Intermolecular ligation mediates efficient cotransformation in Phytophthora infestans.
The processing of DNA molecules during transformation was characterized in the oomycete Phytophthora infestans. Linear and circular forms of non-replicating transformation vectors supported similar rates of stable transformation. Remarkably, digestion of plasmids within the selectable marker genes neomycin phosphotransferase (npt) or hygromycin phosphotransferase (hpt) had little effect on the recovery of drug-resistant transformants, and the cleaved sites were shown to be reconstituted in the transformants. An assay for the transient expression of beta-glucuronidase (GUS) in protoplasts treated with partial or disrupted GUS genes demonstrated that active genes could be reconstituted through intramolecular and/or intermolecular ligation between compatible ends, while incompatible ends were inefficiently joined. Stable transformation studies also demonstrated that complementing portions of incomplete npt or hpt genes joined through homologous recombination. Based on the indication of efficient ligation between DNA molecules during transformation, an efficient procedure for cotransformation was developed. The frequency of cotransformation between vectors expressing selected genes (npt or hpt) and nonselected sequences (GUS, beta-galactosidase, or streptomycin phosphotransferase) approached unity when the plasmids were linearized with the same restriction enzyme before transformation. In contrast, cotransformation between circular plasmids or those cut with different enzymes occurred infrequently (10%). Hybridization analysis of DNA from cotransformants demonstrated that linearized plasmids became colocalized within genomic DNA, while circular plasmids typically inserted at unliked sites.
Judelson
H S
HS
Center for Engineering Plants for Resistance Against Pathogens (CEPRAP), University of California, Davis 95616.
D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism*; D010838:Phytophthora / Q000235:genetics*; D010957:Plasmids; D011995:Recombination, Genetic; D015183:Restriction Mapping; D014170:Transformation, Genetic
D004271:DNA, Fungal