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 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 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 presents the complete enzymatic apparatus required for tryptophan (trp) biosynthesis. The gene encoding a tryptophan synthase -which converts chorismate to trp-was disrupted. The Δ 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 phenotypes and for counter-selection of strains carrying the 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.