Establishment of <i>Agrobacterium tumefaciens</i>-Mediated Transformation of <i>Cladonia macilenta</i>, a Model Lichen-Forming Fungus
Despite the fascinating biology of lichens, such as the symbiotic association of lichen-forming fungi (mycobiont) with their photosynthetic partners and their ability to grow in harsh habitats, lack of genetic tools manipulating mycobiont has hindered studies on genetic mechanisms underpinning liche...
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| Format: | Article |
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MDPI AG
2021-03-01
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| Series: | Journal of Fungi |
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| Online Access: | https://www.mdpi.com/2309-608X/7/4/252 |
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| author | Rundong Liu Wonyong Kim Jaycee Augusto Paguirigan Min-Hye Jeong Jae-Seoun Hur |
| author_facet | Rundong Liu Wonyong Kim Jaycee Augusto Paguirigan Min-Hye Jeong Jae-Seoun Hur |
| author_sort | Rundong Liu |
| collection | DOAJ |
| description | Despite the fascinating biology of lichens, such as the symbiotic association of lichen-forming fungi (mycobiont) with their photosynthetic partners and their ability to grow in harsh habitats, lack of genetic tools manipulating mycobiont has hindered studies on genetic mechanisms underpinning lichen biology. Thus, we established an <i>Agrobacterium tumefaciens</i>-mediated transformation (ATMT) system for genetic transformation of a mycobiont isolated from <i>Cladonia macilenta</i>. A set of combinations of ATMT conditions, such as input biomass of mycobiont, co-cultivation period with <i>Agrobacterium</i> cells, and incubation temperature, were tested to identify an optimized ATMT condition for the <i>C. macilenta</i> mycobiont. As a result, more than 10 days of co-cultivation period and at least 2 mg of input biomass of the mycobiont were recommended for an efficient ATMT, owing to extremely slow growth rate of mycobionts in general. Moreover, we examined T-DNA copy number variation in a total of 180 transformants and found that 88% of the transformants had a single copy T-DNA insertion. To identify precise T-DNA insertion sites that interrupt gene function in <i>C. macilenta</i>, we performed TAIL-PCR analyses for selected transformants. A hypothetical gene encoding ankyrin repeats at its C-terminus was interrupted by T-DNA insertion in a transformant producing dark-brown colored pigment. Although the identification of the pigment awaits further investigation, this proof-of-concept study demonstrated the feasibility of use of ATMT in construction of a random T-DNA insertion mutant library in mycobionts for studying genetic mechanisms behind the lichen symbiosis, stress tolerance, and secondary metabolite biosynthesis. |
| first_indexed | 2024-03-10T12:50:55Z |
| format | Article |
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| institution | Directory Open Access Journal |
| issn | 2309-608X |
| language | English |
| last_indexed | 2024-03-10T12:50:55Z |
| publishDate | 2021-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Journal of Fungi |
| spelling | doaj.art-053c79381c2f4909906073bb798660a22023-11-21T13:00:18ZengMDPI AGJournal of Fungi2309-608X2021-03-017425210.3390/jof7040252Establishment of <i>Agrobacterium tumefaciens</i>-Mediated Transformation of <i>Cladonia macilenta</i>, a Model Lichen-Forming FungusRundong Liu0Wonyong Kim1Jaycee Augusto Paguirigan2Min-Hye Jeong3Jae-Seoun Hur4Korean Lichen Research Institute, Sunchon National University, Suncheon 57922, KoreaKorean Lichen Research Institute, Sunchon National University, Suncheon 57922, KoreaKorean Lichen Research Institute, Sunchon National University, Suncheon 57922, KoreaKorean Lichen Research Institute, Sunchon National University, Suncheon 57922, KoreaKorean Lichen Research Institute, Sunchon National University, Suncheon 57922, KoreaDespite the fascinating biology of lichens, such as the symbiotic association of lichen-forming fungi (mycobiont) with their photosynthetic partners and their ability to grow in harsh habitats, lack of genetic tools manipulating mycobiont has hindered studies on genetic mechanisms underpinning lichen biology. Thus, we established an <i>Agrobacterium tumefaciens</i>-mediated transformation (ATMT) system for genetic transformation of a mycobiont isolated from <i>Cladonia macilenta</i>. A set of combinations of ATMT conditions, such as input biomass of mycobiont, co-cultivation period with <i>Agrobacterium</i> cells, and incubation temperature, were tested to identify an optimized ATMT condition for the <i>C. macilenta</i> mycobiont. As a result, more than 10 days of co-cultivation period and at least 2 mg of input biomass of the mycobiont were recommended for an efficient ATMT, owing to extremely slow growth rate of mycobionts in general. Moreover, we examined T-DNA copy number variation in a total of 180 transformants and found that 88% of the transformants had a single copy T-DNA insertion. To identify precise T-DNA insertion sites that interrupt gene function in <i>C. macilenta</i>, we performed TAIL-PCR analyses for selected transformants. A hypothetical gene encoding ankyrin repeats at its C-terminus was interrupted by T-DNA insertion in a transformant producing dark-brown colored pigment. Although the identification of the pigment awaits further investigation, this proof-of-concept study demonstrated the feasibility of use of ATMT in construction of a random T-DNA insertion mutant library in mycobionts for studying genetic mechanisms behind the lichen symbiosis, stress tolerance, and secondary metabolite biosynthesis.https://www.mdpi.com/2309-608X/7/4/252<i>Cladonia</i>lichen-forming fungi<i>Agrobacterium</i>ATMTgenetic transformation |
| spellingShingle | Rundong Liu Wonyong Kim Jaycee Augusto Paguirigan Min-Hye Jeong Jae-Seoun Hur Establishment of <i>Agrobacterium tumefaciens</i>-Mediated Transformation of <i>Cladonia macilenta</i>, a Model Lichen-Forming Fungus Journal of Fungi <i>Cladonia</i> lichen-forming fungi <i>Agrobacterium</i> ATMT genetic transformation |
| title | Establishment of <i>Agrobacterium tumefaciens</i>-Mediated Transformation of <i>Cladonia macilenta</i>, a Model Lichen-Forming Fungus |
| title_full | Establishment of <i>Agrobacterium tumefaciens</i>-Mediated Transformation of <i>Cladonia macilenta</i>, a Model Lichen-Forming Fungus |
| title_fullStr | Establishment of <i>Agrobacterium tumefaciens</i>-Mediated Transformation of <i>Cladonia macilenta</i>, a Model Lichen-Forming Fungus |
| title_full_unstemmed | Establishment of <i>Agrobacterium tumefaciens</i>-Mediated Transformation of <i>Cladonia macilenta</i>, a Model Lichen-Forming Fungus |
| title_short | Establishment of <i>Agrobacterium tumefaciens</i>-Mediated Transformation of <i>Cladonia macilenta</i>, a Model Lichen-Forming Fungus |
| title_sort | establishment of i agrobacterium tumefaciens i mediated transformation of i cladonia macilenta i a model lichen forming fungus |
| topic | <i>Cladonia</i> lichen-forming fungi <i>Agrobacterium</i> ATMT genetic transformation |
| url | https://www.mdpi.com/2309-608X/7/4/252 |
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