柑橘溃疡病诱导型转录因子高通量筛选系统及其在柑橘溃疡病抗性调控研究中的应用

doi:10.1016/j.jia.2023.11.011

Journal of Integrative Agriculture ›› 2024, Vol. 23 ›› Issue (1): 155-165.DOI: 10.1016/j.jia.2023.11.011

柑橘溃疡病诱导型转录因子高通量筛选系统及其在柑橘溃疡病抗性调控研究中的应用

收稿日期:2022-12-22 接受日期:2023-10-07 出版日期:2024-01-20 发布日期:2024-01-06

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 Chen^{1, 3}, Yongrui He^{1, 3#}, Qiang Li^{1, 3#} #br#

¹Integrative Science Center of Germplasm Creation in Western China (Chongqing) Science City, Citrus Research Institute, Southwest University, Chongqing 400712, China
²School of Advanced Agriculture and Bioengineering, Yangtze Normal University, Chongqing 408100, China
³National Citrus Engineering Research Center, Chongqing 400712, China

Received:2022-12-22 Accepted:2023-10-07 Online:2024-01-20 Published:2024-01-06
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
Supported by:
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).

摘要/Abstract

摘要：

柑橘溃疡病是由地毯草黄单胞杆菌柑橘亚种（Xanthomonas citri subsp. citri，Xcc）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-蛋白质等调控网络。为探究柑橘溃疡病调控网络，本研究构建了一个含有203个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的基因，CsPrx25的上游调节因子进行了筛选，发现转录因子CsDOF5.8与CsPrx25的1-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CsDOF5.8与CsPrx25的相互作用和转录调控。本研究揭示了CsDOF5.8与CsPrx25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 CsDOF5.8–CsPrx25 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.

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

. 柑橘溃疡病诱导型转录因子高通量筛选系统及其在柑橘溃疡病抗性调控研究中的应用[J]. Journal of Integrative Agriculture, 2024, 23(1): 155-165.

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

High-throughput screening system of citrus bacterial canker-associated transcription factors and its application to the regulation of citrus canker resistance [J]. Journal of Integrative Agriculture, 2024, 23(1): 155-165.

参考文献

Bairoch A, Apweiler R. 1999. The SWISS-PROT protein sequence data bank and its supplement TrEMBL in 1999. Nucleic Acids Research, 27, 49–54.

Duan S, Jia H, Pang Z, Teper D, White F, Jones J, Zhou C, Wang N. 2018. Functional characterization of the citrus canker susceptibility gene CsLOB1. Molecular Plant Pathology, 19, 1908–1916.

Fernández-Pérez F, Pomar F, Pedreño M A, Novo-Uzal E. 2015. Suppression of Arabidopsis peroxidase 72 alters cell wall and phenylpropanoid metabolism. Plant Science, 239, 192–199.

Fields S, Song O. 1989. A novel genetic system to detect protein–protein interactions. Nature, 340, 245–246.

Fornes O, Castro-Mondragon J A, Khan A, van der Lee R, Zhang X, Richmond P A, Modi B P, Correard S, Gheorghe M, Baranašić D, Santana-Garcia W, Tan G, Chèneby J, Ballester B, Parcy F, Sandelin A, Lenhard B, Wasserman W W, Mathelier A. 2020. JASPAR 2020: update of the open-access database of transcription factor binding profiles. Nucleic Acids Research, 48, D87–D92.

He Y, Jia R, Qi J, Chen S, Lei T, Xu L, Peng A, Yao L, Long Q, Li Z, Li Q. 2019. Functional analysis of citrus AP2 transcription factors identified CsAP2-09 involved in citrus canker disease response and tolerance. Gene, 707, 178–188.

Hu B, Jin J, Guo A Y, Zhang H, Luo J, Gao G. 2015. GSDS 2.0: An upgraded gene feature visualization server. Bioinformatics, 31, 1296–1297.

Letunic I, Bork P. 2018. 20 years of the SMART protein domain annotation resource. Nucleic Acids Research, 46, D493–D496.

Li J J, Herskowitz I. 1993. Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybrid system. Science, 262, 1870–1874.

Li Q, Dou W F, Qi J J, Qin X J, Chen S C, He Y R. 2020a. Genomewide analysis of the CIII peroxidase family in sweet orange (Citrus sinensis) and expression profiles induced by Xanthomonas citri subsp. citri and hormones. Journal of Genetics and Genomics, 99, 13.

Li Q, Jia R R, Dou W F, Qi J J, Qin X J, Fu Y Y, He Y R, Chen S C. 2019. CsBZIP40, a BZIP transcription factor in sweet orange, plays a positive regulatory role in citrus bacterial canker response and tolerance. PLoS ONE, 14, e0223498.

Li Q, Qi J, Qin X, Dou W, Lei T, Hu A, Jia R, Jiang G, Zou X, Long Q, Xu L, Peng A, Yao L, Chen S, He Y. 2020b. CitGVD: a comprehensive database of citrus genomic variations. Horticulture Research, 7, 12.

Li Q, Qi J, Qin X, Hu A, Fu Y, Chen S, He Y. 2021. Systematic identification of lysin-motif receptor-like kinases (LYKs) in Citrus sinensis, and analysis of their inducible involvements in citrus bacterial canker and phytohormone signaling. Scientia Horticulturae, 276, 109755.

Li Q, Qin X, Qi J, Dou W, Dunand C, Chen S, He Y. 2020c. CsPrx25, a class III peroxidase in Citrus sinensis, confers resistance to citrus bacterial canker through the maintenance of ROS homeostasis and cell wall lignification. Horticulture Research, 7, 192.

Li Q, Yu H, Cao P B, Fawal N, Mathé C, Azar S, Cassan-Wang H, Myburg A A, Grima-Pettenati J, Marque C, Teulières C, Dunand C. 2015. Explosive tandem and segmental duplications of multigenic families in Eucalyptus grandis. Genome Biology and Evolution, 7, 1068–1081.

Li S J, Yin X R, Wang W L, Liu X F, Zhang B, Chen K S. 2017. Citrus CitNAC62 cooperates with CitWRKY1 to participate in citric acid degradation via up-regulation of CitAco3. Journal of Experimental Botany, 68, 3419–3426.

Liszkay A, Kenk B, Schopfer P. 2003. Evidence for the involvement of cell wall peroxidase in the generation of hydroxyl radicals mediating extension growth. Planta, 217, 658–667.

Liu H, Wang X, Liu S, Huang Y, Guo Y X, Xie W Z, Qamar M T U, Xu Q, Chen L L. 2022. Citrus Pan-Genome to Breeding Database (CPBD): A comprehensive genome database for citrus breeding. Molecular Plant, 15, 1503–1505.

Liu L, White M J, MacRae T H. 1999. Transcription factors and their genes in higher plants functional domains, evolution and regulation. European Journal of Biochemistry, 262, 247–257.

Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔCT method. Methods, 25, 402–408.

Long Q, Du M X, Long J H, Xie Y, Zhang J Y, Xu L Z, He Y R, Li Q, Chen S C, Zou X P. 2021. Transcription factor WRKY22 regulates canker susceptibility in sweet orange (Citrus sinensis Osbeck) by enhancing cell enlargement and CsLOB1 expression. Horticulture Research, 8, 15.

Ma X J, Mo C M. 2017. Prospects of molecular breeding in medical plants. China Journal of Chinese Materia Medica, 42, 2021–2031. (in Chinese)

Mafra V, Kubo K S, Alves-Ferreira M, Ribeiro-Alves M, Stuart R M, Boava L P, Rodrigues C M, Machado M A. 2012. Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions. PLoS ONE, 7, e31263.

Mistry J, Chuguransky S, Williams L, Qureshi M, Salazar G A, Sonnhammer E L L, Tosatto S C E, Paladin L, Raj S, Richardson L J, Finn R D, Bateman A. 2021. Pfam: The protein families database in 2021. Nucleic Acids Research, 49, D412–D419.

Mitsuda N, Ikeda M, Takada S, Takiguchi Y, Kondou Y, Yoshizumi T, Fujita M, Shinozaki K, Matsui M, Ohme-Takagi M. 2010. Efficient yeast one-/two-hybrid screening using a library composed only of transcription factors in Arabidopsis thaliana. Plant and Cell Physiology, 51, 2145–2151.

Pandey V P, Dwivedi U N. 2015. A ripening associated peroxidase from papaya having a role in defense and lignification: Heterologous expression and in-silico and in-vitro experimental validation. Gene, 555, 438–447.

Peng A, Chen S, Lei T, Xu L, He Y, Wu L, Yao L, Zou X. 2017. Engineering canker-resistant plants through CRISPR/Cas9-targeted editing of the susceptibility gene CsLOB1 promoter in citrus. Plant Biotechnology Journal, 15, 1509–1519.

Peng A, Xu L, He Y, Lei T, Yao L, Chen S, Zou X. 2015. Efficient production of marker-free transgenic ‘Tarocco’ blood orange (Citrus sinensis osbeck) with enhanced resistance to citrus canker using a Cre/loxP site-recombination system. Plant Cell, Tissue and Organ Culture, 123, 1–13.

Pruneda-Paz J L, Breton G, Nagel D H, Kang S E, Bonaldi K, Doherty C J, Ravelo S, Galli M, Ecker J R, Kay S A. 2014. A genome-scale resource for the functional characterization of Arabidopsis transcription factors. Cell Reports, 8, 622–632.

Pruneda-Paz J L, Breton G, Para A, Kay S A. 2009. A functional genomics approach reveals CHE as a component of the Arabidopsis circadian clock. Science, 323, 1481–1485.

Riechmann J L, Heard J, Martin G, Reuber L, Jiang C, Keddie J, Adam L, Pineda O, Ratcliffe O J, Samaha R R, Creelman R, Pilgrim M, Broun P, Zhang J Z, Ghandehari D, Sherman B K, Yu G. 2000. Arabidopsis transcription factors: Genome-wide comparative analysis among eukaryotes. Science, 290, 2105–2110.

Wang M, Dai W, Du J, Ming R, Dahro B, Liu J H. 2019. ERF109 of trifoliate orange (Poncirus trifoliata (L.) Raf.) contributes to cold tolerance by directly regulating expression of Prx1 involved in antioxidative process. Plant Biotechnology Journal, 17, 1316–1332.

Xiong Y, Liu T, Tian C, Sun S, Li J, Chen M. 2005. Transcription factors in rice: A genome-wide comparative analysis between monocots and eudicots. Plant Molecular Biology, 59, 191–203.

Ye L X, Zhang J X, Hou X J, Qiu M Q, Wang W F, Hu C G, Zhang J Z. 2021. A MADS-box gene CiMADS43 is involved in citrus flowering and leaf development through interaction with CiAGL9. International Journal of Molecular Sciences, 22, 5205.

Zhuang Y, Chen S, Lian W, Xu L, Wang D, Wang C, Meng J, Tang X, Xu H, Wang S, Du L, Zhang Y, Zhou G, Chai G. 2021. A high-throughput screening system for Populus wood-associated transcription factors and its application to lignin regulation. Frontiers in Plant Science, 12, 715809.

柑橘溃疡病诱导型转录因子高通量筛选系统及其在柑橘溃疡病抗性调控研究中的应用

High-throughput screening system of citrus bacterial canker-associated transcription factors and its application to the regulation of citrus canker resistance

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 0

编辑推荐

Metrics