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The interaction between legumes and their microbial partners, termed rhizobia, results in the formation of the symbiotic nodule, wherein rhizobia convert the atmospheric nitrogen into ammonia. Hundreds of genes are specifically induced in the symbiotic nodule, but the role of many of them is still unclear. One of the most effective approaches to studying the function of genes is the analysis of mutants defective in the process. However, mutant plants are not available for many genes, but they can be generated using CRIPSR/Cas9 gene editing. We have targeted several nodule-specific genes by gene editing in the model legume Medicago truncatula., including the Nodulin-25 (MtNod25), which is expressed in the later stages of nodule development. The Nod25 gene was identified decades ago, but its function still to be discovered. We generated several mutant alleles of MtNod25, including one with a large deletion at the beginning of the gene (Figure 1), which likely resulted in a loss-of-function allele. We have analyzed the symbiotic phenotype of Mtnod25 mutant plants, alongside another ineffective symbiotic mutant (dnf7-2) and wild-type controls 3 weeks post inoculation with the compatible rhizobia strain Sinorhizobium meliloti WSM419. To our surprise, we found that plants impaired in MtNod25 showed no symptoms of nitrogen starvation under symbiotic conditions. The symbiotic nodules of Mtnod25 mutant plants were colonized by rhizobia in all zones of indeterminate nodules, as in the wild type, detected by fluorescent confocal imaging and scanning electron microscopy (Figure 2).    

These results indicated that although MtNod25 is expressed in the symbiotic nodules, it is not required for effective symbiotic nitrogen fixation.  

 

Contributors: Péter Kaló, (MATE, Hun-REN BRC, Szeged), János Barnabás Biró (Hun-REN BRC, Szeged), Ágota Domonkos (MATE), Berivan Güngör (MATE) and Elmira Mohammadi (MATE). Scanning electron microscope images were prepared by Attila Farkas (Functional Cell Biology and Immunology Advanced Core Facility (FCBI-ACF), Hungarian Centre of Excellence for Molecular Medicine (HCEMM), University of Szeged, Szeged, Hungary. 

Dr. Péter Kaló 

Research Excellence Programme 

Figure 1. The position of the guide RNA targeting MtNod25 sequence in exon 1. The structure of wild-type and gene-edited mutant version of the MtNod25 gene. Mtnod25 mutant plant shows a similar gross phenotype as the wild-type (wt) but dnf7-2 mutant defective in a late nodulin gene, NCR169, displayed retarded growth. Elongated and pink, leghemoglobin containing nodules are formed on the roots of wt and Mtnod25 mutant plants. Ineffective, round shaped and white nodules are formed on dnf7-2.   

Figure 2. Nodule sections were stained with fluorescent dye SYTO13 to detect rhizobia and analyzed by confocal microscopy. Wild-type and Mtnod25 mutant nodules showed the characteristic zonation of indeterminate nodules and nodules cells in the interzone and nitrogen fixation zone were fully occupied by rhizobia indicating that MtNod25 is not essential for symbiosis between Medicago truncatula and Sinorhizobium medicae WSM419. dnf7-2 mutant nodules did not contain infected cells in the region corresponding to the nitrogen fixation zone of wild-type plants. Nodules are shown on the left and higher magnification of nodule cells on the right of each nodule. ZII: infection zone, IZ, interzone, ZIII: nitrogen fixation zone 

Scanning electron microscopy analysis of symbiotic cells and bacteroid morphology in wt, Mtnod25 and dnf7-2 mutant nodules (two lower panels). Close up images of nodule cells from the nitrogen fixation zones of wt and Mtnod25 nodules showed that elongated bacteroids, orientated towards the vacuoles, completely fill up infected cells, but the cell in the dnf7-2 mutant nodule contained aggregated bacteroids, often tangled with cell debris, indicating the defect of the function of symbiotic nodule. ZIII: nitrogen fixation zone