Molecular Cloning and Identification of NADPH Cytochrome P450 Reductase from Panax ginseng

Ginseng (Panax ginseng C.A. Mey.) is a precious Chinese traditional medicine, for which ginsenosides are the most important medicinal ingredients. Cytochrome P450 enzymes (CYP450) and their primary redox molecular companion NADPH cytochrome P450 reductase (CPR) play a key role in ginsenoside biosynthesis pathway. However, systematic studies of CPR genes in ginseng have not been reported. Numerous studies on ginsenoside synthesis biology still use Arabidopsis CPR (AtCPR1) as a reductase. In this study, we isolated two CPR genes (PgCPR1, PgCPR2) from ginseng adventitious roots. Phylogenetic tree analysis showed that both PgCPR1 and PgCPR2 are grouped in classⅡ of dicotyledonous CPR. Enzyme experiments showed that recombinant proteins PgCPR1, PgCPR2 and AtCPR1 can reduce cytochrome c and ferricyanide with NADPH as the electron donor, and PgCPR1 had the highest enzymatic activities. Quantitative real-time PCR analysis showed that PgCPR1 and PgCPR2 transcripts were detected in all examined tissues of Panax ginseng and both showed higher expression in stem and main root. Expression levels of the PgCPR1 and PgCPR2s were both induced after a methyl jasmonate (MeJA) treatment and its pattern matched with ginsenoside accumulation. The present investigation suggested PgCPR1 and PgCPR2 are associated with the biosynthesis of ginsenoside. This report will assist in future CPR family studies and ultimately improving ginsenoside production through transgenic engineering and synthetic biology.

Contribution of NADPH-cytochrome P450 Reductase to Azole Resistance in Fusarium oxysporum

Fusarium species exhibit significant intrinsic resistance to most antifungal agents and fungicides, resulting in high mortality rates among immunocompromised patients. Consequently, a thorough characterization of the antifungal resistance mechanism is required for effective treatments and for preventing fungal infections and reducing antifungal resistance. In this study, an isolate of Fusarium oxysporum (wild-type) with broadly resistant to commonly antifungal agents was used to generate 1,450 T-DNA random insertion mutants via Agrobacterium tumefaciens-mediated transformation. Antifungal susceptibility test results revealed one mutant with increased sensitivity to azoles. Compared with the resistant wild-type, the mutant exhibited low MICs to KTZ, ITC, VRC, POS, and PCZ (0.125, 1, 0.06, 0.5, and 0.125μg/ml, respectively). The T-DNA insertion site of this mutant was characterized as involving two adjacent genes, one encoding a hypothetical protein with unknown function and the other encoding the NADPH-cytochrome P450 reductase, referred as CPR1. To confirm the involvement of these genes in the altered azole susceptibility, the independent deletion mutants were generated and the Cpr1 deletion mutant displayed the same phenotypes as the T-DNA random mutant. The deletion of Cpr1 significantly decreased ergosterol levels. Additionally, the expression of the downstream Cyp51 gene was affected, which likely contributed to the observed increased susceptibility to azoles. These findings verified the association between Cpr1 and azole susceptibility in F. oxysporum. Furthermore, this gene may be targeted to improve antifungal treatments.

Esterase, Glutathione S-Transferase and NADPH-Cytochrome P450 Reductase Activity Evaluation in Cacopsylla pyri L. (Hemiptera: Psyllidae) Individual Adults

Cacopsylla pyri (L.) (Hemiptera: Psyllidae) is a key pest of pear orchards in Spain. The large number of insecticide treatments necessary for control may be an important contributor to the emergence of resistance. Laboratory toxicity and biochemical assays are necessary to validate the existence of insecticide resistance and establish the underlying mechanisms. All the methodologies developed to evaluate enzyme activity in C. pyri to date have incorporated “pools” of adults to detect minimum activity ranges. In this study, we determined the optimal working conditions for evaluation of the activities of esterase, glutathione S-transferase and NADPH-cytochrome P450 reductase in individual insects via colorimetric methods using a microplate reader. The main factors affecting enzymatic analysis activity, such as enzyme source and substrate concentration, filter wavelength, buffer pH, reaction time and additives, were evaluated for optimization. Determining the frequency of resistant individuals within a population could be used as an indicator for the evolution of insecticide resistance over time. Two laboratory strains, one of them selected with cypermethrin, and two field populations were analyzed for this purpose. The data obtained revealed high values and great variation in the activity ranges of esterase (EST) in the insecticide-selected population as well as in the field populations validating the applied methodology.

Silencing NADPH-Cytochrome P450 reductase affects imidacloprid susceptibility, fecundity, and embryonic development in Leptinotarsa decemlineata

ABSTRACTThe Colorado potato beetle (CPB) is a prominent insect pest of potatoes, tomatoes and eggplants all over the world, however, the management of CPB remains a challenging task for more than one hundred years. We have successfully developed bacteria-expressed dsRNA-mediated feeding RNA interference (RNAi) approach in our previous study. A critical step towards field management of CPB via feeding RNAi is to identify effective and environmentally safe target genes. NADPH-Cytochrome P450 reductase (CPR) plays a central role in cytochrome P450 action. The full length Leptinotarsa decemlineata CPR (LdCPR) cDNA was isolated from an imidacloprid resistant population. The LdCPR gene was ubiquitously expressed in all stages tested but showed an increase in expression during the early stage of embryonic development. The bacteria-expressed dsRNA-mediated feeding RNAi of LdCPR in adults caused systemic knock down expression of the gene coding for LdCPR in both adults and their eggs. Suppression of LdCPR expression increased susceptibility of imidacloprid in resistant beetles, as well as a significant decrease of fecundity in female beetles (29% less eggs/day) and the hatching rate (47%) of their eggs. These data suggest that LdCPR plays important roles in insecticide detoxification and biosynthetic pathways of endogenous compounds and may serve as an essential target to control CPB.HIGHLIGHTSHigh expression of LdCPR was observed in the egg stage.Silencing of LdCPR reduced the CPR enzymatic activities.LdCPR knockdown increased imidacloprid susceptibility.LdCPR knockdown decreased the fecundity and enhanced embryonic lethality.

Haplotype Diversity of NADPH-Cytochrome P450 Reductase Gene of Ophiocordyceps sinensis and the Effect on Fungal Infection in Host Insects

Ophiocordyceps sinensis Berk. is a fungal parasite that parasitizes the larvae of Hepialidae and is used as a traditional Chinese medicine. However, it is not clear how O. sinensis infects its host. The encoding gene haplotype diversity and predicted function of the nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase (CPR) related to the fungal pathogenicity was analyzed for 219 individuals from 47 O. sinensis populations. Two NADPH CPR genes of O. sinensis were detected and their dominant haplotypes were widely distributed throughout the entire distribution range in Western China. Only 5.43% of all O. sinensis individuals possessed the specific private haplotypes of NADPH CPR-1 and CPR-2 genes. Bioinformatic analyses predicted that the phosphorylation sites, motifs, and domains of NADPH CPR of O. sinensis were different between those encoding by the dominant and private gene haplotypes. The one-to-one match fungus–host correspondence of the same individual suggested that the widely distributed O. sinensis with the dominant NADPH CPR gene haplotypes may strongly infect almost all host insects through a random infection by oral or respiratory pores. Conversely, O. sinensis with the specific private NADPH CPR gene haplotypes is likely to infect only a few corresponding host insects by breaching the cuticle, due to the changed NADPH CPR structure and function.