<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
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>-mediate...
| Main Authors: | , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2021-11-01
|
| Series: | Journal of Fungi |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2309-608X/7/11/961 |
| _version_ | 1797509822694817792 |
|---|---|
| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-03-10T05:23:11Z |
| format | Article |
| id | doaj.art-edee7eefbe26449b95f095d5b45e66b8 |
| institution | Directory Open Access Journal |
| issn | 2309-608X |
| language | English |
| last_indexed | 2024-03-10T05:23:11Z |
| publishDate | 2021-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Journal of Fungi |
| spelling | doaj.art-edee7eefbe26449b95f095d5b45e66b82023-11-22T23:56:15ZengMDPI AGJournal of Fungi2309-608X2021-11-0171196110.3390/jof7110961<i>Agrobacterium tumefaciens</i>-Mediated Transformation of NHEJ Mutant <i>Aspergillus nidulans</i> Conidia: An Efficient Tool for Targeted Gene Recombination Using Selectable Nutritional MarkersVirginia Casado-del Castillo0Andrew P. MacCabe1Margarita Orejas2Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), c/Catedrático Agustín Escardino Benlloch 7, 46980 Paterna, Valencia, SpainInstituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), c/Catedrático Agustín Escardino Benlloch 7, 46980 Paterna, Valencia, SpainInstituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), c/Catedrático Agustín Escardino Benlloch 7, 46980 Paterna, Valencia, SpainProtoplast 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.https://www.mdpi.com/2309-608X/7/11/961<i>Aspergillus nidulans</i>conidiafungal transformationATMTpCAMBIA-derived vectorstargeted gene deletion/disruption |
| spellingShingle | 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 Journal of Fungi <i>Aspergillus nidulans</i> conidia fungal transformation ATMT pCAMBIA-derived vectors targeted gene deletion/disruption |
| 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_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_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_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 |
| topic | <i>Aspergillus nidulans</i> conidia fungal transformation ATMT pCAMBIA-derived vectors targeted gene deletion/disruption |
| url | https://www.mdpi.com/2309-608X/7/11/961 |
| work_keys_str_mv | AT virginiacasadodelcastillo iagrobacteriumtumefaciensimediatedtransformationofnhejmutantiaspergillusnidulansiconidiaanefficienttoolfortargetedgenerecombinationusingselectablenutritionalmarkers AT andrewpmaccabe iagrobacteriumtumefaciensimediatedtransformationofnhejmutantiaspergillusnidulansiconidiaanefficienttoolfortargetedgenerecombinationusingselectablenutritionalmarkers AT margaritaorejas iagrobacteriumtumefaciensimediatedtransformationofnhejmutantiaspergillusnidulansiconidiaanefficienttoolfortargetedgenerecombinationusingselectablenutritionalmarkers |