Azole fungicides – understanding resistance mechanisms in agricultural fungal pathogens
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Claire L. Price, Josie E. Parker, Andrew G. S. Warrilow, Diane E. Kelly and Steven L. Kelly Centre for Cytochrome P450 Biodiversity, Institute of Life Science, College of Medicine, Swansea University, Swansea, Wales SA2 8PP, United Kingdom #To whom correspondence should be addressed. Phone: +44 1792 292207 Fax: +44 1792 503430 Email: [email protected] or [email protected] Keywords: CYP51, azole, fungicide, resistance, cytochrome P450
ABSTRACT Plant fungal pathogens can have devastating effects on a wide range of crops, including cereals and fruit (such as wheat and grapes), causing losses in crop yield, which are costly to the agricultural economy and threaten food security. Azole antifungals are the treatment of choice, however, resistance has arisen against these compounds, which could lead to devastating consequences. Therefore, it is important to understand how these fungicides are used and how the resistance arises to fully tackle the problem. Here, we give an overview of the problem and discuss the mechanisms that mediate azole resistance in agriculture (point mutations in the CYP51 amino acid sequence, overexpression of the CYP51 enzyme and overexpression of genes encoding efflux pumps proteins).
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1 INTRODUCTION Plant fungal pathogens can have devastating effects on a wide range of crops. They are responsible for a number of diseases, including septoria leaf blotch, powdery mildews and rusts, which can cause major losses in crop yields and, in turn, result in a financial burden to the agricultural economy. Some fungi, such as Fusarium and Penicillium, are able to synthesise mycotoxins. These are toxic chemical compounds that, if ingested, can have a major negative impact on the health of humans and animals. Therefore, controlling these pathogens is paramount to ensuring food security. Whilst there are a number of antimycotic compounds available to control the spread of these agricultural fungal pathogens (i.e. benzimidazoles, phenylamides, dicarboximides, anilinopyrimidines, quinone outside inhibitors (QoIs) and carboxylic acid amides (CAAs))1, it is azole antifungals that are the preferred treatment due to their relatively low cost and effectiveness against a broad range of fungi. They were first introduced into the clinic in 1958 with the use of the topical agent, chlormidazole, but it was not until the 1970’s that azole fungicides were first used in agriculture with the introduction of imazalil and triadimefon. Currently there are a number of azoles available for use in agriculture with the most recently introduced (2002) being the triazolinethione derivative prothioconazole, which is used in the treatment of the wheat pathogen, Zymoseptoria tritici (formerly known as Mycosphaerella graminicola). Triazoles are the most widely used class of systemic fungicides (20% market share) with prothioconazole, epoxiconazole and tebuconazole being the three most used fungicides in the United Kingdom.2,3 However, prothioconazole has recently been shown to be a pro-fungicide as the antifungal effect observed in studies with Candida albicans was found to be due to prothiconazole being readily metabolised to its desthio form rather than the activity of the fungicide itself.4 Furthermore, in azole binding studies with the CYP51 enzyme from Z. tritici, prothioconazole was shown to exhibit a novel spectrum not associated with inhibition,5 which was further corroborated in biochemical studies where inhibition of enzymatic activity was not observed.6 Azoles work by targeting the sterol 14α-demethylase, CYP51 (a member of the cytochrome P450 family), which is an important regulatory enzyme in the ergosterol biosynthetic pathway. Azole fungicides bind through direct coordination of the triazole N-4 or the imidazole N-3 nitrogen as the sixth ligand of the haem iron.7 The resultant CYP51-azole complex is catalytically inactive, preventing the demethylation of lanosterol and eburicol, and, in turn, the production of ergosterol, which is essential to maintain fungal membrane fluidity and permeability. Treatment with azoles depletes the amount of ergosterol in the cell, which, in conjunction with an accumulation of 14αdemethylated sterols, disrupts the membrane structure, preventing active membrane transport resulting in fungistasis. However, not all 14α-methylated sterols are incompatible with growth and membrane function as seen in mutants growing with 14αmethyl fecosterol8 and even recently lanosterol.9 These observations in medical mycology have yet to be reproduced in plant pathogenic fungi, although it would be surprising were they not eventually. Possibly fitness costs or altered pathogenicity might mediate against them occurring. Whilst azole fungicides are available to control plant pathogens, extensive use and over-exposure of these compounds has led to reduced sensitivity in the field. Therefore, it is imperative that the effectiveness of these fungicides is maintained, as the resultant
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decrease in crop yields would have a detrimental effect on food production and the economy.10 For instance, if the use of azoles was to stop in Europe, there would be a short-term fall in wheat production of ~7% (9.8 million tons), which would increase to ~12% (18.6 million tons) by 2020.10 This fall in production would mean a relative economical loss of 2.4 billion euros in the short-term and 4.6 billion euros by 2020. This would seriously affect Europe’s ability to produce wheat self-sufficiently.10 Therefore, it is important to understand how these fungicides are used and how resistance arises to prolong their effectiveness. 2 MECHANISMS OF RESISTANCE Many years of exposure to azole antifungals has led to increasing rates of resistance being seen in several plant fungal pathogens. The first case was observed in the powdery mildew, Sphaerotheca fuliginea (which affects cucurbits, including pumpkins and cucumbers)11 and since then, resistance has been detected in a number of other fungal pathogens including Puccinia triticina (brown rust in wheat crops), Z. tritici (septoria leaf blotch) and Penicillium digitatum (citrus fruit mould). There are three main mechanisms of resistance in agricultural fungi: • Point mutations in the CYP51 amino acid sequence • Overexpression of the CYP51 enzyme • Overexpression of genes encoding efflux pumps proteins 2.1 Mutations in the CYP51 enzyme The most widely reported mechanism of resistance in field isolates is the presence of mutations in the CYP51 enzyme. These mutations can cause changes in the fungicide’s affinity for the enzyme leading to azole tolerance. CYP51 mutations have been documented in a number of fungal plant pathogens (see table 1 for an overview) with the greatest number being identified in Z. tritici. Over 30 different substitutions and deletions have been reported in field isolates of this organism (up until 2013),12 which have arisen due to the extensive use of azoles (since the early 1980’s) to treat septoria leaf blotch infections in wheat. While many of the reported mutations can cause azole resistance in isolation, some are lethal to the host and require other mutations to accommodate them to restore this impairment. For instance, in Saccharomyces cerevisiae complementation studies, the introduction of Z. tritici CYP51 containing the I381V mutation did not support growth. However, combining I381V with Y461H partially restored the functionality of this CYP51.22 Combinations of mutations can also result in alterations in the level of resistance to different azoles. During the 1990’s, the most prevalent mutation in Z. tritici was Y137F, which conferred resistance to triadimenol. This was superseded by the double mutation Y137F-S542T in the early 2000’s and resulted in increased resistance to the same compound. However, as triadimenol is no longer used to treat septoria leaf blotch and has been replaced by newer azoles, the Y137F and Y137F-S542T mutations are now rarely seen.23
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2.2 Overexpression of CYP51 Overexpression of CYP51 in fungal pathogens is not as extensively documented as the mutations in the amino acid sequence. However, it has been observed in several fungi including: Z. tritici,24 P. tritici,19 Monilinia fructicola (brown rot of stone fruits),25,26 Blumeriella jaapi (cherry leaf spot),27,28 P. digitatum,29,30 Venturia inaequalis (apple scab)31 and P. brassicae.18 Overexpression is caused by changes in the promoter region of the Cyp51 gene either by the insertion of tandem repeats or transposable elements. It is assumed that increase mRNA levels correlate with the resultant increase in cellular CYP51 levels, and therefore drug target levels, causing a reduction in the azole sensitivity exhibited by the fungus. In P. digitatum, overexpression of Cyp51 (due to a 199-bp insert in the promoter region) was related to the imazalil resistance seen in isolates.32 2.3 Reduced intracellular accumulation of azole antifungals Although CYP51 is the main target of azole antifungals, other non-related enzymes can be involved in causing resistance. Efflux transporters (including ATPbinding cassette (ABC) transporters and major facilitator superfamily (MFS) transporters) are essential in eukaryotes to export toxins and fungicides out of the cell. Overexpression of the genes encoding these transporters can lead to resistance against azole antifungals as the intracellular concentration of the fungicide is reduced. While the link between efflux transporters and azole resistance has been widely reported in human fungal pathogens (such as C. albicans, C. glabrata and Cryptococcus neoformans), there is limited information available regarding this phenomenon in plant fungal pathogens. Cases have been reported in Botrytis cinerea (grey mould on a variety of plant species, including grapes)33,34 and P. digitatum.35,36 It has also been suggested as a possible mechanism of resistance in Z. tritici.37,38 In Botrytis cinerea, the ABC transporter, BcatrD has been associated with azole resistance. Overexpression of BcatrD results in increased expression of the transporter and reduced sensitivity to oxpoconazole. Mutants where BcatrD has been replaced have shown increased azole sensitivity and accumulation of high levels of oxpoconazole, further establishing a role for BcatrD in azole resistance.33,34 Whilst the above resistance mechanisms have been identified in field isolates, the true extent of these mechanisms is not fully understood. For instance, inactivation of ERG3 (Δ7 sterol-C-5-desaturase) has been shown to lead to azole resistance in the human pathogen, C. albicans.39 However, similar mechanisms have yet to be identified in field isolates.17 Recently it has been observed, in the field, that Ascomycetes (such as Fusarium spp., P. digitarium and Magnaporthe oryzae) carry multiple copies of CYP51. It is believed that these extra copies contribute to the increased resistance observed in these organisms40 as sterol 14α-demethylase activity can be retained in multiple copies. Additionally, the re-emergence of the CYP51A paralog in the barley pathogen Rhynchosporium commune has been shown to be a novel evolutionary mechanism of resistance.41
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3 UNDERSTANDING THE MECHANISMS OF RESISTANCE There are many experimental techniques available to study the mechanisms of azole resistance. Firstly, DNA sequencing is necessary in order to determine the genotype of resistance strains. DNA sequencing enables the identification of mutations (including SNPs, insertions and duplications) within the coding regions and promoter region of the CYP51 and transporters from field isolates. Coupled with MIC (minimum inhibitory concentration) data, the effect of these changes on azole affinity can be elucidated. In the medical mycology arena whole genome sequencing of resistance isolates has begun to be published42 and this will occur for plant pathogens. In genetically tractable pathogens the correlation of phenotype and genotype can be examined using genetic manipulation. Alternatively, biochemical studies can be undertaken using purified recombinant CYP51 proteins (ligand binding and inhibition studies) to determine the affinity of a particular azole fungicide for a given CYP51 protein (wild type and mutants). More recently in silico modelling of fungal CYP51 enzymes has been used to predict the affinity of azole antifungals for these enzymes, predominantly looking at how mutations within the structure affect azole docking. Fungal CYP51 proteins currently modelled include: Magnaporthe grisea CYP51,43 Ustilago maydis CYP51,44 Aspergillus fumigatus CYP51,45 M. fijiensis CYP51,16 P. digitatum CYP5146 - 48 and Z. tritici CYP51.49 The 3D structures of fungal cytochromes P450 (CYPs) have primarily been based on the crystal structures of bacterial CYPs, which are readily soluble and relatively easy to crystallize, unlike eukaryotic CYPs which tend to aggregate in free solution due to their hydrophobic N-terminal membrane anchors. While this has allowed in silico models of fungal CYPs to be determined using bacterial CYP templates, these models may not give a complete picture of how the fungal enzymes fold. Recently, the crystal structures of a number of truncated eukaryotic CYP51s have been elucidated. These include CYP51s from the parasites Trypanosoma cruzi, T. brucei CYP5150 - 52 and Leishmania infantum CYP51,53 as well as human CYP51.54 In addition, the first full-length eukaryotic CYP51 crystal structure has now been solved for S. cerevisiae.55 The advent of these eukaryotic crystal structures will greatly improve confidence in in silico structural modelling with the recent S. cerevisiae structure having a modest effect on the current models using multi-template methodology. 4 CONCLUSIONS Plant fungal pathogens are a blight on agricultural crops causing devastating losses in crop yields, a financial burden on the economy and threatening food security. Therefore it is imperative that these diseases can be adequately controlled. Currently, the main treatment for these pathogens is azole antifungals due to their broad spectrum of activity. However, time and over-use of the fungicides in the field has led to resistance being seen in a number of fungal species. While the mechanisms of azole resistance are still being successfully elucidated, the complex nature of the resistance makes it a difficult task. Therefore, it is important to maintain the effectiveness of these fungicides, as the resultant decrease in crop yields would have devastating effects. Practical strategies, which could be employed to overcome this problem, include alternating between azole and non-azole fungicides; grow crop cultivars that are inherently more resistant to fungal pathogens and to develop new azole fungicides. However, continuing
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the challenge of understanding how the mechanisms of azole resistance arise is essential to fully tackling the problem of resistance.
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Table 1 – Overview of mutations identified in the CYP51 of field isolates.
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Organism
Crop affected
Alterations in the amino acid sequence L50S, D107V, D134G, V136A, V136C, V136G, Y137F, M145L, N178S, S188N, S208T, N284H, H303Y, A311G, G312A, A379G, I381V, A410T, G412A, Y459C, Y459D, Y459N, Y459P, Y459S, G460D, Y461D, Y461H, Y461S, ΔY459 or ΔG460, ΔY459/G460, V490L, G510C, N513K, S524T
Reference
Zymoseptoria tritici
Wheat
Blumeria graminis f.sp. tritici
Wheat
Y136F
13
Blumeria graminis f.sp. hordei
Barley
Y136F, K147Q
14
Erysiphe necator
Grape
Y136F
15
Mycosphaerella fijiensis
Banana plants and plantain
Y136F, A313G, Y461D, Y463D, Y463H, Y463N
16
Venturia nasicola
Japanese pear
Y133
17
Pyrenopeziza brassicae
Oilseed rape
G460S, S508T
18
Puccinia triticina
Wheat
Y134F
19
Penicillum digitatum
Citrus fruit
V55A, Y136H, M144T, K253E, Q309H, E331A, T432, I440V, K449R, G459S, R462H, F506I, S507P, K508R, G511S
20
Oculimacula yallundae
Wheat
S35T, Q43H, D78Y, E106K, N244S, S505Q
21
Oculimacula acuformis
Wheat
A29P, V37A, Q167H, Y486H, S505Q
21
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12
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Claire L. Price, Josie E. Parker, Andrew G. S. Warrilow, Diane E. Kelly and Steven L. Kelly Centre for Cytochrome P450 Biodiversity, Institute of Life Science, College of Medicine, Swansea University, Swansea, Wales SA2 8PP, United Kingdom #To whom correspondence should be addressed. Phone: +44 1792 292207 Fax: +44 1792 503430 Email: [email protected] or [email protected] Keywords: CYP51, azole, fungicide, resistance, cytochrome P450
ABSTRACT Plant fungal pathogens can have devastating effects on a wide range of crops, including cereals and fruit (such as wheat and grapes), causing losses in crop yield, which are costly to the agricultural economy and threaten food security. Azole antifungals are the treatment of choice, however, resistance has arisen against these compounds, which could lead to devastating consequences. Therefore, it is important to understand how these fungicides are used and how the resistance arises to fully tackle the problem. Here, we give an overview of the problem and discuss the mechanisms that mediate azole resistance in agriculture (point mutations in the CYP51 amino acid sequence, overexpression of the CYP51 enzyme and overexpression of genes encoding efflux pumps proteins).
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1 INTRODUCTION Plant fungal pathogens can have devastating effects on a wide range of crops. They are responsible for a number of diseases, including septoria leaf blotch, powdery mildews and rusts, which can cause major losses in crop yields and, in turn, result in a financial burden to the agricultural economy. Some fungi, such as Fusarium and Penicillium, are able to synthesise mycotoxins. These are toxic chemical compounds that, if ingested, can have a major negative impact on the health of humans and animals. Therefore, controlling these pathogens is paramount to ensuring food security. Whilst there are a number of antimycotic compounds available to control the spread of these agricultural fungal pathogens (i.e. benzimidazoles, phenylamides, dicarboximides, anilinopyrimidines, quinone outside inhibitors (QoIs) and carboxylic acid amides (CAAs))1, it is azole antifungals that are the preferred treatment due to their relatively low cost and effectiveness against a broad range of fungi. They were first introduced into the clinic in 1958 with the use of the topical agent, chlormidazole, but it was not until the 1970’s that azole fungicides were first used in agriculture with the introduction of imazalil and triadimefon. Currently there are a number of azoles available for use in agriculture with the most recently introduced (2002) being the triazolinethione derivative prothioconazole, which is used in the treatment of the wheat pathogen, Zymoseptoria tritici (formerly known as Mycosphaerella graminicola). Triazoles are the most widely used class of systemic fungicides (20% market share) with prothioconazole, epoxiconazole and tebuconazole being the three most used fungicides in the United Kingdom.2,3 However, prothioconazole has recently been shown to be a pro-fungicide as the antifungal effect observed in studies with Candida albicans was found to be due to prothiconazole being readily metabolised to its desthio form rather than the activity of the fungicide itself.4 Furthermore, in azole binding studies with the CYP51 enzyme from Z. tritici, prothioconazole was shown to exhibit a novel spectrum not associated with inhibition,5 which was further corroborated in biochemical studies where inhibition of enzymatic activity was not observed.6 Azoles work by targeting the sterol 14α-demethylase, CYP51 (a member of the cytochrome P450 family), which is an important regulatory enzyme in the ergosterol biosynthetic pathway. Azole fungicides bind through direct coordination of the triazole N-4 or the imidazole N-3 nitrogen as the sixth ligand of the haem iron.7 The resultant CYP51-azole complex is catalytically inactive, preventing the demethylation of lanosterol and eburicol, and, in turn, the production of ergosterol, which is essential to maintain fungal membrane fluidity and permeability. Treatment with azoles depletes the amount of ergosterol in the cell, which, in conjunction with an accumulation of 14αdemethylated sterols, disrupts the membrane structure, preventing active membrane transport resulting in fungistasis. However, not all 14α-methylated sterols are incompatible with growth and membrane function as seen in mutants growing with 14αmethyl fecosterol8 and even recently lanosterol.9 These observations in medical mycology have yet to be reproduced in plant pathogenic fungi, although it would be surprising were they not eventually. Possibly fitness costs or altered pathogenicity might mediate against them occurring. Whilst azole fungicides are available to control plant pathogens, extensive use and over-exposure of these compounds has led to reduced sensitivity in the field. Therefore, it is imperative that the effectiveness of these fungicides is maintained, as the resultant
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decrease in crop yields would have a detrimental effect on food production and the economy.10 For instance, if the use of azoles was to stop in Europe, there would be a short-term fall in wheat production of ~7% (9.8 million tons), which would increase to ~12% (18.6 million tons) by 2020.10 This fall in production would mean a relative economical loss of 2.4 billion euros in the short-term and 4.6 billion euros by 2020. This would seriously affect Europe’s ability to produce wheat self-sufficiently.10 Therefore, it is important to understand how these fungicides are used and how resistance arises to prolong their effectiveness. 2 MECHANISMS OF RESISTANCE Many years of exposure to azole antifungals has led to increasing rates of resistance being seen in several plant fungal pathogens. The first case was observed in the powdery mildew, Sphaerotheca fuliginea (which affects cucurbits, including pumpkins and cucumbers)11 and since then, resistance has been detected in a number of other fungal pathogens including Puccinia triticina (brown rust in wheat crops), Z. tritici (septoria leaf blotch) and Penicillium digitatum (citrus fruit mould). There are three main mechanisms of resistance in agricultural fungi: • Point mutations in the CYP51 amino acid sequence • Overexpression of the CYP51 enzyme • Overexpression of genes encoding efflux pumps proteins 2.1 Mutations in the CYP51 enzyme The most widely reported mechanism of resistance in field isolates is the presence of mutations in the CYP51 enzyme. These mutations can cause changes in the fungicide’s affinity for the enzyme leading to azole tolerance. CYP51 mutations have been documented in a number of fungal plant pathogens (see table 1 for an overview) with the greatest number being identified in Z. tritici. Over 30 different substitutions and deletions have been reported in field isolates of this organism (up until 2013),12 which have arisen due to the extensive use of azoles (since the early 1980’s) to treat septoria leaf blotch infections in wheat. While many of the reported mutations can cause azole resistance in isolation, some are lethal to the host and require other mutations to accommodate them to restore this impairment. For instance, in Saccharomyces cerevisiae complementation studies, the introduction of Z. tritici CYP51 containing the I381V mutation did not support growth. However, combining I381V with Y461H partially restored the functionality of this CYP51.22 Combinations of mutations can also result in alterations in the level of resistance to different azoles. During the 1990’s, the most prevalent mutation in Z. tritici was Y137F, which conferred resistance to triadimenol. This was superseded by the double mutation Y137F-S542T in the early 2000’s and resulted in increased resistance to the same compound. However, as triadimenol is no longer used to treat septoria leaf blotch and has been replaced by newer azoles, the Y137F and Y137F-S542T mutations are now rarely seen.23
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2.2 Overexpression of CYP51 Overexpression of CYP51 in fungal pathogens is not as extensively documented as the mutations in the amino acid sequence. However, it has been observed in several fungi including: Z. tritici,24 P. tritici,19 Monilinia fructicola (brown rot of stone fruits),25,26 Blumeriella jaapi (cherry leaf spot),27,28 P. digitatum,29,30 Venturia inaequalis (apple scab)31 and P. brassicae.18 Overexpression is caused by changes in the promoter region of the Cyp51 gene either by the insertion of tandem repeats or transposable elements. It is assumed that increase mRNA levels correlate with the resultant increase in cellular CYP51 levels, and therefore drug target levels, causing a reduction in the azole sensitivity exhibited by the fungus. In P. digitatum, overexpression of Cyp51 (due to a 199-bp insert in the promoter region) was related to the imazalil resistance seen in isolates.32 2.3 Reduced intracellular accumulation of azole antifungals Although CYP51 is the main target of azole antifungals, other non-related enzymes can be involved in causing resistance. Efflux transporters (including ATPbinding cassette (ABC) transporters and major facilitator superfamily (MFS) transporters) are essential in eukaryotes to export toxins and fungicides out of the cell. Overexpression of the genes encoding these transporters can lead to resistance against azole antifungals as the intracellular concentration of the fungicide is reduced. While the link between efflux transporters and azole resistance has been widely reported in human fungal pathogens (such as C. albicans, C. glabrata and Cryptococcus neoformans), there is limited information available regarding this phenomenon in plant fungal pathogens. Cases have been reported in Botrytis cinerea (grey mould on a variety of plant species, including grapes)33,34 and P. digitatum.35,36 It has also been suggested as a possible mechanism of resistance in Z. tritici.37,38 In Botrytis cinerea, the ABC transporter, BcatrD has been associated with azole resistance. Overexpression of BcatrD results in increased expression of the transporter and reduced sensitivity to oxpoconazole. Mutants where BcatrD has been replaced have shown increased azole sensitivity and accumulation of high levels of oxpoconazole, further establishing a role for BcatrD in azole resistance.33,34 Whilst the above resistance mechanisms have been identified in field isolates, the true extent of these mechanisms is not fully understood. For instance, inactivation of ERG3 (Δ7 sterol-C-5-desaturase) has been shown to lead to azole resistance in the human pathogen, C. albicans.39 However, similar mechanisms have yet to be identified in field isolates.17 Recently it has been observed, in the field, that Ascomycetes (such as Fusarium spp., P. digitarium and Magnaporthe oryzae) carry multiple copies of CYP51. It is believed that these extra copies contribute to the increased resistance observed in these organisms40 as sterol 14α-demethylase activity can be retained in multiple copies. Additionally, the re-emergence of the CYP51A paralog in the barley pathogen Rhynchosporium commune has been shown to be a novel evolutionary mechanism of resistance.41
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3 UNDERSTANDING THE MECHANISMS OF RESISTANCE There are many experimental techniques available to study the mechanisms of azole resistance. Firstly, DNA sequencing is necessary in order to determine the genotype of resistance strains. DNA sequencing enables the identification of mutations (including SNPs, insertions and duplications) within the coding regions and promoter region of the CYP51 and transporters from field isolates. Coupled with MIC (minimum inhibitory concentration) data, the effect of these changes on azole affinity can be elucidated. In the medical mycology arena whole genome sequencing of resistance isolates has begun to be published42 and this will occur for plant pathogens. In genetically tractable pathogens the correlation of phenotype and genotype can be examined using genetic manipulation. Alternatively, biochemical studies can be undertaken using purified recombinant CYP51 proteins (ligand binding and inhibition studies) to determine the affinity of a particular azole fungicide for a given CYP51 protein (wild type and mutants). More recently in silico modelling of fungal CYP51 enzymes has been used to predict the affinity of azole antifungals for these enzymes, predominantly looking at how mutations within the structure affect azole docking. Fungal CYP51 proteins currently modelled include: Magnaporthe grisea CYP51,43 Ustilago maydis CYP51,44 Aspergillus fumigatus CYP51,45 M. fijiensis CYP51,16 P. digitatum CYP5146 - 48 and Z. tritici CYP51.49 The 3D structures of fungal cytochromes P450 (CYPs) have primarily been based on the crystal structures of bacterial CYPs, which are readily soluble and relatively easy to crystallize, unlike eukaryotic CYPs which tend to aggregate in free solution due to their hydrophobic N-terminal membrane anchors. While this has allowed in silico models of fungal CYPs to be determined using bacterial CYP templates, these models may not give a complete picture of how the fungal enzymes fold. Recently, the crystal structures of a number of truncated eukaryotic CYP51s have been elucidated. These include CYP51s from the parasites Trypanosoma cruzi, T. brucei CYP5150 - 52 and Leishmania infantum CYP51,53 as well as human CYP51.54 In addition, the first full-length eukaryotic CYP51 crystal structure has now been solved for S. cerevisiae.55 The advent of these eukaryotic crystal structures will greatly improve confidence in in silico structural modelling with the recent S. cerevisiae structure having a modest effect on the current models using multi-template methodology. 4 CONCLUSIONS Plant fungal pathogens are a blight on agricultural crops causing devastating losses in crop yields, a financial burden on the economy and threatening food security. Therefore it is imperative that these diseases can be adequately controlled. Currently, the main treatment for these pathogens is azole antifungals due to their broad spectrum of activity. However, time and over-use of the fungicides in the field has led to resistance being seen in a number of fungal species. While the mechanisms of azole resistance are still being successfully elucidated, the complex nature of the resistance makes it a difficult task. Therefore, it is important to maintain the effectiveness of these fungicides, as the resultant decrease in crop yields would have devastating effects. Practical strategies, which could be employed to overcome this problem, include alternating between azole and non-azole fungicides; grow crop cultivars that are inherently more resistant to fungal pathogens and to develop new azole fungicides. However, continuing
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the challenge of understanding how the mechanisms of azole resistance arise is essential to fully tackling the problem of resistance.
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Table 1 – Overview of mutations identified in the CYP51 of field isolates.
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Organism
Crop affected
Alterations in the amino acid sequence L50S, D107V, D134G, V136A, V136C, V136G, Y137F, M145L, N178S, S188N, S208T, N284H, H303Y, A311G, G312A, A379G, I381V, A410T, G412A, Y459C, Y459D, Y459N, Y459P, Y459S, G460D, Y461D, Y461H, Y461S, ΔY459 or ΔG460, ΔY459/G460, V490L, G510C, N513K, S524T
Reference
Zymoseptoria tritici
Wheat
Blumeria graminis f.sp. tritici
Wheat
Y136F
13
Blumeria graminis f.sp. hordei
Barley
Y136F, K147Q
14
Erysiphe necator
Grape
Y136F
15
Mycosphaerella fijiensis
Banana plants and plantain
Y136F, A313G, Y461D, Y463D, Y463H, Y463N
16
Venturia nasicola
Japanese pear
Y133
17
Pyrenopeziza brassicae
Oilseed rape
G460S, S508T
18
Puccinia triticina
Wheat
Y134F
19
Penicillum digitatum
Citrus fruit
V55A, Y136H, M144T, K253E, Q309H, E331A, T432, I440V, K449R, G459S, R462H, F506I, S507P, K508R, G511S
20
Oculimacula yallundae
Wheat
S35T, Q43H, D78Y, E106K, N244S, S505Q
21
Oculimacula acuformis
Wheat
A29P, V37A, Q167H, Y486H, S505Q
21
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12
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