A Nodule Protein Mystery: Deleting a Highly Expressed Legume Gene Leaves Nitrogen Fixation Untouched

Some of the most abundant genes in the world’s nitrogen-fixing root nodules have quietly kept their secrets for more than three decades. NOD25, first discovered in alfalfa in 1990 through differential hybridization between root and nodule cDNA libraries, is expressed at levels rivaling leghemoglobin itself, accounting together with two other transcripts for roughly one percent of the entire nodule transcriptome in Medicago. Its protein is shipped to the symbiosome, the membrane-bound compartment where rhizobial bacteria convert atmospheric nitrogen into ammonia for the plant. On every conventional measure, NOD25 looked like a linchpin of symbiotic nitrogen fixation. Yet a new study published in Plant Molecular Biology reports that when researchers deleted the gene using CRISPR/Cas9 genome editing in the model legume Medicago truncatula, the plants barely noticed. Nodules formed normally, bacteria differentiated on schedule, and nitrogen fixation proceeded at full capacity, leaving scientists with a striking evolutionary puzzle.

The legume-rhizobium symbiosis is one of biology’s most intricate cooperative arrangements. After mutual molecular recognition, soil bacteria called rhizobia attach to root hairs and travel down infection threads into the cells of a developing nodule. There, each bacterium is engulfed in a plant-derived membrane, becoming a symbiosome, and then differentiates into a bacteroid, the specialized form capable of reducing atmospheric nitrogen to ammonia. In temperate legumes such as Medicago, pea and clover, nodules are indeterminate, meaning they retain a persistent apical meristem that continuously generates new cells arranged in a spatial gradient of developmental zones. In the infection zone, rhizobia are released into host cells; in the narrow interzone, bacterial division halts and both partners undergo dramatic differentiation, including multiple rounds of genome multiplication without cell division; and in the nitrogen-fixing zone, elongated bacteroids packed around central vacuoles carry out the actual chemistry of nitrogen reduction under the microaerobic conditions maintained by leghemoglobin.

This elaborate program requires the coordinated activation of hundreds to thousands of genes, and the legume-specific transcripts induced during it were christened nodulins in 1980. Early nodulins are switched on before nitrogen fixation begins, while late nodulins dominate the mature, functioning nodule. NOD25 belongs firmly to the late category. In Medicago truncatula, the gene contains thirteen exons, and its expression is first detectable four days after rhizobial inoculation, rising sharply by seven days and remaining elevated throughout the nodule’s active life. Laser-capture microdissection coupled to RNA sequencing showed that MtNOD25 transcripts are most abundant in the interzone and nitrogen-fixing zone, precisely where bacteroid differentiation and nitrogen reduction occur. Quantitatively, its expression exceeds not only several nodule-specific cysteine-rich peptide genes but even that of leghemoglobin 1, long considered the benchmark of nodule abundance.

Structurally, NOD25-like proteins are unlike almost anything else in the plant proteome. They combine conserved N-terminal and C-terminal regions with a central core built from repetitive modules, each encoded by a separate exon, a genomic architecture that arose through exon shuffling. The N-terminal sequence functions as a signal peptide, demonstrated experimentally by the targeting of an MtNOD25-GFP fusion to the symbiosome and by immunogold staining in alfalfa nodules. Proteomic work had also identified MtNOD25 among the proteins of the symbiosome membrane fraction, the very interface between plant and bacterium. Everything about the gene’s expression pattern, localization and evolutionary conservation pointed toward an essential role in the symbiotic conversation between the two partners.

To find out what that role might be, a team at the HUN-REN Biological Research Centre in Szeged, Hungary, led by Péter Kaló, turned to CRISPR/Cas9 editing. Because no mutant alleles of NOD25 existed in any available insertion or deletion collections of Medicago truncatula, the researchers designed a guide RNA targeting the first exon and delivered the editing construct into roots via Agrobacterium rhizogenes-mediated hairy root transformation. From the edited roots they regenerated whole plants and recovered seeds from four independent mutant lines. Three of these, Mtnod25-1, Mtnod25-3 and Mtnod25-4, carried small insertions or deletions that introduced premature stop codons, truncating the protein before its modular core and C-terminal domain. The fourth, Mtnod25-2, was more dramatic: a 1,304-base-pair deletion removed most of the promoter and the first two exons, abolishing transcript production entirely and classifying the line as a true knockout.

What followed was a systematic search for any trace of a symbiotic defect, and the search came up empty. Four weeks after inoculation with either Sinorhizobium meliloti or Sinorhizobium medicae, mutant plants were indistinguishable from wild type in growth habit, producing dark green leaves with no sign of nitrogen starvation and bearing elongated pink nodules of normal size and morphology. Spinning disc confocal microscopy of SYTO13-stained nodule sections revealed the characteristic zonation of mature indeterminate nodules, with infection, interzone and nitrogen-fixing zones all properly organized and densely colonized. Bacterial positioning and morphology matched wild type at every magnification. Scanning electron microscopy of high-pressure-fixed nodules confirmed that cells in the nitrogen-fixing zone were packed with elongated bacteroids oriented toward the central vacuoles, in sharp contrast to the dnf7-2 control mutant, which lacks the NCR169 peptide and whose nodules contain only undifferentiated rod-shaped bacteria and empty symbiotic cells.

The functional assays were equally unambiguous. Shoot dry weight, a standard proxy for symbiotic effectiveness, was essentially identical between Mtnod25 mutants, the Mtncr068 control line and wild-type plants inoculated with S. medicae, and the mutants even slightly outperformed wild type with S. meliloti. Under non-symbiotic conditions with supplemental nitrogen, all lines grew identically, confirming intact nitrogen assimilation. Finally, the team checked whether the loss of NOD25 might trigger hidden stress responses by measuring the expression of MtPR10, a pathogenesis-related marker, and MtCP2, a senescence-associated cysteine protease. Neither gene was activated in mutant nodules, unlike in the nad1-3 control mutant, in which defense-like and senescence pathways run unchecked. By every cellular, ultrastructural, physiological and molecular measure, symbiotic nitrogen fixation remained fully functional without NOD25.

The evolutionary analysis conducted alongside the mutagenesis deepens the mystery. NOD25-like sequences were found exclusively in the Hologalegina clade of cool-season legumes, and the modular repeat architecture of the central core is restricted to the Inverted Repeat-Lacking Clade, or IRLC, the same group of legumes characterized by terminal bacteroid differentiation and by the rapidly evolving family of secreted NCR peptides that drive that differentiation. Nine distinct repeat motifs were identified across the family, with striking clade specificity: Motifs 1 and 2 occur almost exclusively in the Vicioid clade, Motif 3 is confined to Medicago and Trigonella, and Motif 9 appears only in Lathyrus, Pisum and Vicia. More distantly related species such as Lotus japonicus and Anthyllis vulneraria possess homologous sequences lacking any internal repeats, suggesting that the ancestral NOD25-like protein was a simpler, non-repetitive form and that the repeats arose by exon shuffling after the IRLC diverged. AlphaFold 3 structure predictions, with confidence scores below 0.5 outside the signal peptide, indicate that these proteins likely contain substantial intrinsically disordered regions, a feature often associated with flexible interaction surfaces in host-microbe recognition systems.

How can a gene be so highly expressed, so precisely localized and so evolutionarily conserved, yet dispensable for the process it accompanies? The authors point to precedents: the early nodulin ENOD12 was shown thirty years ago to be non-essential in alfalfa, though in that case redundant proline-rich genes could compensate, whereas no homolog of MtNOD25 exists that might take its place. One tantalizing clue lies in the conserved RK/RLLL motif of the C-terminal region, which resembles the canonical cleavage sites recognized by subtilisin-like serine proteases. In Brassicaceae, subtilase-mediated processing of PROSCOOP precursor proteins generates bioactive signaling peptides that regulate defense and root development, and subtilases have been implicated in legume symbioses and found in soybean peribacteroid membrane fractions. The researchers propose that NOD25 may be produced as an inactive proprotein within the symbiosome, activated by proteolytic cleavage only under specific stresses such as pathogen attack, drought, salinity or temperature extremes, conditions absent from standard laboratory growth. Such a context-dependent function would reconcile the protein’s strict conservation with its invisibility in greenhouse assays. For now, NOD25 joins the growing list of nodule proteins whose true roles await the right environmental trigger, a reminder that in symbiosis, as in much of biology, abundance and necessity are not the same thing.

Subject of Research: Functional analysis of the NOD25 nodulin gene in the nitrogen-fixing root nodule symbiosis of Medicago truncatula

Article Title: Loss of Nod25 function does not affect nitrogen-fixing symbiosis in Medicago truncatula

Article References: Biró, J. B., Domonkos, Á., Güngör, B., Farkas, A., Mohammadi Eghbash, E., Kiss, G. B., & Kaló, P. (2026). Loss of Nod25 function does not affect nitrogen-fixing symbiosis in Medicago truncatula. Plant Molecular Biology, 116(5), Article 101. https://doi.org/10.1007/s11103-026-01766-y

Image Credits: AI Generated

DOI: 10.1007/s11103-026-01766-y

Keywords: Medicago truncatula, nitrogen-fixing symbiosis, nodulin-25, CRISPR/Cas9, rhizobia, symbiosome, IRLC legumes, bacteroid differentiation, nodule development, exon shuffling, subtilases, plant molecular biology