Structural model and functional characterization of the Bemisia tabaci CYP6CM1vQ, a cytochrome P450 associated with high levels of imidacloprid resistance

The neonicotinoid imidacloprid is one of the most important insecticides worldwide. It is used extensively against the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae), an insect pest of eminent importance globally, which was also the first pest to develop high levels of resistance against imidacloprid and other neonicotinoids in the field. Recent reports indicated that in both the B and Q biotypes of B. tabaci, the resistant phenotype is associated with over-expression of the cytochrome P450 gene CYP6CM1. In this study, molecular docking and dynamic simulations were used to analyze interactions of imidacloprid with the biotype Q variant of the CYP6CM1 enzyme (CYP6CM1vQ). The binding mode with the lowest energy in the enzyme active site, the key amino acids involved (i.e. Phe-130 and Phe-226), and the putative hydroxylation site (lowest distance to carbon 5 of the imidazolidine ring system of imidacloprid) were predicted. Heterologous expression of the CYP6CM1vQ confirmed the accuracy of our predictions and demonstrated that the enzyme catalyses the hydroxylation of imidacloprid to its less toxic 5-hydroxy form (Kcat = 3.2 pmol/min/pmol P450, Km = 36 μM). The data identify CYP6CM1vQ as a principle target for inhibitor design, aimed at inactivating insecticide-metabolizing P450s in natural insect pest populations. © 2009 Elsevier Ltd. All rights reserved

Targeting detoxification genes by phloem-mediated RNAi: A new approach for controlling phloem-feeding insect pests

Many phloem-feeding insects are considered severe pests of agriculture and are controlled mainly by chemical insecticides. Continued extensive use of these inputs is environmentally undesirable, and also leads to the development of insecticide resistance. Here, we used a plant-mediated RNA interference (RNAi) approach, to develop a new control strategy for phloem-feeding insects. The approach aims to silence “key” detoxification genes, involved in the insect's ability to neutralize defensive and toxic plant chemistry. We targeted a glutathione S-transferase (GST) gene, BtGSTs5, in the phloem-feeding whitefly Bemisia tabaci, a devastating global agricultural pest. We report three major findings. First, significant down regulation of the BtGSTs5 gene was obtained in the gut of B. tabaci when the insects were fed on Arabidopsis thaliana transgenic plants expressing dsRNA against BtGSTs5 under a phloem-specific promoter. This brings evidence that phloem-feeding insects can be efficiently targeted by plant-mediated RNAi. Second, in-silico and in-vitro analyses indicated that the BtGSTs5 enzyme can accept as substrates, hydrolyzed aliphatic- and indolic-glucosinolates, and produce their corresponding detoxified conjugates. Third, performance assays suggested that the BtGSTs5 gene silencing prolongs the developmental period of B. tabaci nymphs. Taken together, these findings suggest that BtGSTs5 is likely to play an important role in enabling B. tabaci to successfully feed on glucosinolate-producing plants. Targeting the gene by RNAi in Brassicaceae cropping systems, will likely not eliminate the pest populations from the fields but will significantly reduce their success over the growing season, support prominent activity of natural enemies, eventually allowing the establishment of stable and sustainable agroecosystem

E3 ligases determine ubiquitination site and conjugate type by enforcing specificity on E2 enzymes.

Ubiquitin-conjugating enzymes (E2s) have a dominant role in determining which of the seven lysine residues of ubiquitin is used for polyubiquitination. Here we show that tethering of a substrate to an E2 enzyme in the absence of an E3 ubiquitin ligase is sufficient to promote its ubiquitination, whereas the type of the ubiquitin conjugates and the identity of the target lysine on the substrate are promiscuous. In contrast, when an E3 enzyme is introduced, a clear decision between mono- and polyubiquitination is made, and the conjugation type as well as the identity of the target lysine residue on the substrate becomes highly specific. These features of the E3 can be further regulated by auxiliary factors as exemplified by MDMX (Murine Double Minute X). In fact, we show that this interactor reconfigures MDM2-dependent ubiquitination of p53. Based on several model systems, we propose that although interaction with an E2 is sufficient to promote substrate ubiquitination the E3 molds the reaction into a specific, physiologically relevant protein modification