Genetic transformation, infection process and qPCR quantification of Verticillium dahliae on smoke-tree Cotinus coggygria
Genetic transformation, infection process and qPCR quantification of Verticillium dahliae on smoke-tree Cotinus coggygria
复制标题
烟树黄栌上大丽黄萎病菌的遗传转化、侵染过程及qPCR定量
DOI：
10.1007/s13313-012-0172-0
复制
发表时间：
2013-01-01
影响因子：
1.4
通讯作者：
Tian, Chengming
中科院分区：
文献类型：
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作者：
Wang, Yonglin;Xiao, Shuxiao;Tian, Chengming
利用peg介导的原生质体成功转化了烟树血管性枯萎菌Verticillium dahliae，并成功表达了eGFP和hygromycin B抗性基因。在选择性和非选择性培养基上交替继代培养6个月后，转化体稳定。转化菌株在生长、形态特征、无性繁殖和致病性方面与野生型菌株无明显差异。荧光观察清楚地显示绿色荧光蛋白在真菌结构中的强表达。利用荧光技术监测了egfp标记的大丽花弧菌在烟树根部的定植和渗透，并清晰地显示了早期侵染过程。接种24小时后，菌丝主要在纤维上而不是在主根上定植，随后在36小时时，菌丝开始沿表皮细胞连接处平行于根的纵轴生长。在48hpi时，细长的菌丝散布在根毛中，在5dpi时首次观察到中心柱定植。在茎6 dpi、枝12 dpi和叶14 dpi中检测到真菌DNA的升高。结果表明，一旦大丽花病菌进入烟树，病原菌在植物中迅速传播和增加，绿色荧光蛋白可作为研究大丽花病菌与木本植物相互作用的重要稳定标记。此外，peg介导的转化体系的建立将有助于烟树黄萎病真菌的功能基因组学研究。
The smoke-tree vascular wilt fungus, Verticillium dahliae, was successfully transformed via PEG-mediated protoplasts using cassettes for eGFP and hygromycin B resistance (hph) gene expression. The transformants were stable after alternative sub-culturing on selective and non-selective medium for 6 months. The transformed isolates did not differ markedly from wild-type strains in terms of growth, morphological characteristics, asexual conidiation and pathogenicity. Fluorescence observations clearly indicated strong expression of the green fluorescent protein in fungal structures. The colonization and penetration of smoke-tree roots by eGFP-tagged V. dahliae was monitored by fluorescence, and the early infection processes were clearly visualized. At 24-hour-post-inoculation (hpi), hyphae were found to be predominantly colonizing the fibers rather than the taproots, and subsequently, hyphae started to grow parallel to the longitudinal axis of the root along the junctions of epidermal cells at 36 hpi. At 48 hpi, the elongated hyphae were interspersed among the root hairs and the first observation of central cylinder colonization occurred at 5 dpi. Elevated amounts of fungal DNA were detectable in stems 6 dpi, in branches 12 dpi, and in leaves 14 dpi. The data indicated that once V. dahliae enters smoke-tree, the pathogen rapidly spreads and increases in planta, and GFP can be used as a vital and stable marker for studying the interaction between V. dahliae and woody plants. Furthermore, the establishment of a PEG-mediated transformation system will contribute to functional genomics studies of smoke-tree Verticillium wilt fungus.