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Heterologous Expression of Mutated Eburicol 14α-Demethylase (CYP51) Proteins of Mycosphaerella graminicola To Assess Effects on Azole Fungicide Sensitivity and Intrinsic Protein Function

H. J. Cools, Josie Parker, Diane Kelly, J. A. Lucas, B. A. Fraaije, Steven Kelly Orcid Logo

Applied and Environmental Microbiology, Volume: 76, Issue: 9, Pages: 2866 - 2872

Swansea University Authors: Josie Parker, Diane Kelly, Steven Kelly Orcid Logo

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DOI (Published version): 10.1128/aem.02158-09

Abstract

<p><span>The recent decrease in the sensitivity of the Western European population of the wheat pathogen </span><em>Mycosphaerella graminicola</em><span> to azole fungicides has been associated with the emergence and subsequent spread of mutations in the </span...

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Published in: Applied and Environmental Microbiology
ISSN: 0099-2240 1098-5336
Published: American Society for Microbiology 2010
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URI: https://cronfa.swan.ac.uk/Record/cronfa6856
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fullrecord <?xml version="1.0"?><rfc1807><datestamp>2021-10-29T10:05:26.7134825</datestamp><bib-version>v2</bib-version><id>6856</id><entry>2012-01-26</entry><title>Heterologous Expression of Mutated Eburicol 14&#x3B1;-Demethylase (CYP51) Proteins of Mycosphaerella graminicola To Assess Effects on Azole Fungicide Sensitivity and Intrinsic Protein Function</title><swanseaauthors><author><sid>e563ed4e1c7db8d1e131fb78a5f8d0d5</sid><firstname>Josie</firstname><surname>Parker</surname><name>Josie Parker</name><active>true</active><ethesisStudent>false</ethesisStudent></author><author><sid>5ccf81e5d5beedf32ef8d7c3d7ac6c8c</sid><firstname>Diane</firstname><surname>Kelly</surname><name>Diane Kelly</name><active>true</active><ethesisStudent>false</ethesisStudent></author><author><sid>b17cebaf09b4d737b9378a3581e3de93</sid><ORCID>0000-0001-7991-5040</ORCID><firstname>Steven</firstname><surname>Kelly</surname><name>Steven Kelly</name><active>true</active><ethesisStudent>false</ethesisStudent></author></swanseaauthors><date>2012-01-26</date><deptcode>FGMHL</deptcode><abstract>&lt;p&gt;&lt;span&gt;The recent decrease in the sensitivity of the Western European population of the wheat pathogen&#xA0;&lt;/span&gt;&lt;em&gt;Mycosphaerella graminicola&lt;/em&gt;&lt;span&gt;&#xA0;to azole fungicides has been associated with the emergence and subsequent spread of mutations in the&#xA0;&lt;/span&gt;&lt;em&gt;CYP51&lt;/em&gt;&lt;span&gt;gene, encoding the azole target sterol 14&#x3B1;-demethylase. In this study, we have expressed wild-type and mutated&#xA0;&lt;/span&gt;&lt;em&gt;M. graminicola&lt;/em&gt;&lt;span&gt;&#xA0;CYP51 (MgCYP51) variants in a&lt;/span&gt;&lt;em&gt;Saccharomyces cerevisiae&lt;/em&gt;&lt;span&gt;&#xA0;mutant carrying a doxycycline-regulatable&#xA0;&lt;/span&gt;&lt;em&gt;tetO&lt;sub&gt;7&lt;/sub&gt;&lt;/em&gt;&lt;span&gt;-&lt;/span&gt;&lt;em&gt;CYC&lt;/em&gt;&lt;span&gt;promoter controlling native&#xA0;&lt;/span&gt;&lt;em&gt;CYP51&lt;/em&gt;&lt;span&gt;&#xA0;expression. We have shown that the wild-type MgCYP51 protein complements the function of the orthologous protein in&#xA0;&lt;/span&gt;&lt;em&gt;S. cerevisiae&lt;/em&gt;&lt;span&gt;. Mutant MgCYP51 proteins containing amino acid alterations L50S, Y459D, and Y461H and the two-amino-acid deletion &#x394;Y459/G460, commonly identified in modern&#xA0;&lt;/span&gt;&lt;em&gt;M. graminicola&lt;/em&gt;&lt;span&gt;&#xA0;populations, have no effect on the capacity of the&lt;/span&gt;&lt;em&gt;M. graminicola&lt;/em&gt;&lt;span&gt;&#xA0;protein to function in&#xA0;&lt;/span&gt;&lt;em&gt;S. cerevisiae&lt;/em&gt;&lt;span&gt;. 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&#xA0;&lt;/span&gt;&lt;em&gt;MgCYP51&lt;/em&gt;&lt;span&gt;&#xA0;to complement the&#xA0;&lt;/span&gt;&lt;em&gt;S. cerevisiae&lt;/em&gt;&lt;span&gt;&#xA0;mutant when introduced alone. However, when I381V is combined with changes between residues Y459 and Y461, the function of the&#xA0;&lt;/span&gt;&lt;em&gt;M. graminicola&lt;/em&gt;&lt;span&gt;&#xA0;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&#xA0;&lt;/span&gt;&lt;em&gt;M. graminicola&lt;/em&gt;&lt;span&gt;&#xA0;population emerged.&lt;/span&gt;&lt;/p&gt;</abstract><type>Journal Article</type><journal>Applied and Environmental Microbiology</journal><volume>76</volume><journalNumber>9</journalNumber><paginationStart>2866</paginationStart><paginationEnd>2872</paginationEnd><publisher>American Society for Microbiology</publisher><placeOfPublication/><isbnPrint/><isbnElectronic/><issnPrint>0099-2240</issnPrint><issnElectronic>1098-5336</issnElectronic><keywords/><publishedDay>1</publishedDay><publishedMonth>5</publishedMonth><publishedYear>2010</publishedYear><publishedDate>2010-05-01</publishedDate><doi>10.1128/aem.02158-09</doi><url>http://dx.doi.org/10.1128/aem.02158-09</url><notes/><college>COLLEGE NANME</college><department>Medicine, Health and Life Science - Faculty</department><CollegeCode>COLLEGE CODE</CollegeCode><DepartmentCode>FGMHL</DepartmentCode><institution>Swansea University</institution><apcterm/><lastEdited>2021-10-29T10:05:26.7134825</lastEdited><Created>2012-01-26T10:14:03.0100000</Created><path><level id="1">Faculty of Medicine, Health and Life Sciences</level><level id="2">Swansea University Medical School - Medicine</level></path><authors><author><firstname>H. J.</firstname><surname>Cools</surname><order>1</order></author><author><firstname>Josie</firstname><surname>Parker</surname><order>2</order></author><author><firstname>Diane</firstname><surname>Kelly</surname><order>3</order></author><author><firstname>J. A.</firstname><surname>Lucas</surname><order>4</order></author><author><firstname>B. A.</firstname><surname>Fraaije</surname><order>5</order></author><author><firstname>Steven</firstname><surname>Kelly</surname><orcid>0000-0001-7991-5040</orcid><order>6</order></author></authors><documents/><OutputDurs/></rfc1807>
spelling 2021-10-29T10:05:26.7134825 v2 6856 2012-01-26 Heterologous Expression of Mutated Eburicol 14α-Demethylase (CYP51) Proteins of Mycosphaerella graminicola To Assess Effects on Azole Fungicide Sensitivity and Intrinsic Protein Function e563ed4e1c7db8d1e131fb78a5f8d0d5 Josie Parker Josie Parker true false 5ccf81e5d5beedf32ef8d7c3d7ac6c8c Diane Kelly Diane Kelly true false b17cebaf09b4d737b9378a3581e3de93 0000-0001-7991-5040 Steven Kelly Steven Kelly true false 2012-01-26 FGMHL <p><span>The recent decrease in the sensitivity of the Western European population of the wheat pathogen </span><em>Mycosphaerella graminicola</em><span> to azole fungicides has been associated with the emergence and subsequent spread of mutations in the </span><em>CYP51</em><span>gene, encoding the azole target sterol 14α-demethylase. In this study, we have expressed wild-type and mutated </span><em>M. graminicola</em><span> CYP51 (MgCYP51) variants in a</span><em>Saccharomyces cerevisiae</em><span> mutant carrying a doxycycline-regulatable </span><em>tetO<sub>7</sub></em><span>-</span><em>CYC</em><span>promoter controlling native </span><em>CYP51</em><span> expression. We have shown that the wild-type MgCYP51 protein complements the function of the orthologous protein in </span><em>S. cerevisiae</em><span>. Mutant MgCYP51 proteins containing amino acid alterations L50S, Y459D, and Y461H and the two-amino-acid deletion ΔY459/G460, commonly identified in modern </span><em>M. graminicola</em><span> populations, have no effect on the capacity of the</span><em>M. graminicola</em><span> protein to function in </span><em>S. cerevisiae</em><span>. 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 </span><em>MgCYP51</em><span> to complement the </span><em>S. cerevisiae</em><span> mutant when introduced alone. However, when I381V is combined with changes between residues Y459 and Y461, the function of the </span><em>M. graminicola</em><span> 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 </span><em>M. graminicola</em><span> population emerged.</span></p> Journal Article Applied and Environmental Microbiology 76 9 2866 2872 American Society for Microbiology 0099-2240 1098-5336 1 5 2010 2010-05-01 10.1128/aem.02158-09 http://dx.doi.org/10.1128/aem.02158-09 COLLEGE NANME Medicine, Health and Life Science - Faculty COLLEGE CODE FGMHL Swansea University 2021-10-29T10:05:26.7134825 2012-01-26T10:14:03.0100000 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Medicine H. J. Cools 1 Josie Parker 2 Diane Kelly 3 J. A. Lucas 4 B. A. Fraaije 5 Steven Kelly 0000-0001-7991-5040 6
title Heterologous Expression of Mutated Eburicol 14α-Demethylase (CYP51) Proteins of Mycosphaerella graminicola To Assess Effects on Azole Fungicide Sensitivity and Intrinsic Protein Function
spellingShingle Heterologous Expression of Mutated Eburicol 14α-Demethylase (CYP51) Proteins of Mycosphaerella graminicola To Assess Effects on Azole Fungicide Sensitivity and Intrinsic Protein Function
Josie Parker
Diane Kelly
Steven Kelly
title_short Heterologous Expression of Mutated Eburicol 14α-Demethylase (CYP51) Proteins of Mycosphaerella graminicola To Assess Effects on Azole Fungicide Sensitivity and Intrinsic Protein Function
title_full Heterologous Expression of Mutated Eburicol 14α-Demethylase (CYP51) Proteins of Mycosphaerella graminicola To Assess Effects on Azole Fungicide Sensitivity and Intrinsic Protein Function
title_fullStr Heterologous Expression of Mutated Eburicol 14α-Demethylase (CYP51) Proteins of Mycosphaerella graminicola To Assess Effects on Azole Fungicide Sensitivity and Intrinsic Protein Function
title_full_unstemmed Heterologous Expression of Mutated Eburicol 14α-Demethylase (CYP51) Proteins of Mycosphaerella graminicola To Assess Effects on Azole Fungicide Sensitivity and Intrinsic Protein Function
title_sort Heterologous Expression of Mutated Eburicol 14α-Demethylase (CYP51) Proteins of Mycosphaerella graminicola To Assess Effects on Azole Fungicide Sensitivity and Intrinsic Protein Function
author_id_str_mv e563ed4e1c7db8d1e131fb78a5f8d0d5
5ccf81e5d5beedf32ef8d7c3d7ac6c8c
b17cebaf09b4d737b9378a3581e3de93
author_id_fullname_str_mv e563ed4e1c7db8d1e131fb78a5f8d0d5_***_Josie Parker
5ccf81e5d5beedf32ef8d7c3d7ac6c8c_***_Diane Kelly
b17cebaf09b4d737b9378a3581e3de93_***_Steven Kelly
author Josie Parker
Diane Kelly
Steven Kelly
author2 H. J. Cools
Josie Parker
Diane Kelly
J. A. Lucas
B. A. Fraaije
Steven Kelly
format Journal article
container_title Applied and Environmental Microbiology
container_volume 76
container_issue 9
container_start_page 2866
publishDate 2010
institution Swansea University
issn 0099-2240
1098-5336
doi_str_mv 10.1128/aem.02158-09
publisher American Society for Microbiology
college_str Faculty of Medicine, Health and Life Sciences
hierarchytype
hierarchy_top_id facultyofmedicinehealthandlifesciences
hierarchy_top_title Faculty of Medicine, Health and Life Sciences
hierarchy_parent_id facultyofmedicinehealthandlifesciences
hierarchy_parent_title Faculty of Medicine, Health and Life Sciences
department_str Swansea University Medical School - Medicine{{{_:::_}}}Faculty of Medicine, Health and Life Sciences{{{_:::_}}}Swansea University Medical School - Medicine
url http://dx.doi.org/10.1128/aem.02158-09
document_store_str 0
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description <p><span>The recent decrease in the sensitivity of the Western European population of the wheat pathogen </span><em>Mycosphaerella graminicola</em><span> to azole fungicides has been associated with the emergence and subsequent spread of mutations in the </span><em>CYP51</em><span>gene, encoding the azole target sterol 14α-demethylase. In this study, we have expressed wild-type and mutated </span><em>M. graminicola</em><span> CYP51 (MgCYP51) variants in a</span><em>Saccharomyces cerevisiae</em><span> mutant carrying a doxycycline-regulatable </span><em>tetO<sub>7</sub></em><span>-</span><em>CYC</em><span>promoter controlling native </span><em>CYP51</em><span> expression. We have shown that the wild-type MgCYP51 protein complements the function of the orthologous protein in </span><em>S. cerevisiae</em><span>. Mutant MgCYP51 proteins containing amino acid alterations L50S, Y459D, and Y461H and the two-amino-acid deletion ΔY459/G460, commonly identified in modern </span><em>M. graminicola</em><span> populations, have no effect on the capacity of the</span><em>M. graminicola</em><span> protein to function in </span><em>S. cerevisiae</em><span>. 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 </span><em>MgCYP51</em><span> to complement the </span><em>S. cerevisiae</em><span> mutant when introduced alone. However, when I381V is combined with changes between residues Y459 and Y461, the function of the </span><em>M. graminicola</em><span> 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 </span><em>M. graminicola</em><span> population emerged.</span></p>
published_date 2010-05-01T03:08:27Z
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score 11.016235

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