Penicillium marneffei (recently renamed Talaromyces marneffei) is well placed as a model experimental system to investigate processes controlling fungal growth and pathogenicity. An opportunistic pathogen of humans, P. marneffei is a dimorphic fungus that displays multicellular hyphal growth and asexual development (conidiation) at 25°C and unicellular fission yeast growth at 37°C and in host cells. We have adopted a forward genetic approach to discover novel genes contributing to the dimorphic switch and pathogenic potential of this fungus. While traditional chemical mutagenesis screens involve extensive genetic mapping and large-scale sequencing or cloning by complementation with whole-genome libraries, insertional mutagenesis systems facilitate isolation of the affected gene since the genetic lesion is tagged with the insertional marker. Transposon-based insertional mutagenesis systems take advantage of mobile genetic elements (DNA transposons) that are able to excise and re-insert in the genome in vivo.
We have developed a two-component transposon mutagenesis system for P. marneffei using the piggyBac transposon of Trichoplusia ni. The key features of this system include: the ability to control the activation of the transposon by induction/repression of transposase gene expression; the capacity to detect transposon movement by positive and negative selection; and the rapid identification of transposon insertion sites using splinkerette PCR/sequencing. Here we present the parameters affecting transposition frequency, the capacity to isolate mutants affected for growth in vitro and in vivo, and high-throughput approaches for identifying transposon insertion events including potential downstream applications.