A simple and efficient method for successful gene silencing of HspA1 in Trametes hirsuta AH28-2

Trametes sp. are among the most extensively studied basidiomycetes due to their importance in biotechnology. However, they are difficult to genetically modify. For instance, the low incidence of homologous integration hampers gene inactivation. To overcome this, we here constructed two post-transcriptional gene-silencing vectors that contain an antisense and a RNA interference mediating hairpin sequence, respectively. These vectors were used to knock down transcription of the heat shock protein 70 family gene HspA1. The two vectors were transformed into Trametes hirsuta AH28-2 by using a PEG/CaCl2 mediated transformation method. Based on Southern blot analysis, target sequences were integrated into the genome as multi-copies. Transcription analysis revealed that the antisense and hairpin sequences reduced the transcript number of HspA1 when compared to the wild type strain. Moreover, the antisense sequence resulted in a higher gene silencing efficiency when compared to the RNA interference vector. Together, antisense methodology provides a simple method for gene silencing in Trametes sp.