Transcriptomic response to glabridin and wighteone
Prenylated (iso)flavonoids are plant metabolites with promising antimicrobial (including antifungal) properties. Recently, it was shown that the isoflavan glabridin and the isoflavone wighteone, which are both mono-prenylated isoflavonoids, are potent antifungal agents against the notorious food spoilage yeast, Zygosaccharomyces parabailii. The mode of antimicrobial action (MoA) of prenylated (iso)flavonoids is usually associated with plasma membrane (PM) disruption which is believed to relate to the high hydrophobicity of these molecules. However, glabridin and wighteone also have some differing molecular properties and distinct effects on membrane disruption in Z. parabailii were reported previously. Transcriptomic profiling with Z. parabailii ATCC 60483 was used here to offer additional insight to responses of the organism to these two molecules. Transcriptome profiling was performed for RNA extracted from exponential phase Z. parabailii cells after 30 and 120 min exposure to doses of glabridin (20 µg/ml) or wighteone (8 µg/ml) that gave a mild effect on yeast outgrowth after 120 min. Triplicate, independent biological replicate samples were prepared for each condition. Indexed sequencing libraries were prepared using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina and the Lexogen i7 6nt Index Set. Libraries were quantified using a Qubit Fluorometer and the Qubit dsDNA HS Kit. Library fragment-length distributions were analyzed using an Agilent TapeStation 4200 and the Agilent High Sensitivity D1000 ScreenTape Assay. Libraries were pooled in equimolar amounts and final library quantification performed using a KAPA Library Quantification Kit for Illumina. The library pool was sequenced on an Illumina NextSeq 500 with a NextSeq 500 High Output Kit v2.5 75 cycle kit, to generate approximately 5 million 75-bp single-end reads per sample. Raw reads were trimmed using Cutadapt v3.0 and aligned to Z.parabailii ATCC60483 (ASM198439v2) reference genome using Star v2.7.6a. Aligned reads were counted using HTSeq v0.12.4. The obtained gene counts were further analysed using the standard analysis protocol with DESeq2 1.30.1. PCA plots revealed a batch effect associated with one (R1) of the three biological-replicate experiments used to produce RNA samples. To account for this, R1 was added to the design formula ('R1 + condition').