Mcleod Adéle, Fry Barbara A, Zuluaga A Paola, Myers Kevin L, Fry William E
Department of Plant Pathology, Cornell University, Ithaca, NY 14853, USA.
J Eukaryot Microbiol. 2008 Mar-Apr;55(2):103-9. doi: 10.1111/j.1550-7408.2008.00304.x.
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.
全球一些最重要的植物病原体是卵菌，每年花费数十亿美元来抑制它们所引发的病害。更高效的病害抑制技术将源于对这些生物体基础生物学的更好理解，但低效的转化目前限制了基础分子研究。在各种方法中，使用聚乙二醇/氯化钙对原生质体进行转化仍然最为成功，但稳定转化的频率仍然很低且不稳定。在此我们报告，对先前用于拟南芥叶肉细胞的方案进行修改后，成功从四种不同的卵菌（柑橘疫霉、致病疫霉、大豆疫霉和瓜果腐霉）中释放出了原生质体。所有卵菌的原生质体都能够摄取DNA并再生，其中瓜果腐霉的原生质体释放和再生效率最高。除了良好的原生质体生产系统外，更有效的转化载体可能会提高稳定转化率。我们构建并评估了17种新型候选转化载体，以检测它们在致病疫霉和瓜果腐霉中驱动β-葡萄糖醛酸酶（GUS）报告基因瞬时表达的能力。其中5种新构建的载体也在大豆疫霉和柑橘疫霉中进行了评估，并且表现出与在致病疫霉和瓜果腐霉中相似的转录活性模式。新构建的载体之一pDBHAMT35G含有嵌合启动子，在致病疫霉和柑橘疫霉中支持最高的GUS表达，可能对未来的研究有用。
