Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, B-9000 Ghent, Belgium.
J Insect Physiol. 2013 Mar;59(3):295-305. doi: 10.1016/j.jinsphys.2012.12.003. Epub 2013 Jan 2.
Whole insect assays where Rhizoctonia solani agglutinin (RSA) was fed to larval stages of the cotton leaf-worm Spodoptera littoralis and the pea aphid Acyrthosiphon pisum demonstrated a high concentration-dependent entomotoxicity, suggesting that this GalNAc/Gal-specific fungal lectin might be a good control agent for different pest insects. RSA at 10 mg/g in the solid diet of 2nd-instar caterpillars caused 84% weight reduction after 8 days with none of the caterpillars reaching the 4th-instar stage. In sucking aphids, 50% mortality was achieved after 3 days with 9 μM of RSA in the liquid diet. Feeding of FITC-labeled RSA to both insect pest species revealed strong lectin binding at the apical/luminal side of the midgut epithelium with the brush border zone, suggesting the insect midgut as a primary insecticide target tissue for RSA. This was also confirmed with cell cultures in vitro, where there was high fluorescence binding at the microvillar zone with primary cultures of larval midgut columnar cells of S. littoralis, and also at the surface with the insect midgut CF-203 cell line without lectin uptake in the midgut cells. In vitro assays using insect midgut CF-203 cells, revealed that RSA was highly toxic with an EC50 of 0.3 μM. Preincubation with GalNAc and saponin indicated that this action of RSA was carbohydrate-binding dependent and happened at the surface of the cells. Intoxicated CF-203 cells showed symptoms of apoptosis as nuclear condensation and DNA fragmentation, and this concurred with an increase of caspase-3/7, -8 and -9 activities. Finally, RSA affinity chromatography of membrane extracts of CF-203 cells followed by LC-MS/MS allowed the identification of 5747 unique peptides, among which four putatively glycosylated membrane proteins that are associated with apoptosis induction, namely Fas-associated factor, Apoptosis-linked gene-2, Neuroglian and CG2076, as potential binding targets for RSA. These data are discussed in relation to the physiological effects of RSA.
将根腐病菌凝集素（RSA）喂食给棉叶虫（Spodoptera littoralis）幼虫和豌豆蚜（Acyrthosiphon pisum）的整体昆虫实验表明，其具有高浓度依赖性的杀虫毒性，这表明这种半乳糖/半乳糖特异性真菌凝集素可能是一种控制不同害虫的良好药剂。在 2 龄幼虫的固体饮食中添加 10mg/g 的 RSA，8 天后会导致 84%的体重减轻，且没有幼虫进入 4 龄阶段。在吸食蚜虫的实验中，在液体饮食中添加 9μM 的 RSA 3 天后，有 50%的蚜虫死亡。将 FITC 标记的 RSA 喂食给这两种害虫，均发现凝集素在中肠上皮细胞的顶端/腔侧与刷状缘区域强烈结合，表明昆虫中肠是 RSA 的主要杀虫剂靶组织。这也通过体外细胞培养得到了证实，在那里，用 RSA 高荧光结合对 S. littoralis 幼虫中肠柱状细胞的原代培养，以及与昆虫中肠 CF-203 细胞系的表面结合，而在中肠细胞中没有凝集素摄取。使用昆虫中肠 CF-203 细胞的体外实验表明，RSA 具有高毒性，EC50 为 0.3μM。用半乳糖和皂角苷进行预孵育表明，RSA 的这种作用依赖于碳水化合物结合，并发生在细胞表面。中毒的 CF-203 细胞表现出凋亡的症状，如核浓缩和 DNA 片段化，这与 caspase-3/7、-8 和 -9 活性的增加一致。最后，通过 LC-MS/MS 对 CF-203 细胞膜提取物进行 RSA 亲和层析，鉴定出 5747 个独特的肽，其中 4 个推测为与凋亡诱导相关的糖基化膜蛋白，即 Fas 相关因子、凋亡相关基因-2、神经胶质和 CG2076，作为 RSA 的潜在结合靶标。这些数据与 RSA 的生理效应有关。
