Simple and efficient recycling of fungal selectable marker genes with the Cre-loxP recombination system via anastomosis

Reverse-genetics analysis has played a significant role in advancing fungal biology, but is limited by the number of available selectable marker genes (SMGs). The Cre-loxP recombination system has been adapted for use in filamentous fungi to overcome this limitation. Expression of the Cre recombinase results in excision of an integrated SMG that is flanked by loxP sites, allowing a subsequent round of transformation with the same SMG. However, current protocols for regulated expression or presentation of Cre require multiple time-consuming steps. During efforts to disrupt four different RNA-dependent RNA polymerase genes in a single strain of the chestnut blight fungus Cryphonectria parasitica, we tested whether Cre could successfully excise loxP-flanked SMGs when provided in trans via anastomosis. Stable Cre-producing donor strains were constructed by transformation of wild-type C. parasitica strain EP155 with the Cre-coding domain under the control of a constitutive promoter. Excision of multiple loxP-flanked SMGs was efficiently achieved by simply pairing the Cre-donor strain and the loxP-flanked SMGs-transformed recipient strain and recovering mycelia from the margin of the recipient colony near the anastomosis zone. This method was shown to be as efficient as and much less time consuming than excision by transformation-mediated expression of Cre. It also allows unlimited recycling of loxP-flanked SMGs and the generation of disruption mutant strains that are free of any foreign gene. The successful application of this method to Metarhizium robertsii suggests potential use for optimizing reverse-genetics analysis in a broad range of filamentous fungi.