柑橘PR4A参与柑橘对溃疡病的防御反应

doi:10.1016/j.jia.2024.11.011

Journal of Integrative Agriculture ›› 2025, Vol. 24 ›› Issue (12): 4643-4655.DOI: 10.1016/j.jia.2024.11.011

柑橘PR4A参与柑橘对溃疡病的防御反应

收稿日期:2024-09-14 修回日期:2024-11-04 接受日期:2024-10-08 出版日期:2025-12-20 发布日期:2025-11-13

Citrus PR4A is involved in the defense responses against Xanthomonas citri subsp. citri.

Chenxing Hao^{1, 2*}, Jian Han^3*, Peihan Yan^{1, 2}, Jia Ouyang^{1, 2}, Ling Sheng^{1, 2}, Guiyou Long², Ziniu Deng², Yunlin Cao^{1, 2}, Xianfeng Ma^{1, 2}

¹Engineering 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
³Hunan Horticultural Research Institute, Hunan Academy of Agricultural Sciences, Changsha 410125, China

Received:2024-09-14 Revised:2024-11-04 Accepted:2024-10-08 Online:2025-12-20 Published:2025-11-13
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.
Supported by:
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).

摘要/Abstract

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-likes（PR4s）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，P_CmPR4A-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CmSMU2或CmPR4A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.

Key words: Citron C-05 , Citrus canker , Pathogenesis-related protein , PR4A , SMU2 , Reactive oxygen species

Chenxing Hao, Jian Han, Peihan Yan, Jia Ouyang, Ling Sheng, Guiyou Long, Ziniu Deng, Yunlin Cao, Xianfeng Ma. 柑橘PR4A参与柑橘对溃疡病的防御反应[J]. Journal of Integrative Agriculture, 2025, 24(12): 4643-4655.

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

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柑橘PR4A参与柑橘对溃疡病的防御反应

Citrus PR4A is involved in the defense responses against Xanthomonas citri subsp. citri.

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