Changes in phenotypic heterogeneity as an adaptation of fungi to environmental stress

Phenotype may vary between genetically identical cells or organisms, despite exposure to an identical environment. This phenomenon is termed phenotypic heterogeneity. The extent of heterogeneity in a particular phenotype may be determined by stochastic or deterministic (including epigenetics) differences arising between cells which may produce cell-to-cell differences, for example in gene expression. The degree of phenotypic heterogeneity associated with a clonal cell population can be determined by its genotype, e.g. specific gene-promoter sequences producing nosier gene expression. A number of single-cell characteristics displaying phenotypic heterogeneity have been identified as fitness-determining. Therefore, heterogeneity is likely to have a bearing on population survival under temporally stable or unstable environmental characteristics (including stress). Previous evidence suggests that environmental stress can select for strains with increased levels of heterogeneity in resistance (heteroresistance) to the given stressors. In the present study, novel and existing methods for studying fungal cell and colony phenotype were developed and assessed, primarily using imaging and computational analysis. Methods for quantifying macroscopic yeast colony growth rate heterogeneity and intra-mycelial hyphal growth rate in filamentous fungi are described. The influence of yeast colony growth on agar versus planktonic growth in broth on copper heteroresistance was also investigated. This revealed that colony-derived cells were surprisingly less copper heteroresistant than planktonically-grown cells, but that some copper heteroresistance is maintained by SOD1 expression. Previous studies have indicated that increased phenotypic heterogeneity might be favoured in environments subject to fluctuations in environmental stressor exposure. However, neither the influence of environmental stability on selection for phenotypic heterogeneity, nor the long-term stability of this trait, are well understood. Therefore, yeast and filamentous fungal isolates from experimental field sites subject to different regimes of copper pollution were studied to provide insights. Metagenomic analysis revealed that copper polluted sites exhibited taxonomic and gene functional differences which were indicative of strong selection by copper, relative to control unpolluted sites. Dose-response gradients, amongst other measures of cell-cell variation, indicated that an elevated, stable level of copper stress may have selected for a decrease in heteroresistance. This was based on results comparing levels of heteroresistance in isolates of the yeast Saitozyma podzolica and the filamentous fungus Purpureocillium lilacinum from polluted versus control sites. This suggested that the basal level of copper heteroresistance (i.e. that occurring in the absence of selective pressures, such as environmental stress) may in fact be intermediate, with the potential to evolve in either direction depending on the stability and predictability of the stressor dose in the environment. Preliminary data from S. podzolica yeast evolved under stable copper stress supported the hypothesis that a stable stressor concentration may favour evolution of a less heteroresistant population.