SignificanceFixed nitrogen is essential for plant growth. Some plants, such as legumes, can host nitrogen-fixing bacteria within cells in root organs called nodules. Nodules are considered to have evolved in parallel in different lineages, but the genetic changes underlying this evolution remain unknown. Based on gene expression in the nitrogen-fixing nonlegume Parasponia andersonii and the legume Medicago truncatula, we find that nodules in these different lineages may share a single origin. Comparison of the genomes of Parasponia with those of related nonnodulating plants reveals evidence of parallel loss of genes that, in legumes, are essential for nodulation. Taken together, this raises the possibility that nodulation originated only once and was subsequently lost in many descendant lineages.
30Rhizobium nitrogen-fixing nodules are a well-known trait of legumes, but nodules also occur 31 in other plant lineages either with rhizobium or the actinomycete Frankia as microsymbiont.
32The widely accepted hypothesis is that nodulation evolved independently multiple times, with 33 only a few losses. However, insight in the evolutionary trajectory of nodulation is lacking. We 34 conducted comparative studies using Parasponia (Cannabaceae), the only non-legume able This finding challenges a long-standing hypothesis on evolution of nitrogen-fixing symbioses,
42and has profound implications for translational approaches aimed at engineering nitrogen-43 fixing nodules in crop plants.
Summary
In the nodules of IRLC legumes, including Medicago truncatula, nitrogen‐fixing rhizobia undergo terminal differentiation resulting in elongated and endoreduplicated bacteroids specialized for nitrogen fixation. This irreversible transition of rhizobia is mediated by host produced nodule‐specific cysteine‐rich (NCR) peptides, of which c. 700 are encoded in the M. truncatula genome but only few of them have been proved to be essential for nitrogen fixation.
We carried out the characterization of the nodulation phenotype of three ineffective nitrogen‐fixing M. truncatula mutants using confocal and electron microscopy, monitored the expression of defence and senescence‐related marker genes, and analysed the bacteroid differentiation with flow cytometry. Genetic mapping combined with microarray‐ or transcriptome‐based cloning was used to identify the impaired genes.
Mtsym19 and Mtsym20 mutants are defective in the same peptide NCR‐new35 and the lack of NCR343 is responsible for the ineffective symbiosis of NF‐FN9363. We found that the expression of NCR‐new35 is significantly lower and limited to the transition zone of the nodule compared with other crucial NCRs. The fluorescent protein‐tagged version of NCR343 and NCR‐new35 localized to the symbiotic compartment.
Our discovery added two additional members to the group of NCR genes essential for nitrogen‐fixing symbiosis in M. truncatula.
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