Functional analysis of a down-regulated transcription factor-SoxNeuroA gene involved in the acaricidal mechanism of scopoletin against spider mites.

Abstract

Insight into the mode of action of plant-derived acaricides will help in the development of sustainable control strategies for mite pests. Scopoletin, a promising plant-derived bioactive compound, displays prominent acaricidal activity against Tetranychus cinnabarinus. The transcription factor SoxNeuroA plays a vital role in maintaining calcium ion (Ca2+ ) homeostasis. Down-regulation of SoxNeuroA gene expression occurs in scopoletin-exposed mites, but the functional role of this gene remains unknown. A SoxNeuroA gene from T. cinnabarinus (TcSoxNeuroA) was first cloned and identified. Reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time polymerase chain reaction (qPCR), and Western blotting assays all confirmed that the gene expression and protein levels of TcSoxNeuroA were significantly reduced under scopoletin exposure. Furthermore, RNA interference silencing of the weakly expressed SoxNeuroA gene significantly enhanced the susceptibility of mites to scopoletin, suggesting that the acaricidal mechanism of scopoletin was mediated by the weakly expressed SoxNeuroA gene. Additionally, yeast one-hybrid (Y1H) and dual-luciferase reporter assays revealed that TcSoxNeuroA was a repressor of Orai1 Ca2+ channel gene transcription, and the key binding sequence was ATCAAAG (positions -361 to -368 of the Orai1 promoter). Importantly, site-directed mutagenesis and microscale thermophoresis assays further indicated that ASP185, ARG189, and LYS217, which were key predicted hydrogen-bonding sites in the molecular docking model, may be the vital binding sites for scopoletin in TcSoxNeuroA. These results demonstrate that the acaricidal mechanism of scopoletin involves inhibition of the transcription factor SoxNeuroA, thus inducing the activation of the Orai1 Ca2+ channel, eventually leading to Ca2+ overload and lethality. Elucidation of the transcription factor-targeted mechanism for this potent plant-derived acaricide has vital implications for the design of next-generation green acaricides with novel targets. © 2023 Society of Chemical Industry.