State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China.
BMC Genomics. 2011 Jan 30;12:87. doi: 10.1186/1471-2164-12-87.
Small 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.
Here, 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.
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.
小非编码 RNA（sRNAs）被认为是原核生物中的重要调控因子，在多种细胞过程中发挥着重要作用。稻黄单胞菌稻致病变种（Xoo）是一种重要的植物病原菌，可引起水稻严重的细菌性条斑病。然而，对于 Xoo 中 sRNAs 的数量、基因组分布和生物学功能知之甚少。
在这里，我们进行了系统筛选，以鉴定 Xoo 菌株 PXO99 中的 sRNAs。通过克隆共鉴定了 850 个源自基因间和基因反义区的假定非编码 RNA 序列，其中 63 个也通过计算预测被鉴定为 sRNA 候选物，因此被认为是 Xoo sRNA 候选物。Northern blot 杂交证实了 6 个 sRNA 候选物和其他 2 个克隆小 RNA 序列的大小和表达，随后将它们添加到 sRNA 候选物列表中。我们进一步检查了 8 个 sRNAs 在 hfq 缺失突变体中的表达谱，发现其中 2 个的表达水平明显降低，另一个表现出 Hfq 依赖性的转录加工模式。获得了 7 个经 Northern 确认的 sRNA 的缺失突变体，但它们均未表现出明显的表型。通过二维凝胶电泳（2-DE）分析比较了三个ΔsRNA 突变体和野生型菌株之间的蛋白质组差异，结果表明这些 sRNAs 参与了多种生理和生化过程。
我们在全基因组筛选中实验验证了 8 个 sRNAs，并在 Xoo 中发现了 3 个依赖 Hfq 的 sRNAs。蛋白质组学分析表明，Xoo sRNAs 可能参与了各种代谢过程。总之，这项工作代表了对水稻病原菌中 sRNAs 的首次全面筛选和功能分析，并为今后研究该重要植物病原菌中 sRNA 介导的调控网络提供了便利。
