Identification and functional characterization of small non-coding RNAs in Xanthomonas oryzae pathovar oryzae

Abstract

BackgroundSmall non-coding RNAs (sRNAs) are regarded as important regulators in prokaryotes and play essential roles in diverse cellular processes. Xanthomonas oryzae pathovar oryzae (Xoo) is an important plant pathogenic bacterium which causes serious bacterial blight of rice. However, little is known about the number, genomic distribution and biological functions of sRNAs in Xoo.ResultsHere, we performed a systematic screen to identify sRNAs in the Xoo strain PXO99. A total of 850 putative non-coding RNA sequences originated from intergenic and gene antisense regions were identified by cloning, of which 63 were also identified as sRNA candidates by computational prediction, thus were considered as Xoo sRNA candidates. Northern blot hybridization confirmed the size and expression of 6 sRNA candidates and other 2 cloned small RNA sequences, which were then added to the sRNA candidate list. We further examined the expression profiles of the eight sRNAs in an hfq deletion mutant and found that two of them showed drastically decreased expression levels, and another exhibited an Hfq-dependent transcript processing pattern. Deletion mutants were obtained for seven of the Northern confirmed sRNAs, but none of them exhibited obvious phenotypes. Comparison of the proteomic differences between three of the ΔsRNA mutants and the wild-type strain by two-dimensional gel electrophoresis (2-DE) analysis showed that these sRNAs are involved in multiple physiological and biochemical processes.ConclusionsWe experimentally verified eight sRNAs in a genome-wide screen and uncovered three Hfq-dependent sRNAs in Xoo. Proteomics analysis revealed Xoo sRNAs may take part in various metabolic processes. Taken together, this work represents the first comprehensive screen and functional analysis of sRNAs in rice pathogenic bacteria and facilitates future studies on sRNA-mediated regulatory networks in this important phytopathogen.

Small non-coding RNAs in plant-pathogenicXanthomonasspp

“…21 The expression of these sRNAs, termed sRNA-Xoo1 to 8, was verified by northern blot. Xoo single mutants deleted in each of the seven sRNA genes (sRNA-Xoo1 to 7) did not exhibit phenotypic changes compared with the wild-type with respect to in vitro growth, secretion of extracellular enzymes, and virulence.…”

“…Xoo single mutants deleted in each of the seven sRNA genes (sRNA-Xoo1 to 7) did not exhibit phenotypic changes compared with the wild-type with respect to in vitro growth, secretion of extracellular enzymes, and virulence. 21 Proteome analyses suggest that sRNA-Xoo1 might be involved in the regulation of amino acid metabolism and transport. 21 Such a role was also demonstrated for the Salmonella sRNA GcvB.…”

“…21 Proteome analyses suggest that sRNA-Xoo1 might be involved in the regulation of amino acid metabolism and transport. 21 Such a role was also demonstrated for the Salmonella sRNA GcvB. 32,33 In addition, the proteome data imply a role of sRNA-Xoo3 in the regulation of oxidation-reduction processes, stress response, and metabolic pathways.…”

“…32,33 In addition, the proteome data imply a role of sRNA-Xoo3 in the regulation of oxidation-reduction processes, stress response, and metabolic pathways. 21 A role in housekeeping processes was suggested for sRNA-Xoo4, which affects the abundance of proteins involved in DNA replication, rRNA processing, and metabolism. 21 It will be interesting to determine whether the impact of Xoo sRNAs on protein abundance is due to base-pairing interactions with corresponding mRNAs.…”

“…tomato, and members of the genera Erwinia and Xanthomonas. [15][16][17][18][19][20][21][22][23][24] This review focuses on sRNAs and the RNA-binding proteins (RBPs) Hfq and CsrA/RsmA in Xanthomonas spp. We will only discuss sRNAs, which have been validated by northern blot analyses and apologize to all colleagues whose data were not discussed in detail for space reasons.…”