title: New developments of RNAi in Paracoccidioides brasiliensis: prospects for high-throughput, genome-wide, functional genomics.
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.
  
  
  RESULTS
  In this paper, we report Agrobacterium tumefaciens mediated transformation of yeast cells for high-throughput applications with which higher transformation frequencies of 150±24 yeast cell transformants per 1×106 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 73.1±22.9% with this approach.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Brasil.
authors.forename: Tercio
authors.initials: T
authors.lastname: Goes
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás (UFG), Goiânia, Goiás, Brasil.
authors.forename: Elisa Flavia L C
authors.initials: EF
authors.lastname: Bailão
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Brasil.
authors.forename: Cristiane R
authors.initials: CR
authors.lastname: Correa
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Brasil.
authors.forename: Adriana
authors.initials: A
authors.lastname: Bozzi
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Brasil.
authors.forename: Luara I
authors.initials: LI
authors.lastname: Santos
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Brasil.
authors.forename: Dawidson A
authors.initials: DA
authors.lastname: Gomes
authors.affiliation: Departamento de Bioquímica e Biologia Molecular, Instituto de Ciências Biológicas, Universidade Federal de Goiás (UFG), Goiânia, Goiás, Brasil.
authors.forename: Celia M A
authors.initials: CM
authors.lastname: Soares
authors.affiliation: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Brasil.
authors.forename: Alfredo M
authors.initials: AM
authors.lastname: Goes
mesh_terms: D055785:Gene Knockdown Techniques; D023281:Genomics; D006801:Humans; D016366:Open Reading Frames; D010228:Paracoccidioides / Q000235:genetics*; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D014170:Transformation, Genetic