High entomotoxicity and mechanism of the fungal GalNAc/Gal-specific Rhizoctonia solani lectin in pest insects

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Whole insect assays where Rhizoctonia solani agglutinin (RSA) was fed to larval stages of the cotton leafworm 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 10mg/g in the solid diet of 2nd-instar caterpillars caused 84% weight reduction after 8days with none of the caterpillars reaching the 4th-instar stage. In sucking aphids, 50% mortality was achieved after 3days with 9μM 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 0.3μM. 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.