MdWRKY75激活MdDEF30表达增强对苹果壳囊孢抗性

doi:10.1016/j.jia.2024.06.001

摘要/Abstract

摘要：

防御素在植物的生长发育和抵御病原菌侵染过程中发挥重要的作用，然而苹果中防御素对苹果树腐烂病菌抗性的作用尚不清楚。本研究中，共鉴定出29个苹果防御素蛋白，它们具有保守的序列特征。基于表达分析，发现苹果防御素在苹果各组织中均有分布，5个防御素基因的表达受到苹果壳囊孢的显著诱导。构建5个防御素的转基因愈伤，过表达防御素基因均能增强对苹果壳囊孢的抗性。其中，MdDEF30表达受苹果壳囊孢菌强烈诱导并显著提高愈伤抗性。进一步的体外活性实验证实MdDEF30能抑制壳囊孢的生长。MdDEF30能够促进活性氧积累和激活防卫相关基因PR4，PR10，CML13及MPK3的表达。通过构建MdDEF30共表达网络，发现转录因子MdWRKY75可能调控MdDEF30的表达。利用酵母单杂、荧光酶素报告基因和染色质免疫共沉淀荧光定量实验证实MdWRKY75能够与MdDEF30启动子直接结合。接种实验表明MdWRKY75正调控对苹果树腐烂病抗性，并且激活MdDEF30的表达。这些结果阐明苹果树通过MdWRKY75正向调控具抗菌活性和诱导抗性的MdDEF30表达抵御壳囊孢菌侵染的分子机理。

Abstract:

Defensin, an essential component of plant development, is indispensable in pathogen resistance. However, the molecular function of defensins under pathological conditions of Cytospora canker has not been characterized in apple plants. The present study exhibits a detailed overview of the phylogeny and structure of 29 defensins (MdDEF) in apple. Expression analysis revealed that MdDEF genes were spatiotemporally diverse across apple tissues. Five MdDEF genes were found to be significantly up-regulated following a challenge with Cytospora mali. The transgenic overexpression of five defensin genes in apple calli enhanced resistance to C. mali. Among them, MdDEF30 was strongly induced and conferred the highest resistance level in vivo. Meanwhile, antifungal activity assays in vitro demonstrated that a recombinant protein produced from MdDEF30 could inhibit the growth of C. mali. Notably, MdDEF30 promoted the accumulation of reactive oxygen species (ROS) and activated defense-related genes such as PR4, PR10, CML13, and MPK3. Co-expression regulatory network analysis showed that MdWRKY75 may regulate the expression of MdDEF30. Further yeast one-hybrid (Y1H), luciferase, and chromatin Immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) assays verified that MdWRKY75 could directly bind to the promoter of MdDEF30. Importantly, pathogen inoculation assays confirmed that MdWRKY75 positively regulates resistance by transcriptionally activating MdDEF30. Overall, these results demonstrated that MdDEF30 promotes resistance to C. mali in apple plants and that MdWRKY75 regulates MdDEF30 expression during the induction of resistance, thereby clarifying biochemical mechanisms of resistance to C. mali in apple trees.

Key words: apple calli , defensin , gene family , Cytospora mali , induced resistance , antifungal activity , weighted correlation network analysis (WGCNA) , transcription factors

Hongchen Jia, Youwei Du, Yuanyuan Liu, Shuanghong Wang, Yan Wang, Sadia Noorin, Mark L. Gleason, Rong Zhang, Guangyu Sun. MdWRKY75激活MdDEF30表达增强对苹果壳囊孢抗性[J]. Journal of Integrative Agriculture, 2025, 24(3): 1108-1125.

Hongchen Jia, Youwei Du, Yuanyuan Liu, Shuanghong Wang, Yan Wang, Sadia Noorin, Mark L. Gleason, Rong Zhang, Guangyu Sun. Transcriptional activation of MdDEF30 by MdWRKY75 enhances apple resistance to Cytospora canker [J]. Journal of Integrative Agriculture, 2025, 24(3): 1108-1125.

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