Primary root growth in the absence or presence of exogenous NO(3)(-) was studied by a quantitative genetic approach in a recombinant inbred line (RIL) population of Medicago truncatula. A quantitative trait locus (QTL) on chromosome 5 appeared to be particularly relevant because it was seen in both N-free medium (LOD score 5.7; R(2)=13.7) and medium supplied with NO(3)(-) (LOD score, 9.5; R(2)=21.1) which indicates that it would be independent of the general nutritional status. Due to its localization exactly at the peak of this QTL, the putative NRT1-NO(3)(-) transporter (Medtr5g093170.1), closely related to Arabidopsis AtNRT1.3, a putative low-affinity nitrate transporter, appeared to be a significant candidate involved in the control of primary root growth and NO(3)(-) sensing. Functional characterization in Xenopus oocytes using both electrophysiological and (15)NO(3)(-) uptake approaches showed that Medtr5g093170.1, named MtNRT1.3, encodes a dual-affinity NO(3)(-) transporter similar to the AtNRT1.1 'transceptor' in Arabidopsis. MtNRT1.3 expression is developmentally regulated in roots, with increasing expression after completion of germination in N-free medium. In contrast to members of the NRT1 superfamily characterized so far, MtNRT1.3 is environmentally up-regulated by the absence of NO(3)(-) and down-regulated by the addition of the ion to the roots. Split-root experiments showed that the increased expression stimulated by the absence of NO(3)(-) was not the result of a systemic signalling of plant N status. The results suggest that MtNRT1.3 is involved in the response to N limitation, which increases the ability of the plant to acquire NO(3)(-) under N-limiting conditions.

Scorpion alpha-neurotoxins can be classified into distinct subgroups according to their sequence and pharmacological properties. Using toxicity tests, binding studies, and electrophysiological recordings, BmK M1, a toxin from the Asian scorpion Buthus martensi Karsch, was experimentally identified as an alpha-like toxin. Being the first alpha-like toxin available in a recombinant form, BmK M1 was then modified by site-directed mutagenesis for investigation of the molecular basis of its activity. The results suggested a functional site which protrudes from the molecular scaffold as a unique tertiary arrangement, constituted by the five-residue reverse turn 8-12 and the C-terminal segment. The C-terminal basic residues Lys62 and His64 together with Lys8 in the turn, which are critical for the bioactivities, may directly interact with the receptor site on the sodium channel. Residues Asn11 and Arg58, indispensable for the activities, are mainly responsible for stabilizing the distinct conformation of the putative bioactive site. Among others, His10 and His64 seem to be involved in the preference of BmK M1 for phylogenetically distinct target sites. The comparison of BmK M1 with Aah2 (classical alpha-toxin) and Lqh(alpha)IT (alpha-insect toxin) showed that the specific orientation of the C-terminus mediated by the reverse turn might be relevant to the preference of alpha-toxin subgroups for phylogenetically distinct yet closely related receptor sites. The Y5G mutation indicated the "conserved hydrophobic surface" might be structurally important for stabilizing the beta-sheet in the alpha/beta-scaffold. The observations in this work shed light on the nature and roles of the residues possibly involved in the biological activity of a scorpion alpha-like toxin.

The Eyguières 42 strain of Drosophila simulans, obtained by laboratory selection, displayed 20 000-fold resistance to the insecticide fipronil. Molecular cloning of the cDNA encoding the RDL GABA receptor subunit of this strain revealed the presence of two mutations: the Rdl mutation (A301G) and an additional mutation in the third transmembrane domain (T350M). In order to assess the individual and combined roles of the two mutations in fipronil resistance, the functional properties of wild-type, A301G, T350M and A301G/T350M homomultimeric RDL receptors were compared by expression in Xenopus oocytes. In wild-type receptors, the inhibition of GABA (EC 30 )-induced currents by fipronil and picrotoxin was enhanced by repeated GABA applications. The A301G mutation nearly abolished this effect, decreased the sensitivity to fipronil and picrotoxin and increased the reversibility of inhibition. The T350M mutation also reduced the sensitivity to both antagonists. Of the four receptor variants tested, the double mutant showed the highest resistance to fipronil, following repeated GABA applications. In conclusion, the present study emphasizes new aspects of the pharmacological alterations induced by the Rdl mutation and shows that resistance to GABA receptor-directed insecticides may implicate a mutation distinct from Rdl. Keywords: Drosophila simulans, fipronil, GABA, insecticide resistance, Rdl, Xenopus oocyte. Ionotropic GABA receptors (GABARs) are widely distributed throughout the nervous and muscular systems of insects and are important targets of several classes of insecticides such as cyclodienes, cyclohexanes and phenylpyrazoles (Bloomquist 2001). All these convulsant antagonists inhibit the flow of Cl -ions through the receptor channel complex by a mixed/non-competitive mechanism that remains under investigation. Although the discovery of their molecular targets is relatively recent, cyclodienes and cyclohexanes have been widely used in agriculture during the last half century, which has led to the development of resistance mechanisms in many pest species. Cyclodiene resistance is usually accompanied by tolerance to other convulsant antagonists like picrotoxin (PTX) or ethynylbicyclo-orthobenzoate (EBOB; Kadous et al. 1983;Bloomquist et al. 1991;Cole et al. 1995). In contrast, insects showing high resistance to dieldrin generally exhibit very low levels of cross-resistance to fipronil (review in Bloomquist 2001), suggesting that the binding site or mechanisms of action of these two compounds may not be identical. This view is strengthened by studies showing that cyclodienes and PTX are competitive inhibitors of [3H]-EBOB, whereas fipronil displaces this radioligand non-competitively (Deng et al. 1993;Gant et al. 1998).The two main mechanisms of insecticide resistance are increased detoxification and decreased affinity for the target

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3 Rat brain membranes were also shown to possess a population of saturable, speci®c [ 3 H]-BIDN binding sites, though of lower a nity than in rootworm and with a di erent pharmacological pro®le. Of the insecticidal GABAergic convulsants that displaced [ 3 H]-BIDN from rootworm, cockroach (Periplaneta americana) and rat brain membranes, many were more e ective in rootworm. 4 Functional GABA-gated chloride channels of rootworm nervous system and of cockroach nerve and muscle were blocked by BIDN, whereas cockroach neuronal GABA B receptors were una ected. 5 Expression in Xenopus oocytes of either rat brain mRNA, or cDNA-derived RNA encoding a GABA receptor subunit (Rdl) that is expressed widely in the nervous system of Drosophila melanogaster resulted in functional, homo-oligomeric GABA receptors that were blocked by BIDN. Thus, BIDN probes a novel site on GABA-gated Cl 7 channels to which a number of insecticidally-active molecules bind.

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Two novel toxins, Lqh6 and Lqh7, isolated from the venom of the scorpion Leiurus quinquestriatus hebraeus, have in their sequence a molecular signature (8Q/KPE10) associated with a recently defined group of α‐toxins that target Na channels, namely the α‐like toxins [reviewed in Gordon, D., Savarin, P., Gurevitz, M. & Zinn‐Justin, S. (1998) J. Toxicol. Toxin Rev. 17, 131–159]. Lqh6 and Lqh7 are highly toxic to insects and mice, and inhibit the binding of α‐toxins to cockroach neuronal membranes. Although they kill rodents by intracerebroventricular injection, they do not inhibit the binding of antimammal α‐toxins (e.g. Lqh2) to rat brain synaptosomes, not even at high concentrations. Furthermore, in voltage‐clamp experiments, rat brain Na channels IIA (rNav1.2A) expressed in Xenopus oocytes are not affected by Lqh6 nor by Lqh7 below 3 µm. In contrast, muscular Na channels (rNav1.4 and hNav1.5) expressed in the same cells respond to nanomolar concentrations of Lqh6 and Lqh7 by slowing of Na current inactivation and a leftward shift of the peak conductance–voltage curve. The structural and pharmacological properties of the new toxins are compared to those of other scorpion α‐toxins in order to re‐examine the hallmarks previously set for the α‐like toxin group.