Commercial biotechnology solutions for controlling lepidopteran and coleopteran insect pests on crops depend on the expression of Bacillus thuringiensis insecticidal proteins, most of which permeabilize the membranes of gut epithelial cells of susceptible insects. However, insect control strategies involving a different mode of action would be valuable for managing the emergence of insect resistance. Toward this end, we demonstrate that ingestion of double-stranded (ds)RNAs supplied in an artificial diet triggers RNA interference in several coleopteran species, most notably the western corn rootworm (WCR) Diabrotica virgifera virgifera LeConte. This may result in larval stunting and mortality. Transgenic corn plants engineered to express WCR dsRNAs show a significant reduction in WCR feeding damage in a growth chamber assay, suggesting that the RNAi pathway can be exploited to control insect pests via in planta expression of a dsRNA.

No abstract

Abstract:The responsiveness towards orally delivered dsRNA and the potency of a subsequent environmental RNA interference (RNAi) response strongly differs between different insect species. While some species are very sensitive to dsRNA delivery through the diet, others are not. The underlying reasons for this may vary, but degradation of dsRNA by nucleases in the gut lumen is believed to play a crucial role. The Colorado potato beetle, Leptinotarsa decemlineata, is a voracious defoliator of potato crops worldwide, and is currently under investigation for novel control methods based on dsRNA treatments. Here we describe the identification and characterization of two nuclease genes exclusively expressed in the gut of this pest species. Removal of nuclease activity in adults increased the sensitivity towards dsRNA and resulted in improved protection of potato plants. A similar strategy in the desert locust, Schistocerca gregaria, for which we show a far more potent nuclease activity in the gut juice, did however not lead to an improvement of the RNAi response.Possible reasons for this are discussed. Taken together, the present data confirm a negative effect of nucleases in the gut on the environmental RNAi response, and further suggest that interfering with this activity is a strategy worth pursuing for improving RNAi efficacy in insect pest control applications.

The leptin receptor is a class I transmembrane protein with either a short or a long cytoplasmic domain. Using chemical cross-linking we have analyzed the binding of leptin to its receptor. Cross-linking of radiolabeled leptin to different isoforms of the leptin receptor expressed on COS-1 cells reveals leptin receptor monomer, homodimer, and oligomer complexes. Cotransfection of the long and short form of the leptin receptor did not provide any evidence for the formation of heterodimer complexes. Soluble forms consisting of either the entire extracellular domain or the two cytokine receptor homologous domains of the leptin receptor were purified to homogeneity from recombinant baculovirus-infected insect cells by leptin affinity chromatography. Gel filtration chromatography showed that these proteins exist in a dimeric form. Analysis of the complex formed between soluble leptin receptor and leptin by native polyacrylamide gel electrophoresis, and data obtained from the amino acid composition of the complex provide direct evidence that the extracellular domain of the leptin receptor binds leptin in a 1:1 ratio.

The beta subunit (beta c) of the receptors for human granulocyte macrophage colony stimulating factor (GM‐CSF), interleukin‐3 (IL‐3) and interleukin‐5 (IL‐5) is essential for high affinity ligand‐binding and signal transduction. An important feature of this subunit is its common nature, being able to interact with GM‐CSF, IL‐3 and IL‐5. Analogous common subunits have also been identified in other receptor systems including gp130 and the IL‐2 receptor gamma subunit. It is not clear how common receptor subunits bind multiple ligands. We have used site‐directed mutagenesis and binding assays with radiolabelled GM‐CSF, IL‐3 and IL‐5 to identify residues in the beta c subunit involved in affinity conversion for each ligand. Alanine substitutions in the region Tyr365‐Ile368 in beta c showed that Tyr365, His367 and Ile368 were required for GM‐CSF and IL‐5 high affinity binding, whereas Glu366 was unimportant. In contrast, alanine substitutions of these residues only marginally reduced the conversion of IL‐3 binding to high affinity by beta c. To identify likely contact points in GM‐CSF involved in binding to the 365‐368 beta c region we used the GM‐CSF mutant eco E21R which is unable to interact with wild‐type beta c whilst retaining full GM‐CSF receptor alpha chain binding. Eco E21R exhibited greater binding affinity to receptor alpha beta complexes composed of mutant beta chains Y365A, H367A and I368A than to those composed of wild‐type beta c or mutant E366A. These results (i) identify the residues Tyr365, His367 and Ile368 as critical for affinity conversion by beta c, (ii) show that high affinity binding of GM‐CSF and IL‐5 can be dissociated from IL‐3 and (iii) suggest that Tyr365, His367 and Ile368 in beta c interact with Glu21 of GM‐CSF.