Construction and analysis of novel strains to examine the genetic regulation of Candida albicans filamentation

Candida albicans is a fungus that lives as a commensal organism on many muscosal surfaces of the human body but can also behave as a pathogen causing both treatable illnesses as well as a more serious disease known as disseminated candidiasis. C. albicans exists in three distinct morphotypes: yeast, pseudohyphae, and hyphae. It has been accepted that the ability of the fungus to switch between the yeast and filamentous types is the main source of its virulence. This transition is governed by a network of signaling pathways involving many transcriptional regulators of hyphal growth one of which being the repressor Nrg1p. Recently, we demonstrated that Brg1p, a positive regulator of filamentation is involved in a feedback mechanism with NRG1, whereby BRG1 overexpression induces hypha formation through the production of an antisense RNA molecule and subsequent destabilization of NRG1 mRNA. Since this is the first known example of antisense RNA production in C. albicans, we sought to determine if the stated transcript destabilization was occurring through the mechanism of RNAi by constructing deletion mutants of Dicer and Argonaute (the main proteins involved in RNAi) and examining their ability to form hyphae. We also sought to investigate the functions of other genes that are down-regulated by NRG1 (like BRG1) as they too could shed light on the yeast to hypha transition. We chose three transcription factors SEF2, ZCF3, and ZCF35 to construct overexpression strains with and test their ability to induce filamentation alone and in the context of Nrg1p-mediated repression.