Deletion of the < i > Candida albicans TLO < /i > gene family using CRISPR-Cas9 mutagenesis allows characterisation of functional differences in α-, β- and γ- < i > TLO < /i > gene function

by Jessica Fletcher, James O ’Connor-Moneley, Dean Frawley, Peter R. Flanagan, Leenah Alaalm, Pilar Menendez-Manjon, Samuel Vega Estevez, Shane Hendricks, Andrew L. Woodruff, Alessia Buscaino, Matthew Z. Anderson, Derek J. Sullivan, Gary P. Moran TheCandida albicans genome contains between ten and fifteen distinctTLO genes that all encode a Med2 subunit of Mediator. In order to investigate the biological role of Med2/Tlo inC.albicans we deleted all fourteenTLO genes using CRISPR-Cas9 mutagenesis. ChIP-seq analysis showed that RNAP II localized to 55% fewer genes in thetloΔ mutant strain compared to the parent, while RNA-seq analysis showed that thetloΔ mutant exhibited differential expression of genes required for carbohydrate metabolism, stress responses, white-opaque switching and filamentous growth. Consequently, thetloΔ mutant grows poorly in glucose- and galactose-containing media, is unable to grow as true hyphae, is more sensitive to oxidative stress and is less virulent in the wax worm infection model. Reintegration of genes representative of the α-, β- and γ-TLO clades resulted in the complementation of the mutant phenotypes, but to different degrees.TLO α1 could restore phenotypes and gene expression patterns similar to wild-type and was the strongest activator of glycolytic and Tye7-regulated gene expression. In contrast, the two γ-TLO genes examined (i.e.,TLOγ5 and TLOγ11) had a far lower impact on complementing phenotypic and transcriptomic changes. ...
Source: PLoS Genetics - Category: Genetics & Stem Cells Authors: Source Type: research