岷江百合WRKY11通过诱导dirigent表达增加木质素/木脂素积累正调控枯萎病抗性

doi:10.1016/j.jia.2023.07.032

摘要/Abstract

摘要：

百合属植物具有极高的观赏价值和经济价值，但其易感主要由尖孢镰刀菌（Fusarium oxysporum）引起的枯萎病。岷江百合（Lilium regale Wilson）是我国特有的百合野生种，对枯萎病具有极强的抗性。本研究从岷江百合中分离了一个WRKY转录因子LrWRKY11，并鉴定其在岷江百合与尖孢镰刀菌分子互作中的功能。LrWRKY11在烟草(Nicotiana tabacum)中的异位表达增强了对尖孢镰刀菌的抗性，而通过RNAi下调岷江百合鳞片中LrWRKY11的表达水平则降低对尖孢镰刀菌的抗性。转录组学分析发现LrWRKY11的异位表达上调了烟草中水杨酸/茉莉酸信号及木质素/木脂素生物合成相关基因的表达，同时UPLC-MS/MS检测结果进一步明确了LrWRKY11过表达烟草的水杨酸甲酯以及茉莉酸甲酯含量均显著高于野生型烟草。值得注意的是，LrWRKY11异位表达显著上调了烟草中木质素/木脂素生物合成相关基因dirigent (NtDIR)的表达水平，相反地，瞬时表达LrWRKY11 RNAi载体抑制了岷江百合中dirigent基因(LrDIR1)的表达。因而从岷江百合中分离获得LrDIR1，并克隆其启动子PLrDIR1，生物信息学分析表明PLrDIR1序列中含有一个WRKY转录因子结合的顺式作用元件W-box。酵母单杂交和凝胶阻滞实验结果显示LrWRKY11重组蛋白特异性结合PLrDIR1的W-box元件，并反式激活LrDIR1的转录。LrWRKY11/PLrDIR1-GUS（β-葡糖醛酸酶基因）转基因烟草的GUS活性显著高于PLrDIR1-GUS转基因烟草的GUS活性。LrDIR1的表达谱分析及PLrDIR1的活性检测表明植物激素、生物及非生物胁迫均能诱导LrDIR1表达水平上升。在大肠杆菌(Escherichia coli)中表达的LrDIR1重组蛋白明显抑制尖孢镰刀菌的菌丝生长。此外，LrDIR1在烟草中异位表达不仅增加了木质素/木脂素的积累，还增强了转基因烟草对尖孢镰刀菌的抗性。综上所述，LrWRKY11通过与水杨酸/茉莉酸信号通路相互作用，上调LrDIR1的表达，促进木质素/木脂素积累，进而正调控岷江百合对枯萎病的抗性。本研究的结果丰富了WRKY转录因子在植物抗病防卫反应中的调控机制，并有助于深入认知岷江百合对枯萎病的抗性分子机制。

Abstract:

Lilium are highly economically valuable ornamental plants that are susceptible to Fusarium wilt caused by Fusarium oxysporum. Lilium regale Wilson, a wild lily native to China, is highly resistant to F. oxysporum. In this study, a WRKY transcription factor, WRKY11, was isolated from L. regale, and its function during the interaction between L. regale and F. oxysporum was characterized. The ectopic expression of LrWRKY11 in tobacco increased the resistance to F. oxysporum, moreover, the transcriptome sequencing and UHPLC-MS/MS analysis indicated that the methyl salicylate and methyl jasmonate levels rose in LrWRKY11 transgenic tobacco, meanwhile, the expression of lignin/lignans biosynthesis-related genes including a dirigent (DIR) was up-regulated. The lignin/lignans contents in LrWRKY11-transgenic tobacco also significantly increased compared with the wild-type tobacco. In addition, the resistance of L. regale scales in which LrWRKY11 expression was silenced by RNAi evidently decreased, and additionally, the expression of lignin/lignans biosynthesis-related genes including LrDIR1 was significantly suppressed. Therefore, LrDIR1 and its promoter (PLrDIR1) sequence containing the W-box element were isolated from L. regale. The interaction assay indicated that LrWRKY11 specifically bound to the W-box element in PLrDIR1 and activated LrDIR1 expression. Additionally, β-glucuronidase activity in the transgenic tobacco co-expressing LrWRKY11/PLrDIR1-β-glucuronidase was higher than that in transgenic tobacco expressing PLrDIR1-β-glucuronidase alone. Furthermore, the ectopic expression of LrDIR1 in tobacco enhanced the resistance to F. oxysporum and increased the lignin/lignans accumulation. In brief, this study revealed that LrWRKY11 positively regulated L. regale resistance to F. oxysporum through interaction with salicylic acid/jasmonic acid signaling pathways and modulating LrDIR1 expression to accumulate lignin/lignans.

Key words: Lilium regale , Fusarium oxysporum , WRKY transcription factor , dirigent

Jie Deng, Zi’e Wang, Wenyun Li, Xiaohua Chen, Diqiu Liu. 岷江百合WRKY11通过诱导dirigent表达增加木质素/木脂素积累正调控枯萎病抗性[J]. Journal of Integrative Agriculture, 2024, 23(8): 2703-2722.

Jie Deng, Zi’e Wang, Wenyun Li, Xiaohua Chen, Diqiu Liu. WRKY11 up-regulated dirigent expression to enhance lignin/lignans accumulation in Lilium regale Wilson during response to Fusarium wilt[J]. Journal of Integrative Agriculture, 2024, 23(8): 2703-2722.

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