-MS/MS, namely GeLC-MS/MS, which combines a size-based protein separation to an in-gel digestion of the resulting fractions. As part of our interest in identifying the AM fungal functions that are expressed in the ERM mycorrhiza, we have explored the efficiency of GeLC-MS/MS to identify proteins from the mycelium of Glomus intraradices developed on root organ cultures, and report on the identification of 92 different proteins. Glomus intraradices (DAOM 181602) was grown at 27°C in the dark with carrot (Daucus carota) hairy roots on modified minimal nutrient medium (Bécard & Fortin, 1988) containing 10 g sucrose l−1 and 0.3% Phytagel. To recover root-free extra-radical hyphae and spores, Petri dishes were incubated vertically after placing a mycorrhizal root organ plug in the upper side of the plate. Areas colonized by the ERM were collected after 12 wk, as previously described (Dumas-Gaudot et al., 2004). Two independent experiments were performed, each consisting of 50 root organ cultures. Protein extraction was performed as previously described (Dumas-Gaudot et al., 2004), except that proteins were solubilized in Laemmli buffer (Laemmli et al., 1970) and boiled for 3 min at 95°C before ultracentrifugation. For each independent experiment, proteins (75 µg) were separated on a linear 12%, pH 8.8, SDS-PAGE gel (Valot et al., 2006), which was stained with Coomassie Brilliant Blue (Mathesius et al., 2001). The whole gel lane from one representative experiment was sliced into 40 bands of equal size. In-gel trypsin digestion of proteins and nano-LC-MS/MS analyses of the digests using a Switchos-Ultimate II capillary LC system (LC Packings/Dionex, Amsterdam, the Netherlands) coupled to a hybrid quadrupole time-of flight mass spectrometer (Q-TOF Global, Micromass/Waters, Manchester, UK) were performed as described in Repetto et al. (2008). MS/MS data were analysed with Mascot (Matrix Science, London, UK) against the ‘Fungi’ subset of Swiss-Prot (release 54.4) and TrEMBL (release 37.4) databases. Trypsin was specified as the proteolytic enzyme and one miss cleavage was allowed. Fixed and variable modifications were carbamidomethylation of cysteine and oxidation of methionine, respectively. Mass tolerance was set at 150 ppm for peptide precursors and at 0.3 Da for fragment ions. Only matches with P < 0.05 for random occurrence were considered to be significant and a minimum of two unique peptides at disparate sites within a protein were required for a positive identification. When less than two proteins could be identified per gel slice, MS/MS spectra were interpreted de novo using Protein Lynx Global Server 2.0 (Micromass/Waters). The MS BLAST search was performed against the nrdb95 protein database following the procedure described by Shevchenko et al. (2001). Hits were considered statistically confident according to the MS BLAST scoring scheme (Habermann et al., 2004). Only proteins identified with at last two peptide sequences were validated. Identified proteins were functionally classified according to Ruepp et al. (2004) using Uniprot annotations at ExPASy (http://www.expasy.org/sprot/). Theoretical molecular weight (Mw) and isoelectric point (pI) were calculated using the Compute pI/Mw tool from ExPASy. Subcellular location of proteins was determined using Uniprot annotations or predicted by Wolf PSORT (http://wolfpsort.seq.cbrc.jp). Transmembrane (TM) domains were predicted using the TMpred server with a minimum score of 1000 (Valot et al., 2006). Proteins were classified as integral membrane proteins when predicted to have at least two TM regions or were annotated as an integral membrane protein in Swiss-Prot (Everberg et al., 2006). The MetaCyc database (Caspi & Karp, 2007) was used to retrieve biochemical pathways. In this GeLC-MS/MS approach, the 40 LC-MS/MS runs first allowed the confident identification of 158 proteins (data not shown). This initial repertoire was manually curated to provide a minimal list of proteins sufficient to explain all observed peptides, resulting in a final record of 92 (54 distinct and 38 differentiable) proteins (Table 1), according to the nomenclature and principles of parsimony described in Nesvizhskii & Aebersold (2005). Most of proteins (86.9%) were identified when using the MASCOT search engine against Uniprot with taxonomies restricted to Fungi in order to minimize the generation of false-positive identifications and measurement time (Table 1). For seven datasets that led to very little conclusive MASCOT identification, spectra were further interpreted de novo. Despite high-quality spectra (data not shown), only 12 additional proteins (Table 1) could be further identified using the MS-BLAST algorithm, probably by reason of significant amino acid variation between AM fungal proteins and their orthologues in other species. Of the 92 proteins identified in the ERM of G. intraradices, only five were inferred from peptides belonging to proteins from this species (Table 1). At the time of data analysis, there were no more than 75 entries for G. intraradices proteins in Uniprot, meaning that the current work, with the identification of 87 additional proteins, represents a significant quantitative breakthrough in the characterization of the AM fungal proteome. As a benchmark for shotgun proteomics of plant-colonizing fungi lacking full genome characterization, the two-dimensional LC-MS/MS analysis performed on fungal uredospores from the obligate pathogen Uromyces appendiculatus led to the identification of 486 proteins (Cooper et al., 2006). Among them, 56 distinct and differentiable proteins were identified with at least two peptides, which is in a range similar to the 92 proteins retrieved in the current work with similar criteria. Compared with the four extra-radical and six in planta-detected proteins of G. intraradices that were previously identified using 2D-PAGE analyses, these GeLC-MS/MS data represent the most comprehensive list of AM fungal proteins so far identified (Dumas-Gaudot et al., 2004; Valot et al., 2005). Additionally, only 75 µg of proteins were used as starting material instead of the 300–600 µg amount required for micro-preparative 2D-PAGE gels. GeLC-MS/MS was reported to sample, in a relatively unbiased manner, proteins with extreme properties. In this work, 15 proteins with pI greater than 9, and 13 proteins with theoretical Mw less than 10 and greater than 80 kDa could be retrieved (Table 1), which are beyond the reported 2D-PAGE separation limits for G. intraradices (Dumas-Gaudot et al., 2004). When analysing the ERM proteins for subcellular location, half of them were localized to the cytoplasm, and nucleus and mitochondrion were the two other cellular components most commonly retrieved (Supporting information, Fig. S1). Regarding the presence of putative integral membrane proteins, 15 proteins were predicted to have at least two TM domains, and three additional proteins (ATP synthase β subunits) lacking putative TM domains were also retrieved as annotated as integral membranes in Swiss-Prot (Table 1). Proteins containing at least two membrane-spanning hydrophobic sequences are estimated to represent c. 18–27% of all proteins in organisms with sequenced genomes (Ward, 2001). In the current work, 19.5% of the identifications were assigned to poorly soluble integral membrane proteins, which is noteworthy in the absence of any specific enrichment for this class of proteins. Of the 92 proteins identified in this study, functional roles for 89 proteins were known or could be predicted from database analysis (Table 1). The FunCat annotation scheme assigned them to nine biological processes, in which energy, protein fate and synthesis were the most prominent retrieved categories (Fig. S2). To obtain an overview of the metabolism of root organ culture-developed ERM, proteins were analysed with the MetaCyc database, which retrieved 11 pathways that span energy, metabolism and cell rescue processes as schematically represented in Fig. 1. Consistent with the use of neutral lipids as the main respiratory substrate in ERM (Requena et al., 1999; Bago et al., 2002), there were enzymes involved in the catabolism of long-chain fatty acids, their subsequent β-oxidation, and energy generation through the TCA cycle coupled to electron transport and oxidative phosphorylation. Additionally, GeLC-MS/MS data pointed to enzymes involved in dark CO2 fixation, glycolysis/gluconeogenesis, pentose phosphate and glutamine biosynthesis-related pathways, the existence of which in AM fungi have so far been inferred from enzymatic, isotopic labelling and/or gene expression studies (Saito, 1995; Pfeffer et al., 1999; Tamasloukht et al., 2003; Breuninger et al., 2004). Of the proteins related to cell redox homeostasis, trans-sulphuration pathway and γ-glutamyl cycle, accumulation in the ERM of AM fungi of the corresponding transcripts has only been reported previously for a superoxide dismutase and an adenosylhomocysteinase (Jun et al., 2002; Lanfranco et al., 2005). Schematic overview of energy, metabolism, and cell rescue pathways retrieved in the extra-radical mycelia (ERM) of Glomus intraradices on the basis of the proteins identified in the current study. Pathways are shown in bold capital letters. Proteins identified in this study are indicated in green and numbered according to the corresponding pathway. Inferred metabolic intermediates are shown in nonbold letters. FA, fatty acid; TCA, tricarboxylic acid cycle; I, II, III, IV, V, complexes I, II, III, IV, V of the electron transport chain, respectively; UQ, ubiquinone; UQH2, ubiquinol; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GS, glutamine synthase; PPP, pentose phosphate pathway; 6PGD, 6-phosphogluconic dehydrogenase; SOD, superoxide dismutase; TrxS2, thioredoxin disulfide; TRx(SH)2, reduced thioredoxin; TrxPx, thioredoxin peroxidase; GSH, reduced glutathione; GSSG, glutathione disulphide; TrxR, thioredoxin reductase; GR, glutathione reductase; GPX, glutathione peroxidase. Similar to other fungi previously classified within the traditional Zygomycota that is no longer considered as a phylum (White et al., 2006), polar growth of extra-radical AM hyphae requires the delivery at the growing apex of secretory vesicles involved in wall and plasma membrane increase (Wessels, 1993). In this study, we identified several proteins related to vesicular trafficking, including the GTP-binding protein Ypt1, the small GTPase SAR1, and three members of the Rab GTPase subfamily (Table 1). Furthermore, signal-transducing proteins calcineurin, Rho1, Cpc2 and Bmh2, for which a role has been demonstrated in fungal morphogenesis (Won et al., 2001; Steinbach et al., 2006; Argimón et al., 2007; Zannis-Hadjopoulos et al., 2008), also accumulated in the ERM of G. intraradices. Bmh proteins are additionally involved in cell cycle regulation as being necessary for the initiation of DNA replication and are positive regulators of rapamycin-sensitive signalling via the TOR kinase pathway, whose expression was previously reported in G. mosseae (Requena et al., 2000). Among proteins playing roles in the cell division cycle, the DNA damage checkpoint protein rad25, two isoforms of the AAA ATPase Cdc48, and a putative prohibitin were concomitantly identified in the ERM of G. intraradices. Currently, the mechanisms underlying AM fungus cell cycle mostly refer to the moving from G0/G1 to S/M during infection and DNA replication occurring during the production of germinating mycelium (Bianciotto & Bonfante, 1993; Bianciotto et al., 1995). GeLC-MS/MS data, together with previous identifications of putative homologues of cell-cycle gene in G. mosseae and G. intraradices (Requena et al., 2000; Jun et al., 2002), suggest that signalling pathways known in model species may also operate in AM fungi. Overall, this GeLC-MS/MS strategy opens the way towards analysing at large-scale fungal responses to environmental cues on the basis of quantitative shotgun protein-profiling experiments. The authors are grateful to Audrey Geairon and Franck Robert for extensive technical assistance and to Benoît Schoefs, Benoît Valot and Natalia Requena for critical reading and helpful suggestions. Fig. S1 Subcellular location of the proteins identified in the ERM of Glomus intraradices following GeLC-MS/MS, as determined using Swiss-Prot/TrEMBL annotations or Wolf PSORT predictions. Fig. S2 Biological process grouping of the proteins identified in the ERM of Glomus intraradices following GeLC-MS/MS according to the MIPS Functional Catalogue Database. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.