Genetics of nodulation in Aeschynomene evenia uncovers mechanisms of the rhizobium–legume symbiosis

Among legumes (Fabaceae) capable of nitrogen-fixing nodulation, several Aeschynomene spp. use a unique symbiotic process that is independent of Nod factors and infection threads. They are also distinctive in developing root and stem nodules with photosynthetic bradyrhizobia. Despite the significance of these symbiotic features, their understanding remains limited. To overcome such limitations, we conduct genetic studies of nodulation in Aeschynomene evenia, supported by the development of a genome sequence for A. evenia and transcriptomic resources for 10 additional Aeschynomene spp. Comparative analysis of symbiotic genes substantiates singular mechanisms in the early and late nodulation steps. A forward genetic screen also shows that AeCRK, coding a receptor-like kinase, and the symbiotic signaling genes AePOLLUX, AeCCamK, AeCYCLOPS, AeNSP2, and AeNIN are required to trigger both root and stem nodulation. This work demonstrates the utility of the A. evenia model and provides a cornerstone to unravel mechanisms underlying the rhizobium–legume symbiosis.

Genetics of nodulation in Aeschynomene evenia uncovers new mechanisms of the rhizobium-legume symbiosis

Among legumes (Fabaceae) capable of nitrogen-fixing nodulation, several Aeschynomene spp. use a unique symbiotic process that is independent of Nod factors and infection threads. They are also distinctive in developing root and stem nodules with photosynthetic bradyrhizobia. Despite the significance of these symbiotic features, their understanding remains limited. To overcome such limitations, we conducted genetic studies of nodulation in Aeschynomene evenia, supported by the development of a genome sequence for A. evenia and transcriptomic resources for 10 additional Aeschynomene spp. Comparative analysis of symbiotic genes substantiated singular mechanisms in the early and late nodulation steps. A forward genetic screen also showed that AeCRK, coding a novel receptor-like kinase, and the symbiotic signaling genes AePOLLUX, AeCCamK, AeCYCLOPS, AeNSP2 and AeNIN, are required to trigger both root and stem nodulation. This work demonstrates the utility of the A. evenia model and provides a cornerstone to unravel new mechanisms underlying the rhizobium-legume symbiosis.

Rhizobial Factors Required for Stem Nodule Maturation and Maintenance in Sesbania rostrata-Azorhizobium caulinodans ORS571 Symbiosis

ABSTRACT The molecular and physiological mechanisms behind the maturation and maintenance of N2-fixing nodules during development of symbiosis between rhizobia and legumes still remain unclear, although the early events of symbiosis are relatively well understood. Azorhizobium caulinodans ORS571 is a microsymbiont of the tropical legume Sesbania rostrata, forming N2-fixing nodules not only on the roots but also on the stems. In this study, 10,080 transposon-inserted mutants of A. caulinodans ORS571 were individually inoculated onto the stems of S. rostrata, and those mutants that induced ineffective stem nodules, as displayed by halted development at various stages, were selected. From repeated observations on stem nodulation, 108 Tn5 mutants were selected and categorized into seven nodulation types based on size and N2 fixation activity. Tn5 insertions of some mutants were found in the well-known nodulation, nitrogen fixation, and symbiosis-related genes, such as nod, nif, and fix, respectively, lipopolysaccharide synthesis-related genes, C4 metabolism-related genes, and so on. However, other genes have not been reported to have roles in legume-rhizobium symbiosis. The list of newly identified symbiosis-related genes will present clues to aid in understanding the maturation and maintenance mechanisms of nodules.

Root nodulation of Sesbania rostrata suppresses stem nodulation by Sinorhizobium teranga but not Azorhizobium caulinodans

Azorhizobium caulinodans ORS571 and Sinorhizobium teranga ORS51 and ORS52 are symbionts of the same host plant Sesbania rostrata. In nature, A. caulinodans nodulates more competitively the stem-located infection sites of Sesbania rostrata. Sinorhizobium strains, although frequently present in root nodules, are seldom found in stem nodules. One probable explanation for this phenomenon is the more abundant presence of Azorhizobium on the leaf and stem surfaces of the host plant. Work presented here hints at other plausible factors that determine the greater "stem specificity" of Azorhizobium. We found that under experimental conditions in which roots are not inoculated, all strains nodulated stems very well. However, ORS51 and ORS52 were much more sensitive than ORS571 to suppression of stem nodulation by previous root inoculation. The introduction of the regulatory nodD gene from A. caulinodans diminished the sensitivity to this suppression, probably by enhanced nod gene expression and subsequent Nod factor production. Our hypothesis is that the greater infectivity of ORS571 is due to a more efficient production of mitogenic Nod factors at stem-located infection sites, thereby more readily overcoming systemic suppression caused by previous root inoculations.Key words: autoregulation, nitrogen fixation, rhizobial ecology, systemic suppression of nodulation.