A Medicago truncatula Tobacco Retrotransposon Insertion Mutant Collection with Defects in Nodule Development and Symbiotic Nitrogen Fixation    

Abstract

A Tnt1-insertion mutant population of Medicago truncatula ecotype R108 was screened for defects in nodulation and symbiotic nitrogen fixation. Primary screening of 9,300 mutant lines yielded 317 lines with putative defects in nodule development and/or nitrogen fixation. Of these, 230 lines were rescreened, and 156 lines were confirmed with defective symbiotic nitrogen fixation.

Validating Genome-Wide Association Candidates Controlling Quantitative Variation in Nodulation

“…4, A-C). The importance of replication also helps to explain why the genes we identified had not been detected in earlier forward-genetic screens conducted on nodulation in M. truncatula (Catoira et al, 2000;Oldroyd and Long, 2003;Schnabel et al, 2005;Pislariu et al, 2012;Domonkos et al, 2013). If the mutations we evaluated had been tested in only a single individual, the mutant phenotype would likely have fallen into the range of phenotypes exhibited by wild-type plants generally and thus would not have been picked up in a standard genetic screen.…”

“…To identify mutants in the 10 candidates selected for validation, we first searched for Tnt1 insertional mutants using the Noble Foundation's Medicago truncatula Mutant Database (http://medicago-mutant.noble.org/mutant/ blast/blast.php; Tadege et al, 2008;Pislariu et al, 2012). Searches of this database, which is comprised of more than 21,000 mutants, identified insertions in five of the seven candidates with the remaining two candidates identified by a PCR reverse screen performed at the Noble Foundation (Supplemental Table S1).…”

“…The ecological and economic importance of this symbiosis has made identifying its genetic basis an important goal for plant biologists . More than 30 plant genes that play central roles in the formation and growth of plant nodules, the site of rhizobial symbiosis, and nitrogen fixation have been identified (Popp and Ott, 2011;Pislariu et al, 2012). These genes were identified primarily through forward genetic screens.…”

“…Here, we report on the application of three reverse genetics mutagenesis tools: Medicago truncatula Tnt1 retrotransposon mutant collection (Tadege et al, 2008;Pislariu et al, 2012), hairpin RNA interference knockdown constructs (Wesley et al, 2001), and CRISPR/Cas9 site-specific nuclease (SSN; Baltes et al, 2014) to empirically evaluate the functional importance of 10 genes that a previous sequence-based GWA study identified as candidates for naturally occurring phenotypic variation in the legume-rhizobia symbiosis (Stanton-Geddes et al, 2013). We used 17 whole-plant, stable mutants (six Tnt1, three hairpin, and eight CRISPR mutants) in well-replicated phenotypic assays to evaluate the function of each of the 10 GWA candidates.…”

Lignin Modification Leads to Increased Nodule Numbers in Alfalfa

“…The wild-type (Wt1-Wt3) and HCT (HCT1-HCT3) antisense cuttings were transferred to small pots containing a sterile substrate of turface and vermiculite (2:1) and were subjected to nitrogen starvation for 6 d with a low-nutrient solution (Broughton and Dilworth, 1971). Five weeks after transplanting, each plant was inoculated with a 50-mL suspension (optical density at 600 nm = 0.03) of Sinorhizobium meliloti wild-type strain Sm1021, as described previously (Ardourel et al, 1994;Pislariu et al, 2012). No further fertilization was applied to inoculated plants prior to harvesting at 8 and 18 dpi, although plants were watered regularly with deionized water.…”