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  • Deletion of the Candida albicans TLO gene family using CRISPR-Cas9 mutagenesis allows characterisation of functional differences in α-, β- and γ- TLO gene function
Title: Deletion of the Candida albicans TLO gene family using CRISPR-Cas9 mutagenesis allows characterisation of functional differences in α-, β- and γ- TLO gene function
TheCandida albicansgenome contains between ten and fifteen distinctTLOgenes that all encode a Med2 subunit of Mediator. In order to investigate the biological role of Med2/Tlo inC.albicanswe deleted all fourteenTLOgenes 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-TLOclades resulted in the complementation of the mutant phenotypes, but to different degrees.TLOα1could 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-TLOgenes examined (i.e.,TLOγ5 and TLOγ11) had a far lower impact on complementing phenotypic and transcriptomic changes. Uniquely, expression ofTLOβ2in thetloΔmutant stimulated filamentous growth in YEPD medium and this phenotype was enhanced when Tloβ2 expression was increased to levels far in excess of Med3. In contrast, expression of reintegratedTLOgenes in atloΔ/med3Δdouble mutant background failed to restore any of the phenotypes tested, suggesting that complementation of these Tlo-regulated processes requires a functional Mediator tail module. Together, these data confirm the importance of Med2/Tlo in a wide range ofC.albicanscellular activities and demonstrate functional diversity within the gene family which may contribute to the success of this yeast as a coloniser and pathogen of humans.  more » « less
Award ID(s):
2046863
PAR ID:
10493688
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ; ; ; ;
Editor(s):
Forche, Anja
Publisher / Repository:
PLoS
Date Published:
Journal Name:
PLOS Genetics
Volume:
19
Issue:
12
ISSN:
1553-7404
Page Range / eLocation ID:
e1011082
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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