RESUMO
Bradyrhizobium elkanii SEMIA587 is a symbiotic nitrogen-fixing bacterium of the group commonly called rhizobia, which induce nodule formation in legumes, and is widely used in Brazilian commercial inoculants of soybean. In response to flavonoid compounds released by plant roots, besides Nod factors, other molecular signals are secreted by rhizobia, such as proteins secreted by type III secretion systems (T3SSs). Rhizobial T3SSs are activated by the transcription regulator TtsI, which binds to sequences present in the promoter regions of T3SS genes via a conserved sequence called the tts box. To study the role of the T3SS of B. elkanii SEMIA587, ttsI was mutated. Protein secretion and flavonoid induction analysis, as well as nodulation tests, were performed with the wild-type and mutant strains. The results obtained showed that B. elkanii SEMIA587 secretes at least two proteins (NopA and NopL, known rhizobial T3SS substrates) after genistein induction, whilst supernatants of the ttsI mutant did not contain these Nops. Unusually for rhizobia, the promoter region of the B. elkanii SEMIA587 ttsI gene contains a tts box, which is responsive to flavonoid induction and to which TtsI can bind. Nodulation tests performed with three different leguminous plants showed that the B. elkanii SEMIA587 ttsI mutant displays host-dependent characteristics; in particular, nodulation of two soybean cultivars, Peking and EMBRAPA 48, was more efficient when TtsI of B. elkanii was functional.
Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Bradyrhizobium/efeitos dos fármacos , Flavonoides/farmacologia , Regulação Bacteriana da Expressão Gênica , Glycine max/microbiologia , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sequência de Bases , Bradyrhizobium/genética , Bradyrhizobium/crescimento & desenvolvimento , Bradyrhizobium/metabolismo , Sequência Conservada , Elementos Facilitadores Genéticos , Flavonoides/metabolismo , Genisteína/metabolismo , Genisteína/farmacologia , Isoflavonas/metabolismo , Isoflavonas/farmacologia , Dados de Sequência Molecular , Mutação , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Glycine max/metabolismo , Simbiose , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
Type III secretion systems (T3SS) have been found in several species of rhizobia. Proteins (termed effectors) secreted by this system are involved in host-range determination and influence nodulation efficiency. Mesorhizobium loti MAFF303099 possesses a functional T3SS in its symbiotic island whose expression is induced by flavonoids. As in other rhizobia, conserved cis-elements (tts box) were found in the promoter regions of genes or operons encoding T3SS components. Using a bioinformatics approach, we searched for other tts-box-controlled genes, and confirmed this transcriptional regulation for some of them using lacZ fusions to the predicted promoter regions. Translational fusions to a reporter peptide were created to demonstrate T3SS-mediated secretion of two new MAFF303099 effectors. Finally, we showed that mutation of the M. loti MAFF303099 T3SS affects its competitiveness on Lotus glaber and investigated, at the molecular level, responses of the model legume L. japonicus to the T3SS.