Impact of mutations in the target-encoding CYP51 gene in Mycosphaerella graminicola populations developing resistance to triazole fungicides

Lead Research Organisation: Rothamsted Research
Department Name: Biological Chemistry & Crop Protection

Abstract

The emergence of resistance to antifungal compounds is an increasing problem for the management of fungal diseases in agriculture and medicine. One of the most important groups of fungicides is the triazoles, used for the treatment of fungal infections of both crop plants and humans. Unfortunately, when fungi are exposed to these chemicals, the treatment can select for strains of the fungus that are less sensitive to the chemical. Over time, resistant strains of the fungus can develop and the chemical no longer controls the disease. This has happened with the human pathogenic yeast Candida, and there is now evidence that some plant pathogenic fungi are also becoming less sensitive to triazoles. The aim of this project is to understand the mechanisms responsible for these changes in sensitivity to triazoles in Mycosphaerella graminicola, the causal agent of Septoria leaf blotch, the most important foliar disease of wheat in the UK. This pathogen has already adapted to many of the fungicides used to control it. Resistance to the strobilurin fungicides, first detected in 2002, is now widespread in UK and European M. graminicola populations, so control of the disease now relies heavily on triazoles. Over the past decade there has been a gradual change in the sensitivity of the fungus to these fungicides so that higher doses are now required to achieve disease control. There is a growing concern, amongst both the agricultural and agrochemical industries, that further shifts in triazole sensitivity will reduce our ability to manage this important disease. There are few new Septoria fungicides available, and the older chemicals used for Septoria control are more damaging to the environment. Several mechanisms are known to contribute to resistance to triazoles. These include changes in the target site protein, as well as participation of other proteins, known as transporters, that are able to pump the fungicide out of the fungus. Therefore, to understand how changes in the target protein may affect triazole sensitivity, the altered forms of the protein must be expressed and studied in isolation. The proposed project aims to determine the effect on sensitivity of mutations in the triazole target protein, a cytochrome P450 called CYP51 involved in sterol 14a-demethylation. The research will assess how such mutations affect interactions between the fungicide and the protein, as well as the activity of the enzyme itself, using several approaches. The mutant proteins will be expressed in another fungus, the yeast Saccharomyces cerevisiae, to see how sensitivity to different triazoles is affected, and to make pure samples of the protein to measure enzyme activity, altered properties and inhibition. In particular the project will concentrate on several mutations that have occurred quite recently in M. graminicola populations exposed to triazole fungicides. We also intend to introduce further changes identified in Candida to the protein by targeted mutation to assess their effects on sensitivity and protein function. The project will provide key information on the contribution of target site mutations to the current status of M. graminicola azole sensitivity in the field and the potential for future resistance development. Information gained from these studies will be used to design novel molecular diagnostics to detect target site changes and monitor the occurrence of such changes in field populations of the fungus. This will show how the fungus evolves in response to treatment by different triazoles and help to devise control strategies to maintain the effectiveness of this group of chemicals against M. graminicola. Results should also aid the design of more effective triazole compounds. Furthermore, determining the relationship between target site changes, fungicide sensitivity and enzyme activity will improve understanding of the molecular evolution of the CYP51 protein in plant pathogenic fungi in response to selection.

Technical Summary

Resistance to antifungal agents is an increasing challenge in the management of fungal diseases in agriculture and medicine. Human pathogens such as Candida have developed resistance to triazoles and the mechanisms involved have been partly characterised. Less is known about resistance in plant pathogens. Septoria leaf blotch, (Mycosphaerella graminicola), is the most important disease of wheat in the UK. Almost all wheat crops are treated with triazoles to control Septoria. Their efficacy in disease control has declined over the past 10 years due to the emergence of strains with reduced sensitivity. The project will determine the contribution of mutations in the CYP51 gene encoding the sterol 14a-demethylase target for triazoles to reduced sensitivity of M. graminicola isolates. Sequencing of the CYP51 genes of recent isolates of M. graminicola will identify which mutations are closely correlated with reduced sensitivity. Key mutations will be tracked by Pyrosequencing the CYP51 gene from M. graminicola DNA in samples from our historical wheat archive, and also introduced into a 'sensitive' CYP51 M. graminicola gene, both as individual changes and in combination, by site directed mutagenesis. The effects of these changes on sensitivity to different triazoles will be determined by expression in an S. cerevisiae cyp51 mutant. Heterologously expressed mutant CYP51 proteins will be solubilised, purified and evaluated for fungicide binding affinity, intrinsic enzyme activity, and other aspects of functionality affecting fitness. Mutations known to confer triazole resistance in Candida will be introduced by targeted mutagenesis. Bioassay of yeast transformants expressing these engineered CYP51 genes, and triazole binding and enzyme activity studies of the purified proteins will provide essential information on the evolution of M. graminicola CYP51 in response to triazoles and the risk of future resistance development.

Publications

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Description Azole sensitivity testing of Mycosphaerella graminicola (Mg) field strains during 2003-2010 revealed an increase in the number of phenotypes and a trend of reduced sensitivity over time. Twenty amino acid alterations at 15 different residues of the sterol 14a-demethylase protein (MgCYP51) were identified. Combinations of alterations were common, with up to six alterations found in the most insensitive strains. Changes at nine residues were linked to azole insensitivity. During 2009-2010, many new MgCYP51 variants were identified, often carrying substitutions V136A and I381V combined, or D134G and S524T. For one MgCYP51 variant, two contrasting azole sensitivity phenotypes were detected and over-expression of MgCYP51 was shown for the least sensitive strains. The impact of MgCYP51 changes on azole sensitivity was studied by heterologous expression in a yeast mutant in which the native CYP51 expression was repressed. Introduction of mutations encoding alterations Y459D, Y461H and deletion of Y459/G460, commonly identified in Mg strains, had no effect on the capacity of MgCYP51 to function in yeast and the sensitivities of transformants expressing these variants to different azoles were reduced. L50S, S188N and N513K had no effect on either protein function, or azole sensitivity when expressed in yeast. Introduction of I381V alone destroyed the capacity of MgCYP51 to complement yeast. However, when I381V was combined with changes at Y459-Y461, MgCYP51 function was partially restored. S524T conferred a reduced sensitivity to all azoles when introduced either alone or in combination with changes at Y459-Y461 and restored the MgCYP51 function in variants carrying the otherwise lethal alterations Y137F and V136A. Sensitivity tests of yeast transformed with a recently emerged MgCYP51 variant (D134G-V136A-Y461S-S524T) showed a substantial reduction in sensitivity to most azoles. Pyrosequencing studies of the temporal sequence of mutational events in the CYP51 gene of Mg populations preserved in archived wheat samples from the Broadbalk experiment at Rothamsted (1844-2010) have proved vital to the retrospective understanding of the evolution of azole resistance. Findings include the rapid emergence and selection of substitution Y137F from 1990 onwards and its subsequent decline in the early 2000s. It also revealed the emergence of I381V (2000), V136A (2002) and A379G (2003), and the subsequent prominence of I381V. Both Broadbalk and functional studies in yeast suggest the appearance of I381V to be contingent on the prior emergence of alterations between Y459-Y461 in the mid 1990s. Recent functional studies have suggested that the previously rare alterations D134G and S524T will impact on future MgCYP51 evolution. Molecular modelling, based on multiple CYP51 homologues, individually identified for each variant rather than using a single structural scaffold, was accurate in predicting the likely binding orientation of an azole molecule in any MgCYP51 variant. The importance of V136A, Y137F, I381V, Y459-Y461 and S524T on the interactions with different azoles was confirmed in an evolutionary context. Wild-type MgCYP51 was successfully expressed in E. coli and purified protein was used to probe the mechanism and affinity of azoles. Treatment of Mg strain IPO323 with azoles resulted in MgCYP51 inhibition, evidenced by accumulation of 14a methylated sterols and ergosterol depletion. Azole binding studies with MgCYP51 revealed that epoxiconazole, tebuconazole and triadimenol all bound tightly, producing spectra indicative of the formation of classical low-spin sixth ligand complexes. Interaction of prothioconazole with MgCYP51 showed a different inhibition mechanism. Prothioconazole bound to MgCYP51 with much lower affinity than epoxiconazole and unlike the other azoles, all non-competitive inhibitors, it was found to be a competitive inhibitor of substrate binding. The desthio metabolite of prothioconazole might be more important for field efficacy.
Exploitation Route Research is important for public knowledge on how diseases are affected by climate change, how they are controlled (host resistance and/or chemical control) and its impact on food security/environment and human health. Outcome of the project is further communicated in University lectures to students, farming press and several exhibitions to the general public (e.g. 2009 'Pest wars' at British Science Festival, University of Guildford and 2011 'Combating the superpests: the battle to save our food' Royal Society's annual Summer Science Exhibition, London, 5-10 July 2011 (both events had press coverage in a range of newspapers)) Interest in the project is very high from the agrochemical industry, growers and regulatory authorities. This is due to the importance of azole fungicide resistance for agriculture and food security, the most important position of Mycosphaerella graminicola as a pathogen (causal agent of Septoria leaf blotch) in this regard and the possible future restriction of azole fungicide use in proposed EU legislation. Several small grants were obtained from HGCA and AgroChem industry after the project was ended.
Sectors Chemicals,Environment

 
Description Improved disease management: Septoria tritici in winter
Geographic Reach National 
Policy Influence Type Influenced training of practitioners or researchers
Impact Advise on how to control Septoria leaf blotch in wheat is very important. Optimal use of cultivars and programmed spray applications (optimal disease control and anti-resistance strategies) will benefit the environment and the profitability of cereal growers.
URL http://cereals.ahdb.org.uk/media/178045/ts113_septoria_tritici_in_winter_wheat.pdf
 
Description AHDB
Amount £150,127 (GBP)
Funding ID RD-2009-3713 
Organisation Agricultural and Horticulture Development Board 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2014 
End 03/2019
 
Description DEFRA
Amount £35,000 (GBP)
Funding ID PS2716 
Organisation Department For Environment, Food And Rural Affairs (DEFRA) 
Sector Public
Country United Kingdom
Start 10/2009 
End 12/2009
 
Description HGCA
Amount £60,000 (GBP)
Funding ID RD-2009-3713 
Organisation Agricultural and Horticulture Development Board 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2011 
End 03/2014
 
Description Annual 'Cereals' events 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Display (small plots of wheat and poster boards) of chemical control and resistance issues controlling Septoria leaf blotch. Hundreds of visitors (agrochem industry, farmers and agronomists).
Year(s) Of Engagement Activity 2011,2014,2015
URL https://cereals.ahdb.org.uk/media/330005/T2F-March-2013-Wheat-diseases.pdf
 
Description Invited speaker at the 2017 APS Annual meeting in San Antonio, Texas, USA (8 August 2017) 'Evolution of multi-fungicide resistance in cereal pathogens and impact on disease control'. 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact APS Annual Meeting 2017, American Phytopathological Society Annual Meeting, Date: 8/5/2017 - 8/9/2017 , Venue: Henry B. Gonzalez Convention Center, San Antonio TX, United States

APS's premier event, the APS Annual Meeting, attracts more than 1,500 participants each year. It's where the latest information on plant pathology in unveiled, where you'll have the opportunity to participate in symposia and discussion sessions, present research results, view hundreds of technical posters, attend special events, learn about new products and services, and connect with others who share your interests.
Year(s) Of Engagement Activity 2017
 
Description Royal Society's Summer Science Exhibition for general public, London 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Display 'Combating the superpests: the battle to save our food'. Engagement with general public to explain how fungal diseases can be controlled by fungicides and how resistance can develop and what can be done to prevent/delay this.
Year(s) Of Engagement Activity 2011
URL http://www.farming.co.uk/articles/view/4417