Skip to main content
Advertisement

< Back to Article

Approaching the Functional Annotation of Fungal Virulence Factors Using Cross-Species Genetic Interaction Profiling

Figure 1

Cross-species genetic interaction mapping to predict the function of C. neoformans genes necessary for murine infection.

A) Cross-species genetic interaction method. We created S. cerevisiae strains expressing each C. neoformans gene of unknown function at the URA3 locus under the control of the S. cerevisiae GPD1 promoter [77] and linked to nourseothricin (NAT) resistance. We used the synthetic genetic analysis (SGA) strain background, which allows for selection of the MATa mating type and, ultimately, the haploid cellular state [16], [17]. We crossed this strain to the S. cerevisiae deletion library of targeted gene deletions marked by kanamycin (G418) resistance [8]. We selected for diploids on YPAD+NAT+G418, then sporulated diploids on sporulation medium, selected for MATa haploids (+thialysine+canavanine), and then selected for the C. neoformans gene expression construct and the knockout mutations (+NAT+G418+thialysine+canavanine). See Methods and references 10–11 for description of the SGA method. B) Analysis of cross-species genetic interaction data. We scanned plates with colonies containing both the C. neoformans expression construct and the S. cerevisiae knockout mutations with a flatbed scanner. We extracted colony size information using ScreenMill [76], then normalized colony size data using the S-score method [20]. We generated an S-score for each double mutant strain (C.n. expression construct combined with knockout mutant), then computed the Pearson correlation between each such profile and genome-wide profiles available for S. cerevisiae gene deletions [19]. We converted the correlations to Z-scores and filtered out hits for which the Z-score of either the vector or the GFP control was ≥1.96 (p = 0.05). We also filtered out hits whose ratios of C. neoformans gene correlation score/control correlation score (either vector or GFP) was between 0.95 and 1.05 (∼60 profiles).

Figure 1

doi: https://doi.org/10.1371/journal.pgen.1003168.g001