Nodule-specific cysteine-rich peptide 343 is required for symbiotic nitrogen fixation in Medicago truncatula

Forward genetics and transcriptomic analyses identified NCR343, a nodule-specific cysteine-rich peptide required for bacteroid differentiation in Medicago truncatula nodules. Symbiotic interactions between legumes and rhizobia lead to the development of root nodules and nitrogen fixation by differentiated bacteroids within nodules. Differentiation of the endosymbionts is reversible or terminal, determined by plant effectors. In inverted repeat lacking clade legumes, nodule-specific cysteine-rich (NCR) peptides control the terminal differentiation of bacteroids. Medicago truncatula contains & SIM;700 NCR-coding genes. However, the role of few NCR peptides has been demonstrated. Here, we report characterization of fast neutron 2106 (FN2106), a symbiotic nitrogen fixation defective (fix(-)) mutant of M. truncatula. Using a transcript-based approach, together with linkage and complementation tests, we showed that loss-of-function of NCR343 results in impaired bacteroid differentiation and/or maintenance and premature nodule senescence of the FN2106 mutant. NCR343 was specifically expressed in nodules. Subcellular localization studies showed that the functional NCR343-YFP fusion protein colocalizes with bacteroids in symbiosomes in infected nodule cells. Transcriptomic analyses identified senescence-, but not defense-related genes, as being significantly upregulated in ncr343 (FN2106) nodules. Taken together, results from our phenotypic and transcriptomic analyses of a loss-of-function ncr343 mutant demonstrate an essential role of NCR343 in bacteroid differentiation and/or maintenance required for symbiotic nitrogen fixation.

Automated summary

Forward genetics and transcriptomic analyses identified NCR343 as a crucial nodule-specific cysteine-rich peptide in Medicago truncatula, playing a key role in the differentiation and/or maintenance of bacteroids. Loss-of-function of NCR343 resulted in impaired bacteroid differentiation and/or maintenance, as well as premature nodule senescence. Subcellular localization studies confirmed the colocalization of NCR343 with bacteroids in symbiosomes within infected nodule cells. Transcriptomic analyses revealed upregulation of senescence-related genes in ncr343 mutant nodules. Overall, these findings demonstrate the essential role of NCR343 in symbiotic nitrogen fixation.