Yin Fei, Xiao Miaomiao, Berestetskiy Alexander, Hu Qiongbo
Key Laboratory of Bio-Pesticide Innovation and Application of Guangdong Province, College of Plant Protection, South China Agricultural University, Guangzhou 510642, China.
Institute of Plant Protection, Guangdong Academy of Agricultural Sciences and Guangdong Provincial Key Laboratory of High Technology for Plant Protection, Guangzhou 510640, China.
J Fungi (Basel). 2021 Jun 8;7(6):460. doi: 10.3390/jof7060460.
Destruxin A (DA), a mycotoxin isolated from the entomopathogenic fungus , has good insecticidal and immune-inhibitory activity, but the action mechanism has not yet been elucidated. In order to identify the DA-binding proteins, we conducted drug affinity responsive target stability (DARTS) experiments, which indicated that the silkworm's () transmembrane protein 214 (BmTEME214) and protein transport protein SEC23A isoform X2 (BmSEC23) are the potential DA-binding proteins. The current research was focused on validation of the interaction between DA and these two proteins via bio-layer interferometry (BLI) in vitro, insect two-hybrid (I2H) in Sf9 cells, and RNAi in the insect. The results of the BLI tests showed that DA has strong affinity to bind BmTEME214 and BmSEC23 proteins with a K value of 0.286 and 0.291 µM, respectively. In the I2H experiments, DA inhibited (at 0.02 µg/mL) and activated (at 0.002-0.0002 µg/mL) the protein interactions of BmSEC23-BmSEC13, but it only inhibited the BmTMEM214-BmSEC13L interaction. Furthermore, in the RNAi tests, an apparent increase in the silkworm's mortality was recorded in the joint treatment of DA with dsBmSEC23 or dsBmTMEM214 when compared with the single treatment of DA (1.5 µg/g body), 40 µg/g body dsBmSEC23, or dsBmTMEM214. This research confirmed that BmSEC23 and BmTMEM214 are the DA-binding proteins and provided new insights to understand the action mechanism of DA.
destruxin A（DA）是一种从昆虫病原真菌中分离出的霉菌毒素，具有良好的杀虫和免疫抑制活性，但其作用机制尚未阐明。为了鉴定DA结合蛋白，我们进行了药物亲和响应靶点稳定性（DARTS）实验，结果表明家蚕跨膜蛋白214（BmTEME214）和蛋白质转运蛋白SEC23A亚型X2（BmSEC23）是潜在的DA结合蛋白。当前的研究集中于通过体外生物层干涉术（BLI）、Sf9细胞中的昆虫双杂交（I2H）以及昆虫体内的RNA干扰来验证DA与这两种蛋白之间的相互作用。BLI测试结果表明，DA与BmTEME214和BmSEC23蛋白具有很强的结合亲和力，K值分别为0.286和0.291 μM。在I2H实验中，DA（0.02 μg/mL时）抑制且（0.002 - 0.0002 μg/mL时）激活了BmSEC23 - BmSEC13的蛋白相互作用，但它仅抑制了BmTMEM214 - BmSEC13L的相互作用。此外，在RNA干扰测试中，与单独使用DA（1.5 μg/g体重）、40 μg/g体重的dsBmSEC23或dsBmTMEM214相比，DA与dsBmSEC23或dsBmTMEM214联合处理时，家蚕死亡率明显增加。本研究证实BmSEC23和BmTMEM214是DA结合蛋白，并为理解DA的作用机制提供了新的见解。
