Li Hong-Yu, Pan Chu-Yi, Chen Han, Zhao Chang-Jiang, Lu Guo-Dong, Wang Zong-Hua
School of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China.
Sheng Wu Gong Cheng Xue Bao. 2003 Jul;19(4):419-23.
The rice blast fungus Magnaporthe grisea causes one of the most destructive diseases of rice around the world. Significant progresses have been made recently in genomics studies of the fungus, opening new era of the functional genomics which requires to generate a large scale of gene knockout mutants. It has been demonstrated that T-DNA insertional mutagenesis is a powerful tool of functional genomics not only for plants but also for fungi. In this paper, we optimized the conditions for T-DNA insertional mutagenesis of M. grisea using Agrobacterium tumefaciens-mediated transformation (ATMT) approach. We employed the binary vector pBHtl constructed by Dr. S. Kang's laboratory at the Pennsylvania State University, which carries the bacterial hygromycin B phosphotransferase gene (hph) under the control of the Aspergillus nidulans trpC promoter as a selectable marker to transform the conidia of M. grisea. We optimized the conditions for T-DNA insertional mutagenesis including the medium, dosage of hygromycin B, cefotaxime and carbenicillin to select the transformants and inhibit the growth of A. tumefaciens after co-culturing. The dosage to inhibit non-transformants could vary from 200-600microg/mL among different M. grisea isolates so that the optimal dosage of the antibiotics should be decided according to isolates. Rice polished agar medium was found the best selection medium which would facilitate the mutant sporulation and minimize the contamination chance. In average, about 500 transformants could be obtained when transforming 1 x 10(6) spores at the optimum condition, among which 85% had T-DNA insertion detected by polymerase chain reaction (PCR) and thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). Fifteen out of 1520 transformants showed mutation in colony morphology. Within 58 randomly selected mutants, it was found that there were 4 sporulation-decreased mutants, 8 less germination mutants and 9 appressorium defective mutants. Several virulent mutants to C101LAC(Pi-1)and 75-1-127(Pi-9)were also obtained which would facilitate cloning the corresponding avirulence genes.
稻瘟病菌Magnaporthe grisea引发了全球范围内对水稻最具毁灭性的病害之一。近期该真菌的基因组学研究取得了重大进展，开启了功能基因组学的新时代，这需要构建大规模的基因敲除突变体。业已证明，T-DNA插入诱变不仅是植物功能基因组学的有力工具，对真菌同样如此。在本文中，我们利用根癌农杆菌介导转化（ATMT）方法优化了稻瘟病菌T-DNA插入诱变的条件。我们采用了宾夕法尼亚州立大学S. Kang博士实验室构建的二元载体pBHtl，该载体携带在构巢曲霉trpC启动子控制下的细菌潮霉素B磷酸转移酶基因（hph）作为选择标记，用于转化稻瘟病菌的分生孢子。我们优化了T-DNA插入诱变的条件，包括培养基、潮霉素B、头孢噻肟和羧苄青霉素的用量，以筛选转化体并在共培养后抑制根癌农杆菌的生长。抑制非转化体生长的用量在不同的稻瘟病菌分离株中可能在200 - 600μg/mL之间变化，因此应根据分离株确定抗生素的最佳用量。发现水稻抛光琼脂培养基是最佳选择培养基，它有利于突变体产孢并减少污染几率。在最佳条件下转化1×10⁶个孢子时，平均可获得约500个转化体，其中85%通过聚合酶链反应（PCR）和热不对称交错聚合酶链反应（TAIL-PCR）检测到有T-DNA插入。1520个转化体中有15个在菌落形态上表现出突变。在随机选择的58个突变体中，发现有4个产孢减少的突变体、8个萌发率降低的突变体和9个附着胞缺陷的突变体。还获得了几个对C101LAC（Pi-1）和75-1-127（Pi-9）具有毒性的突变体，这将有助于克隆相应的无毒基因。
