Background Small non-coding RNAs (sRNAs) are regarded as important regulators in prokaryotes and play essential roles in diverse cellular processes. 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 sRNA 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. Conclusions We 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. Background 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. With the rapid progress of research on bacterial transcriptome, hundreds of sRNAs have been identified. Subsequent functional analyses have revealed that these sRNAs regulate various cellular processes, such as stress responses , quorum sensing , life cycle differentiation  and virulence [4-7]. Systematic screen of sRNAs have been performed in diverse Rabbit Polyclonal to CNOT2 (phospho-Ser101) bacteria, such as Escherichia coli [8-11], Salmonella enterica , Pseudomonas aeruginosa  and many other bacterial species distantly related to E. coli [14-18]. These studies reveal that sRNAs are widely encoded in bacterial genomes, the discovery pace of bacterial sRNAs has continued to accelerate and the functions of increasing sRNAs are being elucidated . Bacterial sRNAs are usually 50-500 nucleotides (nt) in length. Besides binding with proteins to modulate their activities, the majority of sRNAs regulate their target genes by base pairing and function as diffusible molecules . The base pairing sRNAs can be further classified into two subgroups: trans-encoded sRNAs and cis-encoded sRNAs. Of them, trans-encoded sRNAs have been well-studied during the last two decades. These sRNAs are transcribed from the genomic loci which are physically unlinked to their target genes. Trans-encoded sRNAs usually regulate the translation or stability of their target mRNAs through partial and discontinuous complementarities. The trans-encoded sRNAs resemble the eukaryotic microRNAs in their ability to modulate mRNA 989-51-5 manufacture stability and translation [19,20]. In addition, most of the trans-encoded sRNAs require the bacterial Sm-like protein, Hfq, to perform their regulatory functions . Hfq plays important roles in sRNAs-mediated regulation by affecting the stability of sRNAs and facilitating the base-pairing between sRNAs and their target mRNAs . The hfq mutant exhibits various phenotypes in many bacterial species, including reduced growth rate, changed pathogenicity and altered tolerance to stress conditions [23-28]. 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 . Although 989-51-5 manufacture most of the identified cis-encoded sRNAs are encoded by phages, plasmids and transposons , recent studies revealed that bacterial chromosomes also generate a large number of cis-encoded sRNAs. Besides, RNA regulators such as riboswitches and CRISPR (clusters of regularly interspaced 989-51-5 manufacture short palindromic repeats) RNAs also play regulatory roles and exist widely in bacteria . Xanthomonas oryzae pathovar oryzae (Xoo) is a Gram-negative bacterium that belongs to the gamma subdivision of Proteobacteria and is the causal agent of the bacterial blight of rice. Xoo has long been used as a model organism in studying plant pathology. Currently, the complete genomic sequences of three Xoo strains are available [31-33], allowing for genome-scale analysis. During the past few years, a number of regulatory genes were identified in Xoo, especially those involved in virulence and host cell recognition, but very little is known about sRNAs and sRNA-mediated regulations in this bacterium. Bona fide small regulatory RNAs have not yet been described in Xoo, although some house-keeping sRNAs, regulatory RNAs such as riboswitches  and CRISPR RNAs  were reported. In the Xanthomonas genus, only four sRNAs from Xanthomonas campestris pv.campestris (Xcc) , the causal agent of black rot disease of crucifers,.