Department of Agricultural Chemistry, Institute of Environmentally Friendly Agriculture, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 61186, Republic of Korea.
Science for Life Laboratory, Department of Cell and Molecular Biology, Uppsala University, SE-75124 Uppsala, Sweden.
Pestic Biochem Physiol. 2022 Nov;188:105244. doi: 10.1016/j.pestbp.2022.105244. Epub 2022 Sep 19.
Grammicin (Gra) is derived from the endophytic fungus Xylaria grammica EL000614 and shows nematicidal activity against the devastating root-knot nematode Meloidogyne incognita in-vitro, in planta, and in-field experiments. However, the mechanism of the nematicidal action of Gra remains unclear. In this study, Gra exposure to the model genetic organism Caenorhabditis elegans affected its L1, L2/3, L4, and young adult stages. In addition, Gra treatment increased the intracellular reactive oxygen species (ROS) levels of C. elegans and M. incognita. Molecular docking interaction analysis indicated that Gra could bind and interact with GCS-1, GST-4, and DAF-16a in order of low binding energy, followed by SOD-3, SKN-1, and DAF-16b. This implies that the anthelmintic action of Gra is related to the oxidative stress response. To validate this mechanism, we examined the expression of the genes involved in the oxidative stress responses following treatment with Gra using transgenic C. elegans strains such as the TJ356 strain zIs356 [daf-16p::daf-16a/b::GFP + rol-6 (su1006)], LD1 ldIs7 [skn-1p::skn-1b/c::GFP + rol-6 (su1006)], LD1171 ldIs3 [gcs-1p::GFP + rol-6 (su1006)], CL2166 dvIs19 [(pAF15) gst-4p::GFP::NLS], and CF1553 strain muIs84 [(pAD76) sod-3p::GFP + rol-6 (su1006)]. Gra treatment caused nuclear translocation of DAF-16/FoxO and enhanced gst-4::GFP expression, but it had no change in sod-3::GFP expression. These results indicate that Gra induces oxidative stress response via phase II detoxification without reduced cellular redox machinery. Gra treatment also inhibited the nuclear localization of SKN-1::GFP in the intestine, which may lead to a condition in which oxidative stress tolerance is insufficient to protect C. elegans by the inactivation of SKN-1, thus inducing nematode lethality. Furthermore, Gra caused the mortality of two mutant strains of C. elegans, CB113 and DA1316, which are resistant to aldicarb and ivermectin, respectively. This indicates that the mode of action of Gra is different from the traditional nematicides currently in use, suggesting that it could help develop novel approaches to control plant-parasitic nematodes.
格兰菌素（Gra）来源于内生真菌 Xylaria grammica EL000614，对毁灭性的根结线虫 Meloidogyne incognita 在体外、体内和田间试验中具有杀线虫活性。然而，Gra 的杀线虫作用机制尚不清楚。在这项研究中，Gra 暴露于模式遗传生物秀丽隐杆线虫会影响其 L1、L2/3、L4 和幼体成虫阶段。此外，Gra 处理增加了秀丽隐杆线虫和南方根结线虫的细胞内活性氧（ROS）水平。分子对接相互作用分析表明，Gra 可以结合并与 GCS-1、GST-4 和 DAF-16a 相互作用，按结合能从低到高依次排列，其次是 SOD-3、SKN-1 和 DAF-16b。这意味着 Gra 的驱虫作用与氧化应激反应有关。为了验证这一机制，我们使用转基因秀丽隐杆线虫品系，如 TJ356 菌株 zIs356 [daf-16p::daf-16a/b::GFP + rol-6 (su1006)]、LD1 ldIs7 [skn-1p::skn-1b/c::GFP + rol-6 (su1006)]、LD1171 ldIs3 [gcs-1p::GFP + rol-6 (su1006)]、CL2166 dvIs19 [(pAF15) gst-4p::GFP::NLS] 和 CF1553 菌株 muIs84 [(pAD76) sod-3p::GFP + rol-6 (su1006)]，检测了 Gra 处理后与氧化应激反应相关的基因表达。Gra 处理导致 DAF-16/FoxO 的核易位，并增强了 gst-4::GFP 的表达，但 sod-3::GFP 的表达没有变化。这些结果表明，Gra 通过 II 期解毒诱导氧化应激反应，而不会降低细胞内氧化还原机制。Gra 处理还抑制了 SKN-1::GFP 在肠道中的核定位，这可能导致 SKN-1 失活不足以保护秀丽隐杆线虫的氧化应激耐受性，从而诱导线虫死亡。此外，Gra 导致两种突变型秀丽隐杆线虫 CB113 和 DA1316 的死亡，这两种线虫分别对丙硫克百威和伊维菌素具有抗性。这表明 Gra 的作用模式与目前使用的传统杀线虫剂不同，表明它可以帮助开发控制植物寄生线虫的新方法。
