Fluorescence Assisted Selection of Transformants (FAST): Using flow cytometry to select fungal transformants

The availability of drug resistance markers for fungal transformation is often a limiting factor in both fungal genetics research and industrial applications. We describe a new technique using flow cytometry to select fungal transformants using well-known fluorescent proteins as markers for transformation. This new technique, Fluorescence-Assisted Selection of Transformants (FAST), was used for a transformation of Fusarium oxysporum with GFP as a marker targeted at a specific site on chromosome 14. The resulting strain was then transformed again with a gene replacement construct containing both RFP and a gene for drug resistance as markers. By directly comparing FAST with drug resistance selection we show that both methods yield comparable numbers of gene deletion mutants.

Schematic overview of Fluorescence Assisted Selection of Transformants. Fungal spores are co-incubated with A. tumefaciens cells carrying a binary vector encoding a fluorescent protein. Agrobacterium tumefaciens inserts the T-DNA into the fungal spores. Cells positive for the fluorescent protein are selected using flow cytometry. By measuring the signal from the fluorescent protein, successfully transformed spores can be identified and cultured for further analysis. GFP-A and FSC-A represent the area under the curve of the signal for a single cell in the specified detection channel. GFP-A measures the signal for GFP. FSC-A is the signal for the forward scatter, the light that passes straight through the spore; this is directly related to the size of the particle.Figure optionsDownload high-quality image (74 K)Download as PowerPoint slide