Doutoramento em Biologia
Improvement of chickpea rhizobia by genetic transformation with symbiosis genes
José Rodrigo da Silva

Orientação: Solange Oliveira & Ana Alexandre

Rhizobia are soil bacteria able to induce the formation of nodules in leguminous plants and convert atmospheric nitrogen into assimilable forms to these plants. Some Mesorhizobium species establish symbiosis with chickpea and can increase productivity of this culture. Rhizobia symbiosis genes, such as nod and nif, are involved in nodule development and nitrogen fixation. Nevertheless, genes involved in other molecular mechanisms, namely stress response may influence the symbiotic interaction plant-rhizobia. The objective of this study was to evaluate the effects of overexpressing symbiotic and stress response genes in the symbiotic performance of chickpea Mesorhizobium. Mesorhizobium strains were transformed with pRKnifA, pRKnodD, pRKenvZ and pRKgroEL (expression vector pRK415 with nifA, nodD, envZ and groEL genes from M. mediterraneum UPMCa36T, respectively). From the four strains transformed with extra nifA copies, only V15-b was able to increase plant biomass, when compared to wild-type and empty vector strains. Among the four strains transformed with extra nodD copies, ST-2 and PMI-6 showed a higher symbiotic effectiveness compared to wild type and control strains. Additional copies of envZ led to in a higher symbiotic effectiveness when introduced in PMI-6 and EE-7. Evaluation of the symbiotic effectiveness of the four strains overexpressing groEL showed that only ST-2 improved, compared to wild-type and empty vector strains. For all these strains the rate of nodule formation was seen to be higher and further analysis of the infection process and nodule histological analysis were performed. Overall, this study shows that extra copies of a given gene may have different effects in the symbiotic effectiveness, depending on the modified strain. This study contributes to a better understanding of the nodulation and nitrogen fixation processes, namely regarding the contribution of non-symbiotic genes, especially envZ, which was to our knowledge for the first time reported to be involved in the rhizobia-legume symbiosis.