Genome-wide and comparative phylogenetic analysis of senescence-associated NAC transcription factors in sunflower (Helianthus annuus).

BACKGROUND: Leaf senescence delay impacts positively in grain yield by maintaining the photosynthetic area during the reproductive stage and during grain filling. Therefore a comprehensive understanding of the gene families associated with leaf senescence is essential. NAC transcription factors (TF) form a large plant-specific gene family involved in regulating development, senescence, and responses to biotic and abiotic stresses. The main goal of this work was to identify sunflower NAC TF (HaNAC) and their association with senescence, studying their orthologous to understand possible functional relationships between genes of different species. RESULTS: To clarify the orthologous relationships, we used an in-depth comparative study of four divergent taxa, in dicots and monocots, with completely sequenced genomes (Arabidopsis thaliana, Vitis vinifera, Musa acuminata and Oryza sativa). These orthologous groups provide a curated resource for large scale protein sequence annotation of NAC TF. From the 151 HaNAC genes detected in the latest version of the sunflower genome, 50 genes were associated with senescence traits. These genes showed significant differential expression in two contrasting lines according to an RNAseq assay. An assessment of overexpressing the Arabidopsis line for HaNAC001 (a gene of the same orthologous group of Arabidopsis thaliana ORE1) revealed that this line displayed a significantly higher number of senescent leaves and a pronounced change in development rate. CONCLUSIONS: This finding suggests HaNAC001 as an interesting candidate to explore the molecular regulation of senescence in sunflower.

Expression of Babesia bovis rhoptry-associated protein 1 (RAP1) in Brucella abortus S19.

Brucella abortus strain 19 (live vaccine) induces a strong humoral and cellular immune response and therefore, it is an attractive vector for the delivery of heterologous antigens. The objective of the present study was to express the rhoptry-associated protein (RAP1) of Babesia bovis in B. abortus S19, as a model for heterologous expression of immunostimulatory antigens from veterinary pathogens. A plasmid for the expression of recombinant proteins fused to the aminoterminal of the outer membrane lipoprotein OMP19 was created, pursuing the objective of increasing the immunogenicity of the recombinant antigen being expressed by its association to a lipid moiety. Recombinant strains of B. abortus S19 expressing RAP1 as a fusion protein either with the first amino acids of beta-galactosidase (S19pBB-RAP1) or B. abortus OMP19 (S19pBB19-RAP1) were generated. Plasmid stability and the immunogenicity of the heterologous proteins were analyzed. Mice immunized with S19pBB-RAP1 or S19pBB19-RAP1 developed specific humoral immune response to RAP1, IgG2a being the predominant antibody isotype. Furthermore, a specific cellular immune response to recombinant RAP1 was elicited in vitro by lymphocytes from mice immunized with both strains. Therefore, we concluded that B. abortus S19 expressing RAP1 is immunostimulatory and may provide the basis for combined heterologous vaccines for babesiosis and brucellosis.

Expression of MPB83 from Mycobacterium bovis in Brucella abortus S19 induces specific cellular immune response against the recombinant antigen in BALB/c mice.

Immunodominant MPB83 antigen from Mycobacterium bovis was expressed as a chimeric protein fused to either ß-galactosidase, outer membrane lipoprotein OMP19 or periplasmic protein BP26 in gram-negative Brucella abortus S19, in all cases driven by each gene's own promoter. All fusion proteins were successfully expressed and localized in the expected subcellular fraction. Moreover, OMP19-MPB83 was processed as a lipoprotein when expressed in B. abortus. Splenocytes from BALB/c mice immunized with the recombinant S19 strains carrying the genes coding for the heterologous antigens in replicative plasmids, showed equally specific INF-γ production in response to MPB83 stimulation. Association to the lipid moiety of OMP19 presented no advantage in terms of immunogenicity for MPB83. In contrast, fusion to BP26, which was encoded by an integrative plasmid, resulted in a weaker immune response. None of the constructions affected the survival rate or the infection pattern of Brucella. We concluded that B. abortus S19 is an appropriate candidate for the expression of M. bovis antigens both associated to the membrane or cytosolic fraction and may provide the basis for a future combined vaccine for bovine brucellosis and tuberculosis.

The TolC homologue of Brucella suis is involved in resistance to antimicrobial compounds and virulence.

Brucella spp., like other pathogens, must cope with the environment of diverse host niches during the infection process. In doing this, pathogens evolved different type of transport systems to help them survive and disseminate within the host. Members of the TolC family have been shown to be involved in the export of chemically diverse molecules ranging from large protein toxins to small toxic compounds. The role of proteins from the TolC family in Brucella and other alpha-2-proteobacteria has been explored little. The gene encoding the unique member of the TolC family from Brucella suis (BepC) was cloned and expressed in an Escherichia coli mutant disrupted in the gene encoding TolC, which has the peculiarity of being involved in diverse transport functions. BepC fully complemented the resistance to drugs such as chloramphenicol and acriflavine but was incapable of restoring hemolysin secretion in the tolC mutant of E. coli. An insertional mutation in the bepC gene strongly affected the resistance phenotype of B. suis to bile salts and toxic chemicals such as ethidium bromide and rhodamine and significantly decreased the resistance to antibiotics such as erythromycin, ampicillin, tetracycline, and norfloxacin. Moreover, the B. suis bepC mutant was attenuated in the mouse model of infection. Taken together, these results suggest that BepC-dependent efflux processes of toxic compounds contribute to B. suis survival inside the host.