Measuring spatially-dependent gene expression across fungal mycelia by single molecule FISH

Here we describe the use of single-molecule fluorescence in situ hybridization (smFISH) to measure the change in gene expression across fungal mycelia. Fungi can interact with their environment at very fine length scales (microns) resulting in responses varying in intensity across hyphae. For example, by the biosynthetic gene cluster that produces the fungal pigment bikaverin has distinct spatial production in strains of Monosporascus when confronted with a second seemingly antagonistic strain of Monosporascus. When co-cultured, the deep-red phenotype is observed at the interfaces and mycelial boundaries closest to the fungal cultures. This is also accompanied by an exclusion zone that bars growth from the opposing fungus. To better understand the mechanism and triggers for bikaverin production, we are utilizing smFISH of key genes in the biosynthetic gene cluster to measure the transcriptional dynamics across the fungi. Our results agree with the red phenotype observed and indicate high expression of bikaverin producing genes closest to the interface between fungi.