Performance of a tetracycline-responsive transactivator system for regulating transgenes in the oomycete Phytophthora infestans.
The oomycete genus Phytophthora includes many important plant pathogens for which extensive genome data exist, but lacking is an inducible expression system to study contributions of their genes to growth and pathogenicity. Here the adaptation of the reverse tetracycline transactivator (rtTA) system to P. infestans is described. Vectors were developed containing rtTA expressed from an oomycete promoter, and beta-glucuronidase (GUS) controlled by TetR binding sites fused to a minimal oomycete promoter. Transformants were obtained in which GUS was expressed in a dose-dependent manner by the rtTA inducer doxycycline, indicating that the gene switch functions in P. infestans. However, toxicity of rtTA hindered the isolation of transformants if expressed on the same plasmid as the nptII selection marker. Better results were obtained by cotransforming those genes on separate plasmids, with 92% of transformants acquiring both DNAs although only 4% expressed rtTA at detectable levels. Low levels of reporter activity were measured in such transformants, suggesting that rtTA activated transcription weakly. Also, significant variation in the sensitivity of isolates to doxycycline and tetracycline was observed. These results are useful both in terms of developing tools for functional genomics and understanding the fate of DNA during Phytophthora transformation.
Judelson
Howard S
HS
Department of Plant Pathology, University of California, Riverside, CA 92521, USA. howard.judelson@ucr.edu
Narayan
Reena
R
Fong
Audrey M V Ah
AM
Tani
Shuji
S
Kim
Kyoung Su
KS
D017930:Genes, Reporter; D005822:Genetic Vectors; D010838:Phytophthora / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger; D013752:Tetracycline / Q000378:metabolism*; D013754:Tetracyclines / Q000378:metabolism; D015533:Transcriptional Activation; D014170:Transformation, Genetic; D019076:Transgenes
D012333:RNA, Messenger; D013754:Tetracyclines; D013752:Tetracycline