Negative selection using thymidine kinase increases the efficiency of recovery of transformants with targeted genes in the filamentous fungus Leptosphaeria maculans.

A vector system was constructed that is designed to decrease the number of transformants required to be screened when looking for gene disruption events in filamentous fungi. This vector was used to mutate two genes, an ATP-binding cassette transporter ( LmABCt4) and a two-component histidine kinase gene ( LmHK1) in the ascomycete Leptosphaeria maculans. The system uses the thymidine kinase gene from the herpes simplex virus as a negative selectable marker. Thymidine kinase expression is regulated by the TrpC regulatory elements from Aspergillus nidulans and should be applicable to other ascomycetous fungi. When thymidine kinase is expressed in the presence of particular thymidine analogues, these analogues are converted to toxic compounds which kill the cell. We also report the transformation of L. maculans using Agrobacterium tumefaciens-mediated DNA delivery.