Expression Profiling In The European Union Genome Project Medicago Construction Of Arrays Representing The M Truncatula Root Interaction Transcriptome

P. Gamas1, J. Denarie1, L. Brechenmacher2, D. van Tuinen2, V. Gianinazzi-Pearson2,

M. Crespi3, P. Mergaert3, A. Kondorosi3, F. Krajinski4, P. Franken5, A. Pühler6,

^BMPRM, CNRS-INRA, Toulouse, France; 2UMR BBCE-IPM, CMSE-INRA, Dijon, France; 3ISV, CNRS, Gif-sur -Yvette, France; 4LG Molekulargenetik, Universität Hannover, Germany; 5MPI, Marburg, Germany; 6Lehrstuhl fur Genetik, Universität Bielefeld, Germany; 7Zentrum für Genomforschung, Bielefeld, Germany; 8IIT GmbH, Bielefeld, Germany

During the past years, Medicago truncatula emerged as a model legume to study plant-microbe interactions as well as general aspects of plant biology (Cook, Denarie 2000). Thus, this legume is currently analyzed in detail in several international genome projects in the US as well as in Europe (Harris, Frugoli 2001). In order to identify genes expressed in specific symbiotic or pathogenic interactions of this legume as well as genes relevant for the development of plant organs, e.g. flowers, seeds and leaves, expression profiling experiments are beginning to be performed on a larger scale. These experiments capitalize on the ever-increasing collection of ESTs available in public databases, e.g. the TIGR Medicago truncatula Gene Index (http://www.tigr.org/tdb/mtgi/).

Eight European labs participate in the expression profiling part of the European Union genome project MEDICAGO. This project is based on a collection of-15,000 ESTs obtained in the Toulouse and Dijon labs (http://sequence.toulouse.inra.fr/Mtruncatula.html) that cluster into -6000 different groups. This collection representing roots, young nodules and mycorrhiza was enriched by ESTs from roots interacting with pathogenic fungi and by ESTs from an SSH library of mycorrhizal roots. We are currently generating a 6k macroarray (nylon filter) of this root interaction transcriptome (Mt6k-RIT) that also includes well-studied M. truncatula marker genes and non-Medicago control ESTs. Preceding the Mt6k-RIT production, a small 96 clone array containing a representative collection of the root interaction transcriptome was constructed (MtSTAMP) that will serve for controlling probe labeling and for setting up experimental conditions. Both the MtSTAMP and the Mt6k-RIT array are to be made available to the Medicago community after initial experiments have been performed within the MEDICAGO Consortium.

Using the MtSTAMP and the Mt6k-RIT array, we will carry out expression profiling experiments in order to identify genes differentially regulated during symbiotic or pathogenic root interactions as well as during different physiological conditions. We will thus set up a database linking EST sequence with expression data. In the meantime, we started to use this EST collection to establish experimental conditions for transcriptome analysis on microarrays (glass slides). During the project, the Mt6k-RIT set will be enriched with ESTs obtained from different M. truncatula organs or tissues, e.g. roots, nodules under drought and saline stress, roots challenged with pathogenic fungi or treated with Nod factors, flowers and seeds. These ESTs either stem from conventional or from SSH cDNA libraries. After normalization, microarrays (glass slides) will be produced carrying a non-redundant set of ESTs representing the M. truncatula transcriptome from all organs or conditions studied. The results of expression profiling will be entered into a database for a global comparison of regulation patterns of the genes identified in different organs or conditions analyzed.

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