Construction Of An Autonomously Replicating Shuttle Plasmid For Transformation Of Chestnut Blight Fungus, Cryphonectria Parasitica

A 2.1kb fragment, containing a TrpC promoter, hygromycin resistance gene and a TrpC terminator, from the non-replicative plasmid vector pCPXHY2 for the chestnut blight fungus, C.parctsitica, was cloned into a plant-transforming vector pCambia130-35SN to form a new construct pGXH1130. The new plasmid was used to transform C.parasitica strain EP713 and was found to have much higher transformation efficiency than the parental plasmid pCPXHY2(6-fold increased), reaching 60-70 transformants/g plasmid DNA. However, no phenotypic mutation was found in a total of 5868 randomly selected hygromycin-resistant transformants conferred by pGXH1130. Southern blot analysis of the transformants DNA showed that pGXH1130 did not integrate into the chromosome of the host C. parasitica, but rather, existed as an autonomously replicating plasmid in the fungus. The replicating plasmid in the fungus could be rescued by using the total DNA or plasmid fraction from the transformants to transform an E.coli strain. It was further found by RFLP that the rescued plasmid was indistinguishable from the introduced pGXH1130, indicating no modification had occurred in the fungal host. The stability test of drug resistance showed that 90% of transformts lost the marker after 6 generations of passages on PDA medium without hygromycin selection pressure. It is the first report that an efficient self-replicating plasmid was developed for C. parasitica and it has the potential to become a powerful tool in C. parasitica genetic manipulation.