The identification of novel loci required for appropriate nodule development in Medicago truncatula

Abstract

BackgroundThe formation of functional symbiotic nodules is the result of a coordinated developmental program between legumes and rhizobial bacteria. Genetic analyses in legumes have been used to dissect the signaling processes required for establishing the legume-rhizobial endosymbiotic association. Compared to the early events of the symbiotic interaction, less attention has been paid to plant loci required for rhizobial colonization and the functioning of the nodule. Here we describe the identification and characterization of a number of new genetic loci in Medicago truncatula that are required for the development of effective nitrogen fixing nodules.ResultsApproximately 38,000 EMS and fast neutron mutagenized Medicago truncatula seedlings were screened for defects in symbiotic nitrogen fixation. Mutant plants impaired in nodule development and efficient nitrogen fixation were selected for further genetic and phenotypic analysis. Nine mutants completely lacking in nodule formation (Nod-) represented six complementation groups of which two novel loci have been identified. Eight mutants with ineffective nodules (Fix-) represented seven complementation groups, out of which five were new monogenic loci. The Fix- M. truncatula mutants showed symptoms of nitrogen deficiency and developed small white nodules. Microscopic analysis of Fix- nodules revealed that the mutants have defects in the release of rhizobia from infection threads, differentiation of rhizobia and maintenance of persistence of bacteria in nodule cells. Additionally, we monitored the transcriptional activity of symbiosis specific genes to define what transcriptional stage of the symbiotic process is blocked in each of the Fix- mutants. Based on the phenotypic and gene expression analysis a functional hierarchy of the FIX genes is proposed.ConclusionsThe new symbiotic loci of M. truncatula isolated in this study provide the foundation for further characterization of the mechanisms underpinning nodulation, in particular the later stages associated with bacterial release and nodule function.

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.…”

Loss of the nodule-specific cysteine rich peptide, NCR169, abolishes symbiotic nitrogen fixation in the Medicago truncatula dnf7 mutant

“…1T). This observation indicates that although the bacterial nifH gene could be induced in dnf7-2 nodules, its expression was restricted and apparently insufficient for substantial nitrogen fixation (22,24).…”

“…The nitrogen fixation mutant dnf7-2 was identified in a genetic screen of a fast-neutron-bombarded population of M. truncatula cv. Jemalong (22). The dnf7-2 mutant showed symptoms of nitrogen starvation under symbiotic conditions ( Fig.…”

“…A previous study found that nitrogenase was inactive in dnf7 mutant nodules 18 d post inoculation (dpi) (22). To determine whether the nifH gene, coding for a subunit of the nitrogenase complex was expressed in the mutant, WT and dnf7-2 plants were inoculated with S. meliloti 1021 strain carrying the nifH promoter-GUS reporter gene fusion (PnifH::uidA) (24).…”

Translational Genomics in Agriculture: Some Examples in Grain Legumes

“…However, systematic mutant populations have been developed in several species in order to address this issue. TILLING populations are available for L. japonicus, pea, M. truncatula and chickpea (Dalmais et al, 2008;Le-Signor et al, 2009;Perry et al, 2003;Varshney et al, unpublished), Fast Neutron generated deletion mutant populations for M. truncatula, pea and soybean (Bolon et al, 2011;Hofer et al, 2009;Rogers et al, 2009;Smykal et al, 2012) and insertion mutant populations generated using exogenous (Medicago, d 'Erfurth et al, 2003) and endogeneous retrotransposons (Lotus, Urbański et al, 2012) have been generated and have contributed to gene discovery in these species (see Domoney et al, 2013;Domonkos et al, 2013;Urbański et al, 2013).…”