Search tips
Search criteria

Results 1-7 (7)

Clipboard (0)

Select a Filter Below

Year of Publication
Document Types
1.  Molecular screening for avirulence alleles AvrLm1 and AvrLm6 in airborne inoculum of Leptosphaeria maculans and winter oilseed rape (Brassica napus) plants from Poland and the UK 
Journal of Applied Genetics  2014;55(4):529-539.
A combination of staining, light microscopy and SYBR green- and dual-labelled fluorescent probe-based qPCR chemistries with species- and gene-specific primers was employed to evaluate fluctuations in the aerial biomass of Leptosphaeria maculans spores captured by volumetric spore trappings in Poznan, Poland (2006, 2008) and Harpenden, UK (2002, 2006). Arising from these surveys, DNA samples extracted from Burkard spore-trap tapes were screened for fluctuation patterns in the frequencies of AvrLm1 and AvrLm6, the most prominent of the 15 genes that code for avirulence effectors in this Dothideomycete cause of the destructive phoma stem canker disease of oilseed rape worldwide. In Poznan, very low frequencies of AvrLm1 allele were found in the autumn of both 2006 and 2008, reflecting significantly increased cultivation of rape seed with Rlm1-based resistance. In contrast, at least six folds-higher frequencies of AvrLm6, which were also confirmed by end-point PCR bioassays on phoma-infected leaves from the same region of Poland, were obtained during both years. In the UK, however, relatively higher AvrLm1 allele titres were found in L. maculans spores captured in air samples from the autumn of 2002 on the experimental fields of Rothamsted Research, Harpenden, that were historically sown to genetically heterogeneous B. napus cultivars. In the 2006 screen these levels had plummeted, to a 1:4 ratio, in favour of frequencies of the AvrLm6 allele. Patterns of fluctuations in erg11 (CYP51) fragments coding for sterol 14α-demethylase suggest October as the month with the most viable wind-dispersed L. maculans propagules of each season of the screens.
PMCID: PMC4185106  PMID: 25081837
Airborne inoculum; Air sampling; Avirulence genes; Disease forecasting; Leptosphaeria maculans; Phoma stem canker
2.  Paralog Re-Emergence: A Novel, Historically Contingent Mechanism in the Evolution of Antimicrobial Resistance 
Molecular Biology and Evolution  2014;31(7):1793-1802.
Evolution of resistance to drugs and pesticides poses a serious threat to human health and agricultural production. CYP51 encodes the target site of azole fungicides, widely used clinically and in agriculture. Azole resistance can evolve due to point mutations or overexpression of CYP51, and previous studies have shown that fungicide-resistant alleles have arisen by de novo mutation. Paralogs CYP51A and CYP51B are found in filamentous ascomycetes, but CYP51A has been lost from multiple lineages. Here, we show that in the barley pathogen Rhynchosporium commune, re-emergence of CYP51A constitutes a novel mechanism for the evolution of resistance to azoles. Pyrosequencing analysis of historical barley leaf samples from a unique long-term experiment from 1892 to 2008 indicates that the majority of the R. commune population lacked CYP51A until 1985, after which the frequency of CYP51A rapidly increased. Functional analysis demonstrates that CYP51A retains the same substrate as CYP51B, but with different transcriptional regulation. Phylogenetic analyses show that the origin of CYP51A far predates azole use, and newly sequenced Rhynchosporium genomes show CYP51A persisting in the R. commune lineage rather than being regained by horizontal gene transfer; therefore, CYP51A re-emergence provides an example of adaptation to novel compounds by selection from standing genetic variation.
PMCID: PMC4069618  PMID: 24732957
standing variation; gene duplication; resistance; fungicides; triazoles; Rhynchosporium
3.  Prothioconazole and Prothioconazole-Desthio Activities against Candida albicans Sterol 14-α-Demethylase 
Prothioconazole is a new triazolinthione fungicide used in agriculture. We have used Candida albicans CYP51 (CaCYP51) to investigate the in vitro activity of prothioconazole and to consider the use of such compounds in the medical arena. Treatment of C. albicans cells with prothioconazole, prothioconazole-desthio, and voriconazole resulted in CYP51 inhibition, as evidenced by the accumulation of 14α-methylated sterol substrates (lanosterol and eburicol) and the depletion of ergosterol. We then compared the inhibitor binding properties of prothioconazole, prothioconazole-desthio, and voriconazole with CaCYP51. We observed that prothioconazole-desthio and voriconazole bind noncompetitively to CaCYP51 in the expected manner of azole antifungals (with type II inhibitors binding to heme as the sixth ligand), while prothioconazole binds competitively and does not exhibit classic inhibitor binding spectra. Inhibition of CaCYP51 activity in a cell-free assay demonstrated that prothioconazole-desthio is active, whereas prothioconazole does not inhibit CYP51 activity. Extracts from C. albicans grown in the presence of prothioconazole were found to contain prothioconazole-desthio. We conclude that the antifungal action of prothioconazole can be attributed to prothioconazole-desthio.
PMCID: PMC3591943  PMID: 23275516
4.  Impact of Recently Emerged Sterol 14α-Demethylase (CYP51) Variants of Mycosphaerella graminicola on Azole Fungicide Sensitivity▿ 
Applied and Environmental Microbiology  2011;77(11):3830-3837.
The progressive decline in the effectiveness of some azole fungicides in controlling Mycosphaerella graminicola, causal agent of the damaging Septoria leaf blotch disease of wheat, has been correlated with the selection and spread in the pathogen population of specific mutations in the M. graminicola CYP51 (MgCYP51) gene encoding the azole target sterol 14α-demethylase. Recent studies have suggested that the emergence of novel MgCYP51 variants, often harboring substitution S524T, has contributed to a decrease in the efficacy of prothioconazole and epoxiconazole, the two currently most effective azole fungicides against M. graminicola. In this study, we establish which amino acid alterations in novel MgCYP51 variants have the greatest impact on azole sensitivity and protein function. We introduced individual and combinations of identified alterations by site-directed mutagenesis and functionally determined their impact on azole sensitivity by expression in a Saccharomyces cerevisiae mutant YUG37::erg11 carrying a regulatable promoter controlling native CYP51 expression. We demonstrate that substitution S524T confers decreased sensitivity to all azoles when introduced alone or in combination with Y461S. In addition, S524T restores the function in S. cerevisiae of MgCYP51 variants carrying the otherwise lethal alterations Y137F and V136A. Sensitivity tests of S. cerevisiae transformants expressing recently emerged MgCYP51 variants carrying combinations of alterations D134G, V136A, Y461S, and S524T reveal a substantial impact on sensitivity to the currently most widely used azoles, including epoxiconazole and prothioconazole. Finally, we exploit a recently developed model of the MgCYP51 protein to predict that the substantial structural changes caused by these novel combinations reduce azole interactions with critical residues in the binding cavity, thereby causing resistance.
PMCID: PMC3127603  PMID: 21478305
5.  Mechanism of Binding of Prothioconazole to Mycosphaerella graminicola CYP51 Differs from That of Other Azole Antifungals ▿  
Prothioconazole is one of the most important commercially available demethylase inhibitors (DMIs) used to treat Mycosphaerella graminicola infection of wheat, but specific information regarding its mode of action is not available in the scientific literature. Treatment of wild-type M. graminicola (strain IPO323) with 5 μg of epoxiconazole, tebuconazole, triadimenol, or prothioconazole ml−1 resulted in inhibition of M. graminicola CYP51 (MgCYP51), as evidenced by the accumulation of 14α-methylated sterol substrates (lanosterol and eburicol) and the depletion of ergosterol in azole-treated cells. Successful expression of MgCYP51 in Escherichia coli enabled us to conduct spectrophotometric assays using purified 62-kDa MgCYP51 protein. Antifungal-binding studies revealed that epoxiconazole, tebuconazole, and triadimenol all bound tightly to MgCYP51, producing strong type II difference spectra (peak at 423 to 429 nm and trough at 406 to 409 nm) indicative of the formation of classical low-spin sixth-ligand complexes. Interaction of prothioconazole with MgCYP51 exhibited a novel spectrum with a peak and trough observed at 410 nm and 428 nm, respectively, indicating a different mechanism of inhibition. Prothioconazole bound to MgCYP51 with 840-fold less affinity than epoxiconazole and, unlike epoxiconazole, tebuconazole, and triadimenol, which are noncompetitive inhibitors, prothioconazole was found to be a competitive inhibitor of substrate binding. This represents the first study to validate the effect of prothioconazole on the sterol composition of M. graminicola and the first on the successful heterologous expression of active MgCYP51 protein. The binding affinity studies documented here provide novel insights into the interaction of MgCYP51 with DMIs, especially for the new triazolinethione derivative prothioconazole.
PMCID: PMC3067226  PMID: 21169436
6.  Molecular Modelling of the Emergence of Azole Resistance in Mycosphaerella graminicola 
PLoS ONE  2011;6(6):e20973.
A structural rationale for recent emergence of azole (imidazole and triazole) resistance associated with CYP51 mutations in the wheat pathogen Mycosphaerella graminicola is presented, attained by homology modelling of the wild type protein and 13 variant proteins. The novel molecular models of M. graminicola CYP51 are based on multiple homologues, individually identified for each variant, rather than using a single structural scaffold, providing a robust structure-function rationale for the binding of azoles, including important fungal specific regions for which no structural information is available. The wild type binding pocket reveals specific residues in close proximity to the bound azole molecules that are subject to alteration in the variants. This implicates azole ligands as important agents exerting selection on specific regions bordering the pocket, that become the focus of genetic mutation events, leading to reduced sensitivity to that group of related compounds. Collectively, the models account for several observed functional effects of specific alterations, including loss of triadimenol sensitivity in the Y137F variant, lower sensitivity to tebuconazole of I381V variants and increased resistance to prochloraz of V136A variants. Deletion of Y459 and G460, which brings about removal of that entire section of beta turn from the vicinity of the binding pocket, confers resistance to tebuconazole and epoxiconazole, but sensitivity to prochloraz in variants carrying a combination of A379G I381V ΔY459/G460. Measurements of binding pocket volume proved useful in assessment of scope for general resistance to azoles by virtue of their accommodation without bonding interaction, particularly when combined with analysis of change in positions of key amino acids. It is possible to predict the likely binding orientation of an azole molecule in any of the variant CYPs, providing potential for an in silico screening system and reliable predictive approach to assess the probability of particular variants exhibiting resistance to particular azole fungicides.
PMCID: PMC3124474  PMID: 21738598
7.  Finished Genome of the Fungal Wheat Pathogen Mycosphaerella graminicola Reveals Dispensome Structure, Chromosome Plasticity, and Stealth Pathogenesis 
PLoS Genetics  2011;7(6):e1002070.
The plant-pathogenic fungus Mycosphaerella graminicola (asexual stage: Septoria tritici) causes septoria tritici blotch, a disease that greatly reduces the yield and quality of wheat. This disease is economically important in most wheat-growing areas worldwide and threatens global food production. Control of the disease has been hampered by a limited understanding of the genetic and biochemical bases of pathogenicity, including mechanisms of infection and of resistance in the host. Unlike most other plant pathogens, M. graminicola has a long latent period during which it evades host defenses. Although this type of stealth pathogenicity occurs commonly in Mycosphaerella and other Dothideomycetes, the largest class of plant-pathogenic fungi, its genetic basis is not known. To address this problem, the genome of M. graminicola was sequenced completely. The finished genome contains 21 chromosomes, eight of which could be lost with no visible effect on the fungus and thus are dispensable. This eight-chromosome dispensome is dynamic in field and progeny isolates, is different from the core genome in gene and repeat content, and appears to have originated by ancient horizontal transfer from an unknown donor. Synteny plots of the M. graminicola chromosomes versus those of the only other sequenced Dothideomycete, Stagonospora nodorum, revealed conservation of gene content but not order or orientation, suggesting a high rate of intra-chromosomal rearrangement in one or both species. This observed “mesosynteny” is very different from synteny seen between other organisms. A surprising feature of the M. graminicola genome compared to other sequenced plant pathogens was that it contained very few genes for enzymes that break down plant cell walls, which was more similar to endophytes than to pathogens. The stealth pathogenesis of M. graminicola probably involves degradation of proteins rather than carbohydrates to evade host defenses during the biotrophic stage of infection and may have evolved from endophytic ancestors.
Author Summary
The plant-pathogenic fungus Mycosphaerella graminicola causes septoria tritici blotch, one of the most economically important diseases of wheat worldwide and a potential threat to global food production. Unlike most other plant pathogens, M. graminicola has a long latent period during which it seems able to evade host defenses, and its genome appears to be unstable with many chromosomes that can change size or be lost during sexual reproduction. To understand its unusual mechanism of pathogenicity and high genomic plasticity, the genome of M. graminicola was sequenced more completely than that of any other filamentous fungus. The finished sequence contains 21 chromosomes, eight of which were different from those in the core genome and appear to have originated by ancient horizontal transfer from an unknown donor. The dispensable chromosomes collectively comprise the dispensome and showed extreme plasticity during sexual reproduction. A surprising feature of the M. graminicola genome was a low number of genes for enzymes that break down plant cell walls; this may represent an evolutionary response to evade detection by plant defense mechanisms. The stealth pathogenicity of M. graminicola may involve degradation of proteins rather than carbohydrates and could have evolved from an endophytic ancestor.
PMCID: PMC3111534  PMID: 21695235

Results 1-7 (7)