Agrobacterium tumefaciens-Mediated Transformation of NHEJ Mutant Aspergillus nidulans Conidia: An Efficient Tool for Targeted Gene Recombination Using Selectable Nutritional Markers

Protoplast transformation for the introduction of recombinant DNA into Aspergillus nidulans is technically demanding and dependant on the availability and batch variability of commercial enzyme preparations. Given the success of Agrobacterium tumefaciens-mediate...

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Autores principales:	Virginia Casado-del Castillo, Andrew P. MacCabe, Margarita Orejas
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	<i>Aspergillus nidulans</i> conidia fungal transformation ATMT pCAMBIA-derived vectors targeted gene deletion/disruption Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/edee7eefbe26449b95f095d5b45e66b8
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spelling	oai:doaj.org-article:edee7eefbe26449b95f095d5b45e66b82021-11-25T18:06:11Z<i>Agrobacterium tumefaciens</i>-Mediated Transformation of NHEJ Mutant <i>Aspergillus nidulans</i> Conidia: An Efficient Tool for Targeted Gene Recombination Using Selectable Nutritional Markers10.3390/jof71109612309-608Xhttps://doaj.org/article/edee7eefbe26449b95f095d5b45e66b82021-11-01T00:00:00Zhttps://www.mdpi.com/2309-608X/7/11/961https://doaj.org/toc/2309-608XProtoplast transformation for the introduction of recombinant DNA into <i>Aspergillus nidulans</i> is technically demanding and dependant on the availability and batch variability of commercial enzyme preparations. Given the success of <i>Agrobacterium tumefaciens</i>-mediated transformation (ATMT) in diverse pathogenic fungi, we have adapted this method to facilitate transformation of <i>A. nidulans</i>. Using suitably engineered binary vectors, gene-targeted ATMT of <i>A. nidulans</i> non-homologous end-joining (NHEJ) mutant conidia has been carried out for the first time by complementation of a nutritional requirement (uridine/uracil auxotrophy). Site-specific integration in the Δ<i>nkuA</i> host genome occurred at high efficiency. Unlike other transformation techniques, however, cross-feeding of certain nutritional requirements from the bacterium to the fungus was found to occur, thus limiting the choice of auxotrophies available for ATMT. In complementation tests and also for comparative purposes, integration of recombinant cassettes at a specific locus could provide a means to reduce the influence of position effects (chromatin structure) on transgene expression. In this regard, targeted disruption of the <i>wA</i> locus permitted visual identification of transformants carrying site-specific integration events by conidial colour (white), even when auxotrophy selection was compromised due to cross-feeding. The protocol described offers an attractive alternative to the protoplast procedure for obtaining locus-targeted <i>A. nidulans</i> transformants.Virginia Casado-del CastilloAndrew P. MacCabeMargarita OrejasMDPI AGarticle<i>Aspergillus nidulans</i>conidiafungal transformationATMTpCAMBIA-derived vectorstargeted gene deletion/disruptionBiology (General)QH301-705.5ENJournal of Fungi, Vol 7, Iss 961, p 961 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	<i>Aspergillus nidulans</i> conidia fungal transformation ATMT pCAMBIA-derived vectors targeted gene deletion/disruption Biology (General) QH301-705.5
spellingShingle	<i>Aspergillus nidulans</i> conidia fungal transformation ATMT pCAMBIA-derived vectors targeted gene deletion/disruption Biology (General) QH301-705.5 Virginia Casado-del Castillo Andrew P. MacCabe Margarita Orejas <i>Agrobacterium tumefaciens</i>-Mediated Transformation of NHEJ Mutant <i>Aspergillus nidulans</i> Conidia: An Efficient Tool for Targeted Gene Recombination Using Selectable Nutritional Markers
description	Protoplast transformation for the introduction of recombinant DNA into <i>Aspergillus nidulans</i> is technically demanding and dependant on the availability and batch variability of commercial enzyme preparations. Given the success of <i>Agrobacterium tumefaciens</i>-mediated transformation (ATMT) in diverse pathogenic fungi, we have adapted this method to facilitate transformation of <i>A. nidulans</i>. Using suitably engineered binary vectors, gene-targeted ATMT of <i>A. nidulans</i> non-homologous end-joining (NHEJ) mutant conidia has been carried out for the first time by complementation of a nutritional requirement (uridine/uracil auxotrophy). Site-specific integration in the Δ<i>nkuA</i> host genome occurred at high efficiency. Unlike other transformation techniques, however, cross-feeding of certain nutritional requirements from the bacterium to the fungus was found to occur, thus limiting the choice of auxotrophies available for ATMT. In complementation tests and also for comparative purposes, integration of recombinant cassettes at a specific locus could provide a means to reduce the influence of position effects (chromatin structure) on transgene expression. In this regard, targeted disruption of the <i>wA</i> locus permitted visual identification of transformants carrying site-specific integration events by conidial colour (white), even when auxotrophy selection was compromised due to cross-feeding. The protocol described offers an attractive alternative to the protoplast procedure for obtaining locus-targeted <i>A. nidulans</i> transformants.
format	article
author	Virginia Casado-del Castillo Andrew P. MacCabe Margarita Orejas
author_facet	Virginia Casado-del Castillo Andrew P. MacCabe Margarita Orejas
author_sort	Virginia Casado-del Castillo
title	<i>Agrobacterium tumefaciens</i>-Mediated Transformation of NHEJ Mutant <i>Aspergillus nidulans</i> Conidia: An Efficient Tool for Targeted Gene Recombination Using Selectable Nutritional Markers
title_short	<i>Agrobacterium tumefaciens</i>-Mediated Transformation of NHEJ Mutant <i>Aspergillus nidulans</i> Conidia: An Efficient Tool for Targeted Gene Recombination Using Selectable Nutritional Markers
title_full	<i>Agrobacterium tumefaciens</i>-Mediated Transformation of NHEJ Mutant <i>Aspergillus nidulans</i> Conidia: An Efficient Tool for Targeted Gene Recombination Using Selectable Nutritional Markers
title_fullStr	<i>Agrobacterium tumefaciens</i>-Mediated Transformation of NHEJ Mutant <i>Aspergillus nidulans</i> Conidia: An Efficient Tool for Targeted Gene Recombination Using Selectable Nutritional Markers
title_full_unstemmed	<i>Agrobacterium tumefaciens</i>-Mediated Transformation of NHEJ Mutant <i>Aspergillus nidulans</i> Conidia: An Efficient Tool for Targeted Gene Recombination Using Selectable Nutritional Markers
title_sort	<i>agrobacterium tumefaciens</i>-mediated transformation of nhej mutant <i>aspergillus nidulans</i> conidia: an efficient tool for targeted gene recombination using selectable nutritional markers
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/edee7eefbe26449b95f095d5b45e66b8
work_keys_str_mv	AT virginiacasadodelcastillo iagrobacteriumtumefaciensimediatedtransformationofnhejmutantiaspergillusnidulansiconidiaanefficienttoolfortargetedgenerecombinationusingselectablenutritionalmarkers AT andrewpmaccabe iagrobacteriumtumefaciensimediatedtransformationofnhejmutantiaspergillusnidulansiconidiaanefficienttoolfortargetedgenerecombinationusingselectablenutritionalmarkers AT margaritaorejas iagrobacteriumtumefaciensimediatedtransformationofnhejmutantiaspergillusnidulansiconidiaanefficienttoolfortargetedgenerecombinationusingselectablenutritionalmarkers
_version_	1718411654942687232

<i>Agrobacterium tumefaciens</i>-Mediated Transformation of NHEJ Mutant <i>Aspergillus nidulans</i> Conidia: An Efficient Tool for Targeted Gene Recombination Using Selectable Nutritional Markers

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