Optimization of Agrobacterium tumefaciens-Mediated Transformation of Xylaria grammica EL000614, an Endolichenic Fungus Producing Grammicin
An endolichenic fungus Xylaria grammica EL000614 produces grammicin, a potent nematicidal pyrone derivative that can serve as a new control option for root-knot nematodes. We optimized an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for X. grammica to support genetic studies. Tr...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Taylor & Francis
2021
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8583754/ https://www.ncbi.nlm.nih.gov/pubmed/34803437 http://dx.doi.org/10.1080/12298093.2021.1961431 |
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author | Jeong, Min-Hye Kim, Jung A. Kang, Seogchan Choi, Eu Ddeum Kim, Youngmin Lee, Yerim Jeon, Mi Jin Yu, Nan Hee Park, Ae Ran Kim, Jin-Cheol Kim, Soonok Park, Sook-Young |
author_facet | Jeong, Min-Hye Kim, Jung A. Kang, Seogchan Choi, Eu Ddeum Kim, Youngmin Lee, Yerim Jeon, Mi Jin Yu, Nan Hee Park, Ae Ran Kim, Jin-Cheol Kim, Soonok Park, Sook-Young |
author_sort | Jeong, Min-Hye |
collection | PubMed |
description | An endolichenic fungus Xylaria grammica EL000614 produces grammicin, a potent nematicidal pyrone derivative that can serve as a new control option for root-knot nematodes. We optimized an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for X. grammica to support genetic studies. Transformants were successfully generated after co-cultivation of homogenized young mycelia of X. grammica with A. tumefaciens strain AGL-1 carrying a binary vector that contains the bacterial hygromycin B phosphotransferase (hph) gene and the eGFP gene in T-DNA. The resulting transformants were mitotically stable, and PCR analysis showed the integratin of both genes in the genome of transformants. Expression of eGFP was confirmed via fluorescence microscopy. Southern analysis showed that 131 (78.9%) out of 166 transformants contained a single T-DNA insertion. Crucial factors for producing predominantly single T-DNA transformants include 48 h of co-cultivation, pre-treatment of A. tumefaciens cells with acetosyringone before co-cultivation, and using freshly prepared mycelia. The established ATMT protocol offers an efficient tool for random insertional mutagenesis and gene transfer in studying the biology and ecology of X. grammica. |
format | Online Article Text |
id | pubmed-8583754 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Taylor & Francis |
record_format | MEDLINE/PubMed |
spelling | pubmed-85837542021-11-18 Optimization of Agrobacterium tumefaciens-Mediated Transformation of Xylaria grammica EL000614, an Endolichenic Fungus Producing Grammicin Jeong, Min-Hye Kim, Jung A. Kang, Seogchan Choi, Eu Ddeum Kim, Youngmin Lee, Yerim Jeon, Mi Jin Yu, Nan Hee Park, Ae Ran Kim, Jin-Cheol Kim, Soonok Park, Sook-Young Mycobiology Research Articles An endolichenic fungus Xylaria grammica EL000614 produces grammicin, a potent nematicidal pyrone derivative that can serve as a new control option for root-knot nematodes. We optimized an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for X. grammica to support genetic studies. Transformants were successfully generated after co-cultivation of homogenized young mycelia of X. grammica with A. tumefaciens strain AGL-1 carrying a binary vector that contains the bacterial hygromycin B phosphotransferase (hph) gene and the eGFP gene in T-DNA. The resulting transformants were mitotically stable, and PCR analysis showed the integratin of both genes in the genome of transformants. Expression of eGFP was confirmed via fluorescence microscopy. Southern analysis showed that 131 (78.9%) out of 166 transformants contained a single T-DNA insertion. Crucial factors for producing predominantly single T-DNA transformants include 48 h of co-cultivation, pre-treatment of A. tumefaciens cells with acetosyringone before co-cultivation, and using freshly prepared mycelia. The established ATMT protocol offers an efficient tool for random insertional mutagenesis and gene transfer in studying the biology and ecology of X. grammica. Taylor & Francis 2021-08-19 /pmc/articles/PMC8583754/ /pubmed/34803437 http://dx.doi.org/10.1080/12298093.2021.1961431 Text en © 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of the Korean Society of Mycology. https://creativecommons.org/licenses/by-nc/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/ (https://creativecommons.org/licenses/by-nc/4.0/) ), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Articles Jeong, Min-Hye Kim, Jung A. Kang, Seogchan Choi, Eu Ddeum Kim, Youngmin Lee, Yerim Jeon, Mi Jin Yu, Nan Hee Park, Ae Ran Kim, Jin-Cheol Kim, Soonok Park, Sook-Young Optimization of Agrobacterium tumefaciens-Mediated Transformation of Xylaria grammica EL000614, an Endolichenic Fungus Producing Grammicin |
title | Optimization of Agrobacterium tumefaciens-Mediated Transformation of Xylaria grammica EL000614, an Endolichenic Fungus Producing Grammicin |
title_full | Optimization of Agrobacterium tumefaciens-Mediated Transformation of Xylaria grammica EL000614, an Endolichenic Fungus Producing Grammicin |
title_fullStr | Optimization of Agrobacterium tumefaciens-Mediated Transformation of Xylaria grammica EL000614, an Endolichenic Fungus Producing Grammicin |
title_full_unstemmed | Optimization of Agrobacterium tumefaciens-Mediated Transformation of Xylaria grammica EL000614, an Endolichenic Fungus Producing Grammicin |
title_short | Optimization of Agrobacterium tumefaciens-Mediated Transformation of Xylaria grammica EL000614, an Endolichenic Fungus Producing Grammicin |
title_sort | optimization of agrobacterium tumefaciens-mediated transformation of xylaria grammica el000614, an endolichenic fungus producing grammicin |
topic | Research Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8583754/ https://www.ncbi.nlm.nih.gov/pubmed/34803437 http://dx.doi.org/10.1080/12298093.2021.1961431 |
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