title: Towards a molecular genetic system for the pathogenic fungus Paracoccidioides brasiliensis.
abstract: We herein report the development of a molecular toolbox for the dimorphic fungus Paracoccidioides brasiliensis, specifically a more efficient transformation and a gene expression system. We evaluated several parameters that influence Agrobacterium tumefaciens-mediated transformation (ATMT), such as co-cultivation conditions and host cell susceptibility. Our results show that cellular recovery and air drying of A. tumefaciens:P. brasiliensis mixtures are essential for ATMT. Overall, our data indicate a transformation efficiency of 78+/-9 transformants/co-cultivation (5+/-1 transformants/10(6) target cells). P. brasiliensis GFP-expressing isolates were also constructed by insertion of the GFP gene under the control of several fungal promoters. RT-PCR, epifluorescence microscopy and flow cytometry analysis revealed Gfp visualization for all studied promoters but without significant differences in fluorescence and gene expression levels. Moreover, we present evidence for the occurrence of random single gene copy integration per haploid nuclei and the generation of homokaryon progeny, relevant for the future use in targeted mutagenesis and linking mutations to phenotypes.
authors.affiliation: Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal.
authors.forename: A J
authors.initials: AJ
authors.lastname: Almeida
authors.forename: J A
authors.initials: JA
authors.lastname: Carmona
authors.forename: C
authors.initials: C
authors.lastname: Cunha
authors.forename: A
authors.initials: A
authors.lastname: Carvalho
authors.forename: C A
authors.initials: CA
authors.lastname: Rappleye
authors.forename: W E
authors.initials: WE
authors.lastname: Goldman
authors.forename: P J
authors.initials: PJ
authors.lastname: Hooykaas
authors.forename: C
authors.initials: C
authors.lastname: Leão
authors.forename: P
authors.initials: P
authors.lastname: Ludovico
authors.forename: F
authors.initials: F
authors.lastname: Rodrigues
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D001377:Azaserine / Q000494:pharmacology; D015139:Blotting, Southern; D046169:Dermoscopy; D005434:Flow Cytometry; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007555:Isoxazoles / Q000494:pharmacology; D008967:Molecular Biology / Q000379:methods*; D010228:Paracoccidioides / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012263:Ribonucleosides / Q000494:pharmacology; D014170:Transformation, Genetic
chemical_list: D007555:Isoxazoles; D011993:Recombinant Fusion Proteins; D012263:Ribonucleosides; D049452:Green Fluorescent Proteins; C010052:mizoribine; D001377:Azaserine; C006303:acivicin