Perpectives

In our earlier studies, 41 proteins were found newly induced in total extracts of G. mosseae-colonised roots (1, 2). This number is significantly increased when compared to our previous reports for other plant species in interaction with the same strain of AM (3, 4). This has to be related to the better ability of separation and higher resolution on immobilised pH gradients (IPG) combined with the use of the model plant species, M. truncatula which appears particularly suitable for proteomic analysis. However, new proteins related to AM symbiosis are certainly still highly

Sub-fractionation into soluble and membrane proteins has already proved to be very useful for identifying more protein modifications in response to AM symbiosis (5, 6). Proteomics has appeared particularly relevant for studying proteins expressed in particular organelles such as the symbiosome in the rhizobial interaction (7-10). Although the AM symbiosome is much less accessible than that of nodules, further developments combining enrichments in arbuscule-containing cells, optimization of solubilization of membrane proteins (11) and/or development of multidimensional protein identification technology (MudPIT) (12, 13) will bring more insight into the identification of mycorrhiza-specific proteins. Finally, the use of the highest sensitive fluorescent stains compatible with MS analysis (14) could also improve the capacity for proteome identification in the AM symbiosis.

Based on the use of in vitro inoculated Ri T-DNA transformed roots, AM proteomics has been initiated on axenically produced biological material leading up to now to a limited number of protein identifications (15). For the fungus part, the proteomic task will now be easier, in relation particularly to an international Glomus intraradices genome-sequencing project (16).

Efforts are needed in order to focus on more precise stages of the symbiosis. This plant/fungus interaction is typically characterised by the asynchronous occurrence of all stages of the symbiosis, rendering it difficult to identify protein expression patterns specific to any of the different developmental steps. At least two stages can be more accurately studied: (i) the full symbiosis with numerous arbuscules, (ii) the recognition step, with the appressoria formation, considered as the first cellular contact between the two partners (17, 18). It was already stressed above that obtaining subcellular fractions corresponding to arbuscule-containing cells would allow identifying proteins expressed in and around the arbuscules. Another strategy relates to the use of plant mutants deficient for the AM symbiosis (19, 20) This has already been successfully applied both at the protein (3) and gene levels (21, 22). A proteome analysis of differentially displayed proteins in early plant mutants inoculated or not with an AM fungus will help in the identification of proteins involved in the appressoria formation. Similarly, studies of differential protein expression following AM fungal inoculation of late mutants, in comparison to wild type genotypes, will strongly support the identification of proteins essential to the AM symbiosis.

[1] Bestel-Corre, G., Dumas-Gaudot E., Poinsot V., Dieu M., Dierick J. F., van Tuinen D., Remacle J., Gianinazzi-Pearson V., and Gianinazzi S. 2002. Proteome analysis and identification of symbiosis-related proteins from Medicago truncatula Gaertn. by two-dimensional electrophoresis and mass spectrometry. Electrophoresis 23:122-137

[2] Bestel-Corre, G., Dumas-Gaudot E., and Gianinazzi S. 2004. Proteomics as a tool to monitor plant-microbe endosymbioses in the rhizosphere. Mycorrhiza 14:1-10

[3] Samra, A., Dumas-Gaudot E., and Gianinazzi S. 1997. Detection of symbiosis-related polypeptides during the early stages of the establishment of arbuscular mycorrhiza between Glomus mosseae and Pisum sativum roots. New Phytol. 135:711-722

[4] Dassi, B., Samra A., DumasGaudot E., and Gianinazzi S. 1999. Different polypeptide profiles from tomato roots following interactions with arbuscular mycorrhizal (Glomus mosseae) or pathogenic (Phytophthora parasitica) fungi. Symbiosis 26:65-77

[5] Benabdellah, K., Azcon-Aguilar C., and Ferrol N. 1998. Soluble and membrane symbiosis-related polypeptides associated with the development of arbuscular mycorrhizas in tomato (Lycopersicon esculentum). New Phytol 140:135-143

[6] Benabdellah, K., Azcon-Aguilar C., and Ferrol N. 2000. Alterations in the plasma membrane polypeptide pattern of tomato roots (Lycopersicon esculentum) during the development of arbuscular mycorrhiza. J Exp Bot 51:747-754

[7] Panter, S., Thomson R., de Bruxelles G., Laver D., Trevaskis B., and Udvardi M. 2000. Identification with proteomics of novel proteins associated with the peribacteroid membrane of soybean root nodules. 13:325-33

[8] Saalbach, G., Erik P., and Wienkoop S. 2002. Characterisation by proteomics of peribacteroid space and peribacteroid membrane preparations from pea (Pisum sativum) symbiosomes. 2:325-337

[9] Wienkoop, S., and Saalbach G. 2003. Proteome analysis. Novel proteins identified at the peribacteroid membrane from Lotus japonicus root nodules. 131:1080-1090

[10] Catalano, C. M., Lane W. S., and Sherrier D. J. 2004. Biochemical characterization of symbiosome membrane proteins from Medicago truncatula root nodules. Electrophoresis 25:519-531

[11] Santoni, V., Kieffer S., Desclaux D., Masson F., and Rabilloud T. 2000. Membrane proteomics: use of additive main effects with multiplicative interaction model to classify plasma membrane proteins according to their solubility and electrophoretic properties. 21:3329-3344

[12] Whitelegge, J. P. 2002. Plant proteomics: BLASTing out of a MudPIT. 99:11564-11566

[13] Wu, C. C., MacCoss M. J., Howell K. E., and Yates J. R. 2003. A method for the comprehensive proteomic analysis of membrane proteins. 21:532-538

[14] Lopez, M. F., Berggren K., Chernokalskaya E., Lazarev A., Robinson M., and Patton W. F. 2000. A comparison of silver stain and SYPRO Ruby Protein Gel Stain with respect to protein detection in two-dimensional gels and identification by peptide mass profiling. Electrophoresis 21:3673-3683

[15] Dumas-Gaudot, E., Valot B., Bestel-Corre G., Recorbet G., St-Arnaud M., Fontaine B., Dieu M., Raes M., Saravanan R. S., and Gianinazzi S. 2004. Proteomics as a way to identify extra-radicular fungal proteins from Glomus intraradices - RiT-DNA carrot root mycorrhizas. 48:401-411

[16] Martin, F., Tuskan G. A., DiFazio S. P., Lammers P., Newcombe G., and Podila G. K. 2004. Symbiotic sequencing for the Populus mesocosm. New Phytol 161:330-335

[17] Giovannetti, M., Sbrana C., Avio L., Citernesi A. S., and Logi C. 1993. Differential hyphal morphogenesis in arbuscular mycorrhizal fungi during pre-infection stages. New Phytol. 125:587-594

[18] Nagahashi, G., and Douds D. D. 1997. Appressorium formation by AM fungi on isolated cell walls of carrot roots. New Phytologist 136:299-304

[19] Marsh, J. F., and Schultze M. 2001. Analysis of arbuscular mycorrhizas using symbiosis-defective plant mutants. New Phytologist 150:525-532

[20] Parniske, M. 2004. Molecular genetics of the arbuscular mycorrhizal symbiosis. 7:414-421

[21] Weidmann, S., Sanchez L., Descombin J., Chatagnier O., Gianinazzi S., and Gianinazzi-Pearson V. 2004. Fungal elicitation of signal transduction-related plant genes precedes mycorrhiza establishment and requires the dmi3 gene in Medicago truncatula. Mol. Plant-Microbe Interact. 17:1385-1393

[22] Brechenmacher, L., Weidmann S., van Tuinen D., Chatagnier O., Gianinazzi S., Franken P., and Gianinazzi-Pearson V. 2004. Expression profiling of up-regulated plant and fungal genes in early and late stages of Medicago truncatula-Glomus mosseae interactions. Mycorrhiza 14:253-262

[1] Bestel-Corre, G., Dumas-Gaudot E., Poinsot V., Dieu M., Dierick J. F., van Tuinen D., Remacle J., Gianinazzi-Pearson V., and Gianinazzi S. 2002. Proteome analysis and identification of symbiosis-related proteins from Medicago truncatula Gaertn. by two-dimensional electrophoresis and mass spectrometry. Electrophoresis 23:122-137

[2] Bestel-Corre, G., Dumas-Gaudot E., and Gianinazzi S. 2004. Proteomics as a tool to monitor plant-microbe endosymbioses in the rhizosphere. Mycorrhiza 14:1-10

[3] Samra, A., Dumas-Gaudot E., and Gianinazzi S. 1997. Detection of symbiosis-related polypeptides during the early stages of the establishment of arbuscular mycorrhiza between Glomus mosseae and Pisum sativum roots. New Phytol. 135:711-722

[4] Dassi, B., Samra A., DumasGaudot E., and Gianinazzi S. 1999. Different polypeptide profiles from tomato roots following interactions with arbuscular mycorrhizal (Glomus mosseae) or pathogenic (Phytophthora parasitica) fungi. Symbiosis 26:65-77

[5] Benabdellah, K., Azcon-Aguilar C., and Ferrol N. 1998. Soluble and membrane symbiosis-related polypeptides associated with the development of arbuscular mycorrhizas in tomato (Lycopersicon esculentum). New Phytol 140:135-143

[6] Benabdellah, K., Azcon-Aguilar C., and Ferrol N. 2000. Alterations in the plasma membrane polypeptide pattern of tomato roots (Lycopersicon esculentum) during the development of arbuscular mycorrhiza. J Exp Bot 51:747-754

[7] Panter, S., Thomson R., de Bruxelles G., Laver D., Trevaskis B., and Udvardi M. 2000. Identification with proteomics of novel proteins associated with the peribacteroid membrane of soybean root nodules. 13:325-33

[8] Saalbach, G., Erik P., and Wienkoop S. 2002. Characterisation by proteomics of peribacteroid space and peribacteroid membrane preparations from pea (Pisum sativum) symbiosomes. 2:325-337

[9] Wienkoop, S., and Saalbach G. 2003. Proteome analysis. Novel proteins identified at the peribacteroid membrane from Lotus japonicus root nodules. 131:1080-1090

[10] Catalano, C. M., Lane W. S., and Sherrier D. J. 2004. Biochemical characterization of symbiosome membrane proteins from Medicago truncatula root nodules. Electrophoresis 25:519-531

[11] Santoni, V., Kieffer S., Desclaux D., Masson F., and Rabilloud T. 2000. Membrane proteomics: use of additive main effects with multiplicative interaction model to classify plasma membrane proteins according to their solubility and electrophoretic properties. 21:3329-3344

[12] Whitelegge, J. P. 2002. Plant proteomics: BLASTing out of a MudPIT. 99:11564-11566

[13] Wu, C. C., MacCoss M. J., Howell K. E., and Yates J. R. 2003. A method for the comprehensive proteomic analysis of membrane proteins. 21:532-538

[14] Lopez, M. F., Berggren K., Chernokalskaya E., Lazarev A., Robinson M., and Patton W. F. 2000. A comparison of silver stain and SYPRO Ruby Protein Gel Stain with respect to protein detection in two-dimensional gels and identification by peptide mass profiling. Electrophoresis 21:3673-3683

[15] Dumas-Gaudot, E., Valot B., Bestel-Corre G., Recorbet G., St-Arnaud M., Fontaine B., Dieu M., Raes M., Saravanan R. S., and Gianinazzi S. 2004. Proteomics as a way to identify extra-radicular fungal proteins from Glomus intraradices - RiT-DNA carrot root mycorrhizas. 48:401-411

[16] Martin, F., Tuskan G. A., DiFazio S. P., Lammers P., Newcombe G., and Podila G. K. 2004. Symbiotic sequencing for the Populus mesocosm. New Phytol 161:330-335

[17] Giovannetti, M., Sbrana C., Avio L., Citernesi A. S., and Logi C. 1993. Differential hyphal morphogenesis in arbuscular mycorrhizal fungi during pre-infection stages. New Phytol. 125:587-594

[18] Nagahashi, G., and Douds D. D. 1997. Appressorium formation by AM fungi on isolated cell walls of carrot roots. New Phytologist 136:299-304

[19] Marsh, J. F., and Schultze M. 2001. Analysis of arbuscular mycorrhizas using symbiosis-defective plant mutants. New Phytologist 150:525-532

[20] Parniske, M. 2004. Molecular genetics of the arbuscular mycorrhizal symbiosis. 7:414-421

[21] Weidmann, S., Sanchez L., Descombin J., Chatagnier O., Gianinazzi S., and Gianinazzi-Pearson V. 2004. Fungal elicitation of signal transduction-related plant genes precedes mycorrhiza establishment and requires the dmi3 gene in Medicago truncatula. Mol. Plant-Microbe Interact. 17:1385-1393

[22] Brechenmacher, L., Weidmann S., van Tuinen D., Chatagnier O., Gianinazzi S., Franken P., and Gianinazzi-Pearson V. 2004. Expression profiling of up-regulated plant and fungal genes in early and late stages of Medicago truncatula-Glomus mosseae interactions. Mycorrhiza 14:253-262