A novel chemotype insecticide flupyrimin (FLP) [N-[(E)-1-(6-chloro-3-pyridinylmethyl)pyridin-2(1H)-ylidene]-2,2,2-trifluoroacetamide], discovered by Meiji Seika Pharma, has unique biological properties, including outstanding potency to imidacloprid (IMI)-resistant rice pests together with superior safety toward pollinators. Intriguingly, FLP acts as a nicotinic antagonist in American cockroach neurons, and [H]FLP binds to the multiple high-affinity binding components in house fly nicotinic acetylcholine (ACh) receptor (nAChR) preparation. One of the [H]FLP receptors is identical to the IMI receptor, and the alternative is IMI-insensitive subtype. Furthermore, FLP is favorably safe to rats as predicted by the very low affinity to the rat α4β2 nAChR. Structure-activity relationships of FLP analogues in terms of receptor potency, featuring the pyridinylidene and trifluoroacetyl pharmacophores, were examined, thereby establishing the FLP molecular recognition at the Aplysia californica ACh-binding protein, a suitable structural surrogate of the insect nAChR. These FLP pharmacophores account for the excellent receptor affinity, accordingly revealing differences in its binding mechanism from IMI.

Summary Systemic acquired resistance (SAR), a natural disease response in plants, can be induced chemically. Salicylic acid (SA) acts as a key endogenous signaling molecule that mediates SAR in dicotyledonous plants. However, the role of SA in monocotyledonous plants has yet to be elucidated. In this study, the mode of action of the agrochemical protectant chemical probenazole was assessed by microarray‐based determination of gene expression. Cloning and characterization of the most highly activated probenazole‐responsive gene revealed that it encodes UDP‐glucose:SA glucosyltransferase (OsSGT1), which catalyzes the conversion of free SA into SA O‐β‐glucoside (SAG). We found that SAG accumulated in rice leaf tissue following treatment with probenazole or 2,6‐dichloroisonicotinic acid. A putative OsSGT1 gene from the rice cultivar Akitakomachi was cloned and the gene product expressed in Escherichia coli was characterized, and the results suggested that probenazole‐responsive OsSGT1 is involved in the production of SAG. Furthermore, RNAi‐mediated silencing of the OsSGT1 gene significantly reduced the probenazole‐dependent development of resistance against blast disease, further supporting the suggestion that OsSGT1 is a key mediator of development of chemically induced disease resistance. The OsSGT1 gene may contribute to the SA signaling mechanism by inducing up‐regulation of SAG in rice plants.

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Approximately 300 microbial natural products in our library were screened for insecticidal activities against three species of agricultural pests, including aphids. Among the several compounds that showed insecticidal activities, pyripyropene A had high aphicidal activity in vivo. Furthermore, in advanced tests, pyripyropene A applications with foliar sprays and soil drenching controlled aphids on cabbage. On the basis of its unique and promising activities, we selected pyripyropene A as the active component of potential insecticides.

Pyripyropenes are potent inhibitors of acyl-CoA:cholesterol acyltransferase, which were initially discovered to be produced by Aspergillus fumigatus. Recently, Penicillium coprobium PF1169 has also found to produce pyripyropene A (PyA), which exhibits insecticidal properties. Pyripyropenes are natural hybrid products of both terpenoid and polyketide origin. In our research, based on data generated using the Genome Sequencer FLX for P. coprobium PF1169, we predicted the biosynthetic gene cluster of PyA by blast analysis comparing with polyketide synthase and prenyltransferase of other species. By screening the genomic fosmid library, nine open reading frames (ppb1 to ppb9) related to the biosynthesis of PyA were deduced. Among them, two cytochrome P450 monooxygenase genes (ppb3 and ppb4) were separately introduced into the model fungus A. oryzae. Bioconversion of certain predicted intermediates in the transformants has elucidated the manner of hydroxylation in the biosynthetic pathway by the expressed products of these two genes (P450-1 and P450-2). That is, P450-1 exhibits monooxygenase activity and plays the hydroxylation role at C-11 of pyripyropene E. While P450-2 plays an active role in the hydroxylation of C-7 and C-13 of pyripyropene O. Keywords: cytochrome P450 monooxygenase; Penicillium coprobium; polyketide synthase; prenyltransferase; pyripyropene INTRODUCTION Pyripyropene A (PyA) has been found to be produced by Penicillium coprobium PF1169 1 and exhibits insecticidal properties. 2 The isomers of pyripyropenes A to L, initially isolated from the fermentation broth of Aspergillus fumigatus FO-1289FO- in 1993 3), have been regarded as potent inhibitors of acyl-CoA:cholesterol acyltransferase, the enzyme responsible for intracellular esterification of cholesterol. It has been reported that PyA displays insecticidal activity against Helicoverpa zea larvae. 2 The isomers of pyripyropenes A, B and D from a marine-derived fungus Aspergillus sp. GF5 were recently found to exhibit selective anti-growth properties against human umbilical vein endothelial cells. 4 Previous biochemical experiments partially delineated the PyA biosynthetic pathway. 5 Recently, the early steps of its biosynthesis in A. fumigatus FO-1289 were precisely elucidated by applying a transgenic approach with heterologous fungus. Nevertheless, hydroxylation and acetylation mechanisms for the late steps had remained unknown.We focused on cytochrome P450s, which are candidate catalysts at this hydroxylation step. Cytochrome P450 enzymes constitute a superfamily that typically catalyze the oxidation of numerous endogenous and exogenous compounds in bacteria, fungi, plants, insects and vertebrates. [6][7][8] The cytochrome acts as a terminal oxidase accepting electrons from NADPH, through NADPH cytochrome P450 reductase, or from NADH through cytochrome b 5 . The exogenous compounds metabolized in this way include numerous pesticides and insecticides. 9 In this paper, we describe the cloning and structural analysis of a 24 kb genomic DNA regi...