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Open Access Research article

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

Agota Domonkos1, Beatrix Horvath1, John F Marsh2, Gabor Halasz1, Ferhan Ayaydin3, Giles ED Oldroyd2 and Peter Kalo1*

  • * Corresponding author: Peter Kalo kalo@abc.hu

  • † Equal contributors

Author Affiliations

1 Agricultural Biotechnology Center, Gödöllő 2100, Hungary

2 John Innes Centre, Norwich NR4 7UH, UK

3 Cellular Imaging Laboratory, Biological Research Center, Szeged 6726, Hungary

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BMC Plant Biology 2013, 13:157  doi:10.1186/1471-2229-13-157

Published: 11 October 2013

Abstract

Background

The 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.

Results

Approximately 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.

Conclusions

The 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.

Keywords:
Medicago truncatula; Legume; Symbiosis; Mutant screen; Ineffective nitrogen fixation mutant