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Top 5 Resources for Medicago Researchers

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My Ph.D. supervisor once told me of academia, "It is easy to try and pursue individual glory, but we only make progress as a community." Indeed, a lot of exciting discoveries are based on prior research and international efforts that provide groundwork, be it to answer fundamental biological questions or tackle global agricultural challenges.

In legume research, as well, advances often rely on joint efforts to study a few "model" species that are amenable to genetic modifications and are easy to handle in the laboratory.

Medicago truncatula, or the barrel medic, is a sister species of the forage legume alfalfa (Medicago sativa). M. truncatula is used extensively as a model organism to investigate, amongst other things, interactions with beneficial soil microbes that help plants acquire nutrients such as nitrogen and phosphorous from the environment.

In an attempt to consolidate the resources available and to introduce these existing tools to those just starting out with research on this model legume, I've compiled a list of what I see as the top five resources made by and for the Medicago research community.

  1 Medicago Gene Expression and Proteomic Atlas

If you're a researcher in the life sciences, you've probably heard this at some point in your career: "Good in silico analyses can save you a lot of time and effort at the bench!" Undeniably, analyzing gene expression, gene structure, homology and co-regulation can provide many clues toward the function of a gene and thereby help design informed experiments.

Medicago researchers can access transcriptomic data at mtgea.noble.org/v3 which curates more than 250 datasets for 50,000 genes ranging from plant organs to infections with symbionts and pathogens (1)! However, the gene chip currently in use provides information for just 80 percent of the known Medicago genes omitting very short genes and is often unable to distinguish between highly homologous genes.

RNAseq data provides a viable alternative to overcome these challenges as we await the newest release of the Medicago Gene Chip. An expression atlas for all published RNAseq data will soon be made available at mtsspdb.noble.org/project/exp.

Finally, with constant synthesis and degradation of mRNA in response to external cues the steady-state levels of a cellular protein can often be wrongly estimated by looking at mRNA expression levels alone. A proteomic atlas for Medicago and its bacterial partner Sinorhizobium meliloti can be accessed at compendium.medicago.wisc.edu. This allows the user to explore protein abundance and identify associated post translational modifications (2).

Gene Expression AtlasCustomizable output for the Medicago truncatula Gene Expression Atlas provides expression data for probesets of interest.

  2 Tnt1 Insertional Mutant Repository

Mutagenesis is a powerful tool to create genetic diversity in the absence of existing variation and to link gene expression with function. To generate mutants in Medicago truncatula, the tobacco retrotransposon Tnt1 was used to transform the ecotype R108 by scientists at the Noble Research Institute (3). Tnt1 transposition is only active during callus induction thereby causing insertional mutations but is otherwise stable during seed propagation. The genes that are interrupted can be identified by sequencing the ends or the "flanks" of the Tnt1 insertions. These "flanking sequence tags" and the affected genes are available to BLAST in a searchable database at medicago-mutant.noble.org/mutant. To date, 20,000 mutant lines have been generated with 400,000 insertions in the genome! It is important to remember to use the R108 sequence of your gene of interest to get accurate matches. Custom requests can also be made by emailing Jiangqi Wen, Ph.D., at jwen@noble.org.

Wild Type flowerWild Type flowers (top left) compared to mutants identified in community screenings of the Tnt1 insertion lines in Medicago truncatula. Photo courtesy of Jiangqi Wen, Ph.D.

  3 Medicago HapMap (Haplotype Mapping) Collection

Differential ecological distribution and adaptation to diverse climactic conditions even within a species is often caused by genetic variations. They might include underlying SNPs (Single Nucleotide Polymorphisms) INDELs (Insertions/Deletions) or Copy Number Variations (CPVs). At least 262 inbred Medicago truncatula lines collected mostly from the Mediterranean region have been sequenced and their corresponding SNPs are available for Genome Wide Association Studies (GWAS) (4). GWAS is useful to associate genetic markers with desirable traits. The total number of lines is predicted to reach 384, and sequencing for marker information is currently underway. Marker information can be downloaded from medicagohapmap.org, and seeds can be requested by filling out the form on the given website.

HapMap linesOngoing genome-wide association studies (GWAS) using Medicago truncatula HapMap lines conducted by Yun Kang, Ph.D.

  4 Medicago Peptide Library

Since their discovery in plants, peptide hormones have been found to influence diverse growth programs and control a plant's response to its environment. Recent research shows there are not only the nine traditional classes of hormones but at least 1,800 such peptide hormones within a single species (5). Since these peptides tend to be short, they can be synthesized with considerable ease. Additionally, they tend to be active at nanomolar concentrations and therefore can be used to set up a quick moderately sized experiment even with a few microliters of the re-suspended synthetic peptide. The synthetic peptide library housed at the Noble Research Institute is sponsored through a grant (#1444549) made by the National Science Foundation and currently consists of 150 post-translationally modified and unmodified peptides. The number of peptides in the library is continuously growing and is estimated to reach about 400. Peptide aliquots are available upon request through Wolf Scheible, Ph.D., at wrscheible@noble.org. More information can be found at mtsspdb.noble.org.

Peptide treatmentsPeptide treatments cause changes in root architecture. Photo courtesy of Shulan Zhang and Wolf Scheible, Ph.D.

  5 Medicago Fluorescent Protein-tagged Lines for Cell Biology

The subcellular location of a protein often provides valuable information about its function. Furthermore, dynamic changes in organelles and cellular compartments in response to microbial accommodation or invasion provide a lot of information about the process. Two groups have generated an extensive collection of stable Medicago truncatula lines and constructs for detection of intracellular components (6, 7). These include markers for the plasma membrane, actin, microtubules, plastids, golgi network, etc., driven by the Arabidopsis UBIQUITIN promoter or the 35S CaMV promoter for strong constitutive expression. These lines can be requested by emailing Maria Harrison, Ph.D., at mjh78@cornell.edu or Paul A. Nakata, Ph.D., at paul.nakata@ars.usda.gov.

  1. V. A. Benedito et al., A gene expression atlas of the model legume Medicago truncatula. Plant Journal 55, 504-513 (2008).
  2. H. Marx et al., A proteomic atlas of the legume Medicago truncatula and its nitrogen-fixing endosymbiont Sinorhizobium meliloti. Nat Biotechnol 34, 1198-1205 (2016).
  3. M. Tadege et al., Large-scale insertional mutagenesis using the Tnt1 retrotransposon in the model legume Medicago truncatula. Plant Journal 54, 335-347 (2008).
  4. S. J. Curtin et al., Validating genome-wide association candidates controlling quantitative variation in nodulation. Plant physiology 173, 921-931 (2017).
  5. T. de Bang et al., Genome-wide Identification of Medicago Peptides involved in Macronutrient Responses and Nodulation. Plant Physiol (2017).

Sonali Roy, Ph.D.
Former Postdoctoral Fellow