{"title":"作物枯萎病菌基因靶向整合和过表达的新载体体系。","authors":"Mikkel Rank Nielsen, Anna Karolina Rilana Holzwarth, Emmett Brew, Natalia Chrapkova, Samba Evelyne Kabemba Kaniki, Kenneth Kastaniegaard, Trine Sørensen, Klaus Ringsborg Westphal, Reinhard Wimmer, Teis Esben Sondergaard, Jens Laurids Sørensen","doi":"10.1186/s40694-019-0089-2","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>Besides their ability to produce several interesting bioactive secondary metabolites, members of the <i>Fusarium solani</i> species complex comprise important pathogens of plants and humans. One of the major obstacles in understanding the biology of this species complex is the lack of efficient molecular tools for genetic manipulation.</p><p><strong>Results: </strong>To remove this obstacle we here report the development of a reliable system where the vectors are generated through yeast recombinational cloning and inserted into a specific site in <i>F. solani</i> through <i>Agrobacterium tumefaciens</i>-mediated transformation. As proof-of-concept, the enhanced yellow fluorescent protein (eYFP) was inserted in a non-coding genomic position of <i>F. solani</i> and subsequent analyses showed that the resulting transformants were fluorescent on all tested media. In addition, we cloned and overexpressed the Zn(II)<sub>2</sub>Cys<sub>6</sub> transcriptional factor <i>fsr6</i> controlling mycelial pigmentation. A transformant displayed deep red/purple pigmentation stemming from bostrycoidin and javanicin.</p><p><strong>Conclusion: </strong>By creating streamlined plasmid construction and fungal transformation systems, we are now able to express genes in the crop pathogen <i>F. solani</i> in a reliable and fast manner. As a case study, we targeted and activated the fusarubin (<i>PKS3</i>: <i>fsr</i>) gene cluster, which is the first case study of secondary metabolites being directly associated with the responsible gene cluster in <i>F. solani</i> via targeted activation. The system provides an approach that in the future can be used by the community to understand the biochemistry and genetics of the <i>Fusarium solani</i> species complex, and is obtainable from Addgene catalog #133094.</p><p><strong>Graphic abstract: </strong></p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2019-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1186/s40694-019-0089-2","citationCount":"7","resultStr":"{\"title\":\"A new vector system for targeted integration and overexpression of genes in the crop pathogen <i>Fusarium solani</i>.\",\"authors\":\"Mikkel Rank Nielsen, Anna Karolina Rilana Holzwarth, Emmett Brew, Natalia Chrapkova, Samba Evelyne Kabemba Kaniki, Kenneth Kastaniegaard, Trine Sørensen, Klaus Ringsborg Westphal, Reinhard Wimmer, Teis Esben Sondergaard, Jens Laurids Sørensen\",\"doi\":\"10.1186/s40694-019-0089-2\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>Besides their ability to produce several interesting bioactive secondary metabolites, members of the <i>Fusarium solani</i> species complex comprise important pathogens of plants and humans. One of the major obstacles in understanding the biology of this species complex is the lack of efficient molecular tools for genetic manipulation.</p><p><strong>Results: </strong>To remove this obstacle we here report the development of a reliable system where the vectors are generated through yeast recombinational cloning and inserted into a specific site in <i>F. solani</i> through <i>Agrobacterium tumefaciens</i>-mediated transformation. As proof-of-concept, the enhanced yellow fluorescent protein (eYFP) was inserted in a non-coding genomic position of <i>F. solani</i> and subsequent analyses showed that the resulting transformants were fluorescent on all tested media. In addition, we cloned and overexpressed the Zn(II)<sub>2</sub>Cys<sub>6</sub> transcriptional factor <i>fsr6</i> controlling mycelial pigmentation. A transformant displayed deep red/purple pigmentation stemming from bostrycoidin and javanicin.</p><p><strong>Conclusion: </strong>By creating streamlined plasmid construction and fungal transformation systems, we are now able to express genes in the crop pathogen <i>F. solani</i> in a reliable and fast manner. As a case study, we targeted and activated the fusarubin (<i>PKS3</i>: <i>fsr</i>) gene cluster, which is the first case study of secondary metabolites being directly associated with the responsible gene cluster in <i>F. solani</i> via targeted activation. The system provides an approach that in the future can be used by the community to understand the biochemistry and genetics of the <i>Fusarium solani</i> species complex, and is obtainable from Addgene catalog #133094.</p><p><strong>Graphic abstract: </strong></p>\",\"PeriodicalId\":52292,\"journal\":{\"name\":\"Fungal Biology and Biotechnology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2019-12-11\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://sci-hub-pdf.com/10.1186/s40694-019-0089-2\",\"citationCount\":\"7\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Fungal Biology and Biotechnology\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.1186/s40694-019-0089-2\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2019/1/1 0:00:00\",\"PubModel\":\"eCollection\",\"JCR\":\"Q1\",\"JCRName\":\"Agricultural and Biological Sciences\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Fungal Biology and Biotechnology","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1186/s40694-019-0089-2","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2019/1/1 0:00:00","PubModel":"eCollection","JCR":"Q1","JCRName":"Agricultural and Biological Sciences","Score":null,"Total":0}
A new vector system for targeted integration and overexpression of genes in the crop pathogen Fusarium solani.
Background: Besides their ability to produce several interesting bioactive secondary metabolites, members of the Fusarium solani species complex comprise important pathogens of plants and humans. One of the major obstacles in understanding the biology of this species complex is the lack of efficient molecular tools for genetic manipulation.
Results: To remove this obstacle we here report the development of a reliable system where the vectors are generated through yeast recombinational cloning and inserted into a specific site in F. solani through Agrobacterium tumefaciens-mediated transformation. As proof-of-concept, the enhanced yellow fluorescent protein (eYFP) was inserted in a non-coding genomic position of F. solani and subsequent analyses showed that the resulting transformants were fluorescent on all tested media. In addition, we cloned and overexpressed the Zn(II)2Cys6 transcriptional factor fsr6 controlling mycelial pigmentation. A transformant displayed deep red/purple pigmentation stemming from bostrycoidin and javanicin.
Conclusion: By creating streamlined plasmid construction and fungal transformation systems, we are now able to express genes in the crop pathogen F. solani in a reliable and fast manner. As a case study, we targeted and activated the fusarubin (PKS3: fsr) gene cluster, which is the first case study of secondary metabolites being directly associated with the responsible gene cluster in F. solani via targeted activation. The system provides an approach that in the future can be used by the community to understand the biochemistry and genetics of the Fusarium solani species complex, and is obtainable from Addgene catalog #133094.
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