Stable transformation of the oomycete, Phytophthora infestans, using microprojectile bombardment

Abstract

Germinated asexual sporangia, zoospores, and mycelia of Phytophthora infestans were transformed to G418-resistance by microprojectile bombardment. After optimization, an average of 14 transformants/shot were obtained, using 10(6) germinated sporangia and gold particles coated with 1 microg of vector. Transformants displayed tandem or simple insertions of vector sequences within chromosomes. Most primary transformants were heterokaryons of transformed and wild-type nuclei, a state which generally persisted for generations, even with G418 selection. Transgenic homokaryons were easily obtained from primary transformants through G418 selection of zoospores. To facilitate the optimization of transformation, experiments were performed using a vector containing neomycin phosphotransferase (npt) and beta-glucuronidase (GUS) genes fused to oomycete transcriptional regulatory sequences. To indicate which orientations of transgenes would maximize their expression, head-to-head, head-to-tail, or tail-to-tail orientations of npt and GUS were compared. Each yielded similar rates of transformation and levels of GUS activity, indicating little transcriptional interference.