Cargando…
Expanding the Toolbox for Functional Genomics in Fonsecaea pedrosoi: The Use of Split-Marker and Biolistic Transformation for Inactivation of Tryptophan Synthase (trpB) Gene
Chromoblastomycosis (CBM) is a disease caused by several dematiaceous fungi from different genera, and Fonsecaea is the most common which has been clinically isolated. Genetic transformation methods have recently been described; however, molecular tools for the functional study of genes have been sc...
Autores principales: | , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9963410/ https://www.ncbi.nlm.nih.gov/pubmed/36836338 http://dx.doi.org/10.3390/jof9020224 |
_version_ | 1784896246803070976 |
---|---|
author | Favilla, Luísa Dan Herman, Tatiana Sobianski Goersch, Camila da Silva de Andrade, Rosangela Vieira Felipe, Maria Sueli Soares Bocca, Anamélia Lorenzetti Fernandes, Larissa |
author_facet | Favilla, Luísa Dan Herman, Tatiana Sobianski Goersch, Camila da Silva de Andrade, Rosangela Vieira Felipe, Maria Sueli Soares Bocca, Anamélia Lorenzetti Fernandes, Larissa |
author_sort | Favilla, Luísa Dan |
collection | PubMed |
description | Chromoblastomycosis (CBM) is a disease caused by several dematiaceous fungi from different genera, and Fonsecaea is the most common which has been clinically isolated. Genetic transformation methods have recently been described; however, molecular tools for the functional study of genes have been scarcely reported for those fungi. In this work, we demonstrated that gene deletion and generation of the null mutant by homologous recombination are achievable for Fonsecaea pedrosoi by the use of two approaches: use of double-joint PCR for cassette construction, followed by delivery of the split-marker by biolistic transformation. Through in silico analyses, we identified that F. pedrosoi presents the complete enzymatic apparatus required for tryptophan (trp) biosynthesis. The gene encoding a tryptophan synthase trpB —which converts chorismate to trp—was disrupted. The ΔtrpB auxotrophic mutant can grow with external trp supply, but germination, viability of conidia, and radial growth are defective compared to the wild-type and reconstituted strains. The use of 5-FAA for selection of trp(-) phenotypes and for counter-selection of strains carrying the trp gene was also demonstrated. The molecular tools for the functional study of genes, allied to the genetic information from genomic databases, significantly boost our understanding of the biology and pathogenicity of CBM causative agents. |
format | Online Article Text |
id | pubmed-9963410 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-99634102023-02-26 Expanding the Toolbox for Functional Genomics in Fonsecaea pedrosoi: The Use of Split-Marker and Biolistic Transformation for Inactivation of Tryptophan Synthase (trpB) Gene Favilla, Luísa Dan Herman, Tatiana Sobianski Goersch, Camila da Silva de Andrade, Rosangela Vieira Felipe, Maria Sueli Soares Bocca, Anamélia Lorenzetti Fernandes, Larissa J Fungi (Basel) Article Chromoblastomycosis (CBM) is a disease caused by several dematiaceous fungi from different genera, and Fonsecaea is the most common which has been clinically isolated. Genetic transformation methods have recently been described; however, molecular tools for the functional study of genes have been scarcely reported for those fungi. In this work, we demonstrated that gene deletion and generation of the null mutant by homologous recombination are achievable for Fonsecaea pedrosoi by the use of two approaches: use of double-joint PCR for cassette construction, followed by delivery of the split-marker by biolistic transformation. Through in silico analyses, we identified that F. pedrosoi presents the complete enzymatic apparatus required for tryptophan (trp) biosynthesis. The gene encoding a tryptophan synthase trpB —which converts chorismate to trp—was disrupted. The ΔtrpB auxotrophic mutant can grow with external trp supply, but germination, viability of conidia, and radial growth are defective compared to the wild-type and reconstituted strains. The use of 5-FAA for selection of trp(-) phenotypes and for counter-selection of strains carrying the trp gene was also demonstrated. The molecular tools for the functional study of genes, allied to the genetic information from genomic databases, significantly boost our understanding of the biology and pathogenicity of CBM causative agents. MDPI 2023-02-08 /pmc/articles/PMC9963410/ /pubmed/36836338 http://dx.doi.org/10.3390/jof9020224 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/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 (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Favilla, Luísa Dan Herman, Tatiana Sobianski Goersch, Camila da Silva de Andrade, Rosangela Vieira Felipe, Maria Sueli Soares Bocca, Anamélia Lorenzetti Fernandes, Larissa Expanding the Toolbox for Functional Genomics in Fonsecaea pedrosoi: The Use of Split-Marker and Biolistic Transformation for Inactivation of Tryptophan Synthase (trpB) Gene |
title | Expanding the Toolbox for Functional Genomics in Fonsecaea pedrosoi: The Use of Split-Marker and Biolistic Transformation for Inactivation of Tryptophan Synthase (trpB) Gene |
title_full | Expanding the Toolbox for Functional Genomics in Fonsecaea pedrosoi: The Use of Split-Marker and Biolistic Transformation for Inactivation of Tryptophan Synthase (trpB) Gene |
title_fullStr | Expanding the Toolbox for Functional Genomics in Fonsecaea pedrosoi: The Use of Split-Marker and Biolistic Transformation for Inactivation of Tryptophan Synthase (trpB) Gene |
title_full_unstemmed | Expanding the Toolbox for Functional Genomics in Fonsecaea pedrosoi: The Use of Split-Marker and Biolistic Transformation for Inactivation of Tryptophan Synthase (trpB) Gene |
title_short | Expanding the Toolbox for Functional Genomics in Fonsecaea pedrosoi: The Use of Split-Marker and Biolistic Transformation for Inactivation of Tryptophan Synthase (trpB) Gene |
title_sort | expanding the toolbox for functional genomics in fonsecaea pedrosoi: the use of split-marker and biolistic transformation for inactivation of tryptophan synthase (trpb) gene |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9963410/ https://www.ncbi.nlm.nih.gov/pubmed/36836338 http://dx.doi.org/10.3390/jof9020224 |
work_keys_str_mv | AT favillaluisadan expandingthetoolboxforfunctionalgenomicsinfonsecaeapedrosoitheuseofsplitmarkerandbiolistictransformationforinactivationoftryptophansynthasetrpbgene AT hermantatianasobianski expandingthetoolboxforfunctionalgenomicsinfonsecaeapedrosoitheuseofsplitmarkerandbiolistictransformationforinactivationoftryptophansynthasetrpbgene AT goerschcamiladasilva expandingthetoolboxforfunctionalgenomicsinfonsecaeapedrosoitheuseofsplitmarkerandbiolistictransformationforinactivationoftryptophansynthasetrpbgene AT deandraderosangelavieira expandingthetoolboxforfunctionalgenomicsinfonsecaeapedrosoitheuseofsplitmarkerandbiolistictransformationforinactivationoftryptophansynthasetrpbgene AT felipemariasuelisoares expandingthetoolboxforfunctionalgenomicsinfonsecaeapedrosoitheuseofsplitmarkerandbiolistictransformationforinactivationoftryptophansynthasetrpbgene AT boccaanamelialorenzetti expandingthetoolboxforfunctionalgenomicsinfonsecaeapedrosoitheuseofsplitmarkerandbiolistictransformationforinactivationoftryptophansynthasetrpbgene AT fernandeslarissa expandingthetoolboxforfunctionalgenomicsinfonsecaeapedrosoitheuseofsplitmarkerandbiolistictransformationforinactivationoftryptophansynthasetrpbgene |