Toward improvements of oomycete transformation protocols

Some of the most important plant pathogens worldwide are oomycetes, and billions of dollars are expended annually to suppress diseases they cause. More efficient disease suppression technologies will be derived from a better understanding of the basic biology of these organisms, but inefficient transformation currently limits basic molecular investigations. Of the various approaches, transformation of protoplasts using polyethylene glycol/calcium chloride remains most successful, but the frequency of stable transformation remains low and inconsistent. Here we report that modifications of a protocol, previously used for Arabidopsis mesophyll cells, successfully releases protoplasts from four different oomycetes (Phytophthora citricola, Phytophthora infestans, Phytophthora sojae, and Pythium aphanidermatum). The protoplasts of all oomycetes were able to take up DNA and regenerate, with protoplast release as well as regeneration being most efficient in P. aphanidermatum. In addition to a good protoplast production system, more effective transformation vectors may improve stable transformation rates. We constructed, and evaluated 17 novel candidate transformation vectors for their ability to drive transient expression of the beta-glucuronidase (GUS) reporter gene in P. infestans and P. aphanidermatum. Five of the newly constructed vectors were also evaluated in P. sojae and P. citricola, and exhibited a similar pattern of transcriptional activity as in P. infestans and P. aphanidermatum. One of the newly constructed vectors, pDBHAMT35G, containing a chimeric promoter, supported the highest GUS expression in P. infestans and P. citricola, and could potentially be useful for future studies.