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.
Highlights
The formation of functional symbiotic nodules is the result of a coordinated developmental program between legumes and rhizobial bacteria
Plant recognition of Nodulation (Nod) factors produced by the rhizobial partner activates many of the developmental processes in the plant associated with nodule formation [3]
Identification of new nodulation-defective M. truncatula mutants In order to identify additional genetic loci required for symbiotic nitrogen fixation, a large-scale forward genetic screen was undertaken with fast neutron bombarded and ethyl methane sulfonate (EMS) mutagenized M. truncatula jemalong populations
Summary
Identification of new nodulation-defective M. truncatula mutants In order to identify additional genetic loci required for symbiotic nitrogen fixation, a large-scale forward genetic screen was undertaken with fast neutron bombarded and ethyl methane sulfonate (EMS) mutagenized M. truncatula jemalong populations. The nodules of 7Y showed extremely low bacterial occupancy; bacteria could be detected only in a few cells of the infection zone and only a few infection threads were present in the nitrogen fixation zone (Figure 3D). Nodules of wild type plants showed characteristic bacteroid differentiation (Figure 4A-C), with the cells in the interzone and nitrogen fixation zone fully occupied by elongated bacteria (Figure 4B and C). MtNAP2 was expressed at lower level in nodules containing some infected cells in the nitrogen fixation zone but the complete failure of its induction was detected in ipd, dnf, dnf and dnf is related to the absence of nodule zonation or lack of bacterial occupancy in the nitrogen fixation zone (Figure 3). The expression data of the symbiotic marker genes were consistent with the block of the symbiotic nodule development detected by microscopic analysis
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