Abstract
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 absence of efficient classical genetic and molecular techniques. Here we describe a method for gene expression knockdown in P. brasiliensis by antisense RNA (aRNA) technology taking advantage of an Agrobacterium tumefaciens-mediated transformation (ATMT) system. Together, these techniques represent a reliable toolbox that can be employed for functional genetic analysis of putative virulence factors and morphogenic regulators, aiming to the identification of new potential drug targets.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Garcia, A.M., et al., Gene expression analysis of Paracoccidioides brasiliensis transition from conidium to yeast cell. Med Mycol. 48(1): p. 147–54.
Hoekema, A., et al., Delivery of T-DNA from the Agrobacterium tumefaciens chromosome into plant cells. EMBO J, 1984. 3(11): p. 2485–90.
Almeida, A.J., et al., Towards a molecular genetic system for the pathogenic fungus Paracoccidioides brasiliensis. Fungal Genet Biol, 2007. 44(12): p. 1387–98.
Michielse, C.B., et al., Agrobacterium-mediated transformation as a tool for functional genomics in fungi. Curr Genet, 2005. 48(1): p. 1–17.
Almeida, A.J., et al., Cdc42p controls yeast-cell shape and virulence of Paracoccidioides brasiliensis. Fungal Genet Biol, 2009. 46(12): p. 919–26.
da Silva Ferreira, M.E., et al., The akuB(KU80) mutant deficient for nonhomologous end joining is a powerful tool for analyzing pathogenicity in Aspergillus fumigatus. Eukaryot Cell, 2006. 5(1): p. 207–11.
Rappleye, C.A., J.T. Engle, and W.E. Goldman, RNA interference in Histoplasma capsulatum demonstrates a role for alpha-(1,3)-glucan in virulence. Mol Microbiol, 2004. 53(1): p. 153–65.
de Groot, M.J., et al., Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat Biotechnol, 1998. 16(9): p. 839–42.
Beijersbergen, A., et al., Conjugative transfer by the virulence system of Agrobacterium tumefaciens. Science, 1992. 256(5061): p. 1324–7.
Bundock, P., et al., Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO J, 1995. 14(13): p. 3206–14.
Molnár, A., et al., miRNAs control gene expression in the single-cell alga Chlamydomonas reinhardtii. Nature, 2007. 447(7148): p. 1126–1129.
den Dulk-Ras, A. and P.J. Hooykaas, Electroporation of Agrobacterium tumefaciens. Methods Mol Biol, 1995. 55: p. 63–72.
Hernandez, O., et al., A 32-KDa hydrolase plays an important role in Paracoccidioides brasiliensis adherence to host cells and influences pathogenicity. Infect Immun.
Acknowledgments
We thank Dr Mark Sturme for critical reading of this chapter and valuable suggestions. This work was supported by a research grant from Fundação para a Ciência e a Tecnologia, Portugal (Ref. PTDC/BIA-MIC/108309/2008). João Menino was financially supported by a fellowship from Fundação para a Ciência e a Tecnologia, Portugal (Ref. SFRH/BD/33446/2008).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Menino, J.F., Almeida, A.J., Rodrigues, F. (2012). Gene Knockdown in Paracoccidioides brasiliensis Using Antisense RNA. In: Brand, A., MacCallum, D. (eds) Host-Fungus Interactions. Methods in Molecular Biology, vol 845. Humana, Totowa, NJ. https://doi.org/10.1007/978-1-61779-539-8_12
Download citation
DOI: https://doi.org/10.1007/978-1-61779-539-8_12
Published:
Publisher Name: Humana, Totowa, NJ
Print ISBN: 978-1-61779-538-1
Online ISBN: 978-1-61779-539-8
eBook Packages: Springer Protocols