# Riboregulation in Nitrogen-Fixing Endosymbiotic Bacteria
## 7 Conclusions and Perspectives  
RNA-mediated regulation of gene expression, which is ubiquitous in all living organisms, contributes to fine-tune most, if not all, prokaryotic physiological processes. However, genome annotations of the majority of bacterial species do not include sRNA genes yet, so that the structure and function of the non-coding transcriptomes remains unaccountably unexplored. Knowledge gaining on how the transcriptomes shape the functions of bacteria populating diverse niches or even establish complex relationships with other organisms is now timely and technically feasible. In plant-nodalulating rhizobia, most of the knowledge about riboregulation derives from work on the alfalfa symbiont _S. meliloti_. This bacterium expresses hundreds of _trans_-sRNAs, but only a handful of those have been characterized and functionally related to free-living and symbiotic rhizobia traits such as metabolism, cell-cycle, quorum sensing or nodule development (Figure 4). Like in other bacterial species, pervasive antisense transcription also occurs in _S. meliloti_, with a remarkable bias towards symbiotically relevant genes. However, the functional significance of asRNA-mediated regulation has not been investigated further neither at genome-wide scale nor for particular symbiotic genes. Other key players in riboregulation, like RNA-binding proteins and ribonucleases remain also underexplored. Unlike in enterobacteria, the well-characterized RNA-chaperone Hfq seems to have a rather limited role in assisting _trans_-sRNA activity in rhizobia and the catalytic features of the set of predicted ribonucleases and its impact in RNA silencing are essentially unknown. Therefore, much work remains to be done to characterize the content and function of the sRNAs-associated proteomes.  
Figure 4: Cell processes that are riboregulated in rhizobia and the sRNAs involved.  
The knowledge gained in _S. meliloti_ should be necessarily extended to other rhizobia and symbiotic systems. Genomes of other model N-fixing symbionts must be accurately annotated using the most recent RNA-Seq protocols to uncover the primary transcriptome. The resulting catalogs of sRNA genes must be extensively compared and characterized to assess the contribution of riboregulation to the symbiotic diversity. Deciphering the biogenesis, function, and mechanistic principles of all types of sRNAs, i.e., _trans_-sRNAs, asRNAs, riboswitches, 5/3'-UTRs, other mRNA leaders, and tRFs will contribute to explore the plasticity of RNA molecules as regulators of gene expression. Of particular interest would be to investigate the RNA trafficking between rhizobia and the plant host cells as a novel layer of dialogue between the symbiotic partners. Finally, the biotechnological implications of post-transcriptional RNA-mediated regulation for the engineering of symbiosis and other environmentally relevant systems should be also explored.  
This research was funded by the Spanish Ministerio de Ciencia, Innovacion y Universidades, ERDF co-financed grants BFU2013-48282-C2-2-P and BFU2017-82645-P to J.I.J.-Z.  
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.