A key amino acid substitution of vacuolar-type H+-ATPases A subunit (VATP-A) confers selective toxicity of a potential botanical insecticide, periplocoside P (PSP), in Mythimna separata and Spodoptera exigua

IF 3.2 2区 农林科学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Xianxia Zhang , Yayun Zuo , Rui Liu , Shuang Wen , Yakun Pei , Qin Zhao , Baojun Shi , Wenjun Wu , Ding Li , Zhaonong Hu
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Abstract

Periplocosides, extracted from the root bark of Periploca sepium, are plant secondary compounds known to inhibit the V-ATPase enzyme in susceptible insect species, such as Mythimna separata. However, many species, including Spodoptera exigua, show resistance to these compounds. Previous studies identified the V-ATPase subunit A (VATP-A) in the midgut epithelium of M. separata as the putative target of periplocoside P (PSP), but the specific amino acids involved in this interaction remained unclear. In this study, we demonstrate the selective toxicity of PSP and its inhibition effect on V-ATPase. Molecular docking identified potential interactions between PSP and three amino acids (K85, R171, E199) in MsVATP-A, with in vitro binding assays revealing that K85 and R171 serve as the primary binding sites. Notably, sequence alignment revealed that R171 in sensitive species is substituted with K in resistant species. To investigate the functional implications of this substitution, we performed in vitro site-directed mutagenesis to exchange the corresponding amino acids between the VATP-A orthologs of M. separata and S. exigua. The R171K mutation in MsVATP-A reduced binding to PSP, while the K170R mutation in SeVATP-A enhanced it. Furthermore, in vivo genome editing in Drosophila melanogaster, a PSP-sensitive species, revealed that the R168K mutation conferred 15.78-fold resistance to PSP compared to the wild-type strain (w1118). Our findings confirm the role of VATP-A as the target of PSP and elucidate the key amino acids influencing its insecticidal selectivity. This research enhances the understanding of the molecular interactions between natural compounds and insect targets, offering insights for the development of targeted pest control strategies.

Abstract Image

液泡型H+- atp酶A亚基(VATP-A)的一个关键氨基酸取代,赋予了潜在的植物性杀虫剂,periploco苷P (PSP)的选择性毒性。
Periploca sepium根皮中提取的Periploca cosides是一种具有抑制v - atp酶活性的植物次生化合物。然而,许多物种,包括夜蛾,对这些化合物表现出抗性。先前的研究发现,分离田鼠中肠上皮中的v - atp酶亚基A (VATP-A)可能是perilocoside P (PSP)的靶点,但参与这种相互作用的具体氨基酸尚不清楚。在本研究中,我们证明了PSP的选择性毒性及其对v - atp酶的抑制作用。分子对接发现PSP与MsVATP-A中的三个氨基酸(K85、R171、E199)之间存在潜在的相互作用,体外结合实验显示K85和R171是主要的结合位点。值得注意的是,序列比对显示,敏感种的R171被抗性种的K所取代。为了研究这种取代的功能意义,我们在体外进行了位点定向诱变,以交换M. separata和S. exigua的VATP-A同源物之间相应的氨基酸。MsVATP-A的R171K突变减少了与PSP的结合,而SeVATP-A的K170R突变增强了与PSP的结合。此外,对PSP敏感物种黑腹果蝇的体内基因组编辑显示,R171K突变使其对PSP的抗性是野生型菌株的15.78倍(w1118)。我们的研究结果证实了VATP-A作为PSP的靶点的作用,并阐明了影响其杀虫选择性的关键氨基酸。该研究增强了对天然化合物与昆虫靶点之间分子相互作用的认识,为害虫定向防治策略的发展提供了见解。
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来源期刊
CiteScore
7.40
自引率
5.30%
发文量
105
审稿时长
40 days
期刊介绍: This international journal publishes original contributions and mini-reviews in the fields of insect biochemistry and insect molecular biology. Main areas of interest are neurochemistry, hormone and pheromone biochemistry, enzymes and metabolism, hormone action and gene regulation, gene characterization and structure, pharmacology, immunology and cell and tissue culture. Papers on the biochemistry and molecular biology of other groups of arthropods are published if of general interest to the readership. Technique papers will be considered for publication if they significantly advance the field of insect biochemistry and molecular biology in the opinion of the Editors and Editorial Board.
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