New developments of RNAi in Paracoccidioides brasiliensis: prospects for high-throughput, genome-wide, functional genomics
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2014-10
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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.
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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.