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Tipo do documento: Artigo
Título: New developments of RNAi in Paracoccidioides brasiliensis: prospects for high-throughput, genome-wide, functional genomics
Autor: Góes, Tércio
Bailão, Elisa Flávia Luiz Cardoso
Correa, Cristiane Rodrigues
Bozzi, Adriana
Santos, Luara Isabela dos
Gomes, Dawidson Assis
Soares, Célia Maria de Almeida
Goes, Alfredo Miranda de
Abstract: Background: The Fungal Genome Initiative of the Broad Institute, in partnership with the Paracoccidioides research community, has recently sequenced the genome of representative isolates of this human-pathogen dimorphic fungus: Pb18 (S1), Pb03 (PS2) and Pb01. The accomplishment of future high-throughput, genome-wide, functional genomics will rely upon appropriate molecular tools and straightforward techniques to streamline the generation of stable loss-of-function phenotypes. In the past decades, RNAi has emerged as the most robust genetic technique to modulate or to suppress gene expression in diverse eukaryotes, including fungi. These molecular tools and techniques, adapted for RNAi, were up until now unavailable for P. brasiliensis. Methodology/Principal Findings: In this paper, we report Agrobacterium tumefaciens mediated transformation of yeast cells for high-throughput applications with which higher transformation frequencies of 150624 yeast cell transformants per 1610 6 viable yeast cells were obtained. Our approach is based on a bifunctional selective marker fusion protein consisted of the Streptoalloteichus hindustanus bleomycin-resistance gene (Shble) and the intrinsically fluorescent monomeric protein mCherry which was codon-optimized for heterologous expression in P. brasiliensis. We also report successful GP43 gene knock-down through the expression of intron-containing hairpin RNA (ihpRNA) from a Gateway-adapted cassette (cALf) which was purpose-built for gene silencing in a high-throughput manner. Gp43 transcript levels were reduced by 73.1622.9% with this approach. Conclusions/Significance: We have a firm conviction that the genetic transformation technique and the molecular tools herein described will have a relevant contribution in future Paracoccidioides spp. functional genomics research.
País: Estados unidos
Unidade acadêmica: Instituto de Ciências Biológicas - ICB (RG)
Citação: GOES, Tercio; BAILÃO, Elisa Flavia L. C.; CORREA, Cristiane R.; BOZZI, Adriana; SANTOS, Luara I.; GOMES, Dawidson A.; SOARES, Celia M. A.; GOES, Alfredo M. New developments of RNAi in Paracoccidioides brasiliensis: prospects for high-throughput, genome-wide, functional genomics. PLoS Neglected Tropical Diseases, San Francisco, v. 8, n. 10, e3173, Oct. 2014.
Tipo de acesso: Acesso Aberto
Identificador do documento: 10.1371/journal.pntd.0003173
Identificador do documento: 10.1371/journal.pntd.0003173
Data de publicação: Out-2014
Aparece nas coleções:IPTSP - Artigos publicados em periódicos

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