Clonal heterogeneity test

Heterogeneity in colony morphologies (colony phenotype switch) and frequency of petite mitochondrial mutants in packed products were assessed by plating samples directly after the first pre-culturing (as described above) onto YPD agar plates (for colony morphologies) and onto GlyYP (glycerol yeast extract peptone) + 0.1% glucose agar plates with cell densities of approx. 200/plate (after cell counting in a haemocytometer). Plates were incubated for 10 days at 30°C (with agar surface facing down) and were visually scored for various phenotypes on YPD (rough, wrinkled, sectored, stalk-like, and very small colonies) and for potential petite mutants on GlyYP. Presumed petites were transferred to YPD and after overnight culturing, were inoculated onto GlyYP plates without glucose. Subclones unable to grow on glucose-free GlyYP were scored as petites. Finally, YPD colonies were washed under tap water to determine the frequency of invasivitiy into agar. At least 1,000 subclone colonies were counted for each strain and for each assay.Clonal heterogeneity ins tress tolerance was assessed by using the same stress conditions as in the spot plate assays, supplemented with assays on SD and YPD media. Freshly grown cells (as described for spot plates) were counted in a haemocytometer and spread to land about 200 cells/plate. Plates were incubated at 30°C for 2 days (YPD and SD media), or for 2, 3, 4, and 6 days (stress media) as colonies reached sizes that were visible but not yet close to each other. For each condition, three replicate plates were used for each sample. Photographs were taken 4 and 6 days after inoculation with a DSLR camera. Data on colony area was gathered by using the Fiji software package CountPHICS (Brzozowska et al., 2019) with circularity set to 0.8. Pixel to mm ratios were measured and area calculations were randomly verified by manual measurment in ImageJ for altogether ten colonies (Rueden et al., 2017). Plate photographs and all colony area values were uploaded to FigShare.