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The cytochrome P450 sterol 14α-demethylase (CYP51, syn. ERG11) is an essential enzyme in the biosynthesis of sterols, critical components of cell membranes of all eukaryotic organisms required for the regulation of membrane fluidity and permeability (Parks et al., 1995; Lepesheva & Waterman, 2007). Of the many sterols identified in fungi, ergosterol is the most common and is required for fungal growth (Rodriguez et al., 1985; Weete et al., 2010). Consequently, CYP51 is a widely exploited target for the control of fungal pathogens of humans and plants, with the azole (imidazole and triazole) fungicides being the leading class of antifungals for over three decades (Sheehan et al., 1999). Surprisingly, despite their widespread long-term use and single-site mode of action, incidences of resistance to azole fungicides are rare. More commonly, reductions in sensitivity are reported with cross-resistance within the azole class often incomplete. To date, three mechanisms of resistance to azoles predominate in filamentous fungi: mutations in the CYP51 gene encoding amino acid alterations, resulting in decreased affinity of the protein for inhibitors (Loffler et al., 1997; Sanglard et al., 1998; Wyand & Brown, 2005; Cools et al., 2010); over-expression of the target CYP51 gene, most frequently caused by insertions in the predicted promoter regions (Mellado et al., 2007; Cools et al., 2012); and over-expression of genes encoding efflux pumps (Sanglard et al., 1995; Kretschmer et al., 2009). These mechanisms can combine, and therefore resistance levels in fungal strains are often determined by combinations of CYP51 amino acid alterations, CYP51 gene over-expression and/or enhanced efflux (Perea et al., 2001; Mellado et al., 2007; Cools et al., 2012).
Until recently, CYP51 was thought to exist as a single gene in all phyla. Mammalian genomes, for example, contain one CYP51, with some nonfunctional pseudogenes identified (Rozman et al., 1996). However, increasing genome sequence information has shown that this is not the case in all kingdoms. To date, multiple CYP51 genes have been found in plants, including rice (12), oats (two), tobacco (two) and Arabidopsis thaliana (two) (Lepesheva & Waterman, 2007). Filamentous fungi, particularly Ascomycetes, often possess two or more CYP51 paralogues, for example in Penicillium digitatum (two), Aspergillus fumigatus (two), A. nidulans (two), A. flavus (three), Magnaporthe oryzae (two) and species of Fusarium, including F. verticillioides, F. oxysporum f. sp. lycopersici and F. graminearum (three). Molecular phylogenetic analysis has shown that the CYP51 genes of fungi within the subphylum Pezizomycotina fall into three clades, designated A, B and C (Becher et al., 2011). All species possess a CYP51 in clade B (CYP51B). Species with multiple paralogues carry an additional CYP51 in clade A (CYP51A), with duplications of CYP51A or CYP51B generating the third paralogue in some species, for example A. flavus and A. terreus, respectively. Uniquely, the third CYP51 paralogue in Fusarium species forms a distinct clade, CYP51C. The CYP51C gene is found exclusively in Fusarium species, and is ubiquitous across the genus, as demonstrated by its use as a reliable phylogenetic marker (Fernández-Ortuño et al., 2010).
Fungi with multiple CYP51s are intrinsically resistant to some azoles, although some remain effective. For example, A. fumigatus is well controlled by itraconazole and voriconazole, whereas fluconazole is ineffective. Species of Fusarium, for example F. solani, are resistant to commonly used medical azoles, including fluconazole, voriconazole and the recently introduced posaconazole (Nucci & Anaissie, 2007). Deletion of CYP51A increases the intrinsic sensitivity to some azoles in M. oryzae (e.g. tebuconazole and prochloraz), A. fumigatus (e.g. fluconazole and ketoconazole) and F. graminearum (e.g. tebuconazole and prochloraz) (Mellado et al., 2005; Liu et al., 2011; Yan et al., 2011). Furthermore, resistance to effective azoles in fungi with multiple CYP51s is most frequently mediated by changes in the CYP51A paralogue. For example, over-expression of CYP51A has been reported for resistant isolates of P. digitatum (Hamamoto et al., 2000; Ghosoph et al., 2007), and mutation of AfCYP51A is the most common mechanism of resistance in A. fumigatus isolates (Diaz-Guerra et al., 2003; Mellado et al., 2005), which, when combined with AfCYP51A over-expression, confers a multi-azole-resistant phenotype (Mellado et al., 2007; Snelders et al., 2008).
The control of F. graminearum, the most important pathogen causing Fusarium head blight (FHB) or head scab disease on wheat and barley, is primarily dependent on effective azole fungicides (e.g. tebuconazole, metconazole and prothioconazole). Effective application of azoles reduces the content of the harmful trichothecene mycotoxin deoxynivalenol (DON) in wheat grains (Beyer et al., 2006; Paul et al., 2008). However, an increased level of DON has been detected after treatment with sublethal doses of prothioconazole both in vitro and in planta (Audenaert et al., 2010), and trichothecenes accumulate in grain samples after treatment with tebuconazole (Kulik et al., 2012). In addition, a study using the enhanced green fluorescent protein gene (egfp) as a reporter demonstrated activation of the F. graminearum trichothecene biosynthetic TRI5 gene by sublethal concentrations of tebuconazole (Ochiai et al., 2007); both TRI4 and TRI5 transcript levels were higher after tebuconazole treatment in culture (Kulik et al., 2012). Currently, although stress responses and enhanced secondary metabolism have been proposed, the mechanism(s) responsible for altered mycotoxin production after azole treatment is unknown.
Functional analysis of multiple CYP51s has identified roles additional to primary sterol biosynthesis. In oats, a CYP51 homologue, AsCyp51H10, is dispensable for sterol biosynthesis, but is required for the synthesis of avenacins, antimicrobial compounds unique to the genus Avena (Qi et al., 2006). Heterologous expression in yeast identified AsCyp51H10 as the first CYP51 not classified as a sterol demethylase (Kunii et al., 2012). Recent analysis of the 12 CYP51s of rice has differentiated some genes as CYP51H, a group likely to have functions beyond sterol biosynthesis (Inagaki et al., 2011). Previous work on the CYP51 genes of F. graminearum (FgCYP51) has demonstrated that the deletion of individual FgCYP51 genes can reduce conidiation, but otherwise causes no changes in in vitro morphology, mycelial growth rate or ergosterol content (Liu et al., 2011).
In this study, we have determined the competence of the three paralogous FgCYP51 genes to act as sterol 14α-demethylases by heterologous expression in S. cerevisiae strain YUG37:erg11, which carries a regulatable promoter controlling native CYP51 expression, and found that FgCYP51C cannot complement the CYP51 function of the yeast gene. We generated single (ΔFgCYP51A, ΔFgCYP51B and ΔFgCYP51C) and combined (ΔFgCYP51AC and ΔFgCYP51BC) CYP51 deletion mutants and characterized their function in vitro and in planta. We report distinct roles of the three FgCYP51 genes, with the FgCYP51C gene specifically required for full virulence on host wheat ears, but not on Arabidopsis floral tissue or the fruits of apple and tomato.
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The recent increase in genome sequence information has revealed that many fungi, particularly ascomycetes, carry more than one copy of the azole fungicide target encoding gene, CYP51. In the Pezizomycotina, a subphylum of the ascomycetes that includes important pathogens of animals and plants, such as Aspergillus spp., M. oryzae, Mycosphaerella graminicola and Fusarium spp., CYP51 paralogues have been classified into three phylogenetic clades, designated A, B and C (Becher et al., 2011), with the CYP51C paralogue only found in Fusarium species. The CYP51C clade is ubiquitous across the genus Fusarium and, consequently, can be used as a reliable phylogenetic marker for the identification of different species (Fernández-Ortuño et al., 2010). A previous study has reported that the deletion of individual F. graminearum CYP51 genes (FgCYP51A, FgCYP51B or FgCYP51C) has no effect on colony morphology, vegetative growth rate or ergosterol content, although conidiation is reduced in all mutants and deletion of the FgCYP51A and FgCYP51C genes increases azole sensitivity (Liu et al., 2011). In this study, by heterologous expression in yeast and systematic characterization of the impact of individual (FgCYP51A, FgCYP51B, FgCYP51C) and double (FgCYP51AC and FgCYP51BC) gene deletions on in vitro growth, fungicide sensitivity, total sterol composition and virulence on wheat ears and other plants, we describe distinct roles for the FgCYP51 paralogues of F. graminearum.
FgCYP51B encodes the primary sterol 14α-demethylase and is essential for ascospore production
The three FgCYP51 genes of F. graminearum isolate Fg1955 were heterologously expressed in S. cerevisiae strain YUG37::erg11, which has been used previously to assess the impact of CYP51 mutations on Mycosphaerella graminicola azole sensitivity and enzyme function (Cools et al., 2010, 2011) and to analyse the role of AfCYP51A and AfCYP51B genes in A. fumigatus (Martel et al., 2010b). FgCYP51A and FgCYP51B were able to substitute ScCYP51 function, whereas FgCYP51C could not (Fig. 2). In addition, transformants expressing FgCYP51B grew faster than those expressing FgCYP51A (Table 2). This suggests that the FgCYP51B protein is a more effective sterol 14α-demethylase than FgCYP51A in yeast.
FgCYP51 gene deletion did not impact on ergosterol content, in accordance with Liu et al. (2011). However, the abundance of intermediate sterols was different in all mutants relative to the wild-type. The specific accumulation of eburicol in ΔFgCYP51B and ΔFgCYP51BC suggests that the overall eburicol demethylation activity is perturbed in mutants lacking FgCYP51B. This is in contrast with those deficient in FgCYP51A or FgCYP51C activity, although the product of CYP51, 4,4-dimethyl ergosta-8,14,24(28)-trienol, was significantly less abundant in all ΔFgCYP51 mutants relative to the wild-type. These data are consistent with studies of A. fumigatus. Deletions of either AfCYP51A or AfCYP51B blocked C14-demethylation, but far more eburicol accumulated in the AfCYP51B mutant than the AfCYP51A mutant (Alcazar-Fuoli et al., 2008). Furthermore, substrate binding studies of AfCYP51 proteins expressed in Escherichia coli detected strong binding with purified AfCYP51B using eburicol and lanosterol, in contrast with AfCYP51A (Warrilow et al., 2010). As a consequence of accumulated eburicol, two additional novel 14-methylated sterol intermediates (4,4,14-trimethyl ergosta-trienol and 4,4,14-trimethyl ergosta-dienol) were detected in ΔFgCYP51B and ΔFgCYP51BC (Fig. S8). These data suggest that Pezizomycotina CYP51B, including FgCYP51B, is central to effective sterol C14-demethylation.
Ascospores forcibly ejected from mature perithecia, formed by the overwintering fungus on field debris, are the primary source of inoculum for F. graminearum epidemics (Parry et al., 1995; Trail et al., 2005). In this study, no ascospores were formed in ΔFgCYP51B and ΔFgCYP51BC mutants (Fig. 3b), although all the FgCYP51 gene deletion mutants produced superficially normal perithecia. This finding demonstrates that FgCYP51B is specifically required in the development of the sexual stage, a role that cannot be fulfilled by the up-regulation of FgCYP51A. Similarly, the delayed colonization of wheat ears by ΔFgCYP51B suggests that FgCYP51A cannot fully complement FgCYP51B function during wheat infection.
FgCYP51A encodes an inducible sterol 14α-demethylase that determines azole sensitivity
In the absence of fungicide treatment, the relative transcript quantities of FgCYP51A and FgCYP51B were highest at 72 h of incubation, decreasing at 96 h during growth in rich medium (PDB). This pattern of expression is coincident with rapid fungal growth, which is linear between 24 and 60 h of incubation, and into stationary phase after 72 h of incubation (data not shown). The relative transcript quantities of FgCYP51A changed most over this time course. Previous studies have shown an increase in CYP51A gene expression after azole treatment and CYP51B deletion in vitro in F. graminearum (Liu et al., 2010; Becher et al., 2011) and M. oryzae (Yan et al., 2011). We report similar results, with FgCYP51A expression induced over 100-fold by azoles in vitro, and c. 10-fold by FgCYP51B deletion both in vitro and in planta. The enhanced transcription of FgCYP51A on exposure to azoles suggests that this gene is not only responsive to chemical or genetic perturbation of FgCYP51B activity, but also other stresses induced by fungicide treatment. For example, in S. cerevisiae, ScCYP51 expression is higher during growth on glucose, in the presence of haem, under oxygen-limiting growth conditions and during exposure to anaerobic conditions (Turi & Loper, 1992).
Amino acid substitutions and CYP51 over-expression are the most common mechanisms of resistance to azoles in filamentous ascomycetes. In fungi with multiple CYP51s, it is the CYP51A paralogue that is most commonly altered, for example in A. fumigatus and P. digitatum (Ghosoph et al., 2007; Mellado et al., 2007). In addition, single gene deletion has confirmed that AfCYP51A is involved in intrinsic azole resistance, for example to fluconazole in A. fumigatus (Mellado et al., 2005), and azole affinity studies have shown that the AfCYP51A protein has lower affinity than AfCYP51B for a wide range of azoles (Warrilow et al., 2010). In F. graminearum, it has been suggested previously that different azole fungicides target different FgCYP51s (Liu et al., 2011). In this study, deletion of FgCYP51C, either alone or with FgCYP51A (ΔFgCYP51AC), had no effect on azole sensitivity. Deletion of FgCYP51B caused an increase in sensitivity to some azoles, with sensitivities to metconazole and prochloraz particularly affected. These data conflict with those of Liu et al. (2011), who reported increased sensitivity to tebuconazole and prochloraz of ΔFgCYP51C mutants, with FgCYP51B deletion having no impact on azole sensitivity. The reason for this discrepancy is unclear, although the different methodologies used for fungicide sensitivity testing and the different origins of the strains used in gene deletion studies may have contributed. However, clearly, in this study, an interaction of azole fungicides with FgCYP51B is consistent with the assertion that this paralogue is the primary sterol 14α-demethylase in F. graminearum. In both studies, however, the deletion of FgCYP51A increased the sensitivity to all azoles tested and, particularly, epoxiconazole. Considering that F. graminearum isolate Fg1955 is least sensitive to epoxiconazole, FgCYP51A expression is inducible on azole exposure and yeast transformants expressing FgCYP51A are least sensitive to epoxiconazole (Table 3), it can be concluded that the intrinsically lower sensitivity to some azoles in F. graminearum is primarily determined by FgCYP51A.
FgCYP51C is a novel genus-specific CYP51 gene required for full virulence on wheat ears
In the S. cerevisiae heterologous expression system, FgCYP51A and FgCYP51B were able to substitute for ScCYP51 function. By contrast, FgCYP51C could not complement ScCYP51. This suggests that FgCYP51C cannot function as a sterol 14α-demethylase. This loss or diversification of function is probably caused by substitutions in conserved putative substrate recognition sites (SRSs) of FgCYP51C. The predicted FgCYP51 amino acid sequences are sufficiently identical (over 40%) to be considered as members of the same P450 family (Liu et al., 2011). Analysis of residues conserved in eukaryotic CYP51s (Fig. S9, Lepesheva & Waterman, 2011) identified two residues (N304 and T305) unique to FgCYP51C. Although the importance of these residues in the function of FgCYP51C is unknown, substitutions T315N or S316T of rat CYP51, equivalent to N304 and T305, caused significant reductions in lanosterol demethylase activity (Nitahara et al., 2001). Deletion of FgCYP51C had no impact on in vitro fungal morphology, growth rate, conidiation and spore germination at almost all vegetative stages, perithecia production, ascospore formation or azole sensitivity. In addition, there was no difference in eburicol or ergosterol content in ΔFgCYP51C mutants. However, ΔFgCYP51C mutants had less 4,4-dimethyl ergosta-8,14,24(28)-trienol, the product of CYP51, and accumulated the sterol intermediates episterol, ergosta-5,7,24(28)-trienol and ergosta-5,7-dienol, products of ERG2, ERG3 and ERG4 activity, respectively (Fig. S8). This suggests that FgCYP51C can impact indirectly on sterol 14α-demethylation, ERG2, ERG3 and ERG4 activity. There was no difference in ERG2, ERG3A, ERG3B and ERG4 gene expression in single ΔFgCYP51C mutants relative to wild-type PH-1 in vitro. However, the ERG3B gene was expressed less in both ΔFgCYP51AC and ΔFgCYP51BC mutants, which grew more slowly and produced less aerial mycelia on rich medium in the dark and when inoculated on wheat ears and Arabidopsis floral tissues. The CYP51 gene is required for aerobic viability in S. cerevisiae, C. albicans and C. glabrata. In a CYP51-deficient mutant, aerobic growth can be restored by null mutation or deletion of ERG3 (Bard et al., 1993; Kelly et al., 1993, 1995; Geber et al., 1995). However, to date, there is no evidence for ERG3-mediated azole resistance in filamentous fungi.
After inoculation with the ΔFgCYP51C, ΔFgCYP51AC and ΔFgCYP51BC mutants, the number of infected spikelets per wheat ear was reduced significantly relative to inoculations with wild-type PH-1, ΔFgCYP51A and ΔFgCYP51B. In addition, not all the grain in bleached spikelets had a rough, shrivelled appearance, although infected grains were pink in wheat ears inoculated with ΔFgCYP51C, ΔFgCYP51AC and ΔFgCYP51BC, rather than the grey in plants inoculated with PH-1, ΔFgCYP51A and ΔFgCYP51B (Fig. 7c). The rachises of wheat heads infected with ΔFgCYP51C, ΔFgCYP51AC and ΔFgCYP51BC were dark brown, in contrast with the bleached rachises of plants inoculated with PH-1, ΔFgCYP51A and ΔFgCYP51B. The blocking of fungal growth from inoculated spikelets to adjacent spikelets is correlated with an unidentified brown substance deposited in the rachis node in the additional wheat line CS-7EL, which carries resistance to FHB on the long arm of chromosome 7E(7EL) (Miller et al., 2011). These data suggest that hyphal development of ΔFgCYP51C, ΔFgCYP51AC and ΔFgCYP51BC during wheat ear infection is impaired.
In contrast with wheat ear infection, there were no differences in virulence between wild-type PH-1 and ΔFgCYP51C on Arabidopsis, in which the trichothecene mycotoxin DON is not required for fungal infection (Cuzick et al., 2008), although colonization by the double ΔFgCYP51AC and ΔFgCYP51BC mutants was impaired. This suggests that altered DON production is responsible for the decreased virulence of the ΔFgCYP51C mutant on wheat ears. However, unlike mutants unable to produce DON, for example TRI5 gene mutants, which cause only discrete eye-shaped lesions on spikelets and fail to infect the rachis (Cuzick et al., 2008), ΔFgCYP51C, ΔFgCYP51AC and ΔFgCYP51BC mutants are able to infect beyond the inoculated spikelet. In addition, TRI4 and TRI5 gene expression is not altered significantly in wheat ears inoculated with ΔFgCYP51C, ΔFgCYP51AC and ΔFgCYP51BC relative to the wild-type. Therefore, rather than a biosynthetic requirement for DON biosynthesis, FgCYP51C is likely to have an indirect effect. A relationship between the sterol and trichothecene biosynthesis pathways has been reported previously. They share a common precursor, farnesol pyrophosphate, and the global regulator TRI6, located in the core TRI gene cluster. For example ERG9 (FGSG_09381), encoding squalene synthase, the first step of sterol biosynthesis, was down-regulated in the ΔTRI6 strain under nitrogen-deprived conditions, and ERG25 (FGSG_10666) was up-regulated, although there was no impact on FgCYP51C expression (Nasmith et al., 2011).
We have identified distinct functions of the three CYP51 paralogues of F. graminearum. FgCYP51B, as the most conserved CYP51 gene in all fungi, encodes the enzyme primarily responsible for sterol 14α-demethylation, a role essential for ascospore formation. FgCYP51A, found in many human and agricultural pathogens, is induced by azoles and environmental stress, encodes a sterol 14α-demethylase with the capacity to compensate for disruption of FgCYP51B function, and is responsible for intrinsic variation in sensitivity to different azoles. FgCYP51C, a Fusarium-specific CYP51 gene, no longer functions as a sterol 14α-demethylase, but rather is specifically required for full virulence on host wheat ears. This is the first example of functional diversification of a fungal CYP51 gene.