Journal of Integrative Agriculture ›› 2024, Vol. 23 ›› Issue (5): 1580-1592.DOI: 10.1016/j.jia.2023.04.043

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基于Tn-seq和SPR对新型杀菌剂在水稻黄单胞菌中作用靶点筛选与鉴定的体系建立

  

  • 收稿日期:2023-03-06 接受日期:2023-03-27 出版日期:2024-05-20 发布日期:2024-04-23

Establishment of a system for screening and identification of novel bactericide targets in the plant pathogenic bacterium Xanthomonas oryzae pv. oryzae using Tn-seq and SPR

Chaoyue Pang1, 3, 4, 5*, Ling Jin1, 3, 4, 5*, Haoyu Zang2, Damalk Saint-Claire S. Koklannou1, 3, 4, 5, Jiazhi Sun1, 3, 4, 5, Jiawei Yang1, 3, 4, 5, Yongxing Wang6, Liang Xu6, Chunyan Gu2, Yang Sun1, 3, 4, 5, Xing Chen1, 3, 4, 5#, Yu Chen1, 3, 4, 5#    

  1. 1 School of Plant Protection, Anhui Agricultural University, Hefei 230036, China

    2 Institute of Plant Protection and Agro-Products Safety, Anhui Academy of Agricultural Sciences, Hefei 230031, China

    3 Anhui Province Laboratory for Green Pesticide Development and Application, School of Plant Protection, Anhui Agricultural University, Hefei 230036, China

    4 Hefei Research Center, State Key Laboratory for Biology of Plant Diseases and Insect Pests, Hefei 230036, China

    5 Key Laboratory of Integrated Crop Pest Management of Anhui Province, School of Plant Protection, Anhui Agricultural University, Hefei 230036, China

    6 Shandong Vicome Greenland Chemical Co., Ltd., Jinan 250204, China

  • Received:2023-03-06 Accepted:2023-03-27 Online:2024-05-20 Published:2024-04-23
  • About author:Chaoyue Pang, E-mail: 954076937@qq.com; Ling Jin, E-mail: 1102246363@qq.com; #Correspondence Yu Chen, E-mail: chenyu66891@sina.com; Xing Chen, E-mail: chenxing2028@163.com * These authors contributed equally to this study.
  • Supported by:
    The authors would like to thank Dehong Zheng, from Guangxi University (China) for providing the Escherichia coli BW29427 carrying the plasmid pMarC9-R6k.  This study was supported by the National Natural Science Foundation of China (32272587 and 32202342), the Programs for the Scientific Research Activities of Academic and Technical Leaders of Anhui Province, China (2020D251), the Development Fund for Talent Personnel of Anhui Agricultural University, China (rc342006), the University Synergy Innovation Program of Anhui Province, China (GXXT-2021-059), the Key Project of the Natural Science Foundation of Anhui Provincial Department of Education, China (2023AH040129), and Anhui Province Agricultural Eco-Environmental Protection and Quality Safety Industry Technology System, China.

摘要:

黄单胞菌是一类重要的植物病原细菌,可引起严重的植物病害,造成巨大的经济损失。化学防治是防治黄单胞菌属细菌侵染导致病害的最经济有效的措施,但又缺乏高效杀菌剂抑制此类病害的发生。因此,探究黄单胞菌基因组特征、鉴定新型药剂靶点是十分必要的。在本研究中,以引起水稻白叶枯病的病原菌稻黄单胞菌稻致病变种Xanthomonas oryzae pv. oryzaeXoo)为代表,成功构建了Xoo的转座子插入文库。转座子测序(Transposon sequencingTn-seq)数据显示,mariner C9转座子匹配插入的位点数占总潜在插入位点的35.7%-36.4%,同时鉴定到Xoo生长所必需的491个基因。与其他已报道细菌的必需基因相比,Xoo的一些必需基因功能未知,25个基因推测为黄单胞属类群的独有必需基因,且3个基因是黄单胞属细菌所特有的。综上,这些研究结果可为开发广谱型、黄单胞特异型、环境友好型的杀菌剂提供候选靶点。辛菌胺是一种对水稻黄单胞菌具有良好抑制效果的新型杀菌剂,本研究利用表面等离子体共振(Surface Plasmon ResonanceSPR)与高效液相色谱-质谱(High Performance Liquid Chromatography-Mass SpectrometryHPLC-MS)相结合的方法,鉴定得到了其在Xoo中的可能作用靶点,Tn-seq联合分析表明辛菌胺的作用靶点为Xoo的必需基因。这些靶点也可为后期开发抑制黄单胞属细菌的杀菌剂提供依据。本研究还结合实时荧光定量PCRQuantitative Real-time, qRT-PCR)的数据分析了一些杀菌剂的作用机制一直未被阐明的原因,Tn-seqSPRHPLC-MS三者结合来研究和验证杀菌剂的作用机制也是本研究中提出的一种新的方法。本研究构建的转座子插入文库也将为Xoo在寄主植物中的生存策略以及识别与Xoo适应度相关的未知基因等研究提供参考。总之,本研究促进了对黄单胞菌的认识,为防治水稻白叶枯病等由黄单胞菌引起的病害提供了新的视角,为今后对黄单胞菌的研究奠定了基础。

Abstract:

Xanthomonas spp. cause severe bacterial diseases.  However, effective strategies for prevention and management of these diseases are scarce.  Thus, it is necessary to improve the efficiency of control of diseases caused by Xanthomonas.  In this study, Xanthomonas oryzae pv. oryzae (Xoo), which causes rice bacterial leaf blight, has been studied as a representative.  A transposon insertion library of Xoo, comprising approximately 200,000 individual insertion mutants, was generated.  Transposon sequencing data indicated that the mariner C9 transposase mapped at 35.7–36.4% of all potential insertion sites, revealing 491 essential genes required for the growth of Xoo in rich media.  The results show that, compared to the functions of essential genes of other bacteria, the functions of some essential genes of Xoo are unknown, 25 genes might be dangerous for the Xanthomonas group, and 3 are specific to Xanthomonas.  High-priority candidates for developing broad-spectrum, Xanthomonas-specific, and environment-friendly bactericides were identified in this study.  In addition, this study revealed the possible targets of dioctyldiethylenetriamine using surface plasmon resonance (SPR) in combination with high performance liquid chromatography–mass spectrometry (HPLC–MS).  The study also provided references for the research of some certain bactericides with unknown anti-bacterial mode of action.  In conclusion, this study urged a better understanding of Xanthomonas, provided meaningful data for the management of bacterial leaf blight, and disclosed selected targets of a novel bactericide.

Key words: rice bacterial leaf blight ,  Xanthomonas ,  essential genes ,  Tn-seq ,  dioctyldiethylenetriamine