Regulatory nodD1 and nodD2 genes of Rhizobium tropici strain CIAT 899 and their roles in the early stages of molecular signaling and host-legume nodulation

Pablo del Cerro, Amanda Alves Paiva Rolla-Santos, Douglas Fabiano Gomes, Bettina Berquó Marks, Francisco Pérez-Montaño, Miguel Ángel Rodríguez-Carvajal, André Shigueyoshi Nakatani, Antonio Gil-Serrano, Manuel Megías, Francisco Javier Ollero, Mariangela Hungria

Nodulation and symbiotic nitrogen fixation are mediated by several genes, both of the host legume and of the bacterium. The rhizobial regulatory nodD gene plays a critical role, orchestrating the transcription of the other nodulation genes. Rhizobium tropici strain CIAT 899 is an effective symbiont of several legumes-with an emphasis on common bean (Phaseolus vulgaris)-and is unusual in carrying multiple copies of nodD, the roles of which remain to be elucidated. Phenotypes, Nod factors and gene expression of nodD1 and nodD2 mutants of CIAT 899 were compared with those of the wild type strain, both in the presence and in the absence of the nod-gene-inducing molecules apigenin and salt (NaCl). Differences between the wild type and mutants were observed in swimming motility and IAA (indole acetic acid) synthesis. In the presence of both apigenin and salt, large numbers of Nod factors were detected in CIAT 899, with fewer detected in the mutants. nodC expression was lower in both mutants; differences in nodD1 and nodD2 expression were observed between the wild type and the mutants, with variation according to the inducing molecule, and with a major role of apigenin with nodD1 and of salt with nodD2. In the nodD1 mutant, nodulation was markedly reduced in common bean and abolished in leucaena (Leucaena leucocephala) and siratro (Macroptilium atropurpureum), whereas a mutation in nodD2 reduced nodulation in common bean, but not in the other two legumes. Our proposed model considers that full nodulation of common bean by R. tropici requires both nodD1 and nodD2, whereas, in other legume species that might represent the original host, nodD1 plays the major role. In general, nodD2 is an activator of nod-gene transcription, but, in specific conditions, it can slightly repress nodD1. nodD1 and nodD2 play other roles beyond nodulation, such as swimming motility and IAA synthesis.