Gene Knockdown in Paracoccidioides brasiliensis Using Antisense RNA
Paracoccidioides brasiliensis is a thermal dimorphic fungus which in the host environment exhibits a multinucleated and multibudding yeast form. The cellular and molecular mechanisms underlying these phenotypes remain to be clarified, mostly due to the ab
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1. Introduction Paracoccidioides brasiliensis is the etiological agent of Paracoccidioidomycosis, one of the most prevalent systemic mycosis in Latin America. As a thermal dimorphic fungus, P. brasiliensis switches from the environmental mycelial/conidial nonpathogenic form at ambient temperatures to the pathogenic multiple budding yeast form when exposed to temperatures similar to those of the mammalian host (1). The absence of effective molecular techniques has significantly hampered studies in P. brasiliensis relevant for understanding the biology of this fungus as well as the mechanisms that underlie its pathogenicity. We herein report an efficient gene expression knockdown protocol for P. brasiliensis by taking advantage of Agrobacterium tumefaciens-mediated transformation (ATMT) for single-copy genetic integration (PbATMT) developed by our group (3, 5). Alexandra C. Brand and Donna M. MacCallum (eds.), Host-Fungus Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 845, DOI 10.1007/978-1-61779-539-8_12, © Springer Science+Business Media, LLC 2012
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ATMT makes use of a natural transformation process induced by A. tumefaciens, a bacterial plant pathogen that randomly inserts the transfer-DNA (T-DNA) into the plant genome during infection (2). Fungal ATMT presents advantages over other methods (4) as it (1) shows high efficiency and simplicity, (2) avoids timeconsuming steps and specialized equipment, and (3) can be easily applied in Biosafety Level 3 (BSL3) microorganisms. Furthermore, we and others have applied this PbATMT system to modulate gene expression in P. brasiliensis using antisense-RNA (aRNA) technology with high success (5, 13). This has proved to be a proficient genetic system for fungi whose homologous recombination machinery is still poorly described (6), such as P. brasiliensis, as contrary to other allelic replacement strategies, since aRNA “knocksdown” gene expression rather than “knocking-out” an entire gene. PbATMT and aRNA technology merge as important molecular tools to study functional genetics in P. brasiliensis and advancing research in a field that has previously been lacking this type of technology.
2. Materials All solutions must be prepared using ultrapure water and analytical grade reagents and stored at room temperature (unless indicated otherwise). Sterilize all the culture medium by autoclaving for 20 min at 121°C. Dispose of all waste materials according to safety regulations. 2.1. Antisense Plasmid Construction
1. Escherichia coli strain: JM109 competent cells (Promega). 2. E. coli culture medium (1 L) Luria Bertani (LB): 10 g tryptone peptone, 5 g yeast extract, 10 g NaCl. Suspend the reagents in distilled or deionized water. Adjust to pH 7.0 with NaOH. 3. Plasmids: pCR35 plasmid containing the green fluorescent protein (GFP) gene downstream from the Histoplasma capsulatum calcium-binding protein (CBP1) promoter (7); pUR5750 parental vector for the insertion of recombinant transfer DNA (T-DNA) in P. brasiliensis, harboring an
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