Aims The symbiosis between rhizobia and their host legumes is initiated by a complex molecular dialogue in which the activation of bacterial NodD proteins by appropriate plant flavonoids triggers the expression of the bacterial nodulation (nod) genes. These genes are involved in the synthesis and export of the Nodulation factors (NF), which are signal molecules that, when recognized by plant receptors, launch the symbiotic process. The core of NF is synthesized by proteins encoded by the nodABC genes, whereas the rest of the nod genes protein products are involved in the decoration of NF with different chemical substituents and their export to the environment. Rhizobium tropici CIAT 899, the microsymbiont of common bean (Phaseolus vulgaris), is characterized for tolerating multiple physical stresses and for synthesizing a large variety of NF not only in the presence of inducing flavonoids but also when high concentrations of salt are present. In addition, another interesting feature of the R. tropici CIAT 899 genome is the presence of three different nodA genes on the symbiotic plasmid, although their exact roles remain to be elucidated. In this work, we characterize the role of the three NodA proteins of R. tropici CIAT 899 in symbiosis. Methods We have analyzed by quantitative PCR the expression of the nodA1, nodA2 and nodA3 genes of CIAT 899, showing that only nodA1 and nodA2 are inducible by flavonoids. We have also constructed single, double and triple non-polar mutants in these genes in order to study their importance in NF production and in nodulation of four different host legumes of CIAT 899: P. vulgaris, Leucaena leucocephala, Lotus japonicus, and L. burttii. Results The nodA1 and nodA2 genes of CIAT 899 (both inducible by flavonoids) are more related between them than with nodA3 (non-flavonoid inducible). Interestingly, the presence of NodA1 or NodA3 is crucial for nodulation with L. leucocephala and L. japonicus, since both NodA1 or NodA3 guarantee Nod factor production upon apigenin induction. Interestingly, osmotic-stressing conditions increase the quantity and diversity of the Nod factors synthesized by NodA2, being these molecules able to induce the formation of nodule primordia on P. vulgaris. Conclusions We concluded that R. tropici CIAT 899 requires at least the flavonoid-induced nodA1 or the flavonoid-independent nodA3 genes for ensuring symbiotic success in the four assayed host-legumes. The inducible nodA2 gene is sufficient to induce nodulation on P. vulgaris and L. burttii but not in L. leucocephala and L. japonicus. To our knowledge, in this work we report for the first time that a non-flavonoid inducible copy of nodA is enough to induce nodulation on legumes.
