STABLE, SELECTABLE, INTEGRATIVE DNA TRANSFORMATION IN PHYSARUM

The Physarum polycephalum actin promoter, PardC, can drive transient expression of heterologous genes in Physarum amoebae. The hph gene, encoding hygromycin (Hy) phosphotransferase, can confer resistance to Hy on a broad spectrum of organisms. When PardC is translationally fused to hph and transformed into yeasts on high-copy-number vectors, the yeasts become Hy resistant (Hy(R)), showing that PardC-hph is a functional, selectable genetic element. To establish a stable transformation system for Physarum, we electroporated plasmids bearing PardC-hph into Physarum amoebae and then selected for Hy(R) transformants. We show that Hy(R) amoebae arise upon the stable integration of PardC-hph into the nuclear genome in single copy. These results establish a. transformation system that can be used to add plasmid-borne genetic information to Physarum.