Table_4_Functional Characterization of a Global Virulence Regulator Hfq and Identification of Hfq-Dependent sRNAs in the Plant Pathogen Pantoea ananatis.XLSX

To successfully infect plant hosts, the collective regulation of virulence factors in a bacterial pathogen is crucial. Hfq is an RNA chaperone protein that facilitates the small RNA (sRNA) regulation of global gene expression at the post-transcriptional level. In this study, the functional role of Hfq in a broad host range phytopathogen Pantoea ananatis was determined. Inactivation of the hfq gene in P. ananatis LMG 2665T resulted in the loss of pathogenicity and motility. In addition, there was a significant reduction of quorum sensing signal molecule acyl-homoserine lactone (AHL) production and biofilm formation. Differential sRNA expression analysis between the hfq mutant and wild-type strains of P. ananatis revealed 276 sRNAs affected in their abundance by the loss of hfq at low (OD600 = 0.2) and high cell (OD600 = 0.6) densities. Further analysis identified 25 Hfq-dependent sRNAs, all showing a predicted Rho-independent terminator of transcription and mapping within intergenic regions of the P. ananatis genome. These included known sRNAs such as ArcZ, FnrS, GlmZ, RprA, RyeB, RyhB, RyhB2, Spot42, and SsrA, and 16 novel P. ananatis sRNAs. The current study demonstrated that Hfq is an important component of the collective regulation of virulence factors and sets a foundation for understanding Hfq-sRNA mediated regulation in the phytopathogen P. ananatis.

若要成功侵染植物宿主，细菌病原菌的毒力因子集体调控机制至关重要。Hfq是一种RNA分子伴侣蛋白，可在转录后水平协助小RNA（small RNA, sRNA）调控全局基因表达。本研究针对宿主范围宽泛的植物病原菌菠萝泛菌（Pantoea ananatis），明确了Hfq的功能作用。对菠萝泛菌LMG 2665T菌株的hfq基因进行失活处理后，其致病性与运动能力均丧失。此外，该菌株的群体感应信号分子酰基高丝氨酸内酯（acyl-homoserine lactone, AHL）合成量与生物被膜形成能力均显著下降。对菠萝泛菌hfq突变株与野生型菌株开展的sRNA差异表达分析显示，在低细胞密度（OD₆₀₀=0.2）与高细胞密度（OD₆₀₀=0.6）两种培养条件下，共有276个sRNA的丰度因hfq基因缺失而出现显著变化。后续分析共鉴定出25个Hfq依赖型sRNA，所有这些sRNA均预测带有不依赖Rho因子的转录终止子，且定位在菠萝泛菌基因组的基因间区范围内。上述Hfq依赖型sRNA包含已知的ArcZ、FnrS、GlmZ、RprA、RyeB、RyhB、RyhB2、Spot42及SsrA，同时还鉴定出16个全新的菠萝泛菌sRNA。本研究证实Hfq是细菌病原菌毒力因子集体调控机制的重要组成部分，同时为解析植物病原菌菠萝泛菌中Hfq-sRNA介导的调控通路奠定了研究基础。