The evolution of the MgCYP51
gene in response to selection pressure imposed by the widespread use of a series of azole fungicides in Western Europe is ongoing. It is therefore unsurprising that recently a number of MgCYP51 variants carrying combinations of amino acid alterations that were previously rare or nonexistent have emerged (3
). The impact of these novel MgCYP51 variants is unclear although it has been suggested that sensitivity to the two azole fungicides that are currently fully effective against Septoria
leaf blotch, prothioconazole and epoxiconazole, has been affected. In this study, by heterologous expression in yeast strain YUG37::erg11
, which carries a doxycycline-regulatable promoter controlling native CYP51
expression, we have determined the impact of recent MgCYP51 variants, particularly those harboring the substitution S524T, on sensitivity to different azoles.
The addition of S524T substantially impacts both azole fungicide sensitivity and the activity of the MgCYP51 protein in S. cerevisiae
. When the MgCYP51 protein is expressed with the single alteration S524T, it complements the function of the S. cerevisiae
CYP51 as effectively as the wild-type protein. Azole sensitivity testing shows that transformants expressing this variant are least sensitive to prothioconazole. Currently, no isolates of M. graminicola
have been identified that carry S524T alone. In M. graminicola
the specific effect of Y137F on triadimenol sensitivity, a previously reported finding (14
), is enhanced by S524T. When the Y137F substitution was expressed in S. cerevisiae
, the addition of S524T rescued the function of the MgCYP51 protein carrying Y137F alone, and transformants expressing pYES-Mg51Y137F/S524T were resistant to triadimenol and substantially reduced in sensitivity to epoxiconazole, tebuconazole, propiconazole, and prochloraz. Together, these data suggest that the combination of substitutions Y137F and S524T confers both a greater decrease in azole sensitivity and greater enzyme activity than the Y137F change alone, a finding consistent with the sterol content of isolates carrying Y137F (2
). The apparent selection in the early 2000s of isolates of M. graminicola
carrying MgCYP51 variants with both Y137F and S524T substitutions, superseding isolates prevalent in the early 1990s with MgCYP51 variants with Y137F alone, also supports this suggestion (Fraaije et al., unpublished).
Isolates carrying MgCYP51 variants with substitutions L50S and Y461S are reduced in sensitivity to all azoles except prothioconazole. Similar results were obtained for sensitivity tests of S. cerevisiae
transformants expressing this MgCYP51 variant (pYES-Mg51L50S/Y461S), consistent with sensitivity profiles reported for other changes in the Y459-Y461 region (6
). The addition of S524T to this variant (pYES-Mg51L50S/Y461S/S524T) enhances resistance of S. cerevisiae
transformants to all azoles equally. Interestingly, no M. graminicola
isolates carrying MgCYP51 with the combination of L50S, Y461S, and S524T have been reported. Instead, current M. graminicola
isolates often carry MgCYP51 with S524T combined with the three mutations L50S, V136A, and Y461S. Unfortunately, the lethality of the V136A substitution when expressed in S. cerevisiae
alone and the very poor growth of transformants expressing MgCYP51 V136A Y461S S524T preclude analysis by heterologous expression of a L50S V136A Y461S variant or L50S V136A Y461S S524T variant. However, the addition of D134G restores the growth of transformants, enabling the effects of V136A to be investigated.
Comparison of M. graminicola
isolates carrying a MgCYP51 L50S Y461S variant with those carrying a L50S V136A Y461S variant reveals the greatest impact of V136A on epoxiconazole, prochloraz, prothioconazole, and propiconazole sensitivity, with isolates more sensitive to tebuconazole and triadimenol. The addition of S524T (L50S V136A Y461S S524T variant) does not change the azole sensitivity profile of isolates but, rather, enhances resistance to epoxiconazole, prochloraz, prothioconazole, and propiconazole, while maintaining sensitivity to tebuconazole and triadimenol. To date, isolates carrying this MgCYP51 variant have been identified only in Ireland, where tebuconazole-sensitive strains have dominated recent M. graminicola
). Substitution D134G has previously been reported (24
) and, although still rare, has been identified in several recent isolates combined with different amino acid changes. Isolate TAG71-3, carrying MgCYP51 with L50S, D134G, V136A, Y461S, and S524T combined is comparatively sensitive to tebuconazole and triadimenol and reduced in sensitivity to epoxiconazole and propiconazole. However, in comparison to isolates from Ireland, TAG71-3 is markedly reduced in sensitivity to prothioconazole. Sensitivity testing of yeast transformants expressing this variant confirms the impact of this MgCYP51 variant on prothioconazole sensitivity.
Prothioconazole binds to MgCYP51 atypically (19
). Therefore, molecular modeling of the L50S D134G V136A Y461S S524T variant in silico
investigated the interaction with epoxiconazole. The most striking feature of the mutant protein is the opening up of the binding cavity caused by the loss of the β1 and β2 sheets from the vicinity of the heme. This results in isolation of the azole molecule and a reduction in residue interaction. These structural rearrangements explain the loss of sensitivity of this variant and represent a common evolutionary solution to azole inhibition. We have previously shown that the heme cavity is increased in other MgCYP51 variants less sensitive to azoles. This allows the accommodation of azole molecules while reducing interactions with adjacent amino acids and limiting rearrangement of specific side chains important for the function of the enzyme (Mullins et al., unpublished).
In conclusion, we have demonstrated that recently emerged variants of the MgCYP51 protein confer reduced in vitro sensitivity to the previously effective azole fungicides, epoxiconazole and prothioconazole. These variants often carry S524T, a substitution not previously identified as altered in isolates of either human or plant-pathogenic fungi. Although the field performance of these compounds in controlling M. graminicola populations is currently unaffected, this study provides functional evidence for the ongoing selection of MgCYP51 variants less sensitive to the most widely used azoles. Interestingly, isolates carrying these recently emerged variants are often sensitive to some of the older azoles, supporting the assertion that retaining a diversity of azole chemistry is important for the management of resistance in this pathogen.