207 - Microbiología de las Interacciones

ROLE OF NCR-RELATED PEPTIDASES IN THE Rhizobium favelukesii – Medicago SYMBIOSIS

Luchetti, Abril - D’Addona, Catalina - Cabrera, María Delfina - Pérez Giménez, Julieta -Torres Tejerizo, Gonzalo

Instituto de Biotecnología y Biología Molecular (CONICET - UNLP) - La Plata - Buenos Aires - Argentina
Contacto: aluchetti@biol.unlp.edu.ar

Legumes can generate two types of nitrogen-fixing nodules depending on both the host plant and the symbiont. In indeterminate nodules, bacteria undergo terminal differentiation into bacteroids, an irreversible state in which they are able to fix atmospheric nitrogen into ammonia. In Medicago truncatula nodules, the differentiation process is controlled by nodule-specific cysteine-rich peptides (NCRs). The NCRs are secreted by the plant and translocated to the bacterial cytosol, where they can be cleaved by bacterial peptidases, as a defense response to the antimicrobial activity of those peptides. Two peptidases involved in the development of nitrogen-fixing nodules have recently been described: HrrP and SapA. Both proteins showed peptidase activity against NCRs. Expression of hrrP (host range restriction peptidase) in S. meliloti B800 inhibits nitrogen fixation in M. truncatula A20. On the other hand, overexpression of sapA generates plants with lower levels of nitrogen fixation. Rhizobium favelukesii LPU83 was isolated from acid soils of Argentina and can nodulate M. truncatula. Even though this bacterium presents a symbiotic plasmid with the necessary genes to establish an effective symbiosis, it is inefficient in nitrogen fixation. In this work, we sought to identify and analyze the role of homologous genes to hrrP and sapA peptidases in the symbiosis between LPU83 and M. truncatula in order to evaluate their implication in the symbiotic phenotype of the strain. Genes homologous to hrrP and sapA were found in the symbiotic plasmid and chromosome of LPU83. Mutants in these genes were generated and their symbiotic phenotype was evaluated in M. truncatula A20. Our results showed that both single and a double mutant on these genes did not exhibit changes in their symbiotic phenotype compared to controls, with no significant differences in shoot dry weight. Previous research indicated that overexpression of hrrP in S. meliloti negatively affected symbiosis, resulting in ineffective nitrogen fixation in plants with white, small nodules. To study the impact of LPU83 peptidases, the homologous genes were cloned into replicative plasmids in rhizobia under the promoter of the hrrP gene and transferred to S. meliloti. However, overexpression of LPU83 peptidases showed no differences with the strain carrying the empty plasmid, with plants exhibiting green leaves and pink, elongated nodules. Confocal microscopy of these nodules revealed that even though the plants fix nitrogen, there were more dead bacteria inside the nodules infected with overexpressed LPU83 peptidases. Overall, these findings suggest that while the hrrP gene plays a crucial role in nodulation and nitrogen fixation in S. meliloti, the peptidases from LPU83 do not significantly impact on the symbiotic effectiveness. Further research is needed to explore and clarify the special phenotype observed in LPU83.

Palabras clave: Symbiosis – Rhizobia – Nodules