Article ID Journal Published Year Pages File Type
5522151 Journal of Microbiological Methods 2017 6 Pages PDF
Abstract

•Optimizing ATMT for genetic manipulation of Hypholoma fasciculare and H. sublateritium.•Validation of the H. sublateritium gpd promoter for expression of transgenes.•Successful GFP expression in Hypholoma and Clitopilus passeckerianus and required introns in both cases.

The basidiomycete fungi Hypholoma fasciculare and H. sublateritium are both prolific producers of sesquiterpenes and triterpenes, some of which have relevant pharmaceutical properties. Although H. sublateritium has been transformed in the past, the low reported efficiencies highlighted the need for establishing an effective simple transformation system for these valuable species. We have optimized Agrobacterium tumefaciens-mediated transformation through testing various parameters in these two Hypholoma species, showing that a mixture of homogenized mycelia and Agrobacterium (strain LBA4404) co-cultivated for 84 h at 25 °C is optimal for efficient transformation in these basidiomycetes. This study also reveals the requirements for transgene expression, with the first report of GFP expression in these Hypholoma, the need for an intron for such transgene expression, and further demonstrates the functionality of the expression vector by its use in Clitopilus passeckerianus. This development of transformation system and expression constructs, can facilitate further genetic investigation such as gene functionality in these fungi.

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