青枯菌GMI 1000菌株c-di-GMP合成酶Rsp1208的功能

doi:10.3864/j.issn.0578-1752.2026.11.006

摘要/Abstract

摘要：

【背景】青枯病是由青枯劳尔氏菌（Ralstonia solanacearum）引发的毁灭性土传植物病害，c-di-GMP作为细菌保守第二信使调控病原菌致病表型，但其在青枯菌中的核心代谢基因功能仍需深入解析。【目的】探究青枯菌GMI 1000中c-di-GMP代谢相关基因的功能及调控机制，明确关键基因对菌株生理表型和致病力的影响，完善青枯菌c-di-GMP信号调控网络，为青枯病新型防控靶点挖掘与绿色防控策略研发提供理论依据。【方法】以青枯菌GMI 1000为研究对象，采用RT-qPCR检测常规培养与模拟侵染条件下24个潜在c-di-GMP代谢基因的转录表达差异；针对极显著下调的Rsp1208，通过同源重组与电转化技术构建缺失、回补、酶活位点突变及过表达工程菌株；系统测定各菌株的生长动态、运动能力、生物膜形成量、胞外多糖产量等关键生理表型，结合RT-qPCR分析运动与胞外多糖合成相关基因的转录水平；采用LC-MS/MS与噻唑橙荧光反应法检测胞内c-di-GMP含量及体外合成酶活性；采用伤根接种法测定菌株对番茄的致病力。【结果】模拟侵染环境下，24个c-di-GMP代谢相关基因均呈不同程度转录下调，其中Rsp1208下调水平极显著，其编码蛋白同时含有GGDEF与EAL双结构域。Rsp1208缺失后，菌株运动能力与胞外多糖产量分别显著提升26.57%、85.92%，生物膜形成量降低75%，胞内c-di-GMP水平显著下降，对番茄致病力极显著增强；回补菌株表型恢复至野生型水平，过表达菌株则表现为运动能力减弱、生物膜形成微量提升、致病力显著降低。Rsp1208缺失株中运动相关基因（flhC、fliA、fliM、fliC）与胞外多糖合成基因（xpsR、epsB）转录水平均极显著上调。GGDEF酶活位点突变可使Rsp1208基本丧失c-di-GMP合成能力，EAL位点突变无显著影响。【结论】Rsp1208通过GGDEF结构域发挥c-di-GMP合成酶功能，调控青枯菌胞内c-di-GMP稳态，进而介导运动性、生物膜、胞外多糖等表型重塑，最终调控病原菌致病力，研究结果可为深入解析青枯菌的致病机制、研发绿色防控技术提供理论支撑。

关键词: 青枯菌, c-di-GMP, 致病力, 运动性, 生物膜

Abstract:

【Background】Bacterial wilt is a devastating soil-borne vascular disease caused by Ralstonia solanacearum. As a conserved second messenger in bacteria, c-di-GMP orchestrates multiple pathogenic phenotypes of plant bacterial pathogens. However, the functional characteristics of core c-di-GMP metabolic genes in R. solanacearum remain to be fully elucidated.【Objective】This study aimed to investigate the functions and regulatory mechanisms of c-di-GMP metabolism-associated genes in R. solanacearum GMI 1000, clarify the impacts of key genes on bacterial physiological phenotypes and pathogenicity, refine the c-di-GMP signaling regulatory network, and to provide a theoretical foundations for screening novel control targets and developing green management strategies against bacterial wilt.【Method】Using R. solanacearum GMI 1000 as the material, RT-qPCR was performed to compare the transcriptional profiles of 24 putative c-di-GMP metabolic genes under routine culture and simulated infection conditions. For the most significantly downregulated gene Rsp1208, gene deletion, complementation, enzyme active-site mutagenesis and overexpression strains were constructed via homologous recombination and electroporation. Key physiological phenotypes including growth kinetics, motility, biofilm formation and exopolysaccharide (EPS) yield were systematically quantified. Transcriptional levels of motility- and EPS synthesis-related genes were analyzed by RT-qPCR. Intracellular c-di-GMP content and in vitro diguanylate cyclase activity were detected using LC-MS/MS and thiazole orange fluorescence assay, respectively. Pathogenicity assays on tomato seedlings were conducted using the root-wounding inoculation method.【Result】Under simulated infection conditions, all 24 c-di-GMP metabolic genes were transcriptionally downregulated to varying degrees, among which Rsp1208 showed the most extreme downregulation. The protein encoded by Rsp1208 harbors both GGDEF and EAL domains. Deletion of Rsp1208 significantly increased bacterial motility by 26.57% and EPS production by 85.92%, reduced biofilm formation by 75%, decreased intracellular c-di-GMP levels markedly, and extremely enhanced pathogenicity on tomato. The complemented strain restored wild-type phenotypes, whereas the overexpression strain exhibited attenuated motility, mildly elevated biofilm formation and significantly weakened pathogenicity. Transcriptional levels of motility-related genes (flhC, fliA, fliM, fliC) and EPS synthesis genes (xpsR, epsB) were drastically upregulated in the Rsp1208 deletion mutant. Mutation of the GGDEF active site abolished the c-di-GMP synthetic activity of Rsp1208, while EAL site mutation exerted no significant effect.【Conclusion】Rsp1208 functions as a c-di-GMP diguanylate cyclase through its GGDEF domain, regulating intracellular c-di-GMP homeostasis in R. solanacearum, mediating phenotypic remodeling of motility, biofilm formation and EPS production, and ultimately governing the pathogenicity of the pathogen. These findings deepen the mechanistic understanding of R. solanacearum pathogenesis and provide critical theoretical support for the development of green control technologies against bacterial wilt.

Key words: Ralstonia solanacearum, c-di-GMP, pathogenicity, motility, biofilm

范晓涵, 张维军, 袁锦锋, 赵栋霖, 张成省, 张之矾, 徐康文. 青枯菌GMI 1000菌株c-di-GMP合成酶Rsp1208的功能[J]. 中国农业科学, 2026, 59(11): 2374-2386.

FAN XiaoHan, ZHANG WeiJun, YUAN JinFeng, ZHAO DongLin, ZHANG ChengSheng, ZHANG ZhiFan, XU KangWen. Function of c-di-GMP Synthase Rsp1208 of Ralstonia solanacearum Strain GMI 1000[J]. Scientia Agricultura Sinica, 2026, 59(11): 2374-2386.

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引物名称 Primer name	引物序列 Primer sequence	靶标基因 Target gene	退火温度 Annealing temperature (℃)	产物大小 Product size (bp)	用途 Purpose
Rsp1208-F1	gagctcggtacccggggatccAACACGTCGTCGTTGGTGATC	上游 Upstream	56	550	基因克隆 Gene clone
Rsp1208-R1	tatcgtcgatccagCAGCAGCTGGACGAATCGTT	上游 Upstream	56	550	基因克隆 Gene clone
Rsp1208-F2	ctgctgCTGGATCGACGATACGCAGG	下游 Downstream	56	740	基因克隆 Gene clone
Rsp1208-R2	acgacggccagtgccaagcttGAAACCGCCTCCAGCATGG	下游 Downstream	56	740	基因克隆 Gene clone
Rsp1208OE-F	agggaacaaaagctgggtaccGTTGCCTGACCGCGAACG	过表达片段 Overexpression fragment	58	1350	基因克隆 Gene clone
Rsp1208OE-R	caggaattcgatatcaagcttTTACGGCATCAGATCGCGC	过表达片段 Overexpression fragment	58	1350	基因克隆 Gene clone
M13F	TGTAAAACGACGGCCAGT	—	55	—	过表达验证 Overexpression verification
M13R	AACAGCTATGACCATGA	—	55	—	过表达验证 Overexpression verification
Rsp1208-pET30-F	taagaaggagatatacatatgTTGCCTGACCGCGAACGATTCGTC	蛋白表达片段 Protein expression fragment	55	1371	基因克隆 Gene clone
Rsp1208-pET30-R	ctcgagtgcggccgcaagcttCGGCATCAGATCGCGCGCGCGTTCG	蛋白表达片段 Protein expression fragment	55	1371	基因克隆 Gene clone

基因Gene	引物序列Primer sequence
gyrB	F: CAAGGGCAAGATCCTGAACG	R: GATGATGATGCGGTGGTAGC
flhC	F: AAGAGGTGCTGTCGATCACT	R: TCACGGTGTGCAACATCTTC
fliA	F: CATCGCCAACCAGATGATGG	R: GCGGCATAGAACTCGAACTG
fliM	F: CATGCCGTACACCATGATCG	R: TGTTGAGGATGTCGGACACA
fliC	F: ACTACAACGGCAACAAGCTG	R: TGGCCATATTGGAAGGTCGT
xpsR	CCCGCCAGTTCATTCTTCAG	GGACTGATGATCCAGGTGGT
epsB	GTGAGCTTGCTCAACGACAT	GTCAGCTTCTTGGGCTTCAC