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An innovative approach reveals a novel strategy for engineering root nodule symbiosis into important crops for more sustainable agri-food systems

Researchers from Aarhus University have now discovered the receptor complex crucial for initiating symbiotic signaling in legume plants that engage in symbiosis with nitrogen-fixing bacteria. In the study published in Science, the researchers describe a novel technique using nanobodies to investigate and activate receptor complexes. Surprisingly, the technique also identified barley receptors with a similar function. This opens a new perspective for more sustainable agri-food systems by engineering root nodule symbiosis into important crops using their own genes.

The researchers behind the scientific article in the prestigious journal Science (from left): Simona Radutoiu, Kasper Røjkjær Andersen, Jens Stougaard, Henriette Rübsam, Nikolaj Abel, Simon Boje Hansen and Marie Vogel Kolte (photo: Lisbeth Heilesen)

Legume plants do not depend on externally supplied nitrogen, because they can form a symbiosis with nitrogen-fixing bacteria, called rhizobia. The plants recognize the nitrogen-fixing bacteria and allow them to colonize specially formed organs called nodules. It has been known for many years that there are two Nod factor receptors, NFR1 and NFR5, which are responsible for recognizing the signaling molecules secreted by rhizobia. But how the receptors work together in signaling complexes was unknown until now.

 “It is difficult to identify active signaling complexes in living cells, so we had to develop a new approach. For quite a while, we had been working with nanobodies, small single-domain antibodies derived from llamas, for other purposes. So, we wondered if we could use nanobodies to study receptor signaling,” Kasper Røjkjær Andersen explains.

The researchers from the Plant Molecular Biology section developed an approach where they coupled a green fluorescent protein (GFP) to one of the receptors and a nanobody that binds to GFP to the other receptor. This way, the two receptors were brought into close vicinity in the living plant. “To our surprise, just bringing the receptors close together through nanobody binding was enough to initiate the formation of nodules. We also tested other receptors, but nothing happened, so that showed us that NFR1 and NFR5 must be forming a complex to start symbiotic signaling,” Henriette Rübsam explains.

The aim is to decrease the use of nitrogen fertilizers and support the green transition

In the long run, the researchers want to engineer root nodule symbiosis into cereals to decrease the use of nitrogen fertilizers, which have an enormous CO2 footprint, and to enable small-holder farmers with limited access to commercial fertilizer to have stable yields.

“We came up with the rather wild idea that existing barley receptors might be capable of initiating nodule formation in legumes,” Simona Radutoiu explains. Christina Krönauer elaborates: “We tested the signaling capacity of barley receptors by pulling them together using nanobodies and could show that associating the two receptors RLK4 and RLK10 leads to nodule formation in Lotus. This was completely unexpected and very promising, because it might be a major step on the way to engineer crop plants to engage in symbiosis with nitrogen-fixing bacteria.”

The researchers are now looking forward to applying the new technique to study other important receptor systems. They are especially excited to explore and develop the prospects to engineering nitrogen-fixing cereals for a more sustainable agro-food system

Link to the article in Science.


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Study type


External funding

This work was supported by the Bill and Melinda Gates Foundation and the UK government’s Department for International Development (DFID) through the Engineering Nitrogen Symbiosis for Africa project (ENSA; OPP11772165), the Danish Council for Independent Research (9040-00175B), the Molecular Mechanisms and Dynamics of Plant-microbe interactions at the Root-Soil Interface project (InRoot), supported by the Novo Nordisk Foundation grant NNF19SA0059362, and the EuropeanResearch Council (ERC) under the European Union’s Horizon 2020 research and innovation program (834221).

Conflicts of interest

Henriette Rübsam, Christina Krönauer, Jens Stougaard, Simona Radutoiu and Kasper R. Andersen are inventors on a filed patent that captures these discoveries.

Link to scientific paper

Henriette Rübsam, Christina Krönauer, Nikolaj B. Abel, Hongtao Ji, Damiano Lironi, Simon B. Hansen, Marcin Nadzieja, Marie V. Kolte, Dörte Abel, Noor de Jong, Lene H. Madsen, Huijun Liu, Jens Stougaard, Simona Radutoiu, Kasper R. Andersen

Nanobody-driven signaling reveals the core receptor complex in root nodule symbiosis



Contact information

Associate Professor Kasper Røjkjær Andersen
Department of Molecular Biology and Genetics
Aarhus University

Associate Professor Simona Radutoiu

Department of Molecular Biology and Genetics
Aarhus University

After the start of stage 3 of the ENSA project in April 2023, the name ENSA was updated to ‘Enabling Nutrient Symbioses in Agriculture’ to reflect the full vision of the project.