Genome-Wide Identification of Nodule-Specific Transcripts in the Model Legume Medicago truncatula  

Abstract

The Medicago truncatula expressed sequence tag (EST) database (Gene Index) contains over 140,000 sequences from 30 cDNA libraries. This resource offers the possibility of identifying previously uncharacterized genes and assessing the frequency and tissue specificity of their expression in silico. Because M. truncatula forms symbiotic root nodules, unlike Arabidopsis, this is a particularly important approach in investigating genes specific to nodule development and function in legumes. Our analyses have revealed 340 putative gene products, or tentative consensus sequences (TCs), expressed solely in root nodules. These TCs were represented by two to 379 ESTs. Of these TCs, 3% appear to encode novel proteins, 57% encode proteins with a weak similarity to the GenBank accessions, and 40% encode proteins with strong similarity to the known proteins. Nodule-specific TCs were grouped into nine categories based on the predicted function of their protein products. Besides previously characterized nodulins, other examples of highly abundant nodule-specific transcripts include plantacyanin, agglutinin, embryo-specific protein, and purine permease. Six nodule-specific TCs encode calmodulin-like proteins that possess a unique cleavable transit sequence potentially targeting the protein into the peribacteroid space. Surprisingly, 114 nodule-specific TCs encode small Cys cluster proteins with a cleavable transit peptide. To determine the validity of the in silico analysis, expression of 91 putative nodule-specific TCs was analyzed by macroarray and RNA-blot hybridizations. Nodule-enhanced expression was confirmed experimentally for the TCs composed of five or more ESTs, whereas the results for those TCs containing fewer ESTs were variable. , and organ development (Ruan et al., 1998;Girke et al., 2000; Zhu and Wang, 2000). Although Arabidopsis serves as the model system for most plant processes, it suffers from two major weaknesses in consideration of plant-microbe interactions: the absence of symbiotic associations with mycorrhizal fungi and with rhizobia.In recent years, Medicago truncatula and Lotus japonicus have emerged as model systems for genomic approaches to plant-microbe symbiotic associations (Barker et al., 1990;Handberg and Stougaard, 1992; Cook et al., 1997; Cook, 1999;Oldroyd and Geurts, 2001;Thoquet et al., 2002). Both species possess small genomes, are diploid, have fast generation times, and can be transformed with Agrobacterium tumefaciens and regenerated (Handberg and Stougaard, 1992; Blondon et al., 1994;Handberg et al., 1994; Chabaud et al., 1996;Jiang and Gresshoff, 1997;Stiller et al., 1997;Trinh et al., 1998;Trieu et al., 2000). Currently, both functional and structural genomics approaches are being pursued within each of these species. Covitz et al. (1998) reported the sequencing of about 900 cDNA tags from the M. truncatula root hairs. In addition, hundreds more expressed sequence tags (ESTs) have been isolated and characterized from effective root nodules of L. japonicus and M. truncatula, and a ...

Bridging Model and Crop Legumes through Comparative Genomics

“…These results suggested that at least some of the genes involved in nodulation are evolved from broadly conserved pathways of plant development (Parniske and Downie, 2003;Szczyglowski and Amyot, 2003). This view was further supported by Fedorova et al (2002), who identified 340 tentative consensus sequences with nodule-specific expression patterns, approximately 40% of which shared sequence homology to sequences from non-legumes. It will be a challenging task to undertake the discovery and dissection of the function of the genes that are required for nodulation but are conserved across plant families (e.g.…”

“…Motif analysis identified three major gene families from this putative legume-specific gene set, including F-box-related proteins, Pro-rich proteins, and Cys cluster proteins. In particular, the more than 300 Cys cluster proteins, with predicted similarity to defensins, represent approximately 1% of expressed genes in Medicago, primarily from nodules (Fedorova et al, 2002;Mergaert et al, 2003;Graham et al, 2004). Work is under way to characterize these putative legumespecific genes using both forward and reverse genetic tools (K. VandenBosch, personal communication).…”

Aging in Legume Symbiosis. A Molecular View on Nodule Senescence in Medicago truncatula

“…Of the total collection of tags, only 56% were represented in the MTGI by ESTs. Strikingly, for the tags linked to an ESTcluster (tentative consensus sequences), only 23% of them had a corresponding EST in the senescent nodule library (Fedorova et al, 2002), indicating that this library is only a limited representation of the nodule senescence transcriptome. The gene tags were grouped into 19 functional classes derived from the Medicago EST Navigation System (Journet et al, 2002;Fig.…”

Summaries of Legume Genomics Projects from around the Globe. Community Resources for Crops and Models

“…We sorted TCs to identify predicted genes with particular expression patterns, based on the libraries of origin of the ESTs in the TCs. For example, Fedorova et al (2002) identified nodulespecific expressed genes, including some unique classes of genes. We have recently released MtDB, a new database that clusters all public M. truncatula ESTs into about 17,000 contigs plus a similar number of singletons (Lamblin et al, 2003; http://www.…”

“…Some of these sequences appear to be members of gene families, based on clustering analyses. One spectacular example is a group of more than 300 putatively secreted proteins that are Cys rich and were previously identified as nodule specific (Fedorova et al, 2002). Despite the lack of apparent sequence similarity of leguminosins to known sequences from other organisms, motif searching has provided clues to the functions for some members of this group.…”