title: Development of a bipartite ecdysone-responsive gene switch for the oomycete Phytophthora infestans and its use to manipulate transcription during axenic culture and plant infection.
abstract: Conditional expression systems have been proven to be useful tools for the elucidation of gene function in many taxa. Here, we report the development of the first useful inducible promoter system for an oomycete, based on an ecdysone receptor (EcR) and the ecdysone analogue methoxyfenozide. In Phytophthora infestans, the potato late blight pathogen, a monopartite transactivator containing the VP16 activation domain from herpes simplex virus, the GAL4 DNA-binding domain from yeast and the EcR receptor domain from the spruce budworm enabled high levels of expression of a β-glucuronidase (GUS) reporter gene, but unacceptable basal activity in the absence of the methoxyfenozide inducer. Greatly improved performance was obtained using a bipartite system in which transcription is activated by a heterodimer between a chimera of VP16 and the migratory locust retinoid X receptor, and a separate EcR-DNA-binding domain chimera. Transformants were obtained that exhibited >100-fold activation of the reporter by methoxyfenozide, with low basal levels of expression and induced activity approaching that of the strong ham34 promoter. Performance varied between transformants, probably as a result of position effects. The addition of methoxyfenozide enabled strong induction during hyphal growth, zoosporogenesis and colonization of tomato. No significant effects of the inducer or transactivators on growth, development or pathogenicity were observed. The technology should therefore be a useful addition to the arsenal of methods for the study of oomycete plant pathogens.
authors.affiliation: Department of Plant Pathology and Microbiology, University of California, Riverside, CA, 92521, USA.
authors.forename: Heber
authors.initials: H
authors.lastname: Gamboa-Meléndez
authors.forename: Howard S
authors.initials: HS
authors.lastname: Judelson
mesh_terms: D061245:Axenic Culture; D004440:Ecdysone / Q000494:pharmacology*; D017930:Genes, Reporter; D005812:Genes, Switch; D006834:Hydrazines / Q000494:pharmacology; D007605:Juvenile Hormones / Q000494:pharmacology; D018551:Solanum lycopersicum / Q000187:drug effects / Q000382:microbiology*; D008969:Molecular Sequence Data; D055750:Phytophthora infestans / Q000187:drug effects / Q000235:genetics* / Q000472:pathogenicity*; D010957:Plasmids / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects*; D014170:Transformation, Genetic; D020798:Two-Hybrid System Techniques
chemical_list: D006834:Hydrazines; D007605:Juvenile Hormones; D004440:Ecdysone; C406712:methoxyfenozide
keywords: biotechnology; functional genomics; inducible promoter; late blight; transformation