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Appl Environ Microbiol. 2010 May; 76(9): 2866–2872.
Published online 2010 March 19. doi:  10.1128/AEM.02158-09
PMCID: PMC2863451

Heterologous Expression of Mutated Eburicol 14α-Demethylase (CYP51) Proteins of Mycosphaerella graminicola To Assess Effects on Azole Fungicide Sensitivity and Intrinsic Protein Function[down-pointing small open triangle]

Abstract

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.

Mycospherella graminicola (Fuckel) J Schroeter in Cohn (anamorph, Septoria tritici Roberge in Desmaz) is an ascomycete fungus that causes Septoria leaf blotch, the most important foliar disease of winter wheat in the United Kingdom (14). Currently, all the commercially available wheat cultivars are susceptible or only partially resistant to this pathogen. Therefore, control relies on the programmed application of fungicides. Over the past 30 years, M. graminicola has shown a capacity to adapt and has developed resistance to all systemic fungicides introduced for its control. In 2002, resistance to the quinone outside inhibitors (QoIs; strobilurins) emerged and rapidly spread in Western European M. graminicola populations (11). Consequently, QoIs are no longer recommended for the control of this pathogen. Recently, resistance to some azole (imidazole and triazole) fungicides, first introduced for the control of cereal pathogens in the late 1970s, emerged (4), and this resistance is also now widespread in Western European populations.

Azoles, the largest group of demethylation inhibiting (DMI) fungicides, are the most widely used agents for the control of fungal pathogens of humans and plants. They inhibit fungal growth by interfering with the biosynthesis of ergosterol, the predominant sterol in fungal membranes, by the interaction of the azole ring with the heme iron of the cytochrome P450 sterol 14α-demethylase (CYP51) (33). Mutations in the CYP51 gene have been shown to confer resistance to azoles, although generally in combination with other mechanisms (22). For example, in highly resistant strains of the opportunistic human pathogen Candida albicans, which can cause serious clinical problems in immunocompromised patients, CYP51 mutations are commonly identified (21, 22, 27) and the corresponding proteins have been shown biochemically to perturb the affinity of azole binding (17, 18). Unlike other single-site fungicides, however, where a single-amino-acid substitution can confer a highly resistant phenotype, combinations of CYP51 amino acid substitutions are required for high levels of resistance to azoles (27).

In plant pathogens, resistance to azoles is less common, and although some CYP51 mutations have been correlated with a reduced-azole-sensitivity phenotype (8, 9, 31), these have yet to be shown biochemically to affect azole binding. In M. graminicola, CYP51 mutations are common in modern populations (2, 6, 7, 28). To date, 19 different amino acid alterations have been reported and, similar to what was found for fluconazole resistance development in C. albicans, isolates least sensitive to azoles carry multiple CYP51 mutations (19, 28). Furthermore, several encoded amino acid alterations are at positions equivalent to those of highly resistant strains of C. albicans, although some, including V136A/C, A379G, and I381V, have been found only in M. graminicola (7). Interestingly, CYP51 amino acid changes in M. graminicola populations appear to have occurred sequentially. For example, with the exception of a single isolate from 2008, I381V has been found only in combination with changes between Y459 and Y461 (19, 28). Furthermore, recent analysis of the sterol content of M. graminicola isolates with different CYP51 variants suggests that some alterations may affect the capacity of M. graminicola CYP51 (MgCYP51) to metabolize eburicol, the CYP51 substrate in filamentous fungi, although without any apparent effects on isolate fitness (1).

To date, studies of the effects of MgCYP51 changes on azole sensitivity and protein biochemistry have been correlative. In this study, by complementation of a Saccharomyces cerevisiae YUG37:erg11 strain that carries a doxycycline-regulatable tetO7-CYC promoter controlling native CYP51 (ERG11) expression (13, 26), we have functionally characterized the impacts of a number of MgCYP51 mutations on azole sensitivity. In addition, the expression of MgCYP51 variants carrying I381V, a substitution associated with resistance to a number of azoles (12), either alone or combined with other CYP51 changes, illustrates that this alteration affects the intrinsic function of the CYP51 protein.

MATERIALS AND METHODS

Strains and media.

Wild-type Mycosphaerella graminicola CYP51 (MgCYP51) was amplified from IPO323, the isolate selected for the M. graminicola genome sequencing project (http://genome.jgi-psf.org/Mycgr1/Mycgr1.home.html). Saccharomyces cerevisiae strain YUG37:erg11 (MATa ura3-52 trp1-63 LEU2::tTA tetO-CYC1::ERG11 (26) was used for heterologous expression studies with MgCYP51 variants. Escherichia coli XL-1 Blue (Stratagene, La Jolla, CA) was used for routine subcloning. M. graminicola material for RNA extraction was grown for 4 days in yeast extract-peptone-dextrose broth (YPD; Formedium, Norwich, United Kingdom) at 21°C with shaking at 200 rpm, harvested by vacuum filtration, snap-frozen in liquid nitrogen, and stored at −80°C prior to freeze-drying. YPD was also used for routine culturing of S. cerevisiae. S. cerevisiae transformants were grown on synthetic dropout (SD) minimal medium containing 1.6 g liter−1 dropout medium supplement without uracil (Sigma-Aldrich, St. Louis, MO), 2 g liter−1 yeast nitrogen base without amino acids (Difco, Detroit, MI), and 2% glucose (GLU) or 2% galactose-2% raffinose (GAL+RAF) with 2% agar added when needed. For complementation studies, 3 μg ml−1 doxycycline (Sigma-Aldrich), which represses native CYP51 expression, was added to SD GAL+RAF medium. LB broth and agar, amended with 100 μg ml−1 ampicillin, were used for plasmid preparation and plating of E. coli transformations.

RNA extraction and cDNA synthesis.

Total RNA was extracted from freeze-dried M. graminicola isolate IPO323 tissue with TRIzol reagent (Invitrogen, Carlsbad, CA) in accordance with the manufacturer's protocol. A subsequent overnight incubation of extracts in 4 M lithium chloride was used to further purify RNA. Five micrograms of total RNA was reverse transcribed with oligo(dT)20 using the SuperScript III first-strand synthesis system (Invitrogen) according to the supplier's instructions.

Complementation of S. cerevisiae YUG37:erg11 strain with MgCYP51.

The full-length coding sequence of MgCYP51 was amplified from a 1/10 dilution of synthesized cDNA from isolate IPO323 by use of primers pYESMg51F07 (5′-GGTACCATGGGTCTCCTCCAGGAAGTC-3′) and pYESMg51R07 (5′-CTCGAGTTCTTCTCCTCCTTCTCCT-3′), which incorporated KpnI and HindIII restriction sites (underlined), respectively, to enable cloning in yeast expression vector pYES2/CT (Invitrogen). Reactions were carried out with a Biometra T3 thermocycler (Biotron GmbH, Göttingen, Germany), using 1 unit of Phusion high-fidelity DNA polymerase (Finnzymes, Espoo, Finland), 125 μM each deoxynucleoside triphosphate (dNTP), and 0.5 μM each primer in a final volume of 50 μl. The reaction conditions were as follows: initial denaturation at 98°C for 30 s, followed by 35 cycles at 98°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 3 min, with a final extension at 72°C for 6 min. The amplified fragment was digested with KpnI and HindIII and cloned into the pYES2/CT vector, creating the pYES-Mg51wt expression plasmid. The full-length MgCYP51 gene in pYES-Mg51wt was sequenced to ensure the validity of the sequences and transformed into the S. cerevisiae YUG37:erg11 strain by use of an S.c.EasyComp transformation kit (Invitrogen). Transformations were plated out onto SD GAL+RAF medium with or without 3 μg ml−1 doxycycline. The pYES2/CT vector was used as a negative control.

Site-directed mutagenesis.

Mutations at sites altered in isolates of M. graminicola less sensitive to azoles were introduced into pYES-Mg51wt by use of a QuikChange II site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions, using 100 ng of target (pYES-Mg51wt) plasmid and 5 ng of each primer (Table (Table1).1). Full-length MgCYP51 genes of mutated plasmids were sequenced to confirm the introduction of mutations and the validity of sequences. Mutated plasmids were transformed into S. cerevisiae YUG37:erg11 and plated out onto selective medium as described above.

TABLE 1.
Primers used for site-directed mutagenesis

Complementation analysis of S. cerevisiae YUG37:erg11 transformants and comparative growth rate analysis.

The complementation efficiencies of transformants were screened using agar plates and Bioscreen C (Oy Growth Curves Ab, Ltd.). Transformants were grown for 24 h at 30°C in SD GAL+RAF medium to induce MgCYP51 expression. Each transformant was drop inoculated with 5 μl of cell suspensions (six 5-fold dilutions of a starting concentration of 1 × 106 cells ml−1) onto SD GAL+RAF agar plates with or without 3 μg ml−1 doxycycline. Photographs were taken after 72 h of growth at 30°C. For Bioscreen C analysis, four cultures of each transformant strain (duplicate cultures of replicate transformants) were grown in SD GAL+RAF medium overnight at 30°C. One hundred microliters of each overnight culture, diluted to 1 × 105 cells/ml, was used to individually inoculate 3 wells containing 200 μl SD GAL+RAF medium with or without 3 μg ml−1 doxycycline in a honeycomb plate. Cultures were incubated at 30°C, and the optical density at 600 nm (OD600) was measured every 15 min for 5 days. All wells were replicated on a second plate, giving a total of 24 measurements for each transformant. The mean maximum growth rate for each strain with or without doxycycline was determined on the basis of the greatest increase in OD over a 2-h period.

Generation of MgCYP51-specific antisera.

Peptides corresponding to the region encompassing amino acids 428 to 442 (COOH-EPHRWDESPSEKYKH-NH2) of the predicted M. graminicola CYP51 protein (GenBank accession no. AY730587) were used to immunize rabbits. Peptide design and rabbit immunizations were carried out by Eurogentec (Seraing, Belgium).

Protein extraction from crude microsomal preparation.

Crude microsomal extracts for each transformant were prepared from 100 ml of culture grown for 24 h at 30°C in SD GAL+RAF medium. After several washes with sterile distilled water, cells were disrupted by sonication (6 1-min bursts, with 2-min rests) on ice in 2 ml cold extraction buffer (0.1 M Tris, 25% glycerol, pH 8.1). After removal of cell debris by centrifugation for 1 min at 3,000 × g, extracts were centrifuged at 16,000 × g for 30 min to obtain crude microsomal pellets. Pellets were washed twice in extraction buffer and subsequently resuspended in 50 μl of 1× SDS-PAGE loading buffer (250 mM Tris-HCl, 30% glycerol, 10% SDS, 5% β-mercaptoethanol, 10 mg bromophenol blue).

MgCYP51 protein detection.

Approximately 20 μl of crude microsomal protein was separated on 15% SDS-PAGE gels and blotted onto Hybond ECL nitrocellulose membranes (Amersham Pharmacia). Membranes were probed with a 1:2,000 dilution of anti-MgCYP51 antiserum and subsequently a 1:10,000 dilution of anti-mouse IgG alkaline phosphatase (Sigma-Aldrich). Antibody binding was detected using a Sigmafast BCIP (5-bromo-4-chloro-3-indolylphosphate)-Nitro Blue Tetrazolium alkaline phosphatase substrate (Sigma-Aldrich).

Sterol analysis.

Four cultures of each transformant (duplicate cultures of replicate transformants) were grown for 24 h at 30°C in SD GAL+RAF medium. Eight milliliters of each overnight culture, diluted to 1 × 105 cells ml−1, was used individually to inoculate 16 ml of SD GAL+RAF medium and SD GAL+RAF medium containing doxycycline. After growth for 72 h at 30°C, cells were harvested and washed twice with sterile water. Nonsaponifiable lipids were extracted as reported previously (16). Samples were dried in a vacuum centrifuge (Heto) and derivatized by addition of 100 μl of 90% bis(trimethylsilyl)-trifluoroacetamide (BSTFA)-10% trimethylsilyl (TMS) (Sigma-Aldrich) and 50 μl anhydrous pyridine (Sigma-Aldrich) and heating for 2 h at 80°C. Gas chromatography-mass spectrometry was performed using a VG12-250 mass spectrometer (VG Biotech) with splitless injection. Individual sterols were identified by reference to relative retention times, mass ions, and fragmentation patterns.

Yeast transformant fungicide sensitivity assays.

The sensitivity assays of yeast transformants were similar to those described by Fraaije et al. (12), with the following modifications. Wells of flat-bottomed microtiter plates were filled with 100 μl of SD GAL+RAF medium containing 6 μg ml−1 doxycycline, amended with 0.5, 0.17, 0.056, 0.019, 0.0062, 0.0021, 0.00069, 0.00023, 7.62E−05, 2.54E−05, or 8.5E−06 μg ml−1 of epoxiconazole, with 5, 1.67, 0.56, 0.19, 0.062, 0.021, 0.0069, 0.0023, 0.00076, 0.00025, or 8.47E−05 μg ml−1 of tebuconazole or cycloheximide, with 20, 6.67, 2.23, 0.74, 0.25, 0.082, 0.027, 0.0091, 0.003, 0.001, or 0.00034 μg ml−1 of triadimenol, or with 1, 0.33, 0.11, 0.037, 0.012, 0.0041, 0.0013, 0.000457, 0.00015, 5.08E−05, or 1.69E−05 μg ml−1 of prochloraz. One hundred microliters of cell suspension (106 cells ml−1) in SD GAL+RAF medium containing S. cerevisiae YUG37:erg11 transformants, grown for 24 h at 30°C in SD GAL+RAF medium, was added to each well. Plates were incubated for 3 days at 30°C, and growth was measured with a plate reader at 630 nm (BMG Labtech, Offenburg, Germany). Fungicide sensitivities were determined as mean 50% effective concentrations (EC50s) for replicate transformants, calculated on the basis of a dose-response relationship. The resistance factor (RF) of each transformant was calculated as the fold change in EC50 compared to the level for transformants expressing wild-type MgCYP51.

RESULTS

Complementation of S. cerevisiae strain YUG37:erg11.

S. cerevisiae strain YUG37:erg11 transformed with pYES-Mg51wt expressing the wild-type MgCYP51 protein grew on SD GAL+RAF medium in the presence of doxycycline, repressing native CYP51 expression, illustrating that the wild-type M. graminicola CYP51 protein complements the function of the orthologous gene in S. cerevisiae (Fig. (Fig.1).1). Bioscreen C analysis of pYES-Mg51wt transformants revealed a lower growth rate in the presence of doxycycline, suggesting, as expected, that the heterologous protein is less effective than the native CYP51 protein in S. cerevisiae (Table (Table2).2). Introduction of the L50S (pYES-Mg51L50S) or Y461H (pYES-Mg51Y461H) alteration into the wild-type MgCYP51 protein had no effect on the capacity of the M. graminicola enzyme to complement the S. cerevisiae CYP51 protein (Fig. (Fig.1).1). Similarly, transformation of YUG37:erg11 with constructs expressing MgCYP51 variants carrying a combination of these alterations (pYES-Mg51L50S/Y461H) restored the growth of transformants on SD GAL+RAF plates with doxycycline (Fig. (Fig.1).1). MgCYP51 variants with alterations Y459D and ΔY459/G460, either alone or combined with L50S, also complemented YUG37:erg11 (data not shown). In contrast, introduction of the I381V substitution alone (pYES-Mg51I381V) or combined with L50S (pYES-Mg51L50S/I381V) did not support YUG37:erg11 complementation (Fig. (Fig.1).1). These data were confirmed by Bioscreen C analysis (Table (Table2).2). Interestingly, functionality of MgCYP51 in S. cerevisiae was partially restored when I381V was combined with Y461H (Fig. (Fig.11 and Table Table2).2). The growth rates of transformants expressing MgCYP51 variants with a combination of I381V and Y461H or L50S, I381V, and Y461H were reduced around 5- to 7-fold compared to the levels for transformants expressing the wild type or variants carrying Y461H (Table (Table2).2). A similar phenotype was also observed when I381V was combined with Y459D and ΔY459/G460 (data not shown).

FIG. 1.
Complementation of S. cerevisiae strain YUG37:erg11 with wild-type (Mg51wt) and mutated variants of the M. graminicola CYP51 gene. Growth in the absence (−DOX) and presence (+DOX) of doxycycline is shown.
TABLE 2.
Bioscreen analysis of S. cerevisiae YUG37:erg11 transformant growth rates

Protein detection.

Bands of similar intensities, corresponding to the estimated molecular mass of the M. graminicola CYP51 protein (61.5 kDa), were detected in crude microsomal extract of all transformants expressing MgCYP51 variants, including those that did not complement YUG37:erg11 (for example, pYES2-MgI381V). No signal at 61.5 kDa was detected in the YUG37:erg11 strain transformed with the pYES2 vector-alone control, demonstrating that the MgCYP51 antiserum does not cross-react with the endogenous S. cerevisiae CYP51 (Fig. (Fig.22).

FIG. 2.
Detection of the MgCYP51 protein in crude microsomal extracts of S. cerevisiae YUG37:erg11 strains transformed with pYES-Mg51I381V/Y461H (1st lane), pYES-Mg51I381V (2nd lane), pYES-Mg51L50S/Y461H (3rd lane), pYES-Mg51Y461H (4th lane), pYES-Mg51L50S (5th ...

Sterol biosynthesis.

In the absence of doxycycline, similar levels of ergosterol (around 75% of total sterol content) and the CYP51 substrate lanosterol were detected in strains transformed with the vector alone (pYES2) or expressing either wild-type or mutant MgCYP51 variants. No 14α-methylated sterol intermediates were detected in the absence of doxycycline (data not shown). In the presence of doxycycline, the ergosterol content levels of transformants expressing pYES-Mg51wt and pYES-Mg51Y461H were reduced to around 50% (Table (Table3),3), confirming that the M. graminicola protein is not as effective as the native protein in S. cerevisiae. Concomitant with the observed reduction in ergosterol was an accumulation of 14α-methylated sterols, including 4,14-cholesta-8,24-dienol, 14α-methyl fecosterol, and 14α-methyl-3,6-diol (Table (Table3).3). In contrast, levels of ergosterol in transformants expressing MgCYP51 proteins carrying substitution I381V in combination with Y461H (pYES-Mg51I381V/Y461H and pYES-Mg51L50S/I381V/Y461H) were reduced to around 25% compared to the growth of transformants expressing pYES-Mg51wt. A corresponding increase in 14α-methylated sterol was detected, particularly for 14α-methyl-3,6-diol (around 30% of total sterol content). Lanosterol levels were similar for all transformants able to grow in the presence of doxycycline (Table (Table33).

TABLE 3.
Sterol content of YUG37:erg11 transformants in the presence of doxycycline

Azole sensitivities of S. cerevisiae YUG37:erg11 transformants.

Analysis of the azole sensitivities of S. cerevisiae YUG37:erg11 expressing MgCYP51 variants which fully restored growth on doxycycline-amended medium revealed considerable differences between transformants expressing wild-type MgCYP51, those variants with the L50S substitution alone, and those expressing MgCYP51 variants with alterations between codons 459 and 461 (pYES-Mg51Y459D, pYES-Mg51Y461H, pYES-Mg51ΔY459/G460, pYES-Mg51L50S/Y459D, pYES-Mg51L50S/Y461H, and pYES-Mg51L50S/ΔY459/G460) (Table (Table4).4). Epoxiconazole sensitivities of transformants expressing MgCYP51 variants with single alterations were reduced between 8-fold (pYES-Mg51Y459D) and 15-fold (pYES-Mg51Y461H), tebuconazole sensitivities up to 45-fold (pYES-Mg51ΔY459/G460), and triadimenol sensitivity 170-fold in transformants expressing pYES-Mg51Y461H. Sensitivities to prochloraz were also reduced, between 12-fold (pYES-Mg51Y459D) and 24-fold (pYES-Mg51Y461H and pYES-Mg51ΔY459/G460). As expected, there was no effect of single alterations between codons 459 and 461 on cycloheximide sensitivity (Table (Table4).4). Azole sensitivities of transformants were further reduced when Y461H and ΔY459/G460 were combined with substitution L50S. For example, the highest RF values for epoxiconazole (30.4) and triadimenol (192) were recorded for transformants expressing Mg51L50S/Y461H, with the highest values for tebuconazole (80.7) recorded for strains transformed with pYES-Mg51L50S/ΔY459/G460. These strains were also unaffected in cycloheximide sensitivity (Table (Table44).

TABLE 4.
Azole sensitivities of S. cerevisiae YUG37:erg11 transformantsa

DISCUSSION

The recent decrease in the sensitivity of the Western European M. graminicola population to some azole fungicides has been correlated with the emergence of mutations in the CYP51 gene encoding the azole target (5, 12, 19, 28, 34). By heterologous expression in an S. cerevisiae strain with a regulatable promoter controlling native CYP51 expression, we have, for the first time for a plant pathogenic fungus, functionally characterized the impacts of CYP51 mutations present in the M. graminicola population on azole fungicide sensitivity and intrinsic protein function. These studies have demonstrated the extent to which mutations encoding alterations between residues Y459 and Y461 reduce the sensitivities of the CYP51 protein to different azoles and revealed that the I381V substitution, both alone and combined with other CYP51 alterations, prevents or limits (depending on other alterations present) the capacity of the M. graminicola CYP51 protein to function in S. cerevisiae.

The region encompassing amino acids Y459, G460, and Y461 of the MgCYP51 protein is conserved among fungi but, interestingly, absent in other phyla (Fig. (Fig.3).3). Consequently, prediction of the proximity of this region to the azole or substrate-bound ligand based on homology modeling using the Mycobacterium tuberculosis CYP51 crystal structure is not possible. However, in C. albicans, alterations at the residue equivalent to G460 (G448 and G448E [20], G448R [30], and G448V [15]) and the residue equivalent to Y461 (F449 and F449L [21], F449S [22], and F449Y [32]) have been identified in resistant isolates, although their precise contribution to the final resistant phenotype is unknown. Furthermore, substitutions at amino acids corresponding to Y459 and Y461 (Y461D and Y463D/H/N, respectively) have recently been identified in isolates of Mycosphaerella fijiensis with reduced sensitivity to propiconazole (3). Similar to what was found for M. graminicola, alterations in this region of the protein in azole-resistant isolates of C. albicans and M. fijiensis are generally found in combination with other CYP51 changes.

FIG. 3.
Sequence alignment of a fragment of the CYP51 protein family. Conserved residues are shaded in black. The predicted substrate recognition site 5 (SRS-5) is shaded in grey. Arrows indicate residues I381, Y459, G460, and Y461 of the Mycosphaerella graminicola ...

Substitution I381V, which appears unique to M. graminicola, having not previously been reported in studies of azole-resistant fungi, is considered responsible for recent sensitivity shifts to some azoles, particularly to tebuconazole (5, 12, 19), and isolates carrying this substitution (in combination with other CYP51 alterations) currently dominate the Western European population (28). In this study, we have demonstrated that introduction of I381V alone destroys MgCYP51 function in S. cerevisiae. Function of MgCYP51 is partially restored by combining I381V with alterations between Y459 and Y461. Although in this study the function of MgCYP51 proteins was analyzed in a heterologous system, it is probable that alterations would have similar effects on the activity of MgCYP51 in M. graminicola. Sterol analysis of transformants expressing MgCYP51 carrying a combination of L50S, I381V, and Y461H (pYES-51L50S/I381V/Y461H), a common CYP51 variant in Western European M. graminicola populations (28), demonstrated a reduced capacity to produce ergosterol and a corresponding increase in 14α-methylated sterols, including the toxic intermediate 14α-methyl-3,6-diol (29). These data are consistent with the recent report of sterol content analysis of M. graminicola isolates, which suggested altered eburicol demethylation activity in isolates carrying I381V. M. graminicola isolates may be able to accommodate this impaired activity, as, unlike for S. cerevisiae, 14α-methyl-3,6-diol could not be detected (1).

The residue equivalent to I381 in the crystallized M. tuberculosis CYP51 strain, L321, lies within 4 Å of fluconazole (23)-, estriol (24)-, and 4,4′-dihydroxybenzophenone (10)-bound ligands and is suggested to stabilize the 3β-hydroxylated ring of sterol substrates via hydrophobic contacts (10). Consequently, it can be envisaged that alteration of this residue would alter the interaction of CYP51 with both azoles and substrates. Interestingly, in a CYP51-like enzyme of oats (Avena strigosa CYP51H10 [AsCYP51H10] [25]), shown to be dispensable for sterol biosynthesis but required for the synthesis of the antimicrobial compound avenacin, an alanine, rather than the conserved leucine or isoleucine (Fig. (Fig.3),3), is at this position. It is suggested that this residue, in combination with other alterations, is a key determinant modulating active site cavity size and shape and therefore substrate range (25).

The illustration that MgCYP51 proteins carrying I381V require alterations between Y459 and Y461 for function is in accordance with the sequence in which these alterations emerged in the population. Alterations between Y459 and Y461 occurred in the early to mid 1990s (B. A. Fraaije, H. J. Cools, and J. A. Lucas, unpublished data) and subsequently became widespread in the Western European population (2). Reduced sensitivities of S. cerevisiae YUG37:erg11 transformants expressing MgCYP51 proteins with alterations between Y459 and Y461 are consistent with the rapid selection of these variants in the azole-exposed Western European population. I381V emerged in 2000 (6; Fraaije et al., unpublished) and has, with the exception of one recently sequenced isolate, which carries a novel substitution, D107V (28), been found only in combination with changes between Y459 and Y461. The data presented in this study confirm that I381V could not have emerged prior to the accumulation of alterations between Y459 and Y461 in the M. graminicola population and, furthermore, suggest an important role for the fungus-specific region encompassing Y459 to Y461 in the function of the CYP51 active site.

Further studies of M. graminicola CYP51 changes will incorporate other mutations correlated with altered azole sensitivity, including Y137F, V136A/C, and A379G. In addition, ongoing random mutagenesis studies should elucidate the potential for further evolution of MgCYP51, thus providing an insight into future azole sensitivity changes in the M. graminicola population.

Acknowledgments

This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) of the United Kingdom, project numbers BBE02257X1 and BBE0218321. Rothamsted Research receives grant-aided support from the BBSRC.

We thank Jason Rudd for technical assistance.

Footnotes

[down-pointing small open triangle]Published ahead of print on 19 March 2010.

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