双组分信号调控系统AcrK/AcrR对西瓜嗜酸菌运动性调控机制的研究

Previous studies found that a LuxR-type transcriptional regulator, AcrR, participated in the regulation of motility and virulence of Acidovoarax citrulli. RNA-seq of acrR mutant strain disclosed that 34 differentially expressed genes were involved in flagellar assembly pathway of A. citrulli. Chromatin Immunoprecipitation (ChIP) sequencing, combining with RNA-seq, will be employed in this project to analyze the two-component signal transduction system comprised of AcrK and AcrR, and reveal the regulatory mechanism of AcrK/AcrR in downstream genes involved in flagellar assembly and other motility-related genes, and eventually demonstrate the role that the AcrK/AcrR plays in flagellar assembly regulation of A. citrulli. To achieve our goals, this project will carry out studies including: (1) verification and functional characterization of two-component signal transduction system AcrK/AcrR; (2) explication of regulatory mechanism of AcrK/AcrR in flagellar assembly and other motility-related genes regulated by AcrR in A. citrulli; (3) demonstration the role of AcrK/AcrR in motility regulatory cascades of A. citrulli. This project will elucidate the regulatory mechanism of AcrK/AcrR in motility, and offer insight into the pathway of motility regulation of A.citrulli. This project will provide theoretical basis for further studies on the pathogenesis of A. citrulli.

前期研究发现，LuxR类型转录调控因子AcrR参与调控西瓜嗜酸菌的运动能力和致病力；对其突变株转录组测序初步分析，发现34个差异表达基因在鞭毛组装基因簇富集。本项目拟利用染色质免疫共沉淀(ChIP)技术结合转录组测序数据，解析AcrR参与的双组份信号调控系统AcrK/AcrR对下游鞭毛基因及其他运动性相关基因调控方式，阐明其对西瓜嗜酸菌运动性的调控机制。内容包括：(1) 验证双组份信号调控系统AcrK/AcrR及功能；(2) 分析AcrK/AcrR对西瓜嗜酸菌鞭毛形成的调控机制及调控的其它运动性相关基因；(3) 研究AcrK/AcrR对西瓜嗜酸菌运动性的调控机制。本研究目的和意义在于阐明AcrR参与的双组份调控系统AcrK/AcrR对西瓜嗜酸菌运动性的调控机制，揭示调控西瓜嗜酸菌运动性的重要途径，为研究病原菌致病机制提供理论依据。

 西瓜噬酸菌引起瓜类细菌性果斑病是西甜瓜产业的重要病害之一。LuxR类型转录调控因子AcrR参与调控西瓜噬酸菌的运动能力和致病力；对其突变株转录组测序初步分析，发现34个差异表达基因在鞭毛组装基因簇富集。本项目深入解析了AcrR与上游磷酸化激酶AcrK对西瓜噬酸菌致病性和运动性的调控机制。（1）明确了AcrK对西瓜噬酸菌致病力、生物膜形成和运动性的调控作用，发现acrK突变菌株和acrK/acrR突变菌株在西瓜上的致病力较野生型菌株Aac-5均显著下降，互补菌株致病力与野生型相似；ΔacrK与ΔacrK/ΔacrR在透射电镜下均未观察到鞭毛形成，而野生型和互补菌株均可观察到有单根极生鞭毛形成。（2）验证了AcrK与AcrR形成双组份信号传导系统，通过细菌双杂交和GST pull-down验证了AcrK与AcrR互作；通过Phos-tag验证了AcrK可使AcrR磷酸化；（3）构建了西瓜噬酸菌的AcrR-HA菌株，并发现AcrR-HA菌株的致病力、运动性和鞭毛形成与野生型菌株无显著差异，可以用于后续ChIP分析。本研究阐明了AcrR参与的双组份调控系统AcrK/AcrR的功能和对西瓜噬酸菌运动性的调控机制，揭示了调控西瓜噬酸菌运动性的重要途径，为深入研究病原菌致病机制提供理论依据。