Scientists reveal long-standing mystery of ENOD40, a pioneering gene in legume nodulation research that marks nodule identity

A gene identified more than 30 years ago has now revealed its role as a natural microRNA (miRNA) sponge to fine-tune the legume nodulation pathway, thanks to an international collaboration led by Wageningen University and the Sainsbury Laboratory Cambridge University. 

Nodules are specialised organs found on the roots of legumes that provide homes and carbon for soil bacteria, in exchange for nitrogen. 

Early Nodulation Gene (ENOD40) was first identified in 1993 by the collaborators from Wageningen University in soybean (Gylcine max) as being active during the early stages of root nodule formation. 

As one of the earliest responders to the soil bacteria that initiate root nodule formation, ENOD40 has long served as a marker gene for studying plant-microbe root symbiosis. 

Within just hours of inoculation, and before any visible changes occur, ENOD40 is switched on deep within root tissue, marking the plant’s decision to begin building bacterial homes.  

However, its exact role remained unclear. Published in PNAS, researchers have discovered that ENOD40 sequesters a miRNA that usually downregulates a positive regulator of nodulation. MiRNAs are known to regulate gene expression by binding to messenger RNA (mRNA), in this case, the mRNA encodes for a positive regulator, and directing the mRNA for destruction. By sequestering the miRNA, ENOD40 fine-tunes the expression of the positive regulator to form nitrogen-fixing nodules successfully. 

The team generated a mutant of the model legume, Medicago truncatula plants lacking ENOD40 and found their ability to start nodules was greatly reduced. 

“ENOD40’s genetic structure is unusual: one end encodes a tiny peptide of just 12–13 amino acids, while the other contains a highly conserved stretch of RNA known as ‘box2’,” said Dr Nadia Mohd-Radzman, first co-author from the Sainsbury Laboratory at the University of Cambridge. 

“This box2 region mimics the binding site for microRNA169, preventing it from targeting NF-YA1 – a core positive regulator needed at the very start of nodule formation.” 

“This is the first time we’ve found a plant gene that combines these two unusual features where it is encoding a small peptide with a built-in microRNA mimic. 

Remarkably, introducing only the box2 region (or even an artificial mimic of the microRNA target) restored much of this lost capacity. 

“It’s like discovering your trusty old lab tool has been a sophisticated double agent all along.” 

Dr Mohd-Radzman describes the putative dual functionality of ENOD40 as elegant and unusual. 

This dual structure, where a small peptide-coding capacity is embedded within a non-canonical RNA transcript, was the first of its kind discovered in eukaryotes. 

More than a decade later, in 2007, a similar gene called tarsal-less was identified in fruit flies (Drosophila melanogaster). Like ENOD40, it exhibits dual functionality, encoding peptides from a longer mRNA transcript. 

“This work not only solves a long-standing puzzle in plant molecular biology,” said Dr Mohd-Radzman, “it also reveals a new layer of control in how plants fine-tune their symbiotic relationships with microbes.” 

Dr Radzman added: “ENOD40 is truly a multifaceted molecule. Now that we’ve established its target-mimicry function via box2, future work will delve into how its peptide-coding potential and RNA structure further contribute to nodulation and beyond, as how these features are involved in the plant nodules is unclear. Are these features correlating to separate processes, or are they intertwined in order to control the same process? 

“Emerging tools such as single-cell and spatial transcriptomics may soon clarify the cell-specific interplay between ENOD40, miRNA169, and NF-YA1, unlocking finer details of how legumes orchestrate symbiotic organogenesis. 

“The findings represent an important leap in our understanding of plant lncRNAs and peptides, with potential implications for improving legume crop performance and advancing our grasp of RNA-mediated regulation.” 

The research is published in Proceedings of the National Academy of Sciences (PNAS). 

Reference 

Tristan Wijsmana, Nadia A. Mohd-Radzman, Jieyu Liu, Giles E. D. Oldroyd, Wouter Kohlen, Olga Kulikova, Renze Heidstra, Ben Scheres, and Henk J. Franssen (2025) The Medicago truncatula lncRNA ENOD40 is a mediator of microRNA169-controlled NF-YA activity in nodule initiation. PNAS 

Funding

This work was supported by a grant of the Graduate School of Experimental Plant Science (NAMR), the Gatsby Charitable Foundation, the Bill and Melinda Gates Foundation and the UK Foreign, Commonwealth and Development Office through Engineering the Nitrogen Symbiosis for Africa (ENSA) project.