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...

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Autores principales: 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
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Taylor & Francis 2021
Materias:
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.
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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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