K2po4

C. reinhardtii wild type strain T222+ (CC-5101) [39 (link)] was obtained from the Chlamydomonas Resource Center and used as recipient strain for all genetic modifications described in this work. Growth experiments were conducted in a laboratory-scale photobioreactor (Multi-Cultivator MC 100-OD, Photon System Instruments, Drasov, Czech Republic) in mixotrophic conditions using an acetate-supplemented tris-acetate-phosphate rich medium (TAP) [40 (link)] under the following controlled conditions: continuous light at 200 μmol photons m⁻² s⁻¹ irradiance, 22 °C and constant air bubbling. For general purposes, algae were propagated on TAP-agar (1,5 % w/v) plates containing appropriate antibiotics. Selective growth experiments in phosphite were conducted by substituting the phosphorous source sodium phosphate (K₂PO₄) with sodium phosphite (Na₂HPO₃ 5H₂O_, Sigma Aldrich), while maintaining the same ion concentration. Phosphite-containing rich and minimal media are hereinafter referred to as TA-Phi and HS-Phi, respectively. A precultivation step in TAP medium preceded the selective growth experiments until a density of 10⁶ cells/mL was reached. Cultures were subsequently pelleted and re-suspended and cultivated in TA medium (phosphorous devoid) for 4 days to exhaust intracellular phosphate stores (phosphate depletion) before being exchanged to TA-Phi medium.

The expression of the SAP4 gene was evaluated using real-time PCR after treatment with silymarin. The cells of C. albicans ATCC 10231 were exposed to silymarin at the concentration of 15 µg/mL (MIC/2) or 1% DMSO (control) during propagation in liquid culture in Spider medium (1% nutrient broth, 1% mannitol, 0.2% K₂PO₄ (Sigma-Aldrich), pH 7.2) with 10% fetal bovine serum (FBS) (Sigma-Aldrich). After 20-h incubation at 37 °C, the yeasts were collected and total RNA was extracted with the YeaStar RNA kit (Zymo Research, USA), according to the manufacturer’s instructions. cDNA was then synthesized using a Smart First Strand cDNA Synthesis Kit (EurX, Poland) following the manufacturer’s instructions. For PCR detection of transcripts, we used TaqMan gene expression assays (Lot: 170255, designed by manufacturer, Thermo Fisher Scientific, England) and the Probe qPCR Master Mix (EurX, Poland). The cDNA samples were pre-treated at 50 °C for 2 min with uracil-N-glycosylase to degrade any dUMP-containing PCR products and then subjected to initial denaturation at 95 °C for 10 min., followed by 40 amplification cycles with denaturation at 94 °C for 15 s, annealing at 60 °C for 30 s, and extension at 72 °C for 30 s using RotorGene-6000 (Corbett). The relative level of expression of the tested gene was calculated with the 2^-(ΔΔCt) method using ACT1 as a reference gene [38 (link)].