Efficient and versatile transformation systems in entomopathogenic fungus Lecanicillium species

Abstract

Lack of genetic tools in entomopathogenic fungi, especially those for targeted homologous recombination, hindered the advance in this field. To facilitate the genetic study, we constructed a transformation system in entomopathogenic fungus Lecanicillium sp. strain HF627 using the uridine auxotrophic pyrG mutant strain as host and endogenous pyrG as marker. pUC19 harboring endogenous pyrG successfully restored the uridine auxotrophy of the host strain, and the integration of the vector DNA was confirmed by Southern hybridization. An autonomously replicating vector harboring an AMA1 sequence was constructed and applied to the constructed transformation system, which improved the transformation efficiency 16.7-fold. Southern hybridization revealed replication of the AMA1-harboring vector with an average copy number of 2.4. A ku80 knock-out strain was created to improve the efficiency of gene targeting. Deletion of the pyrG locus, which is homologous to the marker gene, from the ku80 knock-out strain achieved a targeting efficiency of 62.5 % against both trp1 and his3; the levels of these genes were 3.2- and 5-fold higher, respectively, than the ku80-intact strain. With the pyrG-deleted and ku80-inactivated strain constructed in this study, transformation and targeted homologous recombination were highly enhanced, by which genetic analysis in Lecanicillium spp. will be performed quickly and efficiently.