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Journal of Integrative Agriculture  2024, Vol. 23 Issue (1): 155-165    DOI: 10.1016/j.jia.2023.11.011
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High-throughput screening system of citrus bacterial canker-associated transcription factors and its application to the regulation of citrus canker resistance

Jia Fu1, Jie Fan1, Chenxi Zhang1, Yongyao Fu2, Baohang Xian1, Qiyuan Yu1, Xin Huang1, Wen Yang1, Shanchun Chen1, 3, Yongrui He1, 3#, Qiang Li1, 3# #br#

1 Integrative Science Center of Germplasm Creation in Western China (Chongqing) Science City, Citrus Research Institute, Southwest University, Chongqing 400712, China

2 School of Advanced Agriculture and Bioengineering, Yangtze Normal University, Chongqing 408100, China

3 National Citrus Engineering Research Center, Chongqing 400712, China

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摘要  

柑橘溃疡病是由地毯草黄单胞杆菌柑橘亚种(Xanthomonas citri subsp. citriXcc)引起的细菌性病害,严重危害全球柑橘产业的发展。抵御柑橘溃疡病是一个复杂过程,包括蛋白质-DNA以及蛋白质-蛋白质网络。为探究柑溃疡病调控网络,本研究构建一个含有203受柑橘溃疡病诱导的转录因子的酵母单/双杂(Y1H/Y2H)高通量筛选系统。通过该系统对一个通过维持活性氧稳态来提高对柑橘溃疡病抗性基因,CsPrx25的上游调节因子进行了筛选,发现转录因子CsDOF5.8CsPrx251-kb启动子片段相互作用。结合电泳迁移率实验双荧光素酶实验以及CsDOF5.8的瞬时过表达,进一步验证了CsDOF5.8CsPrx25相互作用和转录调控。本研究揭示了CsDOF5.8CsPrx25通过相互作用来维持H2O2稳态,进而调控柑橘溃疡病抗性的复杂途径。本研究构建的高通量筛选系统是探究柑橘溃疡病抗性调控路径或互作网络的有效工具



Abstract  

One of the main diseases that adversely impacts the global citrus industry is citrus bacterial canker (CBC), caused by the bacteria Xanthomonas citri subsp. citri (Xcc).  Response to CBC is a complex process, with both protein-DNA as well as protein–protein interactions for the regulatory network.  To detect such interactions in CBC resistant regulation, a citrus high-throughput screening system with 203 CBC-inducible transcription factors (TFs), were developed.  Screening the upstream regulators of target by yeast-one hybrid (Y1H) methods was also performed.  A regulatory module of CBC resistance was identified based on this system.  One TF (CsDOF5.8) was explored due to its interactions with the 1-kb promoter fragment of CsPrx25, a resistant gene of CBC involved in reactive oxygen species (ROS) homeostasis regulation.  Electrophoretic mobility shift assay (EMSA), dual-LUC assays, as well as transient overexpression of CsDOF5.8, further validated the interactions and transcriptional regulation.  The CsDOF5.8CsPrx25 promoter interaction revealed a complex pathway that governs the regulation of CBC resistance via H2O2 homeostasis.  The high-throughput Y1H/Y2H screening system could be an efficient tool for studying regulatory pathways or network of CBC resistance regulation.  In addition, it could highlight the potential of these candidate genes as targets for efforts to breed CBC-resistant citrus varieties.

Keywords:  Citrus bacterial canker (CBC)       high-throughput screening system        transcription factor (TF)        yeast-one hybrid (Y1H)        CsPrx25

  
Received: 22 December 2022   Accepted: 07 October 2023
Fund: 

This study was funded by the National Key Research and Development Program of China (2022YFD1201600), the earmarked fund for the China Agriculture Research System (CARS-26), the Fundamental Research Funds for the Central Universities, China (SWU-XDJH202308), and the Science and Technology Research Program of Chongqing Municipal Education Commission, China (KJQN202001418).

About author:  FU Jia, E-mail: fjduolaimi@email.swu.edu.cn; #Correspondence LI Qiang, E-mail: liqiang@cric.cn; HE Yong-rui, E-mail: heyongrui@cric.cn

Cite this article: 

Jia Fu, Jie Fan, Chenxi Zhang, Yongyao Fu, Baohang Xian, Qiyuan Yu, Xin Huang, Wen Yang, Shanchun Chen, Yongrui He, Qiang Li. 2024.

High-throughput screening system of citrus bacterial canker-associated transcription factors and its application to the regulation of citrus canker resistance . Journal of Integrative Agriculture, 23(1): 155-165.

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