Researchers unlock signal recognition between legumes and rhizobia

Root nodules on legumes such as soybeans and alfalfa host soil bacteria known as rhizobia, which convert atmospheric nitrogen into forms of nitrogen the plant can use. Nodules hosting the "right" rhizobia for their species of plant thus act like a natural "nitrogen fertilizer factory." However, as legume roots are surrounded by a multitude of rhizobia and other bacteria, scientists have wondered how plants and bacteria ensure that only "compatible" rhizobia form nodules.

For a long time, researchers have known that legume roots secrete small signal molecules called flavonoids that are recognized by the protein NodD, a rhizobial transcription factor. Crucially, the species or strains of rhizobia that associate with specific legumes carry their own version of NodD. Although NodD was known to contribute to symbiotic specificity, how it specifically recognizes flavonoid chemical signals has remained an intriguing question.

In a major breakthrough, scientists have elucidated how NodD recognizes flavonoids by resolving, for the first time, the high-resolution crystal structure of the complex formed between the NodD protein of pea rhizobia and the flavonoid compound hesperetin. Through this process, the researchers identified key structural elements in NodD that determine signal specificity.

The study was conducted by teams led by Jeremy Murray and ZHANG Yu from the Center for Excellence in Molecular Plant Sciences (CEMPS) of the Chinese Academy of Sciences, along with collaborators. Results were published in Science on January 9.

In this study, the researchers found that the ligand-binding domain of the NodD protein from pea rhizobia recognizes hesperetin through two binding pockets—one located within a monomer of the NodD protein and the other situated at the dimer interface. This binding conformation is the first of its kind to be observed in the family of transcriptional regulators to which NodD belongs.

Further analysis indicated that the shape of the NodD binding pocket accommodates flavonoid molecules like hesperetin while excluding other classes of flavonoids, such as isoflavones or pterocarpans. This observation provides a structural explanation for why rhizobial NodD is only activated by specific flavonoids.

Furthermore, the researchers compared the NodDs from alfalfa and pea rhizobia. Despite an overall similarity of 80% between the two proteins, their "preferences" for different flavonoids are very different. The NodD of pea rhizobia primarily responds to flavanones/flavones, whereas the NodD of the alfalfa rhizobia mainly responds to chalcones.

Through domain-swapping experiments and extensive point mutation studies, the researchers identified several key amino acids located in critical activation regions that determine the ability of the rhizobia to respond to different types of flavonoids.

So why is this specificity needed in the first place? The researchers suggested that such precise recognition stems from millions of years of co-evolution in overlapping habitats. To ensure successful partnerships, each species accurately identifies its preferred rhizobia strain through a mutual "double-lock and key" system: The bacteria recognize unique flavonoid signals from the plant and the plant in turn recognizes specific rhizobial countersignals. This prevents mix-ups when multiple species grow alongside each other.

This study answers the question of how legume plants and rhizobia achieve signal-specific recognition through flavonoids and NodD and provides a new way to design efficient nitrogen fixation systems. In addition, it paves the way for designing efficient, crop-specific "tailor-made" rhizobia for enhanced nitrogen fixation. In this way, it holds promise for extending nitrogen-fixing symbiosis to non-legume crops such as rice and corn, thereby reducing agricultural reliance on chemical nitrogen fertilizers.