Laboratory of Phytopathology, Wageningen University, and Graduate School Experimental Plant Sciences, Binnenhaven 5, NL-6709 PD Wageningen, the Netherlands.
Mol Plant Pathol. 2003 Nov 1;4(6):459-67. doi: 10.1046/j.1364-3703.2003.00191.x.
SUMMARY Agrobacterium tumefaciens is widely used for plant DNA transformation and, more recently, has also been used to transform yeast and filamentous fungi. Here we present a protocol for Agrobacterium-mediated DNA transformation of the oomycete Phytophthora infestans, the causal agent of potato late blight. Binary T-DNA vectors containing neomycin phosphotransferase (npt) and beta-glucuronidase (gus) fused to oomycete transcriptional regulatory sequences were constructed. Seven days of co-cultivation followed by transfer to a selective medium containing cefotaxim to kill Agrobacterium and geneticin to select for transformants, resulted in geneticin resistant colonies. Under optimal conditions with Agrobacterium supplemented with a ternary plasmid carrying a constitutive virG gene and in the presence of acetosyringone as inducer, up to 30 transformants per 10(7) zoospores could be obtained. The majority of these transformants contained a single T-DNA copy randomly integrated at a chromosomal locus. Using a similar protocol, geneticin resistant transformants of two other oomycetes species were obtained, Phytophthora palmivora and Pythium ultimum.
摘要 根癌农杆菌被广泛应用于植物 DNA 转化，最近也被用于酵母和丝状真菌的转化。本研究提供了一种利用根癌农杆菌介导的卵菌 Phytophthora infestans（马铃薯晚疫病菌）的 DNA 转化方法。构建了含有新霉素磷酸转移酶（npt）和β-葡萄糖醛酸酶（gus）融合到卵菌转录调控序列的二元 T-DNA 载体。经过 7 天的共培养，然后转移到含有头孢噻肟杀死根癌农杆菌和遗传霉素选择转化子的选择性培养基上，得到了遗传霉素抗性菌落。在最佳条件下，用携带组成型 virG 基因的三元质粒补充根癌农杆菌，并以乙酰丁香酮作为诱导物，每 10^7 个游动孢子可获得多达 30 个转化子。这些转化子中的大多数含有一个随机整合在染色体位点上的单个 T-DNA 拷贝。使用类似的方案，还获得了两种其他卵菌物种的遗传霉素抗性转化子，即棕榈疫霉和腐霉。
