From Exclusivity to Flexibility in Legume Rhizobia Association

Although the Earth’s atmosphere contains approximately 78% dinitrogen (N₂), this inert form of nitrogen is inaccessible to most organisms, including plants and animals. Unlike carbon, plants cannot fix atmospheric N₂; instead, they absorb nitrogen in the form of nitrate (NO₃⁻) or ammonium (NH₄⁺) from the soil. The availability of fixed nitrogen often limits crop productivity. To meet the growing global food demand, large quantities of nitrogen fertilizers have been applied to agricultural systems. However, an alternative and more sustainable source of nitrogen is Biological Nitrogen Fixation (BNF), a process carried out by certain bacteria and archaea that possess the enzyme nitrogenase, which converts atmospheric N₂ into ammonia (NH₃). The nitrogen fixed through BNF is primarily utilized by the microorganisms and is less prone to leaching, providing a more sustainable input for agriculture. Among plants, legumes have evolved a unique ability to form symbiotic associations with nitrogen-fixing bacteria, known as rhizobia [1]. This symbiotic interaction is a tightly regulated process that ensures compatibility between the plant and its microbial partner. Although legumes can form nodules with multiple strains of rhizobia, the efficiency of nitrogen fixation varies depending on the bacterial partner and environmental conditions. Therefore, identifying the most effective nitrogen-fixing bacteria is essential for maximizing the benefits of this symbiosis in agricultural systems.

Two subclasses of Proteobacteria-Alpha and Beta Proteobacteria-communicate with legumes to establish symbiotic associations. Among them, Alpha-rhizobia include diverse genera such as Rhizobium, Brady rhizobium, Sino rhizobium, and Meso rhizobium, which account for the majority of known rhizobial species.

In contrast, Beta-rhizobia are less diverse, with only two genera-Cupriavidus and Burkholderia-known to participate in symbiosis. Recent taxonomic revisions have reclassified Burkholderia into Paraburkholderia and Trinickia [2,3]. Alpharhizobia are predominantly found in symbiosis with commonly cultivated legumes such as Phaseolus vulgaris, Cicer arietinum, Glycine max, Pisum sativum, Arachis hypogaea, Medicago sativa and Vigna radiata. In contrast, Beta-rhizobia are mainly associated with tropical legumes like Mimosa and other diverse papilionoid species native to the Fynbos/Cape Core Subregion [4-7]. To date, no other bacterial classes have been known to engage in such symbiotic relationships. The interaction between legumes and rhizobia is species-specific, governed by signals from both partners. Plants secrete flavonoids, which induce the synthesis and secretion of Nodulation Factors (NFs) by rhizobia. NFs are composed of four or five β-1,4-linked N-acetylglucosamine units, with a non-reducing terminal sugar acylated by a fatty acid chain (e.g., C16:0, C18:1). The terminal sugar backbone is differentially modified depending on the rhizobial species, contributing significantly to host specificity [8]. These chemical modifications of NFs are recognized by specific Nod Factor Receptors (NFRs) on the plant’s root hair cells. This recognition mechanism, often described as a “lock and key” model, ensures that only compatible NFs can trigger nodulation in particular legume species. The first NFRs were characterized in the model legume Lotus japonicus and are known as LjNFR1 and LjNFR5. Their orthologs in Medicago truncatula are MtLYK3 (LysM domain receptor-like kinase 3) and Nod Factor Perception (NFP) [9]. Interestingly, the transfer of LjNFR1 and LjNFR5 into M. truncatula extended the host range of Meso rhizobium loti-normally restricted to Lotus and its close relatives-allowing it to nodulate M. truncatula [10].