Small RNA sX13: A Multifaceted Regulator of Virulence in the Plant Pathogen Xanthomonas

Abstract

Small noncoding RNAs (sRNAs) are ubiquitous posttranscriptional regulators of gene expression. Using the model plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv), we investigated the highly expressed and conserved sRNA sX13 in detail. Deletion of sX13 impinged on Xcv virulence and the expression of genes encoding components and substrates of the Hrp type III secretion (T3S) system. qRT-PCR analyses revealed that sX13 promotes mRNA accumulation of HrpX, a key regulator of the T3S system, whereas the mRNA level of the master regulator HrpG was unaffected. Complementation studies suggest that sX13 acts upstream of HrpG. Microarray analyses identified 63 sX13-regulated genes, which are involved in signal transduction, motility, transcriptional and posttranscriptional regulation and virulence. Structure analyses of in vitro transcribed sX13 revealed a structure with three stable stems and three apical C-rich loops. A computational search for putative regulatory motifs revealed that sX13-repressed mRNAs predominantly harbor G-rich motifs in proximity of translation start sites. Mutation of sX13 loops differentially affected Xcv virulence and the mRNA abundance of putative targets. Using a GFP-based reporter system, we demonstrated that sX13-mediated repression of protein synthesis requires both the C-rich motifs in sX13 and G-rich motifs in potential target mRNAs. Although the RNA-binding protein Hfq was dispensable for sX13 activity, the hfq mRNA and Hfq::GFP abundance were negatively regulated by sX13. In addition, we found that G-rich motifs in sX13-repressed mRNAs can serve as translational enhancers and are located at the ribosome-binding site in 5% of all protein-coding Xcv genes. Our study revealed that sX13 represents a novel class of virulence regulators and provides insights into sRNA-mediated modulation of adaptive processes in the plant pathogen Xanthomonas.

Genome-Wide Analysis of Small RNAs Expressed by Yersinia pestis Identifies a Regulator of the Yop-Ysc Type III Secretion System

“…In addition, in the plant pathogen Xanthomonas campestris pv. vesicatoria, the sRNA xS13, which is responsive to high salt concentrations and high temperatures, has been suggested to be an indirect positive regulator of secretion by affecting the levels of a key transcriptional regulator of the T3SS (64). In Shigella dysenteriae, the iron-responsive sRNA RyhB has been implicated in the repression of the transcriptional activator VirB, which leads to reduced transcription of T3SS genes (65).…”

Thermodynamic matchers for the construction of the cuckoo RNA family

“…Furthermore, sRNAs with sequence motifs reminding of the cuckoo motif are sX13 RNA and RepG RNA, which are highly conserved in the Xanthomonadaceae family and in Helicobacter pylori strains, respectively. 26,27 So far homologs of neither family could be discovered in other species that would establish a phylogenetic connection between the above mentioned RNA families and thus no homology seems to exists. This is in concordance with observations that only a small proportion of RNA families are widely distributed.…”

The Spot 42 RNA: A regulatory small RNA with roles in the central metabolism

“…Expression of VSsrna24 is repressed by glucose, and is hypothesized to have roles in the central carbohydrate metabolism. 4 The sRNAs sX13, 17 ErsA 18 and Smr7C, 19,20 are found in Xanthomonadaceae, Pseudomonas and Rhizobialez, respectively, but neither has the same function or structure as Spot 42. sX13 and Smr7C share secondary structure features comprising 3 stem-loops with Crich motifs and are Hfq-independent. 17,21 ErsA is Hfq-mediated and regulated by sigma factor 22, in contrast to Spot 42 that is dependent on sigma factor 70.…”