Background

Light is, on the one hand, essential for life on the planet as a source of energy but, on the other hand, it can also be involved in regulatory processes, as well as cause substantial cellular damage and demise. Studies in later years have suggested that cells from the three domains of life, including most human cells, keep stimulus-independent rhythms of activity to adapt to the prevailing recurring alterations of sunlight and darkness (i.e., circadian rhythms). A key stimulus entraining circadian rhythms is visible light. Characterized clock mechanisms, including those in filamentous fungi, involve cryptochrome, rhodopsin, and melanopsin photoreceptors (Yu and Fischer, 2019; Patke et al., 2020). However, circadian rhythms persist in red blood cells in the absence of canonical clock components (O'Neill and Reddy, 2011; O'Neill et al., 2011), indicating that alternative clock mechanisms must exist. In red blood cells, the unusual anti-oxidants and signaling regulators peroxiredoxins were proposed to constitute conserved and transcription-independent regulators and markers of circadian rhythms (Edgar et al., 2012). On a similar note, baker’s yeast has long been thought to lack light receptors (Idnurm et al., 2010). Recently, however, it was discovered that yeast can sense light via a peroxisomal oxidase, hydrogen peroxide, and the peroxiredoxin Tsa1 (Bodvard et al., 2017). Thus, yeast is a model for understanding responses to light in cells and organisms lacking dedicated light receptors. In the yeast Saccharomyces cerevisiae, high-throughput genetic screening by ordered arrays of mutant libraries has become a central tool in understanding gene function on a genome-wide scale (Giaever et al., 2002).

Several filamentous fungi are able to sense light of different wavelengths through photoreceptors homologous to those in mammals and plants. In fungi, light controls circadian clocks and morphological responses, as well as growth/stress response decisions. Investigations of their responses to light have mainly focused on identifying photoreceptors, circadian clock components, and regulators of morphological responses. In these organisms, large-scale investigations of light responses have so far been limited to transcriptomics (Lewis et al., 2002; Dong et al., 2008; Chen et al., 2009; Wu et al., 2014), forward genetics screens using light-dependent reporters (Yu et al., 2016), and reverse genetic screens of limited gene sets (e.g., genes encoding transcription factors) affecting circadian clock reporters (Munoz-Guzman et al., 2021). Here, we outline a protocol for assessing baker’s yeast functions relevant in responses to visible light (400-700 nm) on a genome-wide scale, by scoring light-dependent growth of mutants. Potentially, by using suitable reporters (Kainth et al., 2009; D'Orazio et al., 2019) and with proper modifications to the setup, this protocol could be used to study other types of responses to light or, depending on the availability of genome-wide mutant collections, also understand light-resistance in other microorganisms.

The procedure we describe here, to score genes involved in the resistance of yeast to visible light, starts at a genome-wide scale, and subsequently narrows down to smaller scale validation tests for selected light-sensitive strains. In our case, the haploid BY4741 deletion collection was examined, in an arrayed format used in the Boone lab for synthetic genetic array analysis (Tong et al., 2001), in which all plate edges are lined by a row of wild-type-like (his3∆) control strains. We describe a detailed protocol for achieving the initial genome-wide screen, despite a strong dependence of yeast light-dependent growth on cell density that complicated analyses. Furthermore, to be able to reliably score light-sensitivity, we next describe appropriate methods for image analysis and growth scoring in detail. Following this, we describe a medium-throughput assay suitable for retesting a hundred or so strains, which should preferably be used on multiple deletion mutant collections, such as those containing the haploid (BY4741) and homozygous diploid (BY4743) gene knockouts. Finally, we outline a serial dilution drop test assay suitable for a smaller subset of genes, which in our case was used to retest gene deletions from the homozygous diploid BY4743 collection.