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Agrobacterium tumefaciens-Mediated Genetic Transformation of the Ect-endomycorrhizal Fungus Terfezia boudieri

Mycorrhizal desert truffles such as Terfezia boudieri, Tirmania nivea, and Terfezia claveryi, form mycorrhizal associations with plants of the Cistaceae family. These valued truffles are still collected from the wild and not cultivated under intensive farming due to the lack of basic knowledge about...

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Autores principales: Satish, Lakkakula, Kamle, Madhu, Keren, Guy, Patil, Chandrashekhar D., Yehezkel, Galit, Barak, Ze’ev, Kagan-Zur, Varda, Kushmaro, Ariel, Sitrit, Yaron
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7693413/
https://www.ncbi.nlm.nih.gov/pubmed/33143066
http://dx.doi.org/10.3390/genes11111293
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author Satish, Lakkakula
Kamle, Madhu
Keren, Guy
Patil, Chandrashekhar D.
Yehezkel, Galit
Barak, Ze’ev
Kagan-Zur, Varda
Kushmaro, Ariel
Sitrit, Yaron
author_facet Satish, Lakkakula
Kamle, Madhu
Keren, Guy
Patil, Chandrashekhar D.
Yehezkel, Galit
Barak, Ze’ev
Kagan-Zur, Varda
Kushmaro, Ariel
Sitrit, Yaron
author_sort Satish, Lakkakula
collection PubMed
description Mycorrhizal desert truffles such as Terfezia boudieri, Tirmania nivea, and Terfezia claveryi, form mycorrhizal associations with plants of the Cistaceae family. These valued truffles are still collected from the wild and not cultivated under intensive farming due to the lack of basic knowledge about their biology at all levels. Recently, several genomes of desert truffles have been decoded, enabling researchers to attempt genetic manipulations to enable cultivation. To execute such manipulations, the development of molecular tools for genes transformation into truffles is needed. We developed an Agrobacterium tumefaciens-mediated genetic transformation system in T. boudieri. This system was optimized for the developmental stage of the mycelia explants, bacterial optical density, infection and co-cultivation durations, and concentrations of the selection antibiotics. The pFPL-Rh plasmid harboring hph gene conferring hygromycin resistance as a selection marker and the red fluorescent protein gene were used as visual reporters. The optimal conditions were incubation with 200 μM of acetosyringone, attaining a bacterial optical density of 0.3 OD(600); transfer time of 45 min; and co-cultivation for 3 days. This is the first report on a transformation system for T. boudieri, and the proposed protocol can be adapted for the transformation of other important desert truffles as well as ectomycorrhizal species.
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spelling pubmed-76934132020-11-28 Agrobacterium tumefaciens-Mediated Genetic Transformation of the Ect-endomycorrhizal Fungus Terfezia boudieri Satish, Lakkakula Kamle, Madhu Keren, Guy Patil, Chandrashekhar D. Yehezkel, Galit Barak, Ze’ev Kagan-Zur, Varda Kushmaro, Ariel Sitrit, Yaron Genes (Basel) Technical Note Mycorrhizal desert truffles such as Terfezia boudieri, Tirmania nivea, and Terfezia claveryi, form mycorrhizal associations with plants of the Cistaceae family. These valued truffles are still collected from the wild and not cultivated under intensive farming due to the lack of basic knowledge about their biology at all levels. Recently, several genomes of desert truffles have been decoded, enabling researchers to attempt genetic manipulations to enable cultivation. To execute such manipulations, the development of molecular tools for genes transformation into truffles is needed. We developed an Agrobacterium tumefaciens-mediated genetic transformation system in T. boudieri. This system was optimized for the developmental stage of the mycelia explants, bacterial optical density, infection and co-cultivation durations, and concentrations of the selection antibiotics. The pFPL-Rh plasmid harboring hph gene conferring hygromycin resistance as a selection marker and the red fluorescent protein gene were used as visual reporters. The optimal conditions were incubation with 200 μM of acetosyringone, attaining a bacterial optical density of 0.3 OD(600); transfer time of 45 min; and co-cultivation for 3 days. This is the first report on a transformation system for T. boudieri, and the proposed protocol can be adapted for the transformation of other important desert truffles as well as ectomycorrhizal species. MDPI 2020-10-30 /pmc/articles/PMC7693413/ /pubmed/33143066 http://dx.doi.org/10.3390/genes11111293 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Technical Note
Satish, Lakkakula
Kamle, Madhu
Keren, Guy
Patil, Chandrashekhar D.
Yehezkel, Galit
Barak, Ze’ev
Kagan-Zur, Varda
Kushmaro, Ariel
Sitrit, Yaron
Agrobacterium tumefaciens-Mediated Genetic Transformation of the Ect-endomycorrhizal Fungus Terfezia boudieri
title Agrobacterium tumefaciens-Mediated Genetic Transformation of the Ect-endomycorrhizal Fungus Terfezia boudieri
title_full Agrobacterium tumefaciens-Mediated Genetic Transformation of the Ect-endomycorrhizal Fungus Terfezia boudieri
title_fullStr Agrobacterium tumefaciens-Mediated Genetic Transformation of the Ect-endomycorrhizal Fungus Terfezia boudieri
title_full_unstemmed Agrobacterium tumefaciens-Mediated Genetic Transformation of the Ect-endomycorrhizal Fungus Terfezia boudieri
title_short Agrobacterium tumefaciens-Mediated Genetic Transformation of the Ect-endomycorrhizal Fungus Terfezia boudieri
title_sort agrobacterium tumefaciens-mediated genetic transformation of the ect-endomycorrhizal fungus terfezia boudieri
topic Technical Note
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7693413/
https://www.ncbi.nlm.nih.gov/pubmed/33143066
http://dx.doi.org/10.3390/genes11111293
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