Andrew J. Corran

Purification and reconstitution of activity of Saccharomyces cerevisiae P450 61, a sterol Δ22-desaturase

P450 was purified from microsomal fractions of a strain of Saccharomyces cerevisiae which contained detectable P450 despite the disruption of CYP51A1. The P450 had a molecular mass of 58 kDa, similar to P450 51A1, and in a reconstituted assay with rabbit NADPH‐P450 reductase and dilauryl phosphotidylcholine exhibited activity for conversion of ergosta‐5,7‐dienol into ergosterol. N‐Terminal amino acid sequencing of the purified protein corresponded to the translated sequence of P450 61 which was recently identified during sequencing of chromosome XIII. This allowed the function of this family of P450 to be identified as sterol Δ 22‐desaturation in the pathway of ergosterol biosynthesis.

Resistant P45051A1 activity in azole antifungal tolerant Cryptococcus neoformans from AIDS patients

Azole antifungal compounds are important in the treatment of Cryptococcosis, a major cause of mortality in AIDS patients. The target of the azole drugs is P450 mediated sterol 14α‐demethylase. We have investigated the P450 system of Cryptococcus neoformans with respect to azole tolerance observed in clinical isolates which were obtained following the failure of fluconazole therapy. The clinical failure was correlated with in vitro tolerance of azole antifungal when compared to wild‐type strains. The microsomal P450 system was typical of yeast and fungi and fluconazole tolerance was not associated with defective sterol biosynthesis. The strains had slightly elevated P450 content and slightly reduced azole levels in the cells, but a clear cause for resistance was the increased level of drug needed to inhibit the sterol 14α‐demethylase in vitro.

Approaches to in-vitro lead generation for fungicide invention

There are increasing opportunities for the development of high-throughput in-vitro screens to aid the discovery of fungicides with novel modes of action. In the past, such screens were developed when biochemical targets were validated by fungicides with defined modes of action. However, genetic information is beginning to have a major impact both on the way in-vitro targets are selected and on the speed at which mode-of-action information is gained on current fungicides having an, as yet, undefined mode of action. This paper discusses issues concerning target selection and high-throughput screening, using examples taken from the current literature and from investigations at Zeneca Agrochemicals, using inhibition of fungal respiration as an example. Saccharomyces cerevisiae is discussed as model for fungicide research, both in terms of its sensitivity to known fungicides and its well defined molecular genetics, which makes it amenable to such techniques as gene dosage for mode of action determination.

Protein kinase C is essential for viability of the rice blast fungus Magnaporthe oryzae

Protein kinase C constitutes a family of serine–threonine kinases found in all eukaryotes and implicated in a wide range of cellular functions, including regulation of cell growth, cellular differentiation and immunity. Here, we present three independent lines of evidence which indicate that protein kinase C is essential for viability of Magnaporthe oryzae. First, all attempts to generate a target deletion of PKC1, the single copy protein kinase C‐encoding gene, proved unsuccessful. Secondly, conditional gene silencing of PKC1 by RNA interference led to severely reduced growth of the fungus, which was reversed by targeted deletion of the Dicer2‐encoding gene, MDL2. Finally, selective kinase inhibition of protein kinase C by targeted allelic replacement with an analogue‐sensitive PKC1AS allele led to specific loss of fungal viability in the presence of the PP1 inhibitor. Global transcriptional profiling following selective PKC inhibition identified significant changes in gene expression associated with cell wall re‐modelling, autophagy, signal transduction and secondary metabolism. When considered together, these results suggest protein kinase C is essential for growth and development of M. oryzae with extensive downstream targets in addition to the cell integrity pathway. Targeting protein kinase C signalling may therefore prove an effective means of controlling rice blast disease.

Malayamycin, a new streptomycete antifungal compound, specifically inhibits sporulation of Stagonospora nodorum (Berk) Castell and Germano, the cause of wheat glume blotch disease

BACKGROUND Malayamycin is a novel perhydrofuropyran C-nucleoside isolated from Streptomyces malaysiensis that shows promising antifungal activity, fully controlling a range of diseases when applied to plants at 100 microg mL(-1). The goal of this study was to determine the mode of action. RESULTS Malayamycin exhibited in vitro antifungal activity against Stagonospora nodorum (Berk) Castell & Germano, the cause of stagonospora nodorum blotch of wheat. Growth in liquid minimum medium was merely delayed at 50 microg mL(-1), but sporulation was suppressed by more than 50% by 10 microg mL(-1) of malayamycin. When applied to wheat seedlings 36 h prior to infection, 10 microg mL(-1) of malayamycin reduced lesion size and significantly reduced pycnidiation to only 5% of the non-treated level. A transcription factor gene, Mrg1 (malayamycin response gene) whose expression was upregulated by application of malayamycin, was identified. Both Mrg1 knockout and overexpression strains were created. These strains were fully pathogenic, suggesting that the expression of Mrg1 did not affect pathogenicity. Interestingly, a strain that expressed Mrg1 50 times more than wild type showed a significant reduction in sporulation. However, all the tested knockout and overexpression strains retained sensitivity to malayamycin. CONCLUSIONS Malayamycin is a new type of antifungal compound that acts primarily by inhibiting sporulation. Although Mrg1 may be involved in the sporulation process, it is not the major contributor for sporulation inhibition caused by malayamycin treatment.

The cytochrome P450 complement (CYPome) of Mycosphaerella graminicola

Mycosphaerella graminicola is a key fungal pathogen of wheat and a major target for azole fungicides, many of whose central mode of action is through inhibition of cytochrome P450 51 (lanosterol 14α‐demethylase) in the ergosterol biosynthetic pathway. The range of activities of other fungal CYPs is thought to be a reflection of the differences between different organisms and their range of secondary metabolic pathways as a response to their niche environments, for example, in the production of mycotoxins. The present study collates information from a range of databases, to classify the CYPs found in M. graminicola and assign them an internationally recognized nomenclature, which, when referenced to the recent publication of the JGI version 2.0 genome model, creates a current, robust model for the CYP complement (CYPome) of M. graminicola. These CYPome data, which examined 82 CYPs and one pseudo‐gene, may be utilized not only to further characterize and describe the physiology of the organism but also to enhance our understanding of CYP function and diversity.