Development Of A Shuttle Expression Vector, PFC9, For Cryphonectria Parasitica

There have been only a few examples of shuttle plasmids for filamentous fungi. Here we report the construction of a shuttle expression vector, pFC9, for chestnut blight fungus, Cryphonectria parasitica, developed from a previously found plasmid, pGXH1130, which is able to autonomously replicate in C. parasitica. pFC9 was about 8.9 kbp in size, its multi-cloning sites flanked with gpd promoter and terminator from C. parasitica, and with hygromycin resistance as selectable marker. High level of expression of laccase was achieved in the transformants of hypovirulent C. parasitica strain, EP713 > which harbored the recombinant plasmid in that cDNA or genomic DNA of LAC-1, encoding laccase, was inserted into the multi-cloning sites of pFC9. Southern hybridization analysis indicated that pFC9 replicated in the cytoplasm of C. parasitica, same as its ancester plasmid, pGXH1130. The plasmid was capable of moving along the hypha through anastomosis similar to that of hypovirus, as marked by conferring hygromycin resistance to the sensitive recipient strain, EP155. To our knowledge, it was the first reported shuttle expression vector with the potential to become a robust tool to study the gene function in C. parasitica.