The cytochrome P-450 dependent monooxygenase activity in a 'wild type' susceptible strain of housefly was studied. Catalytic activities were identified that demonstrated that the housefly cytochrome P-450 was capable of similar catalytic functions as those described for higher animals. Although several specific activities were lower than in mammalian species, benzphetamine N-demethylation was comparable and there was higher constitutive activity toward lauric acid than is observed in rat hepatic microsomes. The inducing agent phenobarbital increased both total cytochrome P-450 content and the benzphetamine N-demethylase specific activity. The high constitutive activity for fatty acids was induced by the hypolipidaemic drug clofibrate, specifically inducing the w-hydroxylase activity. The substrate specificity toward lauric acid extended equally to myristic and palmitic acid. Housefly microsomal cytochrome P-450 also metabolised the unsaturated fatty acid, arachidonic acid, the w-hydroxy-lation again inducible by clofibrate pretreatment. The w-hydroxylation of these fatty acids appeared to be a well-coupled reaction, a property that also appeared to be exhibited by the rat hepatic w-hydroxylase. The housefly fatty acid hydroxylation showed certain similarities to that in the rat, both in the specificity for w-hydroxylation and in the result of induction by clofibrate. Structural comparison to cytochrome P-450IVA1, IIB1 and IA1 was made by Western blot analysis with polyclonal antibodies raised to these rat hepatic isoenzymes. Housefly cytochrome P-450 shared few, if any, common epitopes with these rat isoenzymes, nor did these antibodies inhibit housefly cytochrome P-450 dependent monooxygenase activity. Cytochrome P-450 from clofibrate-pretreated housefly microsomes was partially purified by affinity chromatography. The cytochrome P-450 had a specific content of 5. 7nmol.mg-1 and a monomeric molecular weight of 52,000 daltons and a reduced carbon monoxide difference spectrum absorbance maxima at 448nm. In a reconstituted system, this preparation exhibited activity toward lauric acid and to a lesser extent arachidonic acid in each case w-hydroxylated products predominated. This is the first example of a purification of an insect cytochrome P-450 multiple form with a defined product reaction.

Clarireedia spp. (formerly Sclerotinia homoeocarpaF.T. Bennett) is the causal agent dollar spot, the most economically important turfgrass disease impacting golf courses in North America. The most effective strategy for dollar spot control is repeated application of multiple classes of fungicides. However, reliance on chemical application has led to resistance to four classes of fungicides as well as multidrug resistance (MDR). Fungi are known to detoxify xenobiotics, like fungicides, through transcriptional regulation of three detoxification phases: modification, conjugation and secretion. Little is known, however, of the protein-protein interactions that facilitate these pathways. Following next-generation RNA sequencing of Clarireedia spp., a fungus-specific transcription factor, XDR1, was determined to play a role in constitutive and induced overexpression of phases I and III genes of xenobiotic detoxification. Further, a novel activation domain (AD) on XDR1 that does not directly bind with xenobiotics was confirmed to be highly conserved among fungal species. Therefore, we hypothesize that XDR1 must be activated by interacting with other binding partners at this AD in order to regulate downstream xenobiotic detoxification pathways. The main objective of this study is to identify additional proteins/ co-repressors that activate XDR1 in order to gain a better understanding of how transcriptional regulation of xenobiotic detoxification pathways leads to MDR. In order to test the hypothesis, fungicide sensitive strain (HRS10) and fungicide resistant strain (HRI11) were transformed and tagged with xdr1/XDR1 and the 3xFLAG tag. As a result, four fungal transformants were generated and those are HRS10-XDR1-3xFLAG, HRS10-xdr1- 3xFLAG, HRI11-XDR1-3xFLAG, and HRI11-xdr1-3xFLAG. The total protein extractions (whole cell lysates) were subjected to co-immunoprecipitation and the samples were analyzed using LC-MS/MS. According to the set of results, more than 50 proteins were detected with HRS10-XDR1-3xFLAG with and most of these binding partners having functions related to post translational modification, protein turnover, intracellular trafficking, secretion and vascular transport. Going forward, information gained from this experiment could be used to explore how XDR1 interacts with its binding partners to facilitate the transcription of drug metabolizing genes responsible for multidrug resistance. This information could also help identify additional fungicide metabolism pathways in filamentous fungi.