# Riboregulation in Nitrogen-Fixing Endosymbiotic Bacteria
## 4 Assigning Functions to Rhizbial _Trans_-sRNAs
### Nodule Development and Functioning  
Several studies addressing gene expression in legume root nodules have identified highly expressed sRNAs in endosymbiotic rhizobia, thus anticipating riboregulation of bacterial symbiotic traits [21; 29; 34; 90]. Further, differential accumulation of these transcripts in defined zones of indeterminate alfalfa nodules or in developmentally arrested nodules from _M. truncatula_ mutants could be predictive of specific roles of _S. meliloti_ sRNAs in early, intermediate and late symbiotic stages, e.g., infection, bacteroid differentiation or N fixation [34; 91]. A small subset of these _S. meliloti_ sRNAs is uniquely expressed in nodule tissues, thus suggesting yet unexplored RNA functions in regulation of genuine symbiotic pathways. However, in most cases expression in nodules concurs with stress-induced accumulation of the sRNA in free-living rhizobia. This is the case of some of the functionally characterized _S. meliloti trans_-sRNAs described above, e.g., AbcR1, MmgR, EcpR1 or GspR1, whose loss-of-function has none or modest impact in symbiosis [62; 72; 87; 89]. To date, phenotyping of knock-out mutants has revealed putative symbiotic functions for only one chromosomally encoded _S. meliloti_ regulatory _trans_-sRNA, identified in early genome-wide screens as Smr14C2 or SmelC397, and later renamed NfeR1 (nodule formation efficiency RNA) to reflect this fact [20; 26; 92]. NfeR1 belongs to the so-called \(\alpha\)14 family of \(\alpha\)-proteobacterial sRNAs (Table 1), which is represented in most plant symbionts, phytopathogens and mammal pathogens within Rhizobiaceae, Brucellaceae and Phylobacteriaceae bacterial families [44; 46]. \(\alpha\)14 sRNAs share a predicted secondary structure consisting of three hairpins with hypervariable nucleotide content in the stems and the aSD motif unpaired in all the loops [44; 46]. NfeR1 locus occurs in six copies in the _S. meliloti_ Rm1021 and Rm2011 genomes, but in the conditions tested only one of those is transcribed to levels reliably detected by northern blot probing [92]. NfeR1 transcription is driven by a \(\sigma^{70}\)-dependent promoter containing several nucleotide residues that are well-conserved in promoter regions of \(\alpha\)r14 sRNAs. This conserved motif is the determinant of both differential expression and high transcription rates, which render NfeR1 as one of the transcripts with highest coverage scores among the identified _S. meliloti_ sRNAs by RNA-Seq surveys [26; 27; 29; 34]. Intracellular NfeR1 levels are particularly high when bacteria are grown in defined glutamate/mannitol medium, upon salt shock and in all steps of the symbiotic interaction between _S. meliloti_ and alfalfa, i.e., rhizplane colonization, root hair infection, bacteroid differentiation and symbiotic N fixation [34; 92]. Linked to this expression profile, NfeR1 loss-of-function compromises _S. meliloti_ survival in hypersaline medium, nodule development and overall symbiotic efficiency [92]. Nodules induced by a _S. melidiot nfeR1_ deletion mutant on alfalfa roots are apparently colonized by fully differentiated bacteroids, but look round shaped rather than elongated, and smaller than wild-type indeterminate mature nodules. Their histology resembles that of nodules developed by _M. truncatula_ mutants in marker genes for intermediate and late symbiotic stages [91; 93] or induced by _S. melidiot_ derivatives engineered to overexpress N assimilation pathways concurrently with symbiotic N fixation [94]. In silico searches for NfeR1 mRNA targets predict that most of the regulatory potential of this sRNA resides in the three conserved aSD motifs, which are indistinctly suited for base-pairing with the translation initiation region of multiple mRNAs, most coding for transport proteins. NfeR1-mediated post-transcriptional silencing of some of these targets and the redundant function of the three identical regulatory motifs have been genetically confirmed [92].