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1.  Heterologous Expression of Mutated Eburicol 14α-Demethylase (CYP51) Proteins of Mycosphaerella graminicola To Assess Effects on Azole Fungicide Sensitivity and Intrinsic Protein Function▿  
The recent decrease in the sensitivity of the Western European population of the wheat pathogen Mycosphaerella graminicola to azole fungicides has been associated with the emergence and subsequent spread of mutations in the CYP51 gene, encoding the azole target sterol 14α-demethylase. In this study, we have expressed wild-type and mutated M. graminicola CYP51 (MgCYP51) variants in a Saccharomyces cerevisiae mutant carrying a doxycycline-regulatable tetO7-CYC promoter controlling native CYP51 expression. We have shown that the wild-type MgCYP51 protein complements the function of the orthologous protein in S. cerevisiae. Mutant MgCYP51 proteins containing amino acid alterations L50S, Y459D, and Y461H and the two-amino-acid deletion ΔY459/G460, commonly identified in modern M. graminicola populations, have no effect on the capacity of the M. graminicola protein to function in S. cerevisiae. We have also shown that the azole fungicide sensitivities of transformants expressing MgCYP51 variants with these alterations are substantially reduced. Furthermore, we have demonstrated that the I381V substitution, correlated with the recent decline in the effectiveness of azoles, destroys the capacity of MgCYP51 to complement the S. cerevisiae mutant when introduced alone. However, when I381V is combined with changes between residues Y459 and Y461, the function of the M. graminicola protein is partially restored. These findings demonstrate, for the first time for a plant pathogenic fungus, the impacts that naturally occurring CYP51 alterations have on both azole sensitivity and intrinsic protein function. In addition, we also provide functional evidence underlying the order in which CYP51 alterations in the Western European M. graminicola population emerged.
doi:10.1128/AEM.02158-09
PMCID: PMC2863451  PMID: 20305029
2.  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.
doi:10.1128/AEM.01332-10
PMCID: PMC3067226  PMID: 21169436
3.  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.
doi:10.1371/journal.pone.0020973
PMCID: PMC3124474  PMID: 21738598
4.  ABC Transporters and Azole Susceptibility in Laboratory Strains of the Wheat Pathogen Mycosphaerella graminicola 
Antimicrobial Agents and Chemotherapy  2002;46(12):3900-3906.
Laboratory strains of Mycosphaerella graminicola with decreased susceptibilities to the azole antifungal agent cyproconazole showed a multidrug resistance phenotype by exhibiting cross-resistance to an unrelated chemical, cycloheximide or rhodamine 6G, or both. Decreased azole susceptibility was found to be associated with either decreased or increased levels of accumulation of cyproconazole. No specific relationship could be observed between azole susceptibility and the expression of ATP-binding cassette (ABC) transporter genes MgAtr1 to MgAtr5 and the sterol P450 14α-demethylase gene, CYP51. ABC transporter MgAtr1 was identified as a determinant in azole susceptibility since heterologous expression of the protein reduced the azole susceptibility of Saccharomyces cerevisiae and disruption of MgAtr1 in one specific M. graminicola laboratory strain with constitutive MgAtr1 overexpression restored the level of susceptibility to cyproconazole to wild-type levels. However, the level of accumulation in the mutant with an MgAtr1 disruption did not revert to the wild-type level. We propose that variations in azole susceptibility in laboratory strains of M. graminicola are mediated by multiple mechanisms.
doi:10.1128/AAC.46.12.3900-3906.2002
PMCID: PMC132773  PMID: 12435694
5.  Genetic analysis of resistance to septoria tritici blotch in the French winter wheat cultivars Balance and Apache 
The ascomycete Mycosphaerella graminicola is the causal agent of septoria tritici blotch (STB), one of the most destructive foliar diseases of bread and durum wheat globally, particularly in temperate humid areas. A screening of the French bread wheat cultivars Apache and Balance with 30 M. graminicola isolates revealed a pattern of resistant responses that suggested the presence of new genes for STB resistance. Quantitative trait loci (QTL) analysis of a doubled haploid (DH) population with five M. graminicola isolates in the seedling stage identified four QTLs on chromosomes 3AS, 1BS, 6DS and 7DS, and occasionally on 7DL. The QTL on chromosome 6DS flanked by SSR markers Xgpw5176 and Xgpw3087 is a novel QTL that now can be designated as Stb18. The QTLs on chromosomes 3AS and 1BS most likely represent Stb6 and Stb11, respectively, and the QTLs on chromosome 7DS are most probably identical with Stb4 and Stb5. However, the QTL identified on chromosome 7DL is expected to be a new Stb gene that still needs further characterization. Multiple isolates were used and show that not all isolates identify all QTLs, which clearly demonstrates the specificity in the M. graminicola–wheat pathosystem. QTL analyses were performed with various disease parameters. The development of asexual fructifications (pycnidia) in the characteristic necrotic blotches of STB, designated as parameter P, identified the maximum number of QTLs. All other parameters identified fewer but not different QTLs. The segregation of multiple QTLs in the Apache/Balance DH population enabled the identification of DH lines with single QTLs and multiple QTL combinations. Analyses of the marker data of these DH lines clearly demonstrated the positive effect of pyramiding QTLs to broaden resistance spectra as well as epistatic and additive interactions between these QTLs. Phenotyping of the Apache/Balance DH population in the field confirmed the presence of the QTLs that were identified in the seedling stage, but Stb18 was inconsistently expressed and might be particularly effective in young plants. In contrast, an additional QTL for STB resistance was identified on chromosome 2DS that is exclusively and consistently expressed in mature plants over locations and time, but it was also strongly related with earliness, tallness as well as resistance to Fusarium head blight. Although to date no Stb gene has been reported on chromosome 2D, the data provide evidence that this QTL is only indirectly related to STB resistance. This study shows that detailed genetic analysis of contemporary commercial bread wheat cultivars can unveil novel Stb genes that can be readily applied in marker-assisted breeding programs.
Electronic supplementary material
The online version of this article (doi:10.1007/s00122-011-1623-7) contains supplementary material, which is available to authorized users.
doi:10.1007/s00122-011-1623-7
PMCID: PMC3155673  PMID: 21655994
6.  Modelling Wheat Growth and Yield Losses from Late Epidemics of Foliar Diseases using Loss of Green Leaf Area per Layer and Pre-anthesis Reserves 
Annals of Botany  2007;100(4):777-789.
Background and Aims
Crop protection strategies, based on preventing quantitative crop losses rather than pest outbreaks, are being developed as a promising way to reduce fungicide use. The Bastiaans' model was applied to winter wheat crops (Triticum aestivum) affected by leaf rust (Puccinia triticina) and Septoria tritici blotch (STB; Mycosphaerella graminicola) under a range of crop management conditions. This study examined (a) whether green leaf area per layer accurately accounts for growth loss; and (b) whether from growth loss it is possible to derive yield loss accurately and simply.
Methods
Over 5 years of field experiments, numerous green leaf area dynamics were analysed during the post-anthesis period on wheat crops using natural aerial epidemics of leaf rust and STB.
Key Results
When radiation use efficiency (RUE) was derived from bulk green leaf area index (GLAI), RUEbulk was hardly accurate and exhibited large variations among diseased wheat crops, thus extending outside the biological range. In contrast, when RUE was derived from GLAI loss per layer, RUElayer was a more accurate calculation and fell within the biological range. In one situation out of 13, no significant shift in the RUElayer of diseased crops vs. healthy crops was observed. A single linear relationship linked yield to post-anthesis accumulated growth for all treatments. Its slope, not different from 1, suggests that the allocation of post-anthesis photosynthates to grains was not affected by the late occurring diseases under study. The mobilization of pre-anthesis reserves completely accounted for the intercept value.
Conclusions
The results strongly suggest that a simple model based on green leaf area per layer and pre-anthesis reserves can predict both growth and yield of wheat suffering from late epidemics of foliar diseases over a range of crop practices. It could help in better understanding how crop structure and reserve management contribute to tolerance of wheat genotypes to leaf diseases.
doi:10.1093/aob/mcm163
PMCID: PMC2749629  PMID: 17686762
Triticum aestivum; Puccinia triticina; Mycosphaerella graminicola; leaf rust; Septoria tritici blotch; growth loss; yield loss; green leaf area per layer; pre-anthesis reserves
7.  Control of Mycosphaerella graminicola on Wheat Seedlings by Medical Drugs Known To Modulate the Activity of ATP-Binding Cassette Transporters▿  
Applied and Environmental Microbiology  2007;73(15):5011-5019.
Medical drugs known to modulate the activity of human ATP-binding cassette (ABC) transporter proteins (modulators) were tested for the ability to potentiate the activity of the azole fungicide cyproconazole against in vitro growth of Mycosphaerella graminicola and to control disease development due to this pathogen on wheat seedlings. In vitro modulation of cyproconazole activity could be demonstrated in paper disk bioassays. Some of the active modulators (amitriptyline, flavanone, and phenothiazines) increased the accumulation of cyproconazole in M. graminicola, suggesting that they reversed cyproconazole efflux. However, synergism between cyproconazole and modulators against M. graminicola on wheat seedlings could not be shown. Despite their low in vitro toxicity to M. graminicola, some modulators (amitriptyline, loperamide, and promazine) did show significant intrinsic disease control activity in preventive and curative foliar spray tests with wheat seedlings. The results suggest that these compounds have indirect disease control activity based on modulation of fungal ABC transporters essential for virulence and constitute a new class of disease control agents.
doi:10.1128/AEM.00285-07
PMCID: PMC1951022  PMID: 17545327
8.  Late Foliar Diseases in Wheat Crops Decrease Nitrogen Yield Through N Uptake Rather than Through Variations in N Remobilization 
Annals of Botany  2008;102(4):579-590.
Background and Aims
French wheat grains may be of little value on world markets because they have low and highly variable grain protein concentrations (GPC). This nitrogen-yield to yield ratio depends on crop nitrogen (N) fertilization as well as on crop capacity to use N, which is known to vary with climate and disease severity. Here an examination is made of the respective roles that N remobilization and post-anthesis N uptake play in N yield variations; in particular, when wheat crops (Triticum aestivum) are affected by leaf rust (Puccinia triticina) and Septoria tritici blotch (teleomorph Mycosphaerella graminicola).
Methods
Data from a 4-year field experiment was used to analyse N yield variations in wheat crops grown either with a third or no late N fertilization. Natural aerial epidemics ensured a range of disease severity, and fungicide ensured disease-free control plots. The data set of Gooding et al. (2005, Journal of Agricultural Science 143: 503–518) was incorporated in order to enlarge the range of conditions.
Key Results
Post-anthesis N uptake accounted for a third of N yield whilst N remobilization accounted for two-thirds in all crops whether affected by diseases or not. However, variations in N yield were highly correlated with post-anthesis N uptake, more than with N remobilization, in diseased and also healthy crops. Furthermore, N remobilization did not significantly correlate with N yield in healthy crops. These findings matched data from studies using various wheat genotypes under various management and climatic conditions. Leaf area duration (LAD) accurately predicted N remobilization whether or not crops were diseased; in diseased crops, LAD also accurately predicted N uptake.
Conclusions
Under the experimental conditions, N yield variations were closely associated with post-anthesis N uptake in diseased but also in healthy crops. Understanding the respective roles of N uptake and N remobilization in the case of diseased and healthy crops holds the promise of better modelling of variations in N yield, and thus in GPC.
doi:10.1093/aob/mcn124
PMCID: PMC2701774  PMID: 18660494
Triticum aestivum; Puccinia triticina; leaf rust; Mycosphaerella graminicola; Septoria tritici blotch; N uptake; N remobilization; N yield; Leaf area duration
9.  MVE1, Encoding the Velvet Gene Product Homolog in Mycosphaerella graminicola, Is Associated with Aerial Mycelium Formation, Melanin Biosynthesis, Hyphal Swelling, and Light Signaling ▿  
The ascomycete fungus Mycosphaerella graminicola is an important pathogen of wheat that causes Septoria tritici blotch. Despite the serious impact of M. graminicola on wheat production worldwide, knowledge about its molecular biology is limited. The velvet gene, veA, is one of the key regulators of diverse cellular processes, including development and secondary metabolism in many fungi. However, the species analyzed to date are not related to the Dothideomycetes, the largest class of plant-pathogenic fungi, and the function of veA in this group is not known. To test the hypothesis that the velvet gene has similar functions in the Dothideomycetes, a veA-homologous gene, MVE1, was identified and gene deletion mutations (Δmve1) were generated in M. graminicola. All of the MVE1 mutants exhibited consistent pleiotropic phenotypes, indicating the involvement of MVE1 in multiple signaling pathways. Δmve1 strains displayed albino phenotypes with significant reductions in melanin biosynthesis and less production of aerial mycelia on agar plates. In liquid culture, Δmve1 strains showed abnormal hyphal swelling, which was suppressed completely by osmotic stress or lower temperature. In addition, MVE1 gene deletion led to hypersensitivity to shaking, reduced hydrophobicity, and blindness to light-dependent stimulation of aerial mycelium production. However, pathogenicity was not altered in Δmve1 strains. Therefore, the light-signaling pathway associated with MVE1 does not appear to be important for Septoria tritici blotch disease. Our data suggest that the MVE1 gene plays crucial roles in multiple key signaling pathways and is associated with light signaling in M. graminicola.
doi:10.1128/AEM.01830-10
PMCID: PMC3028728  PMID: 21115702
10.  Whole-Genome and Chromosome Evolution Associated with Host Adaptation and Speciation of the Wheat Pathogen Mycosphaerella graminicola 
PLoS Genetics  2010;6(12):e1001189.
The fungus Mycosphaerella graminicola has been a pathogen of wheat since host domestication 10,000–12,000 years ago in the Fertile Crescent. The wheat-infecting lineage emerged from closely related Mycosphaerella pathogens infecting wild grasses. We use a comparative genomics approach to assess how the process of host specialization affected the genome structure of M. graminicola since divergence from the closest known progenitor species named M. graminicola S1. The genome of S1 was obtained by Illumina sequencing resulting in a 35 Mb draft genome sequence of 32X. Assembled contigs were aligned to the previously sequenced M. graminicola genome. The alignment covered >90% of the non-repetitive portion of the M. graminicola genome with an average divergence of 7%. The sequenced M. graminicola strain is known to harbor thirteen essential chromosomes plus eight dispensable chromosomes. We found evidence that structural rearrangements significantly affected the dispensable chromosomes while the essential chromosomes were syntenic. At the nucleotide level, the essential and dispensable chromosomes have evolved differently. The average synonymous substitution rate in dispensable chromosomes is considerably lower than in essential chromosomes, whereas the average non-synonymous substitution rate is three times higher. Differences in molecular evolution can be related to different transmission and recombination patterns, as well as to differences in effective population sizes of essential and dispensable chromosomes. In order to identify genes potentially involved in host specialization or speciation, we calculated ratios of synonymous and non-synonymous substitution rates in the >9,500 aligned protein coding genes. The genes are generally under strong purifying selection. We identified 43 candidate genes showing evidence of positive selection, one encoding a potential pathogen effector protein. We conclude that divergence of these pathogens was accompanied by structural rearrangements in the small dispensable chromosomes, while footprints of positive selection were present in only a small number of protein coding genes.
Author Summary
The fungal wheat pathogen Mycosphaerella graminicola emerged in the Middle East 11,000 years ago, coinciding with host domestication. We sequenced the genome of the closest known endemic relative of M. graminicola infecting wild grass hosts. A comparative genome analysis allowed us to infer how speciation and host specialization processes have influenced pathogen evolution. The wild grass-adapted pathogen can infect wheat, but M. graminicola shows a significantly higher degree of host specificity and virulence in a detached leaf assay. The genomes of the pathogens are 7% divergent with a high degree of synteny in the 13 essential core chromosomes. However, structural rearrangements have strongly affected eight small dispensable chromosomes. These chromosomes also show altered rates of non-synonymous and synonymous substitutions. We found 43 genes showing evidence of positive selection. As the divergence of species occurred very recently, these genes are likely involved in host specialization or speciation. None of the genes have a known function, although one encodes a signal peptide and is a potential pathogen effector. We conclude that the genomic basis of the rapid emergence of the wheat-specialized pathogen M. graminicola has involved structural changes in the eight dispensable chromosomes and positive selection in a small number of genes.
doi:10.1371/journal.pgen.1001189
PMCID: PMC3009667  PMID: 21203495
11.  Detection of Mycosphaerella graminicola in Wheat Leaves by a Microsatellite Dinucleotide Specific-Primer 
Early detection of infection is very important for efficient management of Mycosphaerella graminicola leaf blotch. To monitor and quantify the occurrence of this fungus during the growing season, a diagnostic method based on real-time PCR was developed. Standard and real-time PCR assays were developed using SYBR Green chemistry to quantify M. graminicola in vitro or in wheat samples. Microsatellite dinucleotide specific-primers were designed based on microsatellite repeats of sequences present in the genome of M. graminicola. Specificity was checked by analyzing DNA of 55 M. graminicola isolates obtained from different geographical origins. The method appears to be highly specific for detecting M. graminicola; no fluorescent signals were observed from 14 other closely related taxa. Primer (CT) 7 G amplified a specific amplicon of 570 bp from all M. graminicola isolates. The primers did not amplify DNA extracted from 14 other fungal species. The approximate melting temperature (Tm) of the (CT) 7 G primer was 84.2 °C. The detection limit of the real-time PCR assay with the primer sets (CT) 7 G is 10 fg/25 μL, as compared to 10 pg/25 μL using conventional PCR technology. From symptomless leaves, a PCR fragment could be generated two days after inoculation. Both conventional and real-time PCR could successfully detect the fungus from artificially inoculated wheat leaves. However, real-time PCR appeared much more sensitive than conventional PCR. The developed quantitative real-time PCR method proved to be rapid, sensitive, specific, cost-effective and reliable for the identification and quantification of M. graminicola in wheat.
doi:10.3390/ijms12010682
PMCID: PMC3039974  PMID: 21340008
Septoria tritici blotch; microsatellite; wheat; Dothidiomycete; molecular diagnostics
12.  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.
doi:10.1371/journal.pgen.1002070
PMCID: PMC3111534  PMID: 21695235
13.  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.
doi:10.1128/AEM.03246-12
PMCID: PMC3591943  PMID: 23275516
14.  Defining the Predicted Protein Secretome of the Fungal Wheat Leaf Pathogen Mycosphaerella graminicola 
PLoS ONE  2012;7(12):e49904.
The Dothideomycete fungus Mycosphaerella graminicola is the causal agent of Septoria tritici blotch, a devastating disease of wheat leaves that causes dramatic decreases in yield. Infection involves an initial extended period of symptomless intercellular colonisation prior to the development of visible necrotic disease lesions. Previous functional genomics and gene expression profiling studies have implicated the production of secreted virulence effector proteins as key facilitators of the initial symptomless growth phase. In order to identify additional candidate virulence effectors, we re-analysed and catalogued the predicted protein secretome of M. graminicola isolate IPO323, which is currently regarded as the reference strain for this species. We combined several bioinformatic approaches in order to increase the probability of identifying truly secreted proteins with either a predicted enzymatic function or an as yet unknown function. An initial secretome of 970 proteins was predicted, whilst further stringent selection criteria predicted 492 proteins. Of these, 321 possess some functional annotation, the composition of which may reflect the strictly intercellular growth habit of this pathogen, leaving 171 with no functional annotation. This analysis identified a protein family encoding secreted peroxidases/chloroperoxidases (PF01328) which is expanded within all members of the family Mycosphaerellaceae. Further analyses were done on the non-annotated proteins for size and cysteine content (effector protein hallmarks), and then by studying the distribution of homologues in 17 other sequenced Dothideomycete fungi within an overall total of 91 predicted proteomes from fungal, oomycete and nematode species. This detailed M. graminicola secretome analysis provides the basis for further functional and comparative genomics studies.
doi:10.1371/journal.pone.0049904
PMCID: PMC3517617  PMID: 23236356
15.  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.
doi:10.1093/molbev/msu134
PMCID: PMC4069618  PMID: 24732957
standing variation; gene duplication; resistance; fungicides; triazoles; Rhynchosporium
16.  The Emergence of Resistance to Fungicides 
PLoS ONE  2014;9(3):e91910.
Many studies exist about the selection phase of fungicide resistance evolution, where a resistant strain is present in a pathogen population and is differentially selected for by the application of fungicides. The emergence phase of the evolution of fungicide resistance - where the resistant strain is not present in the population and has to arise through mutation and subsequently invade the population - has not been studied to date. Here, we derive a model which describes the emergence of resistance in pathogen populations of crops. There are several important examples where a single mutation, affecting binding of a fungicide with the target protein, shifts the sensitivity phenotype of the resistant strain to such an extent that it cannot be controlled effectively (‘qualitative’ or ‘single-step’ resistance). The model was parameterized for this scenario for Mycosphaerella graminicola on winter wheat and used to evaluate the effect of fungicide dose rate on the time to emergence of resistance for a range of mutation probabilities, fitness costs of resistance and sensitivity levels of the resistant strain. We also evaluated the usefulness of mixing two fungicides of differing modes of action for delaying the emergence of resistance. The results suggest that it is unlikely that a resistant strain will already have emerged when a fungicide with a new mode of action is introduced. Hence, ‘anti-emergence’ strategies should be identified and implemented. For all simulated scenarios, the median emergence time of a resistant strain was affected little by changing the dose rate applied, within the range of doses typically used on commercial crops. Mixing a single-site acting fungicide with a multi-site acting fungicide delayed the emergence of resistance to the single-site component. Combining the findings with previous work on the selection phase will enable us to develop more efficient anti-resistance strategies.
doi:10.1371/journal.pone.0091910
PMCID: PMC3962370  PMID: 24658678
17.  Triazole Fungicides Can Induce Cross-Resistance to Medical Triazoles in Aspergillus fumigatus 
PLoS ONE  2012;7(3):e31801.
Background
Azoles play an important role in the management of Aspergillus diseases. Azole resistance is an emerging global problem in Aspergillus fumigatus, and may develop through patient therapy. In addition, an environmental route of resistance development has been suggested through exposure to 14α-demethylase inhibitors (DMIs). The main resistance mechanism associated with this putative fungicide-driven route is a combination of alterations in the Cyp51A-gene (TR34/L98H). We investigated if TR34/L98H could have developed through exposure to DMIs.
Methods and Findings
Thirty-one compounds that have been authorized for use as fungicides, herbicides, herbicide safeners and plant growth regulators in the Netherlands between 1970 and 2005, were investigated for cross-resistance to medical triazoles. Furthermore, CYP51-protein homology modeling and molecule alignment studies were performed to identify similarity in molecule structure and docking modes. Five triazole DMIs, propiconazole, bromuconazole, tebuconazole, epoxiconazole and difenoconazole, showed very similar molecule structures to the medical triazoles and adopted similar poses while docking the protein. These DMIs also showed the greatest cross-resistance and, importantly, were authorized for use between 1990 and 1996, directly preceding the recovery of the first clinical TR34/L98H isolate in 1998. Through microsatellite genotyping of TR34/L98H isolates we were able to calculate that the first isolate would have arisen in 1997, confirming the results of the abovementioned experiments. Finally, we performed induction experiments to investigate if TR34/L98H could be induced under laboratory conditions. One isolate evolved from two copies of the tandem repeat to three, indicating that fungicide pressure can indeed result in these genomic changes.
Conclusions
Our findings support a fungicide-driven route of TR34/L98H development in A. fumigatus. Similar molecule structure characteristics of five triazole DMIs and the three medical triazoles appear the underlying mechanism of cross resistance development. Our findings have major implications for the assessment of health risks associated with the use of triazole DMIs.
doi:10.1371/journal.pone.0031801
PMCID: PMC3291550  PMID: 22396740
18.  New broad-spectrum resistance to septoria tritici blotch derived from synthetic hexaploid wheat 
Septoria tritici blotch (STB), caused by the ascomycete Mycosphaerella graminicola, is one of the most devastating foliar diseases of wheat. We screened five synthetic hexaploid wheats (SHs), 13 wheat varieties that represent the differential set of cultivars and two susceptible checks with a global set of 20 isolates and discovered exceptionally broad STB resistance in SHs. Subsequent development and analyses of recombinant inbred lines (RILs) from a cross between the SH M3 and the highly susceptible bread wheat cv. Kulm revealed two novel resistance loci on chromosomes 3D and 5A. The 3D resistance was expressed in the seedling and adult plant stages, and it controlled necrosis (N) and pycnidia (P) development as well as the latency periods of these parameters. This locus, which is closely linked to the microsatellite marker Xgwm494, was tentatively designated Stb16q and explained from 41 to 71% of the phenotypic variation at seedling stage and 28–31% in mature plants. The resistance locus on chromosome 5A was specifically expressed in the adult plant stage, associated with SSR marker Xhbg247, explained 12–32% of the variation in disease, was designated Stb17, and is the first unambiguously identified and named QTL for adult plant resistance to M. graminicola. Our results confirm that common wheat progenitors might be a rich source of new Stb resistance genes/QTLs that can be deployed in commercial breeding programs.
doi:10.1007/s00122-011-1692-7
PMCID: PMC3249545  PMID: 21912855
19.  Azole fungicides affect mammalian steroidogenesis by inhibiting sterol 14 alpha-demethylase and aromatase. 
Environmental Health Perspectives  2003;111(3):255-261.
Azole compounds play a key role as antifungals in agriculture and in human mycoses and as non-steroidal antiestrogens in the treatment of estrogen-responsive breast tumors in postmenopausal women. This broad use of azoles is based on their inhibition of certain pathways of steroidogenesis by high-affinity binding to the enzymes sterol 14-alpha-demethylase and aromatase. Sterol 14-alpha-demethylase is crucial for the production of meiosis-activating sterols, which recently were shown to modulate germ cell development in both sexes of mammals. Aromatase is responsible for the physiologic balance of androgens and estrogens. At high doses, azole fungicides and other azole compounds affect reproductive organs, fertility, and development in several species. These effects may be explained by inhibition of sterol 14-alpha-demethylase and/or aromatase. In fact, several azole compounds were shown to inhibit these enzymes in vitro, and there is also strong evidence for inhibiting activity in vivo. Furthermore, the specificity of the enzyme inhibition of several of these compounds is poor, both with respect to fungal versus nonfungal sterol 14-alpha-demethylases and versus other P450 enzymes including aromatase. To our knowledge, this is the first review on sterol 14-alpha-demethylase and aromatase as common targets of azole compounds and the consequence for steroidogenesis. We conclude that many azole compounds developed as inhibitors of fungal sterol 14-alpha-demethylase are inhibitors also of mammalian sterol 14-alpha-demethylase and mammalian aromatase with unknown potencies. For human health risk assessment, data on comparative potencies of azole fungicides to fungal and human enzymes are needed.
PMCID: PMC1241380  PMID: 12611652
20.  Inhibition of Efflux Transporter-Mediated Fungicide Resistance in Pyrenophora tritici-repentis by a Derivative of 4′-Hydroxyflavone and Enhancement of Fungicide Activity 
Populations of the causal agent of wheat tan spot, Pyrenophora tritici-repentis, that are collected from fields frequently treated with reduced fungicide concentrations have reduced sensitivity to strobilurin fungicides and azole fungicides (C14-demethylase inhibitors). Energy-dependent efflux transporter activity can be induced under field conditions and after in vitro application of sublethal amounts of fungicides. Efflux transporters can mediate cross-resistance to a number of fungicides that belong to different chemical classes and have different modes of action. Resistant isolates can grow on substrata amended with fungicides and can infect plants treated with fungicides at levels above recommended field concentrations. We identified the hydroxyflavone derivative 2-(4-ethoxy-phenyl)-chromen-4-one as a potent inhibitor of energy-dependent fungicide efflux transporters in P. tritici-repentis. Application of this compound in combination with fungicides shifted fungicide-resistant P. tritici-repentis isolates back to normal sensitivity levels and prevented infection of wheat leaves. These results highlight the role of energy-dependent efflux transporters in fungicide resistance and could enable a novel disease management strategy based on the inhibition of fungicide efflux to be developed.
doi:10.1128/AEM.71.6.3269-3275.2005
PMCID: PMC1151812  PMID: 15933029
21.  Mutagenesis and Functional Studies with Succinate Dehydrogenase Inhibitors in the Wheat Pathogen Mycosphaerella graminicola 
PLoS ONE  2012;7(4):e35429.
A range of novel carboxamide fungicides, inhibitors of the succinate dehydrogenase enzyme (SDH, EC 1.3.5.1) is currently being introduced to the crop protection market. The aim of this study was to explore the impact of structurally distinct carboxamides on target site resistance development and to assess possible impact on fitness.
We used a UV mutagenesis approach in Mycosphaerella graminicola, a key pathogen of wheat to compare the nature, frequencies and impact of target mutations towards five subclasses of carboxamides. From this screen we identified 27 amino acid substitutions occurring at 18 different positions on the 3 subunits constituting the ubiquinone binding (Qp) site of the enzyme. The nature of substitutions and cross resistance profiles indicated significant differences in the binding interaction to the enzyme across the different inhibitors. Pharmacophore elucidation followed by docking studies in a tridimensional SDH model allowed us to propose rational hypotheses explaining some of the differential behaviors for the first time. Interestingly all the characterized substitutions had a negative impact on enzyme efficiency, however very low levels of enzyme activity appeared to be sufficient for cell survival. In order to explore the impact of mutations on pathogen fitness in vivo and in planta, homologous recombinants were generated for a selection of mutation types. In vivo, in contrast to previous studies performed in yeast and other organisms, SDH mutations did not result in a major increase of reactive oxygen species levels and did not display any significant fitness penalty. However, a number of Qp site mutations affecting enzyme efficiency were shown to have a biological impact in planta.
Using the combined approaches described here, we have significantly improved our understanding of possible resistance mechanisms to carboxamides and performed preliminary fitness penalty assessment in an economically important plant pathogen years ahead of possible resistance development in the field.
doi:10.1371/journal.pone.0035429
PMCID: PMC3334918  PMID: 22536383
22.  Three-Dimensional Models of Wild-Type and Mutated Forms of Cytochrome P450 14α-Sterol Demethylases from Aspergillus fumigatus and Candida albicans Provide Insights into Posaconazole Binding 
The cytochrome P450 sterol 14α-demethylase enzyme (CYP51) is the target of azole antifungals. Azoles block ergosterol synthesis, and thereby fungal growth, by binding in the active-site cavity of the enzyme and ligating the iron atom of the heme cofactor through a nitrogen atom of the azole. Mutations in and around the CYP51 active site have resulted in azole resistance. In this work, homology models of the CYP51 enzymes from Aspergillus fumigatus and Candida albicans were constructed based on the X-ray crystal structure of CYP51 from Mycobacterium tuberculosis. Using these models, binding modes for voriconazole (VOR), fluconazole (FLZ), itraconazole (ITZ), and posaconazole (POS) were predicted from docking calculations. Previous work had demonstrated that mutations in the vicinity of the heme cofactor had a greater impact on the binding of FLZ and VOR than on the binding of POS and ITZ. Our modeling data suggest that the long side chains of POS and ITZ occupy a specific channel within CYP51 and that this additional interaction, which is not available to VOR and FLZ, serves to stabilize the binding of these azoles to the mutated CYP51 proteins. The model also predicts that mutations that were previously shown to specifically impact POS susceptibility in A. fumigatus and C. albicans act by interfering with the binding of the long side chain.
doi:10.1128/AAC.48.2.568-574.2004
PMCID: PMC321559  PMID: 14742211
23.  Zymoseptoria gen. nov.: a new genus to accommodate Septoria-like species occurring on graminicolous hosts 
The Mycosphaerella complex is both poly- and paraphyletic, containing several different families and genera. The genus Mycosphaerella is restricted to species with Ramularia anamorphs, while Septoria is restricted to taxa that cluster with the type species of Septoria, S. cytisi, being closely related to Cercospora in the Mycosphaerellaceae. Species that occur on graminicolous hosts represent an as yet undescribed genus, for which the name Zymoseptoria is proposed. Based on the 28S nrDNA phylogeny derived in this study, Zymoseptoria is shown to cluster apart from Septoria. Morphologically species of Zymoseptoria can also be distinguished by their yeast-like growth in culture, and the formation of different conidial types that are absent in Septoria s.str. Other than the well-known pathogens such as Z. tritici, the causal agent of septoria tritici blotch on wheat, and Z. passerinii, the causal agent of septoria speckled leaf blotch of barley, both for which epitypes are designated, two leaf blotch pathogens are also described on graminicolous hosts from Iran. Zymoseptoria brevis sp. nov. is described from Phalaris minor, and Z. halophila comb. nov. from leaves of Hordeum glaucum. Further collections are now required to elucidate the relative importance, host range and distribution of these species.
doi:10.3767/003158511X571841
PMCID: PMC3160802  PMID: 22025804
Hordeum vulgare; ITS; LSU; multilocus sequence typing; Mycosphaerella; Septoria; systematics; Triticum aestivum
24.  CYP51 structures and structure-based development of novel, pathogen-specific inhibitory scaffolds 
Graphical abstract
Highlights
► CYP51s (sterol 14alpha-demethylases) are efficient drug target enzymes. ► CYP51s have a highly rigid substrate binding cavity. ► CYP51 structure-based development of a new inhibitory scaffold is described.
CYP51 (sterol 14α-demethylase) is a cytochrome P450 enzyme essential for sterol biosynthesis and the primary target for clinical and agricultural antifungal azoles. The azoles that are currently in clinical use for systemic fungal infections represent modifications of two basic scaffolds, ketoconazole and fluconazole, all of them being selected based on their antiparasitic activity in cellular experiments. By studying direct inhibition of CYP51 activity across phylogeny including human pathogens Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum, we identified three novel protozoa-specific inhibitory scaffolds, their inhibitory potency correlating well with antiprotozoan activity. VNI scaffold (carboxamide containing β-phenyl-imidazoles) is the most promising among them: killing T. cruzi amastigotes at low nanomolar concentration, it is also easy to synthesize and nontoxic. Oral administration of VNI (up to 400 mg/kg) neither leads to mortality nor reveals significant side effects up to 48 h post treatment using an experimental mouse model of acute toxicity. Trypanosomatidae CYP51 crystal structures determined in the ligand-free state and complexed with several azole inhibitors as well as a substrate analog revealed high rigidity of the CYP51 substrate binding cavity, which must be essential for the enzyme strict substrate specificity and functional conservation. Explaining profound potency of the VNI inhibitory scaffold, the structures also outline guidelines for its further development. First steps of the VNI scaffold optimization have been undertaken; the results presented here support the notion that CYP51 structure-based rational design of more efficient, pathogen-specific inhibitors represents a highly promising direction.
doi:10.1016/j.ijpddr.2012.06.001
PMCID: PMC3596085  PMID: 23504044
Sterol 14α-demethylase; CYP51; Inhibition; Crystal structure
25.  Wild-Type MIC Distribution and Epidemiological Cutoff Values for Aspergillus fumigatus and Three Triazoles as Determined by the Clinical and Laboratory Standards Institute Broth Microdilution Methods▿  
Journal of Clinical Microbiology  2009;47(10):3142-3146.
Antifungal susceptibility testing of Aspergillus species has been standardized by both the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Recent studies suggest the emergence of strains of Aspergillus fumigatus with acquired resistance to azoles. The mechanisms of resistance involve mutations in the cyp51A (sterol demethylase) gene, and patterns of azole cross-resistance have been linked to specific mutations. Studies using the EUCAST broth microdilution (BMD) method have defined wild-type (WT) MIC distributions, epidemiological cutoff values (ECVs), and cross-resistance among the azoles. We tested a collection of 637 clinical isolates of A. fumigatus for which itraconazole MICs were ≤2 μg/ml against posaconazole and voriconazole using the CLSI BMD method. An ECV of ≤1 μg/ml encompassed the WT population of A. fumigatus for itraconazole and voriconazole, whereas an ECV of ≤0.25 μg/ml was established for posaconazole. Our results demonstrate that the WT distribution and ECVs for A. fumigatus and the mold-active triazoles were the same when determined by the CLSI or the EUCAST BMD method. A collection of 43 isolates for which itraconazole MICs fell outside of the ECV were used to assess cross-resistance. Cross-resistance between itraconazole and posaconazole was seen for 53.5% of the isolates, whereas cross-resistance between itraconazole and voriconazole was apparent in only 7% of the isolates. The establishment of the WT MIC distribution and ECVs for the azoles and A. fumigatus will be useful in resistance surveillance and is an important step toward the development of clinical breakpoints.
doi:10.1128/JCM.00940-09
PMCID: PMC2756953  PMID: 19692559

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