Caracterización de genes de Rhizobium tropici CIAT 899 implicados en la biosíntesis de los factores de nodulación independiente de la activación por flavonoides e inducidos por estrés osmótico

Resum

In the establishment of the rhizobia-legume symbiosis, the flavonoids secreted by the plant roots activate a transcriptional regulator (NodD protein) that activates the transcription of the nodulation genes (known as nod genes) by binding to the rhizobia-conserved promoter sequences (nod-boxes). The nod genes encode for proteins that are involved in the biosynthesis and secretion of the Nodulation factors (also known as Nod factors). These Nod factors start a regulatory pathway in the legume that culminates in the formation of structures on the roots called nodules, where the established rhizobia will fix atmospheric nitrogen into a reduced form appropriate for the plant metabolism. Rhizobium tropici CIAT 899 is a rhizobial strain which tolerates different abiotic stresses and it is able to synthesizes Nod factors under salt stress. Due to this fact, in this Thesis it has been studied the regulation of the synthesis of Nod factors under stressful and non-stressful conditions, and their involvement in the establishment of the symbiosis with different legumes including Phaseolus vulgaris, Leucaena leucocephala, Lotus japonicus and Lotus burttii. To achieve this goal, ARNseq studies were carried out. Here, it was observed that the CIAT 899 nod genes are induced both in the presence flavonoid apigenin and under osmotic stress, either salt stress or non-ionic stress produced by mannitol. Then, it has been studied which genes are involved in the activation of the Nod factor synthesis pathway under osmotic stress. Thus, directed mutagenesis determined that NodD2 and a new regulator belonging to the AraC family are essentials in the activation of the nod genes and therefore in the synthesis of Nod factors under osmotic stress. On the other hand, a new transcriptional regulator NrcR was also identified. This protein promoted the activation of the nod genes in the presence of apigenin and salt stress but at the same time, it repressed the number of decorations of the Nod factors. Moreover, NrcR regulates other phenotypes such as surface motility and exopolysaccharides production. In addition, it has been also studied the role of the three copies of the nodA gene which are located in the genome of CIAT 899. Thus, non-polar deletion mutagenesis demonstrated that NodA1 and NodA3 (nodA3 gene lacks nod-box upstream) were both required for the synthesis of Nod factors in the presence of apigenin and salt stress, while NodA2 is not able to synthesize these molecules in the presence of apigenin but it does slightly under salt stress. Finally, the symbiotic phenotypes of the mutants obtained before were studied. Here, it was observed that NodD1 is essential to establish symbiosis with L. leucocephala and L. japonicus, while both NodD1 and NodD2 are necessary for the establishment of the symbiosis in the other two legumes of this study: P. vulgaris and L. burttii. In addition, the presence of NodA1 or NodA3 is sufficient to establish symbiosis with all the legumes tested, while NodA2 by itself does not nodulate L. leucocephala and L. japonicus. All these data indicate that the activation of the synthesis of the Nod factors under osmotic stress plays a role in the establishment of the symbiosis with certain legumes. Future jobs will provide more information about this fact.