Banca de DEFESA: Luisa Dan Favilla

Uma banca de DEFESA de MESTRADO foi cadastrada pelo programa.
STUDENT : Luisa Dan Favilla
DATE: 14/07/2023
TIME: 09:00
LOCAL: Videoconferência
TITLE:

Expanding the Toolbox for Functional Genomics in Fonsecaea pedrosoi: The Use of Split-Marker and Biolistic Transformation for Inactivation of Tryptophan Synthase (trpB)

KEY WORDS:

gene replacement by homologous recombination; double-joint PCR; biolistic transformation; knockout mutant; trpB; tryptophan biosynthesis; Fonsecaea; chromoblastomycosis

PAGES: 61
BIG AREA: Ciências Biológicas
AREA: Biologia Geral
SUMMARY:

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.

COMMITTEE MEMBERS:
Externa à Instituição - RENATA CASTIGLIONI PASCON - UNIFESP
Presidente - 2223020 - ANAMELIA LORENZETTI BOCCA
Externa ao Programa - 1335413 - FABIANA BRANDAO ALVES SILVA - nullExterna ao Programa - 1583080 - MARCIA CRISTINA GONCALVES MACIEL - null