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.
Highlights
Small non-coding RNAs are regarded as important regulators in prokaryotes and play essential roles in diverse cellular processes
As the expression of some bacterial sRNAs has been reported to be up-regulated around the stationary phase [8,11,38], we used total RNA extracted from the Xanthomonas oryzae pathovar oryzae (Xoo) cells in the stationary phase (OD600 = 1.5) in this study
Deletion of hfq in several animal bacteria pathogens resulted in attenuation of virulence [44], mutation in hfq in Xoo PXO99 did not cause detectable decrease of virulence after inoculation into the host plant rice cultivar IR24. Both the wild-type and Δhfq mutant strains caused blight disease symptoms on rice leaves and had no difference in mean lesion lengths. These results suggested that Hfq is not involved in the virulence of Xoo under the experimental conditions used in this study, and its biological roles need to be investigated in further studies
Summary
Small non-coding RNAs (sRNAs) are regarded as important regulators in prokaryotes and play essential roles in diverse cellular processes. Little is known about the number, genomic distribution and biological functions of sRNAs in Xoo. As an emerging class of gene expression modulators, small non-coding RNAs (sRNAs) have been detected in almost all kingdoms of life and are gaining increasing attention because of their important roles in various physiological processes. The hfq mutant exhibits various phenotypes in many bacterial species, including reduced growth rate, changed pathogenicity and altered tolerance to stress conditions [23,24,25,26,27,28] Another subgroup of antisense sRNAs is the cis-encoded sRNAs which are transcribed from the opposite strand of their target genes and regulate their target genes through complete complementarities [29]. RNA regulators such as riboswitches and CRISPR (clusters of regularly interspaced short palindromic repeats) RNAs play regulatory roles and exist widely in bacteria [20]
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