Department of Plants and Crops, Faculty of Bioscience Engineering, Ghent University, Belgium.
Department of Biotechnology, Faculty of Bioscience Engineering, Ghent University, Belgium.
Pestic Biochem Physiol. 2021 Jun;175:104853. doi: 10.1016/j.pestbp.2021.104853. Epub 2021 Apr 14.
Lepidopteran insects are highly refractory to oral RNA interference (RNAi). Degradation, impaired cellular uptake and intracellular transport of double-stranded RNA (dsRNA) are considered the major factors responsible for the reduced RNAi efficiency in these insects. In this study, the potential of lectins to improve dsRNA delivery and RNAi efficacy was evaluated. First, a fusion protein consisting of the Galanthus nivalis agglutinin (GNA) and a dsRNA binding domain was developed, further referred to as GNA:dsRBD (GNAF). Then, its ability to increase dsRNA uptake and transfection efficiency in lepidopteran midgut cells was evaluated, as well as its ability to protect and promote the RNAi response in the beet armyworm Spodoptera exigua. Confocal microscopy analysis showed that GNAF-complexed dsRNA was internalized faster in Choristoneura fumiferana midgut CF1 cells (1 min) compared to naked dsRNA (>1 h). The faster uptake was also correlated with an increased RNAi efficiency in these CF1 cells. In vivo feeding bioassays with GNAF-complexed dsRNA led to an increased mortality in S. exigua compared to the controls. By targeting the essential gene V-ATPase A, we observed that the mortality increased to 48% in the GNAF-dsRNA treatment compared to only 8.3% and 6.6% in the control treatments with the naked dsRNA and the GNAF, respectively.
鳞翅目昆虫对口服 RNA 干扰（RNAi）具有很强的抗性。双链 RNA（dsRNA）的降解、细胞摄取受损和细胞内转运被认为是导致这些昆虫中 RNAi 效率降低的主要因素。在本研究中，评估了凝集素提高 dsRNA 递呈和 RNAi 效率的潜力。首先，开发了一种由雪花莲凝集素（GNA）和 dsRNA 结合结构域组成的融合蛋白，进一步称为 GNA：dsRBD（GNAF）。然后，评估了它在鳞翅目昆虫中肠细胞中增加 dsRNA 摄取和转染效率的能力，以及它在甜菜夜蛾 Spodoptera exigua 中保护和促进 RNAi 反应的能力。共聚焦显微镜分析表明，与裸露的 dsRNA（>1 小时）相比，GNAF 复合物化的 dsRNA 在 Choristoneura fumiferana 中肠 CF1 细胞（1 分钟）中更快地被内化。更快的摄取也与这些 CF1 细胞中 RNAi 效率的提高相关。体内喂食用 GNAF 复合物化的 dsRNA 导致甜菜夜蛾的死亡率增加，与对照组相比。通过靶向必需基因 V-ATPase A，我们观察到与裸露 dsRNA 和 GNAF 的对照处理相比，GNAF-dsRNA 处理的死亡率增加到 48%，而仅分别为 8.3%和 6.6%。
