Characterization of VdASP F2 Secretory Factor from Verticillium dahliae by a Fast and Easy Gene Knockout System
The vascular wilt fungus Verticillium dahliae produces persistent resting structures known as microsclerotia, which enable long-term survival of this plant pathogen in soil. The completed genome sequence of V. dahliae has facilitated large-scale investigations of individual gene functions using gene...
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| Format: | Article |
| Language: | English |
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The American Phytopathological Society
2017-06-01
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| Series: | Molecular Plant-Microbe Interactions |
| Online Access: | https://apsjournals.apsnet.org/doi/10.1094/MPMI-01-17-0007-R |
| _version_ | 1828739812801118208 |
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| author | Chengjian Xie Qiaoling Li Xingyong Yang |
| author_facet | Chengjian Xie Qiaoling Li Xingyong Yang |
| author_sort | Chengjian Xie |
| collection | DOAJ |
| description | The vascular wilt fungus Verticillium dahliae produces persistent resting structures known as microsclerotia, which enable long-term survival of this plant pathogen in soil. The completed genome sequence of V. dahliae has facilitated large-scale investigations of individual gene functions using gene-disruption strategies based on Agrobacterium tumefaciens-mediated transformation. However, the construction of gene-deletion vectors and screening of deletion mutants have remained challenging in V. dahliae. In this study, we developed a fast and easy gene knockout system for V. dahliae using ligation-independent cloning and fluorescent screening. We identified secretory factor VdASP F2 in a T-DNA insertion library of V. dahliae and deleted the VdASP F2 gene using the developed knockout system. Phenotypic analysis suggests that VdASP F2 is not necessary for V. dahliae growth on potato dextrose agar under various stress conditions. However, on semisynthetic medium or under limited nutrient conditions at lower temperatures, the VdASP F2 deletion mutant exhibited vigorous mycelium growth, less branching, and a significant delay in melanized microsclerotial formation. Further assessment revealed that VdASP F2 was required for the expression of VDH1 and VMK1, two genes involved in microsclerotial formation. Cotton inoculated with the VdASP F2 deletion mutant wilted, demonstrating that VdASP F2 is not associated with pathogenicity under normal conditions. However, after inducing microsclerotial formation and incubation at low temperatures, cotton infected with the VdASP F2 deletion mutant did not exhibit wilt symptoms. In conclusion, our results show that VdASP F2 plays an important role in the response of V. dahliae to adverse environmental conditions and is involved in a transition to a dormant form for prolonged survival. |
| first_indexed | 2024-04-13T00:28:58Z |
| format | Article |
| id | doaj.art-887ac8c9f2f545069971a3e4896228fa |
| institution | Directory Open Access Journal |
| issn | 0894-0282 1943-7706 |
| language | English |
| last_indexed | 2024-04-13T00:28:58Z |
| publishDate | 2017-06-01 |
| publisher | The American Phytopathological Society |
| record_format | Article |
| series | Molecular Plant-Microbe Interactions |
| spelling | doaj.art-887ac8c9f2f545069971a3e4896228fa2022-12-22T03:10:31ZengThe American Phytopathological SocietyMolecular Plant-Microbe Interactions0894-02821943-77062017-06-0130644445410.1094/MPMI-01-17-0007-RCharacterization of VdASP F2 Secretory Factor from Verticillium dahliae by a Fast and Easy Gene Knockout SystemChengjian Xie0Qiaoling Li1Xingyong Yang2School of Life Sciences and;Chongqing Institute of Medicinal Plant Cultivation, Chongqing 408435, ChinaSchool of Life Sciences and;The vascular wilt fungus Verticillium dahliae produces persistent resting structures known as microsclerotia, which enable long-term survival of this plant pathogen in soil. The completed genome sequence of V. dahliae has facilitated large-scale investigations of individual gene functions using gene-disruption strategies based on Agrobacterium tumefaciens-mediated transformation. However, the construction of gene-deletion vectors and screening of deletion mutants have remained challenging in V. dahliae. In this study, we developed a fast and easy gene knockout system for V. dahliae using ligation-independent cloning and fluorescent screening. We identified secretory factor VdASP F2 in a T-DNA insertion library of V. dahliae and deleted the VdASP F2 gene using the developed knockout system. Phenotypic analysis suggests that VdASP F2 is not necessary for V. dahliae growth on potato dextrose agar under various stress conditions. However, on semisynthetic medium or under limited nutrient conditions at lower temperatures, the VdASP F2 deletion mutant exhibited vigorous mycelium growth, less branching, and a significant delay in melanized microsclerotial formation. Further assessment revealed that VdASP F2 was required for the expression of VDH1 and VMK1, two genes involved in microsclerotial formation. Cotton inoculated with the VdASP F2 deletion mutant wilted, demonstrating that VdASP F2 is not associated with pathogenicity under normal conditions. However, after inducing microsclerotial formation and incubation at low temperatures, cotton infected with the VdASP F2 deletion mutant did not exhibit wilt symptoms. In conclusion, our results show that VdASP F2 plays an important role in the response of V. dahliae to adverse environmental conditions and is involved in a transition to a dormant form for prolonged survival.https://apsjournals.apsnet.org/doi/10.1094/MPMI-01-17-0007-R |
| spellingShingle | Chengjian Xie Qiaoling Li Xingyong Yang Characterization of VdASP F2 Secretory Factor from Verticillium dahliae by a Fast and Easy Gene Knockout System Molecular Plant-Microbe Interactions |
| title | Characterization of VdASP F2 Secretory Factor from Verticillium dahliae by a Fast and Easy Gene Knockout System |
| title_full | Characterization of VdASP F2 Secretory Factor from Verticillium dahliae by a Fast and Easy Gene Knockout System |
| title_fullStr | Characterization of VdASP F2 Secretory Factor from Verticillium dahliae by a Fast and Easy Gene Knockout System |
| title_full_unstemmed | Characterization of VdASP F2 Secretory Factor from Verticillium dahliae by a Fast and Easy Gene Knockout System |
| title_short | Characterization of VdASP F2 Secretory Factor from Verticillium dahliae by a Fast and Easy Gene Knockout System |
| title_sort | characterization of vdasp f2 secretory factor from verticillium dahliae by a fast and easy gene knockout system |
| url | https://apsjournals.apsnet.org/doi/10.1094/MPMI-01-17-0007-R |
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