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Journal of Integrative Agriculture  2025, Vol. 24 Issue (12): 4643-4655    DOI: 10.1016/j.jia.2024.11.011
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Citrus PR4A is involved in the defense responses against Xanthomonas citri subsp. citri.

Chenxing Hao1, 2*, Jian Han3*, Peihan Yan1, 2, Jia Ouyang1, 2, Ling Sheng1, 2, Guiyou Long2, Ziniu Deng2, Yunlin Cao1, 2, Xianfeng Ma1, 2

1 Engineering Research Center for Horticultural Crop Germplasm Creation and New Variety Breeding of Ministry of Education, Hunan Agricultural University, Changsha 410128, China

2 National Center for Citrus Improvement, College of Horticulture, Hunan Agricultural University, Changsha 410128, China

3 Hunan Horticultural Research Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, China

 Highlights 
Overexpression of CmPR4A improved resistance to Xanthomonas citri subsp. citri (Xcc) in sweet orange and resistance to Pseudomonas syringae pv. tomato DC3000 in Arabidopsis.
The major effector pthA4 secreted by Xcc interacts with CmWRKY75, a positive regulator of CmPR4A, and upregulates CmPR4A expression in Citron C-05 leaves.
CmSMU2 interacts with CmPR4A to increase ROS accumulation, thereby enhancing resistance to Xcc in citrus.
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摘要  
柑橘溃疡病是由黄单胞菌属柑橘致病亚种Xanthomonas citri subsp. citri(Xcc)引起的一种毁灭性病害,几乎会感染所有柑橘栽培品种。值得注意的是,我们前期研究鉴定到枸橼C-05(Citrus medica L.)对Xcc的侵染表现出稳定的抗病性,然而其抗病机理仍不明晰。本研究中,我们在甜橙基因组的第8号染色体上发现了一个病程相关蛋白Pathogenesis-related 4-likesPR4s)基因簇,由5个PR4s基因组成。其中,PR4A在枸橼C-05叶片中响应Xcc侵染后上调表达最高,且不同抗病种质的叶片中PR4A的表达量均高于感病种质,表明PR4A的表达可能与柑橘对溃疡病的抗性有关。双分子荧光互补及Split-Luc试验表明,CmPR4A的正调控因子CmWRKY75与Xcc的主要致病效应子pthA4具有相互作用,且pthA4正调控CmPR4A的表达,进一步证明了CmPR4A的上调表达与Xcc侵染的关联性。GUS染色结果显示,PCmPR4A-P-516是受Xcc诱导PR4A表达的主要启动子区段。与此同时,瞬时过表达CmPR4A提高了甜橙对Xcc的抗性,而且OE-CmPR4A拟南芥转基因系也表现出对Pseudomonas syringae pv. tomato DC3000更强的抗性。此外,以CmPR4A为诱饵,通过酵母双杂交文库鉴定到CmSMU2与CmPR4A具有相互作用,并通过BiFC及Split-Luc验证了它们之间的相互作用。甜橙中瞬时过表达CmSMU2增加了其对Xcc的抗性,值得一提的是,与CmSMU2CmPR4A单独过表达相比,二者共表达会增强枸橼C-05叶片中活性氧积累,这些结果表明它们可能会协同增强柑橘对Xcc的抗性。本文为解析枸橼C-05对溃疡病的抗病机制奠定了理论基础,为柑橘抗病育种提供了重要的基因资源。




Abstract  

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a globally quarantine disease infecting nearly all Citrus cultivars.  Citron C-05 has been identified with complete and active resistance to Xcc.  However, the mechanism underlying Citron C-05’s resistance to Xcc remains elusive.  We identified a gene cluster on chromosome 8 of the citrus genome comprising five pathogenesis-related 4-like genes.  PR4A was upregulated in Citron C-05 leaves under Xcc infection, exhibiting the highest expression among these PR4-like genes.  In addition, PR4A expression was higher in leaves of disease-resistant genotypes than susceptible genotypes under Xcc invasion.  Bimolecular fluorescence complementation (BiFC) and Split-Luc assays indicated that CmWRKY75, a positive regulator of PR4A, interacted with pthA4 and upregulated expression of PR4A in Citron C-05 leaves.  Regulatory function for the expression of CmPR4A was localized to a 516-nucleotide region upstream of the translational start site, which was designated ProCmPR4A-P516.  Transient overexpression of CmPR4A improved resistance to Xcc in sweet orange, and three transgenic lines of OE-CmPR4A exhibited resistance to  (Pst DC3000) in Arabidopsis.  Furthermore, CmSMU2 was identified through yeast two-hybrid library using CmPR4A as bait, BiFC and Split-Luc assays further verified their interaction.  Transient overexpression of CmSMU2 in sweet orange increased resistance to Xcc.  Co-expression of CmSMU2 and CmPR4A enhanced accumulation of reactive oxygen species compared to CmSMU2 or CmPR4A, indicating that they may synergistically enhance resistance to Xcc in citrus.  These findings lay the groundwork for a theoretical analysis of the mechanism underlying the resistance of Citron C-05 against citrus canker.

Keywords:  Citron C-05       Citrus canker        Pathogenesis-related protein        PR4A        SMU2        Reactive oxygen species  
Received: 14 September 2024   Accepted: 08 October 2024 Online: 04 November 2024  
Fund: This work was supported by the National Natural Science Foundation of China (32402500 and 31741110) and the Scientific Research Fund of Hunan Provincial Education Department, China (21B0220).
About author:  #Correspondence Xianfeng Ma, E-mail: ma8006@hunau.edu.cn; Yunlin Cao, E-mail: yunlincao@hunau.edu.cn * These authors contributed equally to this study.

Cite this article: 

Chenxing Hao, Jian Han, Peihan Yan, Jia Ouyang, Ling Sheng, Guiyou Long, Ziniu Deng, Yunlin Cao, Xianfeng Ma. 2025. Citrus PR4A is involved in the defense responses against Xanthomonas citri subsp. citri.. Journal of Integrative Agriculture, 24(12): 4643-4655.

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