Transformation of the oomycete pathogen, Phytophthora infestans.
A stable transformation procedure has been developed for Phytophthora infestans, an oomycete fungus that causes the late blight diseases of potato and tomato. This is the first description of reliable methods for transformation in an oomycete pathogen. Drug-resistant transformants were obtained by using vectors that contained bacterial genes for resistance to hygromycin B or G418 fused to promoters and terminators from the Hsp70 and Ham34 genes of the oomycete, Bremia lactucae. Using polyethylene glycol and CaCl2, vector DNA was introduced into protoplasts as a complex with cationic liposomes or with carrier DNA only. Transformants were obtained at similar frequencies with each combination of promoter and selectable marker and were confirmed by DNA and RNA hybridization and phosphotransferase assays. Transformation occurred through the integration of single or tandemly repeated copies of the plasmids into genomic DNA, conferring mitotically stable drug-resistant phenotypes. The sizes of the marker gene mRNAs in each transformant and the results of transcript mapping studies were consistent with the function of the B. lactucae regulatory sequences in P. infestans. A hygromycin-resistant transformant was tested and found to maintain pathogenicity, indicating that the gene transfer procedure will be useful for the molecular analysis of genes relevant to disease.
Judelson
H S
HS
Department of Vegetable Crops, University of California, Davis 95616.
Tyler
B M
BM
Michelmore
R W
RW
D001483:Base Sequence; D002934:Cinnamates; D004247:DNA; D004351:Drug Resistance / Q000235:genetics; D005798:Genes, Bacterial; D005819:Genetic Markers; D005839:Gentamicins / Q000494:pharmacology; D006921:Hygromycin B / Q000031:analogs & derivatives / Q000494:pharmacology; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009868:Oomycetes / Q000235:genetics; D010838:Phytophthora / Q000235:genetics*; D010944:Plants / Q000382:microbiology; D010957:Plasmids; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic; D014170:Transformation, Genetic
D002934:Cinnamates; D005819:Genetic Markers; D005839:Gentamicins; D006921:Hygromycin B; C026273:hygromycin A; D004247:DNA; C010680:antibiotic G 418