Dicer2蛋白的R1005D和E1007H突变导致亚洲镰孢菌对核酸农药的抗性

doi:10.1016/j.jia.2023.10.024

摘要/Abstract

摘要：

基于RNA干扰(RNAi)技术开发的核酸农药是一类不同于传统化学农药的绿色、高效、环保的新型农药，其在农业病虫害防治、农业可持续发展以及抗药性治理上表现出极大的潜力。为了核酸农药更高效合理地应用，病原菌对其是否具有抗性风险是值得关注的问题。亚洲镰孢菌（Fusarium asiaticum）是引起小麦赤霉病的主要病原菌，同时会产生真菌毒素威胁人畜健康，研究表明RNAi技术可防治小麦赤霉病。本文首次揭示了亚洲镰孢菌对核酸农药的抗性机制。通过紫外诱变，从500株干扰菌株中分离获得了5株核酸农药的抗性菌株，其中两株抗性菌株在Dicer2基因的RNase III结构域产生了R1005D或E1007H突变。进化分析显示，这两个位点均位于强烈的纯化选择压力条件下。为了进一步分析R1005D或E1007H突变是否是导致亚洲镰孢菌对核酸农药抗性的关键原因，利用同源双交换将敏感菌株与抗性菌株的Dicer2基因互换后发现，亚洲镰孢菌Dicer2蛋白的R1005D和E1007H突变降低了胞内Myosin5基因的干扰效率，影响菌株的抗病毒防御反应。核酸农药Faβ₂Tub-3 dsRNA对Dicer2^R1005D和Dicer2^E1007H突变体的离体和活体抑菌活性均显著降低。进一步分析发现，R1005D和E1007H突变改变了Dicer2蛋白分子内的相互作用。小RNA测序结果表明，Dicer2蛋白的R1005D和E1007H突变不仅降低了sRNAs的产量，还改变了sRNAs长度的分布比例，但不影响sRNAs 5’端第一个碱基的偏好性，说明Dicer2蛋白的R1005D和E1007H突变改变了Dicer2的切割活性。综上所述，Dicer2蛋白的R1005D和E1007H突变改变了亚洲镰孢菌中Dicer2蛋白的切割活性，从而导致菌株对核酸农药产生抗性。本文的发现对RNAi功能进化的研究具有启示意义，同时也对未来核酸农药的应用具有警示意义。

Abstract:

The use of RNA interference (RNAi) technology to control pests is explored by researchers globally. Even though RNA is a new class of pest control compound unlike conventional chemical pesticides, the evolution of pest resistance needs to be considered. Here, we first investigate RNAi-based biopesticide resistance of Fusarium asiaticum, which is responsible for devastating diseases of plants, for example, Fusarium head blight. Five resistant strains were isolated from 500 strains that treated with UV-mutagenesis. The mutation common to all of the five resistant mutants occurred in the gene encoding Dicer2 (point mutations at codon 1005 and 1007), which were under strong purifying selection pressure. To confirm whether the mutations in Dicer2 confer resistance to RNAi, we exchanged the Dicer2 locus between the sensitive strain and the resistant strain by homologous double exchange. The transformed mutants, Dicer2^R1005D and Dicer2^E1007H, exhibited resistance to dsRNA in vitro. Further study showed that mutations of R1005D and E1007H affected the intramolecular interactions of Dicer2, resulting in the dysfunction of RNase III domain of Dicer2. The amount of sRNAs produced by Dicer2^R1005D and Dicer2^E1007H was extremely reduced along with variation of sRNA length. Together, these findings revealed a new potential mechanism of RNAi resistance and provided insight into RNAi-related biopesticide deployment for fungal control.

Key words: RNA interference , dsRNA , Dicer2 , point mutation , resistance , Fusarium asiaticum

Kaixin Gu, Ran Wei, Yidan Sun, Xiaoxin Duan, Jing Gao, Jianxin Wang, Yiping Hou, Mingguo Zhou, Xiushi Song. Dicer2蛋白的R1005D和E1007H突变导致亚洲镰孢菌对核酸农药的抗性[J]. Journal of Integrative Agriculture, 2025, 24(2): 623-637.

Kaixin Gu, Ran Wei, Yidan Sun, Xiaoxin Duan, Jing Gao, Jianxin Wang, Yiping Hou, Mingguo Zhou, Xiushi Song. Point mutations of Dicer2 conferred Fusarium asiaticum resistance to RNAi-related biopesticide[J]. Journal of Integrative Agriculture, 2025, 24(2): 623-637.

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